Control of transcription presides over a vast array of biological processes, including those mediated by gene regulatory circuits that exhibit multistability. Within these circuits, two- and three-gene network motifs are particularly critical to the repertoire of metabolic and developmental pathways. Theoretical models of these circuits, however, often vary parameters such as dissociation constants, transcription rates, and degradation rates without specifying precisely how these parameters are controlled biologically. In this study, we examine the role of effector molecules, which can alter the concentrations of the active transcription factors that control regulation, and are ubiquitous in regulatory processes across many biological settings. We specifically consider allosteric regulation in the context of extending the standard bistable switch to three-gene networks, and explore the rich multistable dynamics exhibited in these architectures as a function of effector concentrations. We then analyze how the dynamics evolve under various interpretations of regulatory circuit mechanics, underlying inducer activity, and perturbations thereof. Notably, the biological mechanism by which we model effector control over dual-function proteins transforms not only the phenotypic trend of dynamic tuning but also the set of available dynamic regimes. In this way, we determine key parameters and regulatory features that drive phenotypic decisions, and offer an experimentally tunable structure for encoding inducible multistable behavior arising from both single- and dual-function allosteric transcription factors.
", "doi": "10.1103/vkqj-nszx", "issn": "2470-0045", "publisher": "American Physical Society", "publication": "Physical Review E", "publication_date": "2026-06-17", "series_number": "6", "volume": "113", "issue": "6", "pages": "064408" }, { "id": "authors:vvsjx-rpm50", "collection": "authors", "collection_id": "vvsjx-rpm50", "cite_using_url": "https://authors.library.caltech.edu/records/vvsjx-rpm50", "type": "article", "title": "Biological Processes as Exploratory Dynamics", "author": [ { "family_name": "Kondev", "given_name": "Jane", "orcid": "0000-0001-7522-7144" }, { "family_name": "Kirschner", "given_name": "Marc", "orcid": "0000-0001-6540-6130" }, { "family_name": "Garcia", "given_name": "Hernan G.", "orcid": "0000-0002-5212-3649" }, { "family_name": "Salmon", "given_name": "Gabriel L.", "orcid": "0000-0003-2163-8399", "clpid": "Salmon-Gabriel-Luke" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Many biological processes can be thought of as the result of an underlying dynamics in which the system repeatedly undergoes distinct and abortive trajectories with the dynamical process only ending when some specific process, purpose, structure or function is achieved. A classic example is the way in which microtubules attach to kinetochores as a prerequisite for chromosome segregation and cell division. In this example, the dynamics is characterized by apparently futile time histories in which microtubules repeatedly grow and shrink without chromosomal attachment. We hypothesize that for biological processes for which it is not the initial conditions that matter, but rather the final state, this kind of exploratory dynamics is biology's unique and necessary solution to achieving these functions with high fidelity. This kind of cause and effect relationship can be contrasted to examples from physics and chemistry where the initial conditions determine the outcome. In this paper, we examine the similarities of many biological processes that depend upon random trajectories starting from the initial state and the selection of subsets of these trajectories to achieve some desired functional final state. We begin by reviewing the long history of the principles of dynamics, first in the context of physics, and then in the context of the study of life. These ideas are then stacked up against the broad categories of biological phenomenology that exhibit exploratory dynamics. We then build on earlier work by making a quantitative examination of a succession of increasingly sophisticated models for exploratory dynamics, all of which share the common feature of being a series of repeated trials that ultimately end in a \"winning\" trajectory. We also explore the ways in which microscopic parameters can be tuned to alter exploratory dynamics as well as the energetic burden of performing such processes. It is a great privilege to take part in this special volume dedicated to the life and work of Prof. Erich Sackmann (1934-2024). For one of us (RP), at the time of making a switch from traditional condensed matter physics to a life engaged in the study of life, he went to a meeting near Munich which completely opened his eyes to the ways in which the approach of physics could be brought to bear on the study of the living. Sackmann's work was an inspiring presence at that meeting. One of the hallmarks of his work was a principled approach to dissecting biological processes over a range of scales and phenomena. One common thread to much of his work was that it acknowledged the dynamical character of living organisms. The present paper attempts to follow in the tradition of Sackmann's studies of dynamics by suggesting a new way of looking at many biological processes all through the unifying perspective of what we will call exploratory dynamics.", "doi": "10.1016/j.bpj.2025.09.009", "issn": "0006-3495", "publisher": "Cell Press", "publication": "Biophysical Journal", "publication_date": "2026-06-16", "series_number": "12", "volume": "125", "issue": "12", "pages": "2812-2837" }, { "id": "authors:tez64-75e51", "collection": "authors", "collection_id": "tez64-75e51", "cite_using_url": "https://authors.library.caltech.edu/records/tez64-75e51", "type": "article", "title": "Human biomass movement exceeds the biomass movement of all land animals combined", "author": [ { "family_name": "Rosenberg", "given_name": "Yuval", "orcid": "0000-0002-6681-8329", "clpid": "Rosenberg-Yuval" }, { "family_name": "Wiedenhofer", "given_name": "Dominik", "orcid": "0000-0001-7418-3477", "clpid": "Wiedenhofer-Dominik" }, { "family_name": "Vir\u00e1g", "given_name": "Doris", "clpid": "Vir\u00e1g-Doris" }, { "family_name": "Bar-Sella", "given_name": "Gabriel", "clpid": "Bar-Sella-Gabriel" }, { "family_name": "Greenspoon", "given_name": "Lior", "orcid": "0000-0001-8385-4033", "clpid": "Greenspoon-Lior" }, { "family_name": "Herrnstadt", "given_name": "Barr", "clpid": "Herrnstadt-Barr" }, { "family_name": "Akenji", "given_name": "Lewis", "orcid": "0000-0003-4422-8819", "clpid": "Akenji-Lewis" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" } ], "abstract": "Earth is teeming with life on the move, shaping ecosystems and human civilizations alike. However, the magnitude of movement by humans and other animals has yet to be assessed holistically. Here we quantify the movement of biomass across all animal life and in comparison to humanity. We show that the combined biomass movement of all wild birds, land arthropods and wild land mammals is about one-sixth that of humans walking and about 40 times smaller than all the biomass movement of humans. The biomass movement of marine animals, which we find to be the living world's largest, has been halved since 1850 due to industrial fishing and whaling, while human biomass movement has increased by about 40-fold. This study gives a quantitative perspective on global mobility in the Anthropocene and sharpens our perception regarding the extent of human versus animal activity.
", "doi": "10.1038/s41559-025-02863-9", "issn": "2397-334X", "publisher": "Nature Publishing Group", "publication": "Nature Ecology & Evolution", "publication_date": "2025-12", "volume": "16", "pages": "8338" }, { "id": "authors:xy4s8-1w188", "collection": "authors", "collection_id": "xy4s8-1w188", "cite_using_url": "https://authors.library.caltech.edu/records/xy4s8-1w188", "type": "article", "title": "Approximating the living", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Is a herd of wildebeest better thought of as a series of individual animals, each with its own glorious and unmanageable volition, or as a field of moving arrows? Are the morphogen gradients that set up the coordinate systems for embryonic anterior–posterior patterning a smooth and continuous concentration field or instead a chaotic collection of protein molecules each jiggling about in the haphazard way first described by Robert Brown in his microscopical observations of pollen? Is water, the great liquid ether of the living world, a collection of discrete molecules or instead a perfectly continuous medium with a density of 1000 kg/m? In this article, I will argue that these questions pose a false dichotomy since there are many different and powerful representations of the world around us. Different representations suit us differently at different times and it is often useful to be able to hold these seemingly contradictory notions in our heads simultaneously. Indeed, mathematics is not only the language of representation, but often is also the engine of reconciliation of such disparate views. In a letter to Alfred Russel Wallace on 14 April 1869, Charles Darwin noted that Lord Kelvin’s “views on the recent age of the world have been for some time one of my sorest troubles”. Here, I will argue that one of the highest attainments of the scientific enterprise is a coherent picture of the world, a picture in which our stories about the geological age of the Earth are coherent with our stories of how whales populated the oceans, our understanding of the living jibes with our understanding of the inanimate, our insights into the dynamics of genes and molecular structures are consonant with our physical understanding of the laws of statistical physics. The underpinnings of such coherency are often best revealed when viewed through the lens of mathematics.
", "doi": "10.1016/j.semcdb.2025.103646", "issn": "1084-9521", "publisher": "Elsevier", "publication": "Seminars in Cell and Developmental Biology", "publication_date": "2025-11", "volume": "175", "pages": "103646" }, { "id": "authors:3n4ah-x3781", "collection": "authors", "collection_id": "3n4ah-x3781", "cite_using_url": "https://authors.library.caltech.edu/records/3n4ah-x3781", "type": "article", "title": "Geometry-dependent defect merging induces bifurcated dynamics in active networks", "author": [ { "family_name": "Yang", "given_name": "Fan", "orcid": "0000-0002-2248-2026", "clpid": "Yang-Fan" }, { "family_name": "Liu", "given_name": "Shichen", "clpid": "Liu-Shichen" }, { "family_name": "Wang", "given_name": "Hao", "orcid": "0000-0001-9976-9247", "clpid": "Wang-Hao" }, { "family_name": "Lee", "given_name": "Heun Jin", "orcid": "0009-0002-4615-9271", "clpid": "Lee-Heun-Jin" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "abstract": "Cytoskeletal networks can repair defects to maintain structural integrity. However, the mechanisms and dynamics of defect merging remain poorly understood. Here, we report a geometry-tunable merging mechanism in microtubule-motor networks initiated by active crosslinking. We directly generate defects using a light-controlled microtubule-motor system in O-shaped and V-shaped networks, and observe that the defects can self-close. Combining theory and experiment, we find that the V-shaped networks must overcome internal elastic resistance in order to zip up cracks, giving rise to a bifurcation of dynamics dependent on the initial opening angle of the crack: the crack merges below a critical angle and opens up at larger angles. Simulation of a continuum model reproduces the bifurcation dynamics, revealing the importance of overlapping boundary layers where free motors and microtubules can actively crosslink and thereby merge the defects. We also formulate a simple elastic-rod model that can qualitatively predict the critical angle, which is tunable by the network geometry.", "doi": "10.1103/m8cb-gf1q", "issn": "2643-1564", "publisher": "American Physical Society", "publication": "Physical Review Research", "publication_date": "2025-10-03", "series_number": "4", "volume": "7", "issue": "4", "pages": "L042006" }, { "id": "authors:dkwem-9s246", "collection": "authors", "collection_id": "dkwem-9s246", "cite_using_url": "https://authors.library.caltech.edu/records/dkwem-9s246", "type": "article", "title": "Motor-driven microtubule diffusion in a photobleached dynamical coordinate system", "author": [ { "family_name": "Hirokawa", "given_name": "Soichi", "orcid": "0000-0001-5584-2676", "clpid": "Hirokawa-Soichi" }, { "family_name": "Lee", "given_name": "Heun Jin", "orcid": "0009-0002-4615-9271", "clpid": "Lee-Heun-Jin" }, { "family_name": "Banks", "given_name": "Rachel A.", "orcid": "0000-0003-2028-2925", "clpid": "Banks-Rachel-A" }, { "family_name": "Duarte", "given_name": "Ana I.", "orcid": "0000-0003-3726-3018", "clpid": "Duarte-Ana-I" }, { "family_name": "Najma", "given_name": "Bibi", "clpid": "Najma-Bibi" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "A hallmark feature of active matter systems is the ability of individual elements to interact and organize over length scales exceeding that of the constituent molecular players. However, the nature of internal redistribution that occurs in the bulk of the collective is less clear. Using light-dimerizable kinesin motors to spatially control the formation and contraction of a microtubule network, we deliberately photobleach a grid pattern onto the filament network serving as a transient and dynamic coordinate system to observe the deformation and translation of the remaining fluorescent squares of microtubules. We find that the network contracts at a rate set by motor speed but is accompanied by a diffusive-like spread throughout the bulk of the contracting network with effective diffusion constant two orders of magnitude lower than that for freely diffusing microtubules. We further find that on micron scales, the diffusive timescale is only a factor of ≈3 slower than that of advection regardless of conditions, showing that the global contraction and long-time relaxation from this diffusive behavior are both motor-driven but exhibit local competition within the network bulk.
", "doi": "10.1073/pnas.2417020122", "pmcid": "PMC12184364", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences", "publication_date": "2025-06-17", "series_number": "24", "volume": "122", "issue": "24", "pages": "e2417020122" }, { "id": "authors:308tx-01374", "collection": "authors", "collection_id": "308tx-01374", "cite_using_url": "https://authors.library.caltech.edu/records/308tx-01374", "type": "article", "title": "Glycolysis model shows that allostery maintains high ATP and limits accumulation of intermediates", "author": [ { "family_name": "Choe", "given_name": "Mangyu", "orcid": "0000-0002-8889-7494" }, { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-Tal" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Titov", "given_name": "Denis V.", "orcid": "0000-0001-5677-0651" } ], "abstract": "Glycolysis is a conserved metabolic pathway that produces ATP and biosynthetic precursors. It is not well understood how the control of mammalian glycolytic enzymes through allosteric feedback and mass action accomplishes various tasks of ATP homeostasis, such as controlling the rate of ATP production, maintaining high and stable ATP levels, ensuring that ATP hydrolysis generates a net excess of energy, and maintaining glycolytic intermediate concentrations within physiological levels. To investigate these questions, we developed a biophysical model of glycolysis based on enzyme rate equations derived from in vitro kinetic data. This is the first biophysical model of human glycolysis that successfully recapitulates the above tasks of ATP homeostasis and predicts absolute concentrations of glycolytic intermediates and isotope tracing kinetics that align with experimental measurements in human cells. We use the model to show that mass action alone is sufficient to control the ATP production rate and maintain the high energy of ATP hydrolysis. Meanwhile, allosteric regulation of hexokinase (HK) and phosphofructokinase (PFK) by ATP, ADP, inorganic phosphate, and glucose-6-phosphate is required to maintain high ATP levels and to prevent uncontrolled accumulation of phosphorylated intermediates of glycolysis. Allosteric feedback achieves the latter by maintaining HK and PFK enzyme activity at one-half of ATP demand and, thus, inhibiting the reaction of Harden and Young, which otherwise converts glucose to supraphysiological levels of phosphorylated glycolytic intermediates at the expense of ATP. Our methodology provides a roadmap for a quantitative understanding of how metabolic homeostasis emerges from the activities of individual enzymes.", "doi": "10.1016/j.bpj.2025.03.037", "issn": "0006-3495", "publisher": "Cell Press", "publication": "Biophysical Journal", "publication_date": "2025-05-20", "series_number": "10", "volume": "124", "issue": "10", "pages": "1562-1586" }, { "id": "authors:bt3x6-cjk89", "collection": "authors", "collection_id": "bt3x6-cjk89", "cite_using_url": "https://authors.library.caltech.edu/records/bt3x6-cjk89", "type": "article", "title": "The proteome is a terminal electron acceptor", "author": [ { "family_name": "Flamholz", "given_name": "Avi I.", "orcid": "0000-0002-9278-5479", "clpid": "Flamholz-Avi-I" }, { "family_name": "Goyal", "given_name": "Akshit", "orcid": "0000-0002-9425-8269", "clpid": "Goyal-Akshit" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Microbial metabolism is impressively flexible, enabling growth even when available nutrients differ greatly from biomass in redox state. Escherichia coli, for example, rearranges its physiology to grow on reduced and oxidized carbon sources through several forms of fermentation and respiration. To understand the limits on and evolutionary consequences of this metabolic flexibility, we developed a coarse-grained mathematical framework coupling redox chemistry with principles of cellular resource allocation. Our models inherit key qualities from both of their antecedents: i) describing diverse metabolic chemistries and ii) enforcing the simultaneous balancing of atom (e.g., carbon), electron, and energy (adenosine triphosphate) flows, as in redox models, while iii) treating biomass as both the product and catalyst of the growth process, as in resource allocation models. Assembling integrated models of respiration, fermentation, and photosynthesis clarified key microbiological phenomena, including demonstrating that autotrophs grow more slowly than heterotrophs because of constraints imposed by the intracellular production of reduced carbon. Our model further predicted that heterotrophic growth is improved by matching the redox state of biomass to the nutrient environment. Through analysis of ≈60,000 genomes and diverse proteomic datasets, we found evidence that proteins indeed accumulate amino acid substitutions promoting redox matching. We therefore propose an unexpected mode of genome evolution where substitutions neutral or even deleterious to the individual biochemical or structural functions of proteins can nonetheless be selected due to a redox-chemical benefit to the population.
", "doi": "10.1073/pnas.2404048121", "pmcid": "PMC11725909", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences", "publication_date": "2025-01-07", "series_number": "1", "volume": "122", "issue": "1", "pages": "e2404048121" }, { "id": "authors:5ahgv-2v151", "collection": "authors", "collection_id": "5ahgv-2v151", "cite_using_url": "https://authors.library.caltech.edu/records/5ahgv-2v151", "type": "article", "title": "Machine learning models can identify individuals based on a resident oral bacteriophage family", "author": [ { "family_name": "Mahmoudabadi", "given_name": "Gita" }, { "family_name": "Homyk", "given_name": "Kelsey" }, { "family_name": "Catching", "given_name": "Adam B." }, { "family_name": "Mahmoudabadi", "given_name": "Ana" }, { "family_name": "Foley", "given_name": "Helen Bermudez" }, { "family_name": "Tadmor", "given_name": "Arbel D." }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Metagenomic studies have revolutionized the study of novel phages. However these studies trade depth of coverage for breadth. We show that the targeted sequencing of a small region of a phage terminase family can provide sufficient sequence diversity to serve as an individual-specific barcode or a \"phageprint'', defined as the relative abundance profile of the variants within a terminase family. By collecting ~700 oral samples from ~100 individuals living on multiple continents, we found a consistent trend wherein each individual harbors one or two dominant variants that coexist with numerous low-abundance variants. By tracking phageprints over the span of a month across ten individuals, we observed that phageprints were generally stable, and found instances of concordant temporal fluctuations of variants shared between partners. To quantify these patterns further, we built machine learning models that, with high precision and recall, distinguished individuals even when we eliminated the most abundant variants and further downsampled phageprints to 2% of the remaining variants. Except between partners, phageprints are dissimilar between individuals, and neither country-of-residence, genetics, diet nor cohabitation seem to play a role in the relatedness of phageprints across individuals. By sampling from six different oral sites, we were able to study the impact of millimeters to a few centimeters of separation on an individual's phageprint and found that such limited spatial separation results in site-specific phageprints.
", "doi": "10.3389/frmbi.2024.1408203", "issn": "2813-4338", "publisher": "Frontiers Media SA", "publication": "Frontiers in Microbiomes", "publication_date": "2024-09-03", "volume": "3", "pages": "1408203" }, { "id": "authors:nbfep-58z27", "collection": "authors", "collection_id": "nbfep-58z27", "cite_using_url": "https://authors.library.caltech.edu/records/nbfep-58z27", "type": "article", "title": "Wildebeest herds on rolling hills: Flocking on arbitrary curved surfaces", "author": [ { "family_name": "Hueschen", "given_name": "Christina L.", "orcid": "0000-0002-3437-2895", "clpid": "Hueschen-Christina-L" }, { "family_name": "Dunn", "given_name": "Alexander R.", "orcid": "0000-0001-6096-4600", "clpid": "Dunn-Alexander-R" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The collective behavior of active agents, whether herds of wildebeest or microscopic actin filaments propelled by molecular motors, is an exciting frontier in biological and soft matter physics. Almost three decades ago, Toner and Tu developed a continuum theory of the collective action of flocks, or herds, that helped launch the modern field of active matter. One challenge faced when applying continuum active matter theories to living phenomena is the complex geometric structure of biological environments. Both macroscopic and microscopic herds move on asymmetric curved surfaces, like undulating grass plains or the surface layers of cells or embryos, which can render problems analytically intractable. In this paper, we present a formulation of the Toner-Tu flocking theory that uses the finite element method to solve the governing equations on arbitrary curved surfaces. First, we test the developed formalism and its numerical implementation in channel flow with scattering obstacles and on cylindrical and spherical surfaces, comparing our results to analytical solutions. We then progress to surfaces with arbitrary curvature, moving beyond previously accessible problems to explore herding behavior on a variety of landscapes. This approach allows the investigation of transients and dynamic solutions not revealed by analytic methods. It also enables versatile incorporation of new geometries and boundary conditions and efficient sweeps of parameter space. Looking forward, the paper presented here lays the groundwork for a dialogue between Toner-Tu theory and data on collective motion in biologically relevant geometries, from drone footage of migrating animal herds to movies of microscopic cytoskeletal flows within cells.
", "doi": "10.1103/physreve.108.024610", "issn": "2470-0045", "publisher": "American Physical Society", "publication": "Physical Review E", "publication_date": "2023-08", "series_number": "2", "volume": "108", "issue": "2", "pages": "024610" }, { "id": "authors:694bn-4vx45", "collection": "authors", "collection_id": "694bn-4vx45", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230510-982977000.2", "type": "article", "title": "Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies", "author": [ { "family_name": "Banks", "given_name": "Rachel A.", "orcid": "0000-0003-2028-2925", "clpid": "Banks-Rachel-A" }, { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-Vahe" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Hirokawa", "given_name": "Soichi", "orcid": "0000-0001-5584-2676", "clpid": "Hirokawa-Soichi" }, { "family_name": "Ierokomos", "given_name": "Athena", "clpid": "Ierokomos-Athena" }, { "family_name": "Ross", "given_name": "Tyler D.", "orcid": "0000-0002-7872-3992", "clpid": "Ross-Tyler-D" }, { "family_name": "Bryant", "given_name": "Zev", "clpid": "Bryant-Zev" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Active matter systems can generate highly ordered structures, avoiding equilibrium through the consumption of energy by individual constituents. How the microscopic parameters that characterize the active agents are translated to the observed mesoscopic properties of the assembly has remained an open question. These active systems are prevalent in living matter; for example, in cells, the cytoskeleton is organized into structures such as the mitotic spindle through the coordinated activity of many motor proteins walking along microtubules. Here, we investigate how the microscopic motor-microtubule interactions affect the coherent structures formed in a reconstituted motor-microtubule system. This question is of deeper evolutionary significance as we suspect motor and microtubule type contribute to the shape and size of resulting structures. We explore key parameters experimentally and theoretically, using a variety of motors with different speeds, processivities, and directionalities. We demonstrate that aster size depends on the motor used to create the aster, and develop a model for the distribution of motors and microtubules in steady-state asters that depends on parameters related to motor speed and processivity. Further, we show that network contraction rates scale linearly with the single-motor speed in quasi-one-dimensional contraction experiments. In all, this theoretical and experimental work helps elucidate how microscopic motor properties are translated to the much larger scale of collective motor-microtubule assemblies.", "doi": "10.7554/elife.79402", "pmcid": "PMC10014072", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2023-02-08", "volume": "12", "pages": "Art. No. e79402" }, { "id": "authors:atmme-a8250", "collection": "authors", "collection_id": "atmme-a8250", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230316-182632000.47", "type": "monograph", "title": "Data-driven model of glycolysis identifies the role of allostery in maintaining ATP homeostasis", "author": [ { "family_name": "Choe", "given_name": "Mangyu", "orcid": "0000-0002-8889-7494", "clpid": "Choe-Mangyu" }, { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-Tal" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Titov", "given_name": "Denis V.", "orcid": "0000-0001-5677-0651", "clpid": "titov-Denis-V" } ], "abstract": "The specific roles of allostery in regulating metabolism are not well understood. Here, we develop a data-driven mathematical model of mammalian glycolysis that uses enzyme rate equations and coupled ordinary differential equations. The key components of our model are the rate equations for allosterically regulated enzymes based on the Monod-Wyman-Changeux model that we derive using a rigorous analysis of thousands ofin vitrokinetic measurements. The resulting model recapitulates the properties of glycolysis observed in live cells and shows that the specific function of allosteric regulation is to maintain high and stable concentrations of ATP, while glycolysis without allosteric regulation is fully capable of producing ATP and ensuring that ATP hydrolysis generates energy. Our data-based modeling approach provides a roadmap for a better understanding of the role of allostery in metabolism regulation.One-Sentence SummaryThe glycolysis model based on allosteric enzyme rate equations recapitulates properties of glycolysis observed in live cells.", "doi": "10.1101/2022.12.28.522046", "publication_date": "2022-12-30" }, { "id": "authors:vwjfd-nee80", "collection": "authors", "collection_id": "vwjfd-nee80", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20221128-494241100.20", "type": "article", "title": "Modeling and mechanical perturbations reveal how spatially regulated anchorage gives rise to spatially distinct mechanics across the mammalian spindle", "author": [ { "family_name": "Suresh", "given_name": "Pooja", "orcid": "0000-0002-7793-4827", "clpid": "Suresh-Pooja" }, { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-Vahe" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Dumont", "given_name": "Sophie", "orcid": "0000-0002-8283-1523", "clpid": "Dumont-Sophie" } ], "abstract": "During cell division, the spindle generates force to move chromosomes. In mammals, microtubule bundles called kinetochore-fibers (k-fibers) attach to and segregate chromosomes. To do so, k-fibers must be robustly anchored to the dynamic spindle. We previously developed microneedle manipulation to mechanically challenge k-fiber anchorage, and observed spatially distinct response features revealing the presence of heterogeneous anchorage (Suresh et al., 2020). How anchorage is precisely spatially regulated, and what forces are necessary and sufficient to recapitulate the k-fiber's response to force remain unclear. Here, we develop a coarse-grained k-fiber model and combine with manipulation experiments to infer underlying anchorage using shape analysis. By systematically testing different anchorage schemes, we find that forces solely at k-fiber ends are sufficient to recapitulate unmanipulated k-fiber shapes, but not manipulated ones for which lateral anchorage over a 3 \u03bcm length scale near chromosomes is also essential. Such anchorage robustly preserves k-fiber orientation near chromosomes while allowing pivoting around poles. Anchorage over a shorter length scale cannot robustly restrict pivoting near chromosomes, while anchorage throughout the spindle obstructs pivoting at poles. Together, this work reveals how spatially regulated anchorage gives rise to spatially distinct mechanics in the mammalian spindle, which we propose are key for function.", "doi": "10.7554/elife.79558", "pmcid": "PMC9642996", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2022-11-08", "volume": "11", "pages": "Art. No. e79558" }, { "id": "authors:m408r-81q53", "collection": "authors", "collection_id": "m408r-81q53", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20221031-575177800.5", "type": "article", "title": "Anthroponumbers.org: A quantitative database of human impacts on Planet Earth", "author": [ { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-Griffin-D" }, { "family_name": "Banks", "given_name": "Rachel A.", "orcid": "0000-0003-2028-2925", "clpid": "Banks-Rachel-A" }, { "family_name": "Flamholz", "given_name": "Avi I.", "orcid": "0000-0002-9278-5479", "clpid": "Flamholz-Avi-I" }, { "family_name": "Sarai", "given_name": "Nicholas S.", "orcid": "0000-0002-4655-0038", "clpid": "Sarai-Nicholas-S" }, { "family_name": "Kamb", "given_name": "Mason", "orcid": "0000-0001-8036-7221", "clpid": "Kamb-Mason" }, { "family_name": "Lopez-Gomez", "given_name": "Ignacio", "orcid": "0000-0002-7255-5895", "clpid": "Lopez-Gomez-Ignacio" }, { "family_name": "Bar-On", "given_name": "Yinon", "orcid": "0000-0001-8477-609X", "clpid": "Bar-On-Yinon-M" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Over the last 10,000 years, human activities have transformed Earth through farming, forestry, mining, and industry. The complex results of these activities are now observed and quantified as \"human impacts\" on Earth's atmosphere, oceans, biosphere, and geochemistry. While myriad studies have explored facets of human impacts on the planet, they are necessarily technical and often highly focused. Thus, finding reliable quantitative information requires a significant investment of time to assess each quantity and associated uncertainty. We present the Human Impacts Database (www.anthroponumbers.org), which houses a diverse array of such quantities. We review a subset of these values and how they help build intuition for understanding the Earth-human system. While collation alone does not tell us how to best ameliorate human impacts, we contend that any future plans should be made in light of a quantitative understanding of the interconnected ways in which humans influence the planet.", "doi": "10.1016/j.patter.2022.100552", "pmcid": "PMC9481956", "issn": "2666-3899", "publisher": "Elsevier", "publication": "Patterns", "publication_date": "2022-09", "series_number": "9", "volume": "3", "issue": "9", "pages": "Art. No. 100552" }, { "id": "authors:rbgyb-nsz70", "collection": "authors", "collection_id": "rbgyb-nsz70", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220706-965496000", "type": "monograph", "title": "Wildebeest Herds on Rolling Hills: Flocking on Arbitrary Curved Surfaces", "author": [ { "family_name": "Hueschen", "given_name": "Christina L.", "orcid": "0000-0002-3437-2895", "clpid": "Hueschen-Christina-L" }, { "family_name": "Dunn", "given_name": "Alexander R.", "orcid": "0000-0001-6096-4600", "clpid": "Dunn-Alexander-R" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The collective behavior of active agents, whether herds of wildebeest or microscopic actin filaments propelled by molecular motors, is an exciting frontier in biological and soft matter physics. Almost three decades ago, Toner and Tu developed a hydrodynamic theory of the collective action of flocks, or herds, that helped launch the modern field of active matter. One challenge faced when applying continuum active matter theories to living phenomena is the complex geometric structure of biological environments. Both macroscopic and microscopic herds move on asymmetric curved surfaces, like undulating grass plains or the surface layers of cells or embryos, which can render problems analytically intractable. In this work, we present a formulation of the Toner-Tu flocking theory that uses the finite element method to solve the governing equations on arbitrary curved surfaces. First, we test the developed formalism and its numerical implementation in channel flow with scattering obstacles and on cylindrical and spherical surfaces, comparing our results to analytical solutions. We then progress to surfaces with arbitrary curvature, moving beyond previously accessible problems to explore herding behavior on a variety of landscapes. Our approach allows the investigation of transients and dynamic solutions not revealed by analytic methods. It also enables versatile incorporation of new geometries and boundary conditions and efficient sweeps of parameter space. Looking forward, the work presented here lays the groundwork for a dialogue between Toner-Tu theory and data on collective motion in biologically-relevant geometries, from drone footage of migrating animal herds to movies of microscopic cytoskeletal flows within cells.", "doi": "10.1101/2022.06.22.497052", "publication_date": "2022-06-26" }, { "id": "authors:2hvt2-zqd35", "collection": "authors", "collection_id": "2hvt2-zqd35", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220614-222259000", "type": "monograph", "title": "Emergent Actin Flows Explain Diverse Parasite Gliding Modes", "author": [ { "family_name": "Hueschen", "given_name": "Christina L.", "orcid": "0000-0002-3437-2895", "clpid": "Hueschen-Christina-L" }, { "family_name": "Segev Zarko", "given_name": "Li-av", "orcid": "0000-0003-4151-5513", "clpid": "Segev-Zarko-Li-av-T" }, { "family_name": "Chen", "given_name": "Jian-Hua", "orcid": "0000-0002-7998-0878", "clpid": "Chen-Jian-Hua" }, { "family_name": "LeGros", "given_name": "Mark A.", "clpid": "LeGros-Mark-A" }, { "family_name": "Larabell", "given_name": "Carolyn A.", "orcid": "0000-0002-6262-4789", "clpid": "Larabell-Carolyn-A" }, { "family_name": "Boothroyd", "given_name": "John C.", "orcid": "0000-0001-9719-745X", "clpid": "Boothroyd-John-C" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Dunn", "given_name": "Alexander R.", "orcid": "0000-0001-6096-4600", "clpid": "Dunn-Alexander-R" } ], "abstract": "During host infection, single\u2013celled apicomplexan parasites like Plasmodium and Toxoplasma use a motility mechanism called gliding, which differs fundamentally from other known mechanisms of eukaryotic cell motility. Gliding is thought to be powered by a thin layer of flowing filamentous (F)\u2013actin sandwiched between the plasma membrane and a myosin\u2013coated inner membrane complex. How this surface actin layer drives the diverse apicomplexan gliding modes observed experimentally \u2013 helical, circular, and twirling, and patch, pendulum, or rolling \u2013 presents a rich biophysical puzzle. Here, we use single\u2013molecule imaging to track individual actin filaments and myosin complexes in live Toxoplasma gondii. Based on these data, we hypothesize that F\u2013actin flows arise by self\u2013organization, rather than following a microtubule-based template as previously believed. We develop a continuum model of emergent F\u2013actin flow within the unusual confines provided by parasite geometry. In the presence of F\u2013actin turnover, our model predicts the emergence of a steady\u2013state mode in which actin transport is largely rearward. Removing actin turnover leads to actin patches that recirculate up and down the cell, a \t\u2033cyclosis\u2033 that we observe experimentally for drug\u2013stabilized actin bundles in live parasites. These findings provide a mechanism by which actin turnover governs a transition between distinct self\u2013organized F\u2013actin states, whose properties can account for the diverse gliding modes known to occur. More broadly, we illustrate how different forms of gliding motility can emerge as an intrinsic consequence of the self\u2013organizing properties of F\u2013actin flow in a confined geometry.", "doi": "10.1101/2022.06.08.495399", "publication_date": "2022-06-09" }, { "id": "authors:y7f1y-5j131", "collection": "authors", "collection_id": "y7f1y-5j131", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220412-265586000", "type": "monograph", "title": "Modeling and mechanical perturbations reveal how spatially regulated anchorage gives rise to spatially distinct mechanics across the mammalian spindle", "author": [ { "family_name": "Suresh", "given_name": "Pooja", "orcid": "0000-0002-7793-4827", "clpid": "Suresh-Pooja" }, { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-Vahe" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Dumont", "given_name": "Sophie", "orcid": "0000-0002-8283-1523", "clpid": "Dumont-Sophie" } ], "abstract": "During cell division, the spindle generates force to move chromosomes. In mammals, microtubule bundles called kinetochore-fibers (k-fibers) attach to and segregate chromosomes. To do so, k-fibers must be robustly anchored to the dynamic spindle. We previously developed microneedle manipulation to mechanically challenge k-fiber anchorage, and observed spatially distinct response features revealing the presence of heterogeneous anchorage (Suresh et al. 2020). How anchorage is precisely spatially regulated, and what forces are necessary and sufficient to recapitulate the k-fiber's response to force remain unclear. Here, we develop a coarse-grained k-fiber model and combine with manipulation experiments to infer underlying anchorage using shape analysis. By systematically testing different anchorage schemes, we find that forces solely at k-fiber ends are sufficient to recapitulate unmanipulated k-fiber shapes, but not manipulated ones for which lateral anchorage over a 3 \u03bcm length scale near chromosomes is also essential. Such anchorage robustly preserves k-fiber orientation near chromosomes while allowing pivoting around poles. Anchorage over a shorter length scale cannot robustly restrict pivoting near chromosomes, while anchorage throughout the spindle obstructs pivoting at poles. Together, this work reveals how spatially regulated anchorage gives rise to spatially distinct mechanics in the mammalian spindle, which we propose are key for function.", "doi": "10.1101/2022.04.08.487649", "publication_date": "2022-04-08" }, { "id": "authors:776rz-7z975", "collection": "authors", "collection_id": "776rz-7z975", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220308-543776000", "type": "monograph", "title": "Anthroponumbers.org: A Quantitative Database Of Human Impacts on Planet Earth", "author": [ { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-Griffin-D" }, { "family_name": "Banks", "given_name": "Rachel A.", "orcid": "0000-0003-2028-2925", "clpid": "Banks-Rachel-A" }, { "family_name": "Flamholz", "given_name": "Avi I.", "orcid": "0000-0002-9278-5479", "clpid": "Flamholz-Avi-I" }, { "family_name": "Sarai", "given_name": "Nicholas S.", "orcid": "0000-0002-4655-0038", "clpid": "Sarai-Nicholas-S" }, { "family_name": "Kamb", "given_name": "Mason", "orcid": "0000-0001-8036-7221", "clpid": "Kamb-Mason" }, { "family_name": "Lopez-Gomez", "given_name": "Ignacio", "orcid": "0000-0002-7255-5895", "clpid": "Lopez-Gomez-Ignacio" }, { "family_name": "Bar-On", "given_name": "Yinon", "orcid": "0000-0001-8477-609X", "clpid": "Bar-On-Yinon-M" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Over the last 10,000 years, human activities have transformed the Earth through farming, forestry, mining and industry. The complex results of these activities are now observed and quantified as \"human impacts\" on Earth's atmosphere, oceans, biosphere and geochemistry. While myriad studies have explored facets of human impacts on the planet, they are necessarily technical and often tightly-focused. Thus, finding reliable quantitative information requires a significant investment of time to assess each quantity, its methods of determination, and associated uncertainty. We present the Human Impacts Database (www.anthroponumbers.org), which houses a diverse array of such quantities. We review a subset of these values and how they help build intuition for understanding the Earth-human system. While collation alone does not tell us how to best ameliorate human impacts, we contend that any future plans should be made in light of a quantitative understanding of the interconnected ways in which humans impact the planet.", "doi": "10.1101/2022.03.04.483053", "publication_date": "2022-03-08" }, { "id": "authors:6cbkr-5dk50", "collection": "authors", "collection_id": "6cbkr-5dk50", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220422-231443172", "type": "article", "title": "Connections distributed within 3 \u03bcm of chromosomes are necessary and sufficient for the kinetochore-fiber's robust anchorage in the mammalian spindle", "author": [ { "family_name": "Suresh", "given_name": "Pooja", "orcid": "0000-0002-7793-4827", "clpid": "Suresh-Pooja" }, { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-Vahe" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Dumont", "given_name": "Sophie", "clpid": "Dumont-Sophie" } ], "abstract": "In mammalian cells, kinetochore-fibers are responsible for moving chromosomes within the spindle. To perform this task, they must generate and respond to forces while maintaining their connection to the spindle. Yet, how and where along their length they are anchored in the spindle, and which connections are necessary and sufficient remains unknown. Recent advances using microneedle manipulation opened new avenues in directly challenging kinetochore-fiber anchorage and revealed that anchorage near chromosomes restricts kinetochore-fiber pivoting there. To determine the components of this anchorage necessary and sufficient to locally restrict pivoting and maintain kinetochore-fiber orientation in the spindle center under force, we develop a theoretical framework using Euler-Bernoulli beam theory. We determine that highly localized anchorage just at the chromosome end fails to preserve kinetochore-fiber orientation in the spindle center. Similarly, global uniform anchorage fails to capture the localized resistance to pivoting by uniformly preserving structure everywhere. Instead, we show that local anchorage distributed within 3 \u03bcm of chromosomes is both necessary and sufficient for kinetochore-fibers to locally preserve their orientation under force. Together, our work indicates that while kinetochore-fibers have connections all along their length, not all connections are mechanically equivalent. Our model establishes the relationship between spindle architecture and the mechanics underlying kinetochore-fiber anchorage, and can be expanded to diverse spindle architectures across evolution.", "doi": "10.1016/j.bpj.2021.11.536", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2022-02-11", "series_number": "3", "volume": "121", "issue": "3", "pages": "448a-449a" }, { "id": "authors:twv8k-e4262", "collection": "authors", "collection_id": "twv8k-e4262", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220422-225704323", "type": "article", "title": "Reconstituting the nonequilibrium biophysics of the LECA cytoskeleton in active matter", "author": [ { "family_name": "Larios", "given_name": "David A.", "clpid": "Larios-David-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "abstract": "The eukaryotic cytoskeleton is a dense network of filaments and motor proteins that enables nearly all cellular force-generating behaviors, including movement, internal transport, and genome segregation through the mitotic spindle. Even though prokaryotes contain homologous force-generating filaments that drive processes like motion and cell division, no motor proteins have been found in bacterial and archaeal genomes. Current models suggest that eukaryotes emerged from the endosymbiosis and coevolution of two prokaryotic organisms; however, bioinformatic reconstructions reveal a LECA (Last Eukaryotic Common Ancestor) with multiple families of filaments and motor proteins. In this project, we aim to explore how the cytoskeleton underwent such a radical functional expansion during eukaryotic evolution, as well as to reconstruct a minimal active matter model of the LECA cytoskeleton. To do so, we built a computational-experimental platform to identify and synthesize cytoskeletal components in vitro and characterize their self-organizing properties with functional assays. As a proof of concept, we generated two synthetic protozoan motors, one of which produces a novel phase with ballistic microtubule motion. With this approach, our future work will expand our platform to a broader range of cytoskeletal components, including prokaryotic filaments. By exploring diverse eukaryotic and hybrid (eukaryote-prokaryote) cytoskeletal systems under multiple physiological conditions, we aim to provide insights into the evolution of eukaryotic active matter.", "doi": "10.1016/j.bpj.2021.11.2744", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2022-02-11", "series_number": "3", "volume": "121", "issue": "3", "pages": "521a" }, { "id": "authors:fx4m6-pcg27", "collection": "authors", "collection_id": "fx4m6-pcg27", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220425-141009622", "type": "article", "title": "Distinct self-organized actin patterns explain diverse parasite gliding modes", "author": [ { "family_name": "Hueschen", "given_name": "Christina", "orcid": "0000-0002-3437-2895", "clpid": "Hueschen-Christina-L" }, { "family_name": "Segev Zarko", "given_name": "Li-av", "orcid": "0000-0003-4151-5513", "clpid": "Segev-Zarko-Li-av-T" }, { "family_name": "Chen", "given_name": "Jian-Hua", "orcid": "0000-0002-7998-0878", "clpid": "Chen-Jian-Hua" }, { "family_name": "LeGros", "given_name": "Mark", "clpid": "LeGros-Mark" }, { "family_name": "Larabell", "given_name": "Carolyn A.", "orcid": "0000-0002-6262-4789", "clpid": "Larabell-Carolyn-A" }, { "family_name": "Boothroyd", "given_name": "John C.", "orcid": "0000-0001-9719-745X", "clpid": "Boothroyd-John-C" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Dunn", "given_name": "Alexander R.", "orcid": "0000-0001-6096-4600", "clpid": "Dunn-Alexander-R" } ], "abstract": "During host infection, single-celled apicomplexan parasites like Plasmodium and Toxoplasma use a unique form of locomotion called gliding that differs fundamentally from the swim-or-crawl paradigm of eukaryotic cell motility. Gliding is powered by a thin layer of actin and a specialized myosin sandwiched between the plasma membrane and an inner membranous scaffold. How is this actomyosin network organized to generate coherent traction forces, and drive the diverse cell movements observed during gliding? Here, we used single-molecule imaging to track individual actin filaments and myosin complexes in living Toxoplasma gondii. Based on these data, we drew on flocking theory to develop a continuum model of actin self-organization in the unusual confines provided by parasite geometry. Deriving a parameterization-free surface formulation of our governing equations enabled finite element method simulations on detailed reconstructions of the Toxoplasma cell surface. In the presence of rapid actin filament depolymerization, our model predicts the emergence of rearward steady-state actin flows. By contrast, at low depolymerization rates, emergent stable actin patches recirculate up and down the cell in a \"cyclosis\" that we observed experimentally for drug-stabilized actin bundles in live parasites. These findings indicate that actin turnover governs a transition between distinct self-organized actin states, whose different properties can account for the disparate gliding modes observed experimentally: unidirectional (helical, circular, twirling) and bidirectional (patch, pendulum, rolling). More broadly, our experimental observations and theoretical model illustrate how different forms of gliding motility can emerge as an intrinsic consequence of the self-organizing properties of actin filament flow in a complex confined geometry.", "doi": "10.1016/j.bpj.2021.11.1183", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2022-02-11", "series_number": "3", "volume": "121", "issue": "3", "pages": "314a" }, { "id": "authors:0fp8r-q9q88", "collection": "authors", "collection_id": "0fp8r-q9q88", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220422-296339700", "type": "article", "title": "Measuring energy consumption through space and time in an active matter system of cytoskeletal motors and filaments", "author": [ { "family_name": "Duarte", "given_name": "Ana I.", "clpid": "Duarte-Ana-I" }, { "family_name": "Jin Lee", "given_name": "Heun", "clpid": "Jin-Lee-Heun" }, { "family_name": "Banks", "given_name": "Rachel A.", "orcid": "0000-0003-2028-2925", "clpid": "Banks-Rachel-A" }, { "family_name": "Hirokawa", "given_name": "Soichi", "orcid": "0000-0001-5584-2676", "clpid": "Hirokawa-Soichi" }, { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-Vahe" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Active matter systems consume energy from their environment to form organized, dynamical patterns and structures. These ordered states do not exist in the absence of an energy source. As such, a more general description of energy-driven behavior than is offered by near-equilibrium approaches is required to describe active matter. To gain insight into the energetic cost to form and maintain order, we investigate the assembly of an ordered aster from a disordered, homogeneous mixture of microtubules. This formation occurs due to optogenetically controllable cross-linking of the molecular motors that walk on the microtubules and hydrolyze ATP. Theoretical reaction-diffusion models predict a non-equilibrium ATP distribution resulting from a corresponding motor profile; we test these models via fluorescent readouts of ATP and motor concentrations in both space and time. Comparing the theoretical versus measured ATP profiles provides insight into the dynamics of structure formation and properties of the motors, such as if motors operate cooperatively. Our experiments revealed an ATP depletion gradient that is maximized at the aster core, where the concentration of the motors is highest. This work is a first step in understanding the role of energy consumption through space and time in the formation and maintenance of organization in this active matter system. More broadly, our work provides a case study towards the larger effort of developing generalized theories of non-equilibrium systems.", "doi": "10.1016/j.bpj.2021.11.2745", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2022-02-11", "series_number": "3", "volume": "121", "issue": "3", "pages": "521a-522a" }, { "id": "authors:h8qyd-87818", "collection": "authors", "collection_id": "h8qyd-87818", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220422-225130615", "type": "article", "title": "Reproducibility, stochasticity, and geometry in a lattice of cross-feeding microbial cells", "author": [ { "family_name": "Salmon", "given_name": "Gabe", "clpid": "Salmon-Gabriel" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Bacteria often exchange metabolites to achieve together what they cannot alone. Chemically-exchanging cells can survive, even as metabolic specialists in poor environments; perform decentralized chemistry; and regulate phenotypic heterogeneity. Connecting the microscopic features of transport and physiology\u2013often stochastic and heterogeneous\u2013with reproducible, large-scale behaviors is key to advancing microbial ecology. Here we investigate the growth of cross-feeding strains at single-cell, stochastic resolution. Using this framework, we reanalyze recently-published microscopy data on two strains of E. coli that leak an amino acid the other needs to grow. These cells thus participate in a spatially-localized mutualism. Earlier work has reported that in such a close-packed setting, a cell's mean growth rate increases with the fraction of cells of complementary type within a neighborhood of a few cell lengths. Underneath this average behavior, we find that cells grow at rates with pronounced variability over time. Part of this variability is \"intrinsic;\" part is registrable to the turnover of a cell's microenvironment. We study how a cell's variability scales with its typical growth rate, geometric context, and biophysical parameters. While precise trajectories of cells in growth rate (or space) are often noisy and contingent, many display canonical regularities. We reckon with these patterns, and strive to link single-cell dynamics to emergent geometric ordering in microcolonies, from the lens of simple models of timescales, lengthscales, and noise under cross-feeding. This analysis helps dissect how simple, ubiquitous forces in consortia\u2013coupled growth, diffusion, and variability\u2013conspire to shape complex assemblies.", "doi": "10.1016/j.bpj.2021.11.2086", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2022-02-11", "series_number": "3", "volume": "121", "issue": "3", "pages": "120a" }, { "id": "authors:nefnj-f1a55", "collection": "authors", "collection_id": "nefnj-f1a55", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220201-508256000", "type": "article", "title": "MCRL: using a reference library to compress a metagenome into a non-redundant list of sequences, considering viruses as a case study", "author": [ { "family_name": "Tadmor", "given_name": "Arbel D.", "orcid": "0000-0003-1617-2010", "clpid": "Tadmor-Arbel-D" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Motivation: Metagenomes offer a glimpse into the total genomic diversity contained within a sample. Currently, however, there is no straightforward way to obtain a non-redundant list of all putative homologs of a set of reference sequences present in a metagenome. \n\nResults: To address this problem, we developed a novel clustering approach called 'metagenomic clustering by reference library' (MCRL), where a reference library containing a set of reference genes is clustered with respect to an assembled metagenome. According to our proposed approach, reference genes homologous to similar sets of metagenomic sequences, termed 'signatures', are iteratively clustered in a greedy fashion, retaining at each step the reference genes yielding the lowest E values, and terminating when signatures of remaining reference genes have a minimal overlap. The outcome of this computation is a non-redundant list of reference genes homologous to minimally overlapping sets of contigs, representing potential candidates for gene families present in the metagenome. Unlike metagenomic clustering methods, there is no need for contigs to overlap to be associated with a cluster, enabling MCRL to draw on more information encoded in the metagenome when computing tentative gene families. We demonstrate how MCRL can be used to extract candidate viral gene families from an oral metagenome and an oral virome that otherwise could not be determined using standard approaches. We evaluate the sensitivity, accuracy and robustness of our proposed method for the viral case study and compare it with existing analysis approaches.", "doi": "10.1093/bioinformatics/btab703", "pmcid": "PMC10060711", "issn": "1367-4803", "publisher": "Oxford University Press", "publication": "Bioinformatics", "publication_date": "2022-02-01", "series_number": "3", "volume": "38", "issue": "3", "pages": "631-647" }, { "id": "authors:80kbs-6ys29", "collection": "authors", "collection_id": "80kbs-6ys29", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220512-144706094", "type": "article", "title": "Actin Organization and Dynamics in Motile Toxoplasma Gondii Parasites", "author": [ { "family_name": "Hueschen", "given_name": "Christina", "orcid": "0000-0002-3437-2895", "clpid": "Hueschen-Christina-L" }, { "family_name": "Segev Zarko", "given_name": "Li-Av", "orcid": "0000-0003-4151-5513", "clpid": "Segev-Zarko-Li-Av-T" }, { "family_name": "Chen", "given_name": "Jian-Hua", "orcid": "0000-0002-7998-0878", "clpid": "Chen-Jian-Hua" }, { "family_name": "LeGros", "given_name": "Mark", "clpid": "LeGros-Mark" }, { "family_name": "Larabell", "given_name": "Carolyn", "orcid": "0000-0002-6262-4789", "clpid": "Larabell-Carolyn-A" }, { "family_name": "Boothroyd", "given_name": "John", "orcid": "0000-0001-9719-745X", "clpid": "Boothroyd-John-C" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Dunn", "given_name": "Alexander R.", "orcid": "0000-0001-6096-4600", "clpid": "Dunn-Alexander-R" } ], "abstract": "[no abstract]", "doi": "10.4269/ajtmh.abstract2021", "issn": "0002-9637", "publisher": "American Society of Tropical Medicine and Hygiene", "publication": "American Journal of Tropical Medicine and Hygiene", "publication_date": "2021-11", "series_number": "5", "volume": "105", "issue": "5", "pages": "448-449" }, { "id": "authors:4cdt6-s8y82", "collection": "authors", "collection_id": "4cdt6-s8y82", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211102-171349069", "type": "monograph", "title": "Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies", "author": [ { "family_name": "Banks", "given_name": "Rachel A.", "orcid": "0000-0003-2028-2925", "clpid": "Banks-Rachel-A" }, { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-Vahe" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Hirokawa", "given_name": "Soichi", "clpid": "Hirokaw-Soichi" }, { "family_name": "Ierokomos", "given_name": "Athena", "clpid": "Ierokomos-Athena" }, { "family_name": "Ross", "given_name": "Tyler D.", "orcid": "0000-0002-7872-3992", "clpid": "Ross-Tyler-D" }, { "family_name": "Bryant", "given_name": "Zev", "orcid": "0000-0001-6751-3693", "clpid": "Bryant-Zev" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Active matter systems can generate highly ordered structures, avoiding equilibrium through the consumption of energy by individual constituents. How the microscopic parameters that characterize the active agents are translated to the observed mesoscopic properties of the assembly has remained an open question. These active systems are prevalent in living matter; for example, in cells, the cytoskeleton is organized into structures such as the mitotic spindle through the coordinated activity of many motor proteins walking along microtubules. Here, we investigate how the microscopic motor-microtubule interactions affect the coherent structures formed in a reconstituted motor-microtubule system. This question is of deeper evolutionary significance as we suspect motor and microtubule type contribute to the shape and size of resulting structures. We explore key parameters experimentally and theoretically, using a variety of motors with different speeds, processivities, and directionalities. We demonstrate that aster size depends on the motor used to create the aster, and develop a model for the distribution of motors and microtubules in steady-state asters that depends on parameters related to motor speed and processivity. Further, we show that network contraction rates scale linearly with the single-motor speed in quasi one-dimensional contraction experiments. In all, this theoretical and experimental work helps elucidate how microscopic motor properties are translated to the much larger scale of collective motor-microtubule assemblies.", "doi": "10.1101/2021.10.22.465381", "publication_date": "2021-10-23" }, { "id": "authors:s1dw8-apr33", "collection": "authors", "collection_id": "s1dw8-apr33", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201021-080615473", "type": "article", "title": "Fundamental limits on the rate of bacterial growth and their influence on proteomic composition", "author": [ { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-Nathan-M" }, { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-Griffin-D" }, { "family_name": "Hueschen", "given_name": "Christina L.", "orcid": "0000-0002-3437-2895", "clpid": "Hueschen-Christina-L" }, { "family_name": "Garcia", "given_name": "Hernan G.", "orcid": "0000-0002-5212-3649", "clpid": "Garcia-Hernan-G" }, { "family_name": "Kondev", "given_name": "Jane", "orcid": "0000-0001-7522-7144", "clpid": "Kondev-Jane" }, { "family_name": "Fisher", "given_name": "Daniel S.", "clpid": "Fisher-Daniel-S" }, { "family_name": "Theriot", "given_name": "Julie A.", "orcid": "0000-0002-2334-2535", "clpid": "Theriot-Julie-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Despite abundant measurements of bacterial growth rate, cell size, and protein content, we lack a rigorous understanding of what sets the scale of these quantities and when protein abundances should (or should not) depend on growth rate. Here, we estimate the basic requirements and physical constraints on steady-state growth by considering key processes in cellular physiology across a collection of Escherichia coli proteomic data covering \u22484,000 proteins and 36 growth rates. Our analysis suggests that cells are predominantly tuned for the task of cell doubling across a continuum of growth rates; specific processes do not limit growth rate or dictate cell size. We present a model of proteomic regulation as a function of nutrient supply that reconciles observed interdependences between protein synthesis, cell size, and growth rate and propose that a theoretical inability to parallelize ribosomal synthesis places a firm limit on the achievable growth rate. A record of this paper's transparent peer review process is included in the supplemental information.", "doi": "10.1016/j.cels.2021.06.002", "pmcid": "PMC8460600", "issn": "2405-4712", "publisher": "Cell Press", "publication": "Cell Systems", "publication_date": "2021-09-22", "series_number": "9", "volume": "12", "issue": "9", "pages": "924-944" }, { "id": "authors:ks7h3-tf644", "collection": "authors", "collection_id": "ks7h3-tf644", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201110-143708446", "type": "article", "title": "Persistent fluid flows defined by active matter boundaries", "author": [ { "family_name": "Qu", "given_name": "Zijie", "orcid": "0000-0003-1500-3207", "clpid": "Qu-Zijie" }, { "family_name": "Schildknecht", "given_name": "Dominik", "orcid": "0000-0002-0678-0904", "clpid": "Schildknecht-Dominik" }, { "family_name": "Shadkhoo", "given_name": "Shahriar", "orcid": "0000-0003-3582-0634", "clpid": "Shadkhoo-Shahriar" }, { "family_name": "Amaya", "given_name": "Enrique", "clpid": "Amaya-Enrique" }, { "family_name": "Jiang", "given_name": "Jialong", "clpid": "Jiang-Jialong" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Larios", "given_name": "David", "clpid": "Larios-David" }, { "family_name": "Yang", "given_name": "Fan", "orcid": "0000-0002-2248-2026", "clpid": "Yang-Fan" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "abstract": "Biological systems control ambient fluids through the self-organization of active protein structures, including flagella, cilia, and cytoskeletal networks. Self-organization of protein components enables the control and modulation of fluid flow fields on micron scales, however, the physical principles underlying the organization and control of active-matter-driven fluid flows are poorly understood. Here, we use an optically-controlled active-matter system composed of microtubule filaments and light-switchable kinesin motor proteins to analyze the emergence of persistent flow fields. Using light, we form contractile microtubule networks of varying size and shape, and demonstrate that the geometry of microtubule flux at the corners of contracting microtubule networks predicts the architecture of fluid flow fields across network geometries through a simple point force model. Our work provides a foundation for programming microscopic fluid flows with controllable active matter and could enable the engineering of versatile and dynamic microfluidic devices.", "doi": "10.1038/s42005-021-00703-3", "issn": "2399-3650", "publisher": "Nature Publishing Group", "publication": "Communications Physics", "publication_date": "2021-08-27", "volume": "4", "pages": "Art. No. 198" }, { "id": "authors:z9b4x-mah61", "collection": "authors", "collection_id": "z9b4x-mah61", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201118-151353187", "type": "article", "title": "The total number and mass of SARS-CoV-2 virions", "author": [ { "family_name": "Sender", "given_name": "Ron", "orcid": "0000-0002-1165-9818", "clpid": "Sender-Ron" }, { "family_name": "Bar-On", "given_name": "Yinon M.", "orcid": "0000-0001-8477-609X", "clpid": "Bar-On-Yinon-M" }, { "family_name": "Gleizer", "given_name": "Shmuel", "orcid": "0000-0001-9774-0504", "clpid": "Gleizer-Shmuel" }, { "family_name": "Bernsthein", "given_name": "Biana", "orcid": "0000-0002-7957-2575", "clpid": "Bernsthein-Biana" }, { "family_name": "Flamholz", "given_name": "Avi", "orcid": "0000-0002-9278-5479", "clpid": "Flamholz-Avi" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" } ], "abstract": "Quantitatively describing the time course of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection within an infected individual is important for understanding the current global pandemic and possible ways to combat it. Here we integrate the best current knowledge about the typical viral load of SARS-CoV-2 in bodily fluids and host tissues to estimate the total number and mass of SARS-CoV-2 virions in an infected person. We estimate that each infected person carries 109 to 1011 virions during peak infection, with a total mass in the range of 1 \u03bcg to 100 \u03bcg, which curiously implies that all SARS-CoV-2 virions currently circulating within human hosts have a collective mass of only 0.1 kg to 10 kg. We combine our estimates with the available literature on host immune response and viral mutation rates to demonstrate how antibodies markedly outnumber the spike proteins, and the genetic diversity of virions in an infected host covers all possible single nucleotide substitutions.", "doi": "10.1073/pnas.2024815118", "pmcid": "PMC8237675", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2021-06-22", "series_number": "25", "volume": "118", "issue": "25", "pages": "Art. No. e2024815118" }, { "id": "authors:952jf-sjk92", "collection": "authors", "collection_id": "952jf-sjk92", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210629-200149422", "type": "article", "title": "Schr\u00f6dinger's What Is Life? at 75", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "2019 marked the 75th anniversary of the publication of Erwin Schr\u00f6dinger's What Is Life?, a short book described by Roger Penrose in his preface to a reprint of this classic as \"among the most influential scientific writings of the 20th century.\" In this article, I review the long argument made by Schr\u00f6dinger as he mused on how the laws of physics could help us understand \"the events in space and time which take place within the spatial boundary of a living organism.\" Though Schr\u00f6dinger's book is often hailed for its influence on some of the titans who founded molecular biology, this article takes a different tack. Instead of exploring the way the book touched biologists such as James Watson and Francis Crick, as well as its critical reception by others such as Linus Pauling and Max Perutz, I argue that Schr\u00f6dinger's classic is a timeless manifesto, rather than a dated historical curiosity. What Is Life? is full of timely outlooks and approaches to understanding the mysterious living world that includes and surrounds us and can instead be viewed as a call to arms to tackle the great unanswered challenges in the study of living matter that remain for 21st century science.", "doi": "10.1016/j.cels.2021.05.013", "issn": "2405-4712", "publisher": "Elsevier", "publication": "Cell Systems", "publication_date": "2021-06-16", "series_number": "6", "volume": "12", "issue": "6", "pages": "465-476" }, { "id": "authors:2rn67-dgd32", "collection": "authors", "collection_id": "2rn67-dgd32", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210503-101747249", "type": "article", "title": "Understanding the Mechanisms that Give Rise to the Mammalian Spindle's Response to Force using Theory and Microneedle Manipulation", "author": [ { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-Vahe" }, { "family_name": "Suresh", "given_name": "Pooja", "clpid": "Suresh-Pooja" }, { "family_name": "Dumont", "given_name": "Sophie", "clpid": "Dumont-Sophie" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The spindle is the force-generating structure that drives chromosome segregation at cell division. In mammalian spindles, bundles of spindle microtubules called kinetochore-fibers (k-fibers) pull on chromosomes to move them. While we know nearly all components necessary for spindle function, how k-fibers respond to force and maintain themselves under force remains poorly understood. Our recent ability to exert local force on the mammalian spindle with microneedles provides key information to answering this question. Here, we use a modeling approach based on Euler-Bernoulli beam theory to identify the minimal mechanical features of the spindle necessary to recapitulate how k-fibers deform under external load. First, we find that force and moment generation at spindle poles are needed to recapitulate observed k-fiber shapes, both with and without external load. Then, we find that crosslinking near kinetochores, which has been experimentally observed, is necessary and sufficient to recapitulate observed k-fiber shapes, assuming no moment generation at kinetochores. By probing the limits of our model under large k-fiber deformations, we infer conditions under high external load beyond which the mechanical integrity of the k-fiber appears compromised, suggesting that structural changes occur under such forces. Finally, we assess the possibility of using our modeling formalism to learn about the applied loads based purely on k-fiber shape analysis. The modeling framework we developed not only helps us understand the mechanisms underlying the spindle's response to force, but will serve as a quantitative framework for probing how the architecture and dynamics of the k-fiber and its surrounding network give rise to mechanics and function.", "doi": "10.1016/j.bpj.2020.11.2158", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2021-02-12", "series_number": "3", "volume": "120", "issue": "3", "pages": "347A" }, { "id": "authors:twn6t-ab519", "collection": "authors", "collection_id": "twn6t-ab519", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210302-081003409", "type": "monograph", "title": "The Anthropocene by the Numbers: A Quantitative Snapshot of Humanity's Influence on the Planet", "author": [ { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-Griffin-D" }, { "family_name": "Banks", "given_name": "Rachel A.", "orcid": "0000-0003-2028-2925", "clpid": "Banks-Rachel-A" }, { "family_name": "Flamholz", "given_name": "Avi I.", "orcid": "0000-0002-9278-5479", "clpid": "Flamholz-Avi-I" }, { "family_name": "Sarai", "given_name": "Nicholas S.", "orcid": "0000-0002-4655-0038", "clpid": "Sarai-Nicholas-S" }, { "family_name": "Kamb", "given_name": "Mason", "clpid": "Kamb-Mason" }, { "family_name": "Lopez-Gomez", "given_name": "Ignacio", "orcid": "0000-0002-7255-5895", "clpid": "Lopez-Gomez-Ignacio" }, { "family_name": "Bar-On", "given_name": "Yinon M.", "orcid": "0000-0001-8477-609X", "clpid": "Bar-On-Yinon-M" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The presence and action of humans on Earth has exerted a strong influence on the evolution of the planet over the past \u2248 10,000 years, the consequences of which are now becoming broadly evident. Despite a deluge of tightly-focused and necessarily technical studies exploring each facet of \"human impacts\" on the planet, their integration into a complete picture of the human-Earth system lags far behind. Here, we quantify twelve dimensionless ratios which put the magnitude of human impacts in context, comparing the magnitude of anthropogenic processes to their natural analogues. These ratios capture the extent to which humans alter the terrestrial surface, hydrosphere, biosphere, atmosphere, and biogeochemistry of Earth. In almost all twelve cases, the impact of human processes rivals or exceeds their natural counterparts. The values and corresponding uncertainties for these impacts at global and regional resolution are drawn from the primary scientific literature, governmental and international databases, and industry reports. We present this synthesis of the current \"state of affairs\" as a graphical snapshot designed to be used as a reference. Furthermore, we establish a searchable database termed the Human Impacts Database (this http URL) which houses all quantities reported here and many others with extensive curation and annotation. While necessarily incomplete, this work collates and contextualizes a set of essential numbers summarizing the broad impacts of human activities on Earth's atmosphere, land, water, and biota.", "doi": "10.48550/arXiv.2101.09620", "publisher": "arXiv", "publication_date": "2021-01-24" }, { "id": "authors:3yzaq-fw923", "collection": "authors", "collection_id": "3yzaq-fw923", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210217-132137942", "type": "monograph", "title": "Programming Boundary Deformation Patterns in Active Networks", "author": [ { "family_name": "Qu", "given_name": "Zijie", "clpid": "Qu-Zijie" }, { "family_name": "Jiang", "given_name": "Jialong", "clpid": "Jiang-Jialong" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Shadkhoo", "given_name": "Shahriar", "clpid": "Shadkhoo-Shahriar" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "abstract": "Active materials take advantage of their internal sources of energy to self-organize in an automated manner. This feature provides a novel opportunity to design micron-scale machines with minimal required control. However, self-organization goes hand in hand with predetermined dynamics that are hardly susceptible to environmental perturbations. Therefore utilizing this feature of active systems requires harnessing and directing the macroscopic dynamics to achieve specific functions; which in turn necessitates understanding the underlying mechanisms of active forces. Here we devise an optical control protocol to engineer the dynamics of active networks composed of microtubules and light-activatable motor proteins. The protocol enables carving activated networks of different shapes, and isolating them from the embedding solution. Studying a large set of shapes, we observe that the active networks contract in a shape-preserving manner that persists over the course of contraction. We formulate a coarse-grained theory and demonstrate that self-similarity of contraction is associated with viscous-like active stresses. These findings help us program the dynamics of the network through manipulating the light intensity in space and time, and maneuver the network into bending in specific directions, as well as temporally alternating directions. Our work improves understanding the active dynamics in contractile networks, and paves a new path towards engineering the dynamics of a large class of active materials.", "doi": "10.48550/arXiv.2101.08464", "publisher": "arXiv", "publication_date": "2021-01-21" }, { "id": "authors:rdxhn-0d521", "collection": "authors", "collection_id": "rdxhn-0d521", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200615-130249775", "type": "article", "title": "Reconciling kinetic and thermodynamic models of bacterial transcription", "author": [ { "family_name": "Morrison", "given_name": "Muir", "orcid": "0000-0002-0768-7234", "clpid": "Morrison-Muir-J" }, { "family_name": "Razo-Mejia", "given_name": "Manuel", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-Manuel" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The study of transcription remains one of the centerpieces of modern biology with implications in settings from development to metabolism to evolution to disease. Precision measurements using a host of different techniques including fluorescence and sequencing readouts have raised the bar for what it means to quantitatively understand transcriptional regulation. In particular our understanding of the simplest genetic circuit is sufficiently refined both experimentally and theoretically that it has become possible to carefully discriminate between different conceptual pictures of how this regulatory system works. This regulatory motif, originally posited by Jacob and Monod in the 1960s, consists of a single transcriptional repressor binding to a promoter site and inhibiting transcription. In this paper, we show how seven distinct models of this so-called simple-repression motif, based both on thermodynamic and kinetic thinking, can be used to derive the predicted levels of gene expression and shed light on the often surprising past success of the thermodynamic models. These different models are then invoked to confront a variety of different data on mean, variance and full gene expression distributions, illustrating the extent to which such models can and cannot be distinguished, and suggesting a two-state model with a distribution of burst sizes as the most potent of the seven for describing the simple-repression motif.", "doi": "10.1371/journal.pcbi.1008572", "pmcid": "PMC7845990", "issn": "1553-7358", "publisher": "Public Library of Science", "publication": "PLOS Computational Biology", "publication_date": "2021-01-19", "series_number": "1", "volume": "17", "issue": "1", "pages": "Art. No. e1008572" }, { "id": "authors:ghwed-3p358", "collection": "authors", "collection_id": "ghwed-3p358", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200203-092359628", "type": "article", "title": "Multiplexed characterization of rationally designed promoter architectures deconstructs combinatorial logic for IPTG-inducible systems", "author": [ { "family_name": "Yu", "given_name": "Timothy C.", "clpid": "Yu-Timothy-C" }, { "family_name": "Liu", "given_name": "Winnie L.", "clpid": "Liu-Winnie-L" }, { "family_name": "Brinck", "given_name": "Marcia S.", "orcid": "0000-0001-8149-0555", "clpid": "Brinck-Marcia-S" }, { "family_name": "Davis", "given_name": "Jessica E.", "clpid": "Davis-Jessica-E" }, { "family_name": "Shek", "given_name": "Jeremy", "orcid": "0000-0002-5419-060X", "clpid": "Shek-Jeremy" }, { "family_name": "Bower", "given_name": "Grace", "clpid": "Bower-Grace" }, { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-Tal" }, { "family_name": "Insigne", "given_name": "Kimberly D.", "clpid": "Insigne-Kimberly-D" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Kosuri", "given_name": "Sriram", "orcid": "0000-0002-4661-0600", "clpid": "Kosuri-Sriram" }, { "family_name": "Urtecho", "given_name": "Guillaume", "orcid": "0000-0003-1045-1721", "clpid": "Urtecho-Guillaume" } ], "abstract": "A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.", "doi": "10.1038/s41467-020-20094-3", "pmcid": "PMC7804116", "issn": "2041-1723", "publisher": "Nature Publishing Group", "publication": "Nature Communications", "publication_date": "2021-01-12", "volume": "12", "pages": "Art. No. 325" }, { "id": "authors:hs5zp-9ww46", "collection": "authors", "collection_id": "hs5zp-9ww46", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200526-121021095", "type": "article", "title": "Proofreading through spatial gradients", "author": [ { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-Vahe" }, { "family_name": "Husain", "given_name": "Kabir", "clpid": "Husain-Kabir" }, { "family_name": "Xiao", "given_name": "Fangzhou", "orcid": "0000-0002-5001-5644", "clpid": "Xiao-Fangzhou" }, { "family_name": "Murugan", "given_name": "Arvind", "orcid": "0000-0001-5464-917X", "clpid": "Murugan-Arvind" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Key enzymatic processes use the nonequilibrium error correction mechanism called kinetic proofreading to enhance their specificity. The applicability of traditional proofreading schemes, however, is limited because they typically require dedicated structural features in the enzyme, such as a nucleotide hydrolysis site or multiple intermediate conformations. Here, we explore an alternative conceptual mechanism that achieves error correction by having substrate binding and subsequent product formation occur at distinct physical locations. The time taken by the enzyme\u2013substrate complex to diffuse from one location to another is leveraged to discard wrong substrates. This mechanism does not have the typical structural requirements, making it easier to overlook in experiments. We discuss how the length scales of molecular gradients dictate proofreading performance, and quantify the limitations imposed by realistic diffusion and reaction rates. Our work broadens the applicability of kinetic proofreading and sets the stage for studying spatial gradients as a possible route to specificity.", "doi": "10.7554/eLife.60415", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2020-12-24", "volume": "9", "pages": "Art. No. e60415" }, { "id": "authors:99bn3-pg365", "collection": "authors", "collection_id": "99bn3-pg365", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200121-102719123", "type": "article", "title": "Deciphering the regulatory genome of Escherichia coli, one hundred promoters at a time", "author": [ { "family_name": "Ireland", "given_name": "William T.", "orcid": "0000-0003-0971-2904", "clpid": "Ireland-W-T" }, { "family_name": "Beeler", "given_name": "Suzannah M.", "orcid": "0000-0002-1930-4827", "clpid": "Beeler-S-M" }, { "family_name": "Flores-Bautista", "given_name": "Emanuel", "clpid": "Flores-Bautista-E" }, { "family_name": "McCarty", "given_name": "Nicholas S.", "orcid": "0000-0003-4479-284X", "clpid": "McCarty-N-S" }, { "family_name": "R\u00f6schinger", "given_name": "Tom", "orcid": "0000-0002-4900-3216", "clpid": "R\u00f6schinger-T" }, { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-N-M" }, { "family_name": "Sweredoski", "given_name": "Michael J.", "orcid": "0000-0003-0878-3831", "clpid": "Sweredoski-M-J" }, { "family_name": "Moradian", "given_name": "Annie", "orcid": "0000-0002-0407-2031", "clpid": "Moradian-A" }, { "family_name": "Kinney", "given_name": "Justin B.", "orcid": "0000-0003-1897-3778", "clpid": "Kinney-J-B" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for \u2248 65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively-parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than 100 E. coli promoters in 12 growth conditions. We demonstrate that the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulatory information. In many cases, we also identify which transcription factors mediate their regulation. This method clears a path for highly multiplexed investigations of the regulatory genome of model organisms, with the potential of moving to an array of microbes of ecological and medical relevance.", "doi": "10.7554/eLife.55308", "pmcid": "PMC7567609", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2020-09-21", "series_number": "9", "volume": "2020", "issue": "9", "pages": "Art. No. e55308" }, { "id": "authors:5mnm9-ry303", "collection": "authors", "collection_id": "5mnm9-ry303", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190402-080512452", "type": "article", "title": "First-principles prediction of the information processing capacity of a simple genetic circuit", "author": [ { "family_name": "Razo-Mejia", "given_name": "Manuel", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-M" }, { "family_name": "Marzen", "given_name": "Sarah", "orcid": "0000-0001-5386-1101", "clpid": "Marzen-S" }, { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-G" }, { "family_name": "Taubman", "given_name": "Rachel", "orcid": "0000-0002-7779-5185", "clpid": "Taubman-R" }, { "family_name": "Morrison", "given_name": "Muir", "orcid": "0000-0002-0768-7234", "clpid": "Morrison-Muir" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Given the stochastic nature of gene expression, genetically identical cells exposed to the same environmental inputs will produce different outputs. This heterogeneity has been hypothesized to have consequences for how cells are able to survive in changing environments. Recent work has explored the use of information theory as a framework to understand the accuracy with which cells can ascertain the state of their surroundings. Yet the predictive power of these approaches is limited and has not been rigorously tested using precision measurements. To that end, we generate a minimal model for a simple genetic circuit in which all parameter values for the model come from independently published data sets. We then predict the information processing capacity of the genetic circuit for a suite of biophysical parameters such as protein copy number and protein-DNA affinity. We compare these parameter-free predictions with an experimental determination of protein expression distributions and the resulting information processing capacity of E. coli cells. We find that our minimal model captures the scaling of the cell-to-cell variability in the data and the inferred information processing capacity of our simple genetic circuit up to a systematic deviation.", "doi": "10.1103/PhysRevE.102.022404", "issn": "2470-0045", "publisher": "American Physical Society", "publication": "Physical Review E", "publication_date": "2020-08", "series_number": "2", "volume": "102", "issue": "2", "pages": "Art. No. 022404" }, { "id": "authors:3v9sr-9f182", "collection": "authors", "collection_id": "3v9sr-9f182", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191007-074542895", "type": "article", "title": "Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination", "author": [ { "family_name": "Hirokawa", "given_name": "Soichi", "orcid": "0000-0001-5584-2676", "clpid": "Hirokawa-Soichi" }, { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-G" }, { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-N-M" }, { "family_name": "Lovely", "given_name": "Geoffrey A.", "clpid": "Lovely-G-A" }, { "family_name": "Anaya", "given_name": "Michael", "orcid": "0000-0002-6944-3614", "clpid": "Anaya-Michael-A" }, { "family_name": "Schatz", "given_name": "David G.", "orcid": "0000-0002-5669-1176", "clpid": "Schatz-D-G" }, { "family_name": "Baltimore", "given_name": "David", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigen\u2013receptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG\u2013RSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAG\u201312RSS\u201323RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with Ca\u00b2\u207a leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAG\u2013RSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination.", "doi": "10.1093/nar/gkaa418", "pmcid": "PMC7337519", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "2020-07-09", "series_number": "12", "volume": "48", "issue": "12", "pages": "6726-6739" }, { "id": "authors:7b0js-pmt39", "collection": "authors", "collection_id": "7b0js-pmt39", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191202-142214486", "type": "article", "title": "Theoretical investigation of a genetic switch for metabolic adaptation", "author": [ { "family_name": "Laxhuber", "given_name": "Kathrin S.", "orcid": "0000-0002-8104-3525", "clpid": "Laxhuber-K-S" }, { "family_name": "Morrison", "given_name": "Muir J.", "orcid": "0000-0002-0768-7234", "clpid": "Morrison-M-J" }, { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-G" }, { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-N-M" }, { "family_name": "Strandkvist", "given_name": "Charlotte", "clpid": "Strandkvist-C" }, { "family_name": "Naughton", "given_name": "Kyle L.", "clpid": "Naughton-K-L" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Membrane transporters carry key metabolites across the cell membrane and, from a resource standpoint, are hypothesized to be produced when necessary. The expression of membrane transporters in metabolic pathways is often upregulated by the transporter substrate. In E. coli, such systems include for example the lacY, araFGH, and xylFGH genes, which encode for lactose, arabinose, and xylose transporters, respectively. As a case study of a minimal system, we build a generalizable physical model of the xapABR genetic circuit, which features a regulatory feedback loop via membrane transport (positive feedback) and enzymatic degradation (negative feedback) of an inducer. Dynamical systems analysis and stochastic simulations show that the membrane transport makes the model system bistable in certain parameter regimes. Thus, it serves as a genetic \"on-off\" switch, enabling the cell to only produce a set of metabolic enzymes when the corresponding metabolite is present in large amounts. We find that the negative feedback from the degradation enzyme does not significantly disturb the positive feedback from the membrane transporter. We investigate hysteresis in the switching and discuss the role of cooperativity and multiple binding sites in the model circuit. Fundamentally, this work explores how a stable genetic switch for a set of enzymes is obtained from transcriptional auto-activation of a membrane transporter through its substrate.", "doi": "10.1371/journal.pone.0226453", "pmcid": "PMC7205307", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLoS ONE", "publication_date": "2020-05-07", "series_number": "5", "volume": "15", "issue": "5", "pages": "Art. No. e0226453" }, { "id": "authors:rzcf0-hpf84", "collection": "authors", "collection_id": "rzcf0-hpf84", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200331-215659202", "type": "article", "title": "SARS-CoV-2 (COVID-19) by the numbers", "author": [ { "family_name": "Bar-On", "given_name": "Yinon M.", "orcid": "0000-0001-8477-609X", "clpid": "Bar-On-Yinon-M" }, { "family_name": "Flamholz", "given_name": "Avi", "orcid": "0000-0002-9278-5479", "clpid": "Flamholz-Avi" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" } ], "abstract": "The current SARS-CoV-2 pandemic is a harsh reminder of the fact that, whether in a single human host or a wave of infection across continents, viral dynamics is often a story about the numbers. In this snapshot, our aim is to provide a one-stop, curated graphical source for the key numbers that help us understand the virus driving our current global crisis. The discussion is framed around two broad themes: 1) the biology of the virus itself and 2) the characteristics of the infection of a single human host. Our one-page summary provides the key numbers pertaining to SARS-CoV-2, based mostly on peer-reviewed literature. The numbers reported in summary format are substantiated by the annotated references below. Readers are urged to remember that much uncertainty remains and knowledge of this pandemic and the virus driving it is rapidly evolving. In the paragraphs below we provide 'back of the envelope' calculations that exemplify the insights that can be gained from knowing some key numbers and using quantitative logic. These calculations serve to improve our intuition through sanity checks, but do not replace detailed epidemiological analysis.", "doi": "10.7554/elife.57309", "pmcid": "PMC7224694", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2020-03-31", "volume": "9", "pages": "Art. No. e57309" }, { "id": "authors:cwq3v-94138", "collection": "authors", "collection_id": "cwq3v-94138", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200210-085326975", "type": "article", "title": "Kinetic Proofreading using Substrate Gradients and Enzyme Diffusion", "author": [ { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-V" }, { "family_name": "Husain", "given_name": "Kabir", "clpid": "Husain-K" }, { "family_name": "Xiao", "given_name": "Fangzhou", "orcid": "0000-0002-5001-5644", "clpid": "Xiao-Fangzhou" }, { "family_name": "Murugan", "given_name": "Arvind", "clpid": "Murugan-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Low error rates in many of the enzymatic processes in biology, such as translation or DNA replication, are attained by employing the nonequilibrium error correction mechanism called kinetic proofreading. Kinetic proofreading operates by delaying product formation through the introduction of additional biochemical intermediates, giving the enzyme more than one chance to discard the non-cognate substrate before reaching its catalytically active state. The requirements of having multiple biochemically distinct intermediates and direct coupling to NTP hydrolysis, however, limit the flexibility and performance scope of traditional proofreading schemes. In our work, we propose an alternative conceptual mechanism of enzymatic error correction where the necessary delay between initial substrate binding and product formation events is achieved by having them occur at distinct physical locations. Specifically, if the substrates are localized primarily at one end of the physical compartment and enzyme activation takes place only at the other end, then the time that it takes for the enzyme to diffuse the distance between these ends can be leveraged to perform efficient proofreading. Importantly, this mechanism requires neither multiple biochemical intermediates nor the presence of an ATP binding site on the enzyme, making its performance easily adaptable through the tuning of the system's characteristic length scales. We discuss how the tuning of these length scales changes the fidelity, speed and energy dissipation of the mechanism, and propose biological examples of its possible implementation in the cell.", "doi": "10.1016/j.bpj.2019.11.3118", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2020-02-07", "series_number": "3", "volume": "118", "issue": "3", "pages": "573a-574a" }, { "id": "authors:qn9e9-4jm25", "collection": "authors", "collection_id": "qn9e9-4jm25", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200210-095852686", "type": "article", "title": "Genetic Barcodes Enable Quantitative Mapping of Operator Mutants to Gene Expression", "author": [ { "family_name": "McCarty", "given_name": "Nicholas S.", "clpid": "McCarty-N-S" }, { "family_name": "Razo-Mejia", "given_name": "Manuel", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Every organism has intricate regulatory networks that enable them to sense, move, and interact with complex environments. In E. coli, transcription factors (TFs) bind to short sequences upstream of genes, called operators, to repress or activate gene expression. Our lab has previously derived and validated a biophysical model of transcription regulation -the repression of a gene by LacI - demonstrating that repressorcopy number, the energy of TF:operator binding, and genome size all play crucial roles in determining the quantitative features of gene expression. But LacI is a small fish in the cellular pond; we must develop experimental methods that enable us to map how any TF:operator pair imparts predictable gene expression, and do so in a high-throughput manner without sacrificing quantitation. We report a high-throughput method to randomly mutagenize large libraries of operator sequences and quantify their resulting gene expression. Briefly, mutagenized binding sites for a TF are \"mapped\" to a random DNA barcode using next-generation sequencing. The native gene regulated by that TF is then inserted between the mutated operators and barcodes, and these libraries are genomically-integrated into E. coli. The relative abundance of cells carrying each operator mutant can be determined via DNA-sequencing of the barcodes, while their gene expression can be measured by quantitative RNA-sequencing of barcodes using a mixture of Unique Molecular Identifiers and other methods to minimize bias. These data will inform mathematical models for the de novo prediction of gene expression from regulatory sequences.", "doi": "10.1016/j.bpj.2019.11.3298", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2020-02-07", "series_number": "3", "volume": "118", "issue": "3", "pages": "611a" }, { "id": "authors:s9b9v-5r123", "collection": "authors", "collection_id": "s9b9v-5r123", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200210-111806006", "type": "article", "title": "Bridging Kinesin Properties with System-scale Characteristics of Microtubule-Motor Assemblies", "author": [ { "family_name": "Banks", "given_name": "Rachel", "clpid": "Banks-R-A" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Ross", "given_name": "Tyler", "orcid": "0000-0002-7872-3992", "clpid": "Ross-T-D" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "In a dividing cell, multiple subfamilies of kinesin and dynein motors work together to form the mitotic spindle out of microtubule filaments. This complex structure has incited much study into how it is formed and regulated. Knockdown experiments in cells yielded a number of insights, however, the complex environment of the cell prevents a detailed quantitative picture connecting biophysical motor properties to the resulting mesoscopic phenomena. However, in vitro, reconstituted systems of few purified components strip away the other reactions occuring within the cell, creating the opportunity to work from the bottom up. We use an optogenetic system wherein motor interactions are controlled by light to form microtubule structures such as asters. We connect length and speed scales of microtubule structures to motor properties such as speed and processivity by performing experiments with various kinesin motors. With this approach, we develop and test a model explaining how contraction speed varies with network size and motor speed. We also observe how the motors are distributed within microtubule structures and how that varies depending on the motor used. Further, we arrange competition between kinesins that walk towards opposite ends of the microtubules and demonstrate how this competition yields unique structures with properties that depend on both motors. This work is a step towards bridging microscopic motor properties to mesoscopic system-scale characteristics and answering the question of how cooperation between various kinesins can result in the generation of cellular structures.", "doi": "10.1016/j.bpj.2019.11.1081", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2020-02-07", "series_number": "3", "volume": "118", "issue": "3", "pages": "176a-177a" }, { "id": "authors:2ek6r-wqp60", "collection": "authors", "collection_id": "2ek6r-wqp60", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200210-111428049", "type": "article", "title": "Modeling Microbial Interactions across Nutritional Environments using Maximum Entropy", "author": [ { "family_name": "Salmon", "given_name": "Gabe", "clpid": "Salmon-G" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Microbial communities have exponential capacity for complexity. (In principle, among k species, each of (2\u2227k - 1) subsets (every pair, triplet, or larger group up to size k) could modulate the consortium's macroscopic dynamics.) Indeed, recent studies on synthetic communities have argued that high-dimensional (many-species) interactions dominate communal function. Here we explore a contrasting view. Despite the combinatorial potential for multiscale interactions, two features of microbial social life\u2014localized diffusion of intercellular signals and prevalent evolutionary incentives for trophic specialization\u2014provide some physical grounds for relatively low-dimensional behavior. We show how a commonly-used null model used to interpret consortial function can misidentify low-order dynamics as high-dimensional. As an alternative, we develop a mechanism-free model of microbial interactions based on the principle of maximum entropy. The model encodes the effects of potential species interactions via correlated random variables governed by a probability distribution informed by measurements. Our model is statistically-coherent, mathematically-flexible, and compatible with (though not limited to) plausible dynamics of bacterial growth. We test the model using data on microbial growth across carbon sources from a recent droplet microfluidics technology that enables wide sampling over the space of microbial compositions. Over hundreds of synthetic small-scale synthetic communities, our model better generalizes over carbon sources in capturing more nonlinear structure of interactions relative to a set of alternative models. The model's failures identify intriguing ecological behavior. These results highlight the potential predictability of some microbial ecosystems from limited measurements. These ideas also show a framework to identify genuinely emergent interactions in a statistically-principled fashion.", "doi": "10.1016/j.bpj.2019.11.2004", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2020-02-07", "series_number": "3", "volume": "118", "issue": "3", "pages": "347a" }, { "id": "authors:085cp-w5j88", "collection": "authors", "collection_id": "085cp-w5j88", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200210-093408528", "type": "article", "title": "The Molecular Biophysics of Adaptation", "author": [ { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-G" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "From molecular conformations to mass migrations, adaptation is a defining feature of life that is found across time and length scales. Here, we present a theory-experiment dialogue examining how adaptive behavior is manifested at the molecular level in the context of transcriptional regulation in bacteria. Using a statistical mechanical rendering of the Monod-Wyman-Changeux model of allostery, we draw parameter-free predictions of the level of gene expression from a regulated reporter as a function of an extracellular signal across a wide variety of strains. We then derive an expression for the free energy, enabling full data collapse of our experimental measurements onto a single master curve. With this arsenal of analytic ammunition, we then explore how physiological changes (rewiring carbon metabolism and varying temperature) and evolutionary perturbations (mutations with the regulatory protein) tune the level of gene expression and ultimately, the free energy of the system. We demonstrate that the adaptive response of the system can be mapped onto changes in the free energy, providing a set of diagnostic tools that identify which biophysical parameters are modified to yield the observed adaptation by examining the phenomenology of the data alone. These results illustrate that the free energy is the \"natural variable\" of the system and can be treated as a quantitative trait upon which evolutionary forces act, allowing for one to reduce the high-dimensional parameter space to a single-parameter description.", "doi": "10.1016/j.bpj.2019.11.2983", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2020-02-07", "series_number": "3", "volume": "118", "issue": "3", "pages": "545a" }, { "id": "authors:30h7q-80k62", "collection": "authors", "collection_id": "30h7q-80k62", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200210-092745829", "type": "article", "title": "Schrodinger's \"What is Life\" At 75: The Physical Aspects of the Living Cell Revisited", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "In this talk, after a brief review of Schrodinger's essay as a historical document in the rise of modern molecular biology, I will take a different tack and ask to what extent instead his short piece should be seen as a manifesto for the future of physical biology. Schrodinger mused about the need for new ideas from physics to describe biological systems with two primary sets of questions to focus the discussion: how is genetic information transferred from one generation to the next and what gives rise to the reproducible physical structures seen within cells, tissues and organisms? I will argue that to a large extent, these questions remain unanswered from a physics perspective and will describe our own efforts as well as those of many others to understand the rules governing how genomes are regulated and how active matter structures such as the mitotic spindle are organized.", "doi": "10.1016/j.bpj.2019.11.204", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2020-02-07", "series_number": "3", "volume": "118", "issue": "3", "pages": "2a" }, { "id": "authors:vqpdb-45y23", "collection": "authors", "collection_id": "vqpdb-45y23", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191202-151154014", "type": "article", "title": "Allostery and Kinetic Proofreading", "author": [ { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-Vahe" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Kinetic proofreading is an error correction mechanism present in the processes of the central dogma and beyond and typically requires the free energy of nucleotide hydrolysis for its operation. Though the molecular players of many biological proofreading schemes are known, our understanding of how energy consumption is managed to promote fidelity remains incomplete. In our work, we introduce an alternative conceptual scheme called \"the piston model of proofreading\" in which enzyme activation through hydrolysis is replaced with allosteric activation achieved through mechanical work performed by a piston on regulatory ligands. Inspired by Feynman's ratchet and pawl mechanism, we consider a mechanical engine designed to drive the piston actions powered by a lowering weight, whose function is analogous to that of ATP synthase in cells. Thanks to its mechanical design, the piston model allows us to tune the \"knobs\" of the driving engine and probe the graded changes and trade-offs between speed, fidelity, and energy dissipation. It provides an intuitive explanation of the conditions necessary for optimal proofreading and reveals the unexpected capability of allosteric molecules to beat the Hopfield limit of fidelity by leveraging the diversity of states available to them. The framework that we have built for the piston model can also serve as a basis for additional studies of driven biochemical systems.", "doi": "10.1021/acs.jpcb.9b08380", "pmcid": "PMC6995354", "issn": "1520-6106", "publisher": "American Chemical Society", "publication": "Journal of Physical Chemistry B", "publication_date": "2019-12-26", "series_number": "51", "volume": "123", "issue": "51", "pages": "10990-11002" }, { "id": "authors:8mp48-93523", "collection": "authors", "collection_id": "8mp48-93523", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191224-093207952", "type": "monograph", "title": "Physiological Adaptability and Parametric Versatility in a Simple Genetic Circuit", "author": [ { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-G" }, { "family_name": "Kaczmarek", "given_name": "Zofii A.", "orcid": "0000-0002-4887-3121", "clpid": "Kaczmarek-Z-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The intimate relationship between the environment and cellular growth rate has remained a major topic of inquiry in bacterial physiology for over a century. Now, as it becomes possible to understand how the growth rate dictates the wholesale reorganization of the intracellular molecular composition, we can interrogate the biophysical principles underlying this adaptive response. Regulation of gene expression drives this adaptation, with changes in growth rate tied to the activation or repression of genes covering enormous swaths of the genome. Here, we dissect how physiological perturbations alter the expression of a circuit which has been extensively characterized in a single physiological state. Given a complete thermodynamic model, we map changes in physiology directly to the biophysical parameters which define the expression. Controlling the growth rate via modulating the available carbon source or growth temperature, we measure the level of gene expression from a LacI-regulated promoter where the LacI copy number is directly measured in each condition, permitting parameter-free prediction of the expression level. The transcriptional output of this circuit is remarkably robust, with expression of the repressor being largely insensitive to the growth rate. The predicted gene expression quantitatively captures the observations under different carbon conditions, indicating that the bio-physical parameters are indifferent to the physiology. Interestingly, temperature controls the expression level in ways that are inconsistent with the prediction, revealing temperature-dependent effects that challenge current models. This work exposes the strengths and weaknesses of thermodynamic models in fluctuating environments, posing novel challenges and utility in studying physiological adaptation. \n\nSignificance. Cells adapt to changing environmental conditions by repressing or activating gene expression from enormous fractions of their genome, drastically changing the molecular composition of the cell. This requires the concerted adaptation of transcription factors to the environmental signals, leading to binding or releasing of their cognate sequences. Here, we dissect a well characterized genetic circuit in a number of physiological states, make predictions of the response, and measure how the copy number of a regulator and its gene target are affected. We find the parameters defining the regulators behavior are remarkably robust to changes in the nutrient availability, but are susceptible to temperature changes. We quantitatively explore these two effects and discuss how they challenge current models of transcriptional regulation.", "publisher": "Caltech Library", "publication_date": "2019-12-20" }, { "id": "authors:q6jbr-zad07", "collection": "authors", "collection_id": "q6jbr-zad07", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191023-142258650", "type": "article", "title": "Harnessing Avidity: Quantifying the Entropic and Energetic Effects of Linker Length and Rigidity for Multivalent Binding of Antibodies to HIV-1", "author": [ { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-Tal" }, { "family_name": "Yazdi", "given_name": "Shahrzad", "orcid": "0000-0003-1988-4320", "clpid": "Yazdi-Shahrzad" }, { "family_name": "Coey", "given_name": "Aaron", "clpid": "Coey-Aaaron" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "IgG antibodies increase their apparent affinities by using both of their Fabs to simultaneously attach to antigens. HIV-1 foils this strategy by having few, and highly separated, Envelope (Env) spike targets for antibodies, forcing most IgGs to bind monovalently. Here, we develop a statistical mechanics model of synthetic diFabs joined by DNA linkers of different lengths and flexibilities. This framework enables us to translate the energetic and entropic effects of the linker into the neutralization potency of a diFab. We demonstrate that the strongest neutralization potencies are predicted to require a rigid linker that optimally spans the distance between two Fab binding sites on an Env trimer and that avidity can be further boosted by incorporating more Fabs into these constructs. These results inform the design of multivalent anti-HIV-1 therapeutics that utilize avidity effects to remain potent against HIV-1 in the face of the rapid mutation of Env spikes.", "doi": "10.1016/j.cels.2019.09.007", "pmcid": "PMC6892280", "issn": "2405-4712", "publisher": "Cell Press", "publication": "Cell Systems", "publication_date": "2019-11-27", "series_number": "5", "volume": "9", "issue": "5", "pages": "466-474" }, { "id": "authors:c9geb-11h23", "collection": "authors", "collection_id": "c9geb-11h23", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190826-153157241", "type": "article", "title": "Predictive shifts in free energy couple mutations to their phenotypic consequences", "author": [ { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-G" }, { "family_name": "Razo-Mejia", "given_name": "Manuel", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-M" }, { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-N-M" }, { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-T" }, { "family_name": "Kaczmarek", "given_name": "Zofii A.", "orcid": "0000-0002-4887-3121", "clpid": "Kaczmarek-Z-A" }, { "family_name": "Barnes", "given_name": "Stephanie L.", "orcid": "0000-0002-5237-603X", "clpid": "Barnes-S-L" }, { "family_name": "Lewis", "given_name": "Mitchell", "clpid": "Lewis-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find that the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.", "doi": "10.1073/pnas.1907869116", "pmcid": "PMC6744869", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2019-09-10", "series_number": "37", "volume": "116", "issue": "37", "pages": "18275-18284" }, { "id": "authors:kp5yc-avm39", "collection": "authors", "collection_id": "kp5yc-avm39", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190102-092232993", "type": "article", "title": "Controlling Organization and Forces in Active Matter Through Optically-Defined Boundaries", "author": [ { "family_name": "Ross", "given_name": "Tyler D.", "orcid": "0000-0002-7872-3992", "clpid": "Ross-Tyler-D" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Qu", "given_name": "Zijie", "orcid": "0000-0003-1500-3207", "clpid": "Qu-Zijie" }, { "family_name": "Banks", "given_name": "Rachel A.", "orcid": "0000-0003-2028-2925", "clpid": "Banks-Rachel-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" } ], "abstract": "Living systems are capable of locomotion, reconfiguration and replication. To perform these tasks, cells spatiotemporally coordinate the interactions of force-generating, 'active' molecules that create and manipulate non-equilibrium structures and force fields of up to millimetre length scales. Experimental active-matter systems of biological or synthetic molecules are capable of spontaneously organizing into structures and generating global flows. However, these experimental systems lack the spatiotemporal control found in cells, limiting their utility for studying non-equilibrium phenomena and bioinspired engineering. Here we uncover non-equilibrium phenomena and principles of boundary-mediated control by optically modulating structures and fluid flow in an engineered system of active biomolecules. Our system consists of purified microtubules and light-activatable motor proteins that crosslink and organize the microtubules into distinct structures upon illumination. We develop basic operations\u2014defined as sets of light patterns\u2014to create, move and merge the microtubule structures. By combining these operations, we create microtubule networks that span several hundred micrometres in length and contract at speeds up to an order of magnitude higher than the speed of an individual motor protein. We manipulate these contractile networks to generate and sculpt persistent fluid flows. The principles of boundary-mediated control that we uncover may be used to study emergent cellular structures and forces and to develop programmable active-matter devices.", "doi": "10.1038/s41586-019-1447-1", "pmcid": "PMC6719720", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2019-08-08", "series_number": "7768", "volume": "572", "issue": "7768", "pages": "224-229" }, { "id": "authors:2gxy3-7a647", "collection": "authors", "collection_id": "2gxy3-7a647", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190405-094702005", "type": "article", "title": "How the avidity of polymerase binding to the \u201335/\u201310 promoter sites affects gene expression", "author": [ { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-Tal" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Although the key promoter elements necessary to drive transcription in Escherichia coli have long been understood, we still cannot predict the behavior of arbitrary novel promoters, hampering our ability to characterize the myriad sequenced regulatory architectures as well as to design new synthetic circuits. This work builds upon a beautiful recent experiment by Urtecho et al. [G. Urtecho, et al., Biochemistry, 68, 1539\u20131551 (2019)] who measured the gene expression of over 10,000 promoters spanning all possible combinations of a small set of regulatory elements. Using these data, we demonstrate that a central claim in energy matrix models of gene expression\u2014that each promoter element contributes independently and additively to gene expression\u2014contradicts experimental measurements. We propose that a key missing ingredient from such models is the avidity between the \u201335 and \u201310 RNA polymerase binding sites and develop what we call a multivalent model that incorporates this effect and can successfully characterize the full suite of gene expression data. We explore several applications of this framework, namely, how multivalent binding at the \u201335 and \u201310 sites can buffer RNA polymerase (RNAP) kinetics against mutations and how promoters that bind overly tightly to RNA polymerase can inhibit gene expression. The success of our approach suggests that avidity represents a key physical principle governing the interaction of RNA polymerase to its promoter.", "doi": "10.1073/pnas.1905615116", "pmcid": "PMC6613100", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2019-07-02", "series_number": "27", "volume": "116", "issue": "27", "pages": "13340-13345" }, { "id": "authors:2evqa-s3c27", "collection": "authors", "collection_id": "2evqa-s3c27", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190529-152829578", "type": "article", "title": "Torque-dependent remodeling of the bacterial flagellar motor", "author": [ { "family_name": "Wadhwa", "given_name": "Navish", "clpid": "Wadhwa-Navish" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Berg", "given_name": "Howard C.", "clpid": "Berg-Howard-C" } ], "abstract": "Multisubunit protein complexes are ubiquitous in biology and perform a plethora of essential functions. Most of the scientific literature treats such assemblies as static: their function is assumed to be independent of their manner of assembly, and their structure is assumed to remain intact until they are degraded. Recent observations of the bacterial flagellar motor, among others, bring these notions into question. The torque-generating stator units of the motor assemble and disassemble in response to changes in load. Here, we used electrorotation to drive tethered cells forward, which decreases motor load, and measured the resulting stator dynamics. No disassembly occurred while the torque remained high, but all of the stator units were released when the motor was spun near the zero-torque speed. When the electrorotation was turned off, so that the load was again high, stator units were recruited, increasing motor speed in a stepwise fashion. A model in which speed affects the binding rate and torque affects the free energy of bound stator units captures the observed torque-dependent stator assembly dynamics, providing a quantitative framework for the environmentally regulated self-assembly of a major macromolecular machine.", "doi": "10.1073/pnas.1904577116", "pmcid": "PMC6576217", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2019-06-11", "series_number": "24", "volume": "116", "issue": "24", "pages": "11764-11769" }, { "id": "authors:j1eym-8np70", "collection": "authors", "collection_id": "j1eym-8np70", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190611-161448645", "type": "monograph", "title": "Defining the Energetic Costs of Cellular Structures", "author": [ { "family_name": "Mahmoudabadi", "given_name": "Gita", "orcid": "0000-0002-8812-7246", "clpid": "Mahmoudabadi-Gita" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Lynch", "given_name": "Michael", "clpid": "Lynch-Michael" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" } ], "abstract": "All cellular structures are assembled from molecular building blocks, and molecular building blocks incur energetic costs to the cell. In an energy-limited environment, the energetic cost of a cellular structure imposes a fitness cost and impacts a cell's evolutionary trajectory. While the importance of energetic considerations was realized for decades, the distinction between direct energetic costs expended by the cell and potential energy that the cell diverts into cellular biomass components, which we define as the opportunity cost, was not explicitly made, leading to large differences in values for energetic costs of molecular building blocks used in the literature. We describe a framework that defines and separates various components relevant for estimating the energetic costs of molecular building blocks and the resulting cellular structures. This distinction among energetic costs is an essential step towards discussing the conversion of an energetic cost to a corresponding fitness cost.", "doi": "10.1101/666040", "publication_date": "2019-06-10" }, { "id": "authors:y47b9-pmn34", "collection": "authors", "collection_id": "y47b9-pmn34", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190516-074953419", "type": "monograph", "title": "The Energetics of Molecular Adaptation in Transcriptional Regulation", "author": [ { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-G" }, { "family_name": "Razo-Mejia", "given_name": "Manuel", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-M" }, { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-N-M" }, { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-T" }, { "family_name": "Kaczmarek", "given_name": "Zofii A.", "orcid": "0000-0002-4887-3121", "clpid": "Kaczmarek-Z-A" }, { "family_name": "Barnes", "given_name": "Stephanie L.", "orcid": "0000-0002-5237-603X", "clpid": "Barnes-S-L" }, { "family_name": "Lewis", "given_name": "Mitchell", "clpid": "Lewis-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only a subset of the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions.", "doi": "10.48550/arXiv.1905.06360", "publication_date": "2019-05-15" }, { "id": "authors:fyyyd-cyr53", "collection": "authors", "collection_id": "fyyyd-cyr53", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190520-085650396", "type": "article", "title": "Figure 1 Theory Meets Figure 2 Experiments in the Study of Gene Expression", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-N-M" }, { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-G" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Razo-Mejia", "given_name": "Manuel", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-M" }, { "family_name": "Scholes", "given_name": "Clarissa", "clpid": "Scholes-C" } ], "abstract": "It is tempting to believe that we now own the genome. The ability to read and rewrite it at will has ushered in a stunning period in the history of science. Nonetheless, there is an Achilles' heel exposed by all of the genomic data that has accrued: We still do not know how to interpret them. Many genes are subject to sophisticated programs of transcriptional regulation, mediated by DNA sequences that harbor binding sites for transcription factors, which can up- or down-regulate gene expression depending upon environmental conditions. This gives rise to an input\u2013output function describing how the level of expression depends upon the parameters of the regulated gene\u2014for instance, on the number and type of binding sites in its regulatory sequence. In recent years, the ability to make precision measurements of expression, coupled with the ability to make increasingly sophisticated theoretical predictions, has enabled an explicit dialogue between theory and experiment that holds the promise of covering this genomic Achilles' heel. The goal is to reach a predictive understanding of transcriptional regulation that makes it possible to calculate gene expression levels from DNA regulatory sequence. This review focuses on the canonical simple repression motif to ask how well the models that have been used to characterize it actually work. We consider a hierarchy of increasingly sophisticated experiments in which the minimal parameter set learned at one level is applied to make quantitative predictions at the next. We show that these careful quantitative dissections provide a template for a predictive understanding of the many more complex regulatory arrangements found across all domains of life.", "doi": "10.1146/annurev-biophys-052118-115525", "issn": "1936-122X", "publisher": "Annual Reviews", "publication": "Annual Review of Biophysics", "publication_date": "2019-05", "volume": "48", "pages": "121-163" }, { "id": "authors:cz0xm-bfr24", "collection": "authors", "collection_id": "cz0xm-bfr24", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190102-135256302", "type": "article", "title": "Combinatorial Control through Allostery", "author": [ { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-Vahe" }, { "family_name": "Funk", "given_name": "Luke", "clpid": "Funk-Luke" }, { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-Tal" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Many instances of cellular signaling and transcriptional regulation involve switch-like molecular responses to the presence or absence of input ligands. To understand how these responses come about and how they can be harnessed, we develop a statistical mechanical model to characterize the types of Boolean logic that can arise from allosteric molecules following the Monod\u2013Wyman\u2013Changeux (MWC) model. Building upon previous work, we show how an allosteric molecule regulated by two inputs can elicit AND, OR, NAND, and NOR responses but is unable to realize XOR or XNOR gates. Next, we demonstrate the ability of an MWC molecule to perform ratiometric sensing\u2014a response behavior where activity depends monotonically on the ratio of ligand concentrations. We then extend our analysis to more general schemes of combinatorial control involving either additional binding sites for the two ligands or an additional third ligand and show how these additions can cause a switch in the logic behavior of the molecule. Overall, our results demonstrate the wide variety of control schemes that biological systems can implement using simple mechanisms.", "doi": "10.1021/acs.jpcb.8b12517", "pmcid": "PMC6467274", "issn": "1520-6106", "publisher": "American Chemical Society", "publication": "Journal of Physical Chemistry B", "publication_date": "2019-04-04", "series_number": "13", "volume": "123", "issue": "13", "pages": "2792-2800" }, { "id": "authors:sketg-0qt49", "collection": "authors", "collection_id": "sketg-0qt49", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190311-132254175", "type": "article", "title": "Measuring the Energetic Costs of Embryonic Development", "author": [ { "family_name": "Foster", "given_name": "Peter J.", "clpid": "Foster-P-J" }, { "family_name": "Razo-Mejia", "given_name": "Manuel", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "What are the thermodynamic costs of development? In this issue of Developmental Cell, Rodenfels et al. (2019) demonstrate that the high energetic cost of coordinated cell division that is regulated by phospho-signaling gives rise to a measurable periodicity in the heat dissipated during zebrafish embryogenesis.", "doi": "10.1016/j.devcel.2019.02.016", "issn": "1534-5807", "publisher": "Cell Press", "publication": "Developmental Cell", "publication_date": "2019-03-11", "series_number": "5", "volume": "48", "issue": "5", "pages": "591-592" }, { "id": "authors:fd4mj-eqg74", "collection": "authors", "collection_id": "fd4mj-eqg74", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190207-155651541", "type": "article", "title": "Microtubule End-Clustering Maintains a Steady-State Spindle Shape", "author": [ { "family_name": "Hueschen", "given_name": "Christina L.", "clpid": "Hueschen-C-L" }, { "family_name": "Galstyan", "given_name": "Vahe", "orcid": "0000-0001-7073-9175", "clpid": "Galstyan-V" }, { "family_name": "Amouzgar", "given_name": "Meelad", "clpid": "Amouzgar-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Dumont", "given_name": "Sophie", "clpid": "Dumont-S" } ], "abstract": "Each time a cell divides, the microtubule cytoskeleton self-organizes into the metaphase spindle: an ellipsoidal steady-state structure that holds its stereotyped geometry despite microtubule turnover and internal stresses [1, 2, 3, 4, 5, 6]. Regulation of microtubule dynamics, motor proteins, microtubule crosslinking, and chromatid cohesion can modulate spindle size and shape, and yet modulated spindles reach and hold a new steady state [7, 8, 9, 10, 11]. Here, we ask what maintains any spindle steady-state geometry. We report that clustering of microtubule ends by dynein and NuMA is essential for mammalian spindles to hold a steady-state shape. After dynein or NuMA deletion, the mitotic microtubule network is \"turbulent\"; microtubule bundles extend and bend against the cell cortex, constantly remodeling network shape. We find that spindle turbulence is driven by the homotetrameric kinesin-5 Eg5, and that acute Eg5 inhibition in turbulent spindles recovers spindle geometry and stability. Inspired by in vitro work on active turbulent gels of microtubules and kinesin [12, 13], we explore the kinematics of this in vivo turbulent network. We find that turbulent spindles display decreased nematic order and that motile asters distort the nematic director field. Finally, we see that turbulent spindles can drive both flow of cytoplasmic organelles and whole-cell movement\u2014analogous to the autonomous motility displayed by droplet-encapsulated turbulent gels [12]. Thus, end-clustering by dynein and NuMA is required for mammalian spindles to reach a steady-state geometry, and in their absence Eg5 powers a turbulent microtubule network inside mitotic cells.", "doi": "10.1016/j.cub.2019.01.016", "pmcid": "PMC6383811", "issn": "0960-9822", "publisher": "Cell Press", "publication": "Current Biology", "publication_date": "2019-02-18", "series_number": "4", "volume": "29", "issue": "4", "pages": "700-708" }, { "id": "authors:5jkgs-7fr18", "collection": "authors", "collection_id": "5jkgs-7fr18", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20181030-132608570", "type": "article", "title": "Mapping DNA sequence to transcription factor binding energy in vivo", "author": [ { "family_name": "Barnes", "given_name": "Stephanie L.", "orcid": "0000-0002-5237-603X", "clpid": "Barnes-S-L" }, { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-N-M" }, { "family_name": "Ireland", "given_name": "William T.", "orcid": "0000-0003-0971-2904", "clpid": "Ireland-W-T" }, { "family_name": "Kinney", "given_name": "Justin B.", "orcid": "0000-0003-1897-3778", "clpid": "Kinney-J-B" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Despite the central importance of transcriptional regulation in biology, it has proven difficult to determine the regulatory mechanisms of individual genes, let alone entire gene networks. It is particularly difficult to decipher the biophysical mechanisms of transcriptional regulation in living cells and determine the energetic properties of binding sites for transcription factors and RNA polymerase. In this work, we present a strategy for dissecting transcriptional regulatory sequences using in vivo methods (massively parallel reporter assays) to formulate quantitative models that map a transcription factor binding site's DNA sequence to transcription factor-DNA binding energy. We use these models to predict the binding energies of transcription factor binding sites to within 1 k_BT of their measured values. We further explore how such a sequence-energy mapping relates to the mechanisms of trancriptional regulation in various promoter contexts. Specifically, we show that our models can be used to design specific induction responses, analyze the effects of amino acid mutations on DNA sequence preference, and determine how regulatory context affects a transcription factor's sequence specificity.", "doi": "10.1371/journal.pcbi.1006226", "pmcid": "PMC6375646", "issn": "1553-734X", "publisher": "Public Library of Science", "publication": "PLoS Computational Biology", "publication_date": "2019-02-04", "series_number": "2", "volume": "15", "issue": "2", "pages": "Art. No. e1006226" }, { "id": "authors:2mpqj-ev319", "collection": "authors", "collection_id": "2mpqj-ev319", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190110-132257326", "type": "monograph", "title": "Human Phageprints: A high-resolution exploration of oral phages reveals globally-distributed phage families with individual-specific and temporally-stable community compositions", "author": [ { "family_name": "Mahmoudabadi", "given_name": "Gita", "orcid": "0000-0002-8812-7246", "clpid": "Mahmoudabadi-Gita" }, { "family_name": "Homyk", "given_name": "Kelsey", "clpid": "Homyk-Kelsey" }, { "family_name": "Catching", "given_name": "Adam", "clpid": "Catching-Adam" }, { "family_name": "Foley", "given_name": "Helen", "clpid": "Foley-Helen" }, { "family_name": "Tadmor", "given_name": "Arbel", "clpid": "Tadmor-A-D" }, { "family_name": "Mahmoudabadi", "given_name": "Ana", "clpid": "Mahmoudabadi-Ana" }, { "family_name": "Cheung", "given_name": "Allison", "clpid": "Cheung-Allison" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Metagenomic studies have revolutionized the study of novel phages. However these studies trade the depth of coverage for breadth. In this study we show that the targeted sequencing of a phage genomic region as small as 200-300 base pairs, can provide sufficient sequence diversity to serve as an individual-specific barcode or Phageprint. The targeted approach reveals a high-resolution view of phage communities that is not available through metagenomic datasets. By creating instructional videos and collection kits, we enabled citizen scientists to gather ~700 oral samples spanning ~100 individuals residing in different parts of the world. In examining phage communities at 6 different oral sites, and by comparing phage communities of individuals living across the globe, we were able to study the effect of spatial separation, ranging from several millimeters to thousands of kilometers. We found that the spatial separation of just a few centimeters (the distance between two oral sites) can already result in highly distinct phage community compositions. For larger distances, spanning the phage communities of different individuals living in different parts of the world, we did not observe any correlation between spatial distance and phage community composition as individuals residing in the same city did not have any more similar phage communities than individuals living on different continents. Additionally, we found that neither genetics nor cohabitation seem to play a role in the relatedness of phage community compositions across individuals. Cohabitating siblings and even identical twins did not have phage community compositions that were any more similar than those of unrelated individuals. The primary factor contributing to phage community composition relatedness is direct contact between two habitats, as is demonstrated by the similarity between oral phage community compositions of partners. Furthermore, by exploring phage communities across the span of a month, and in some cases several years, we observed highly stable community compositions. These studies consistently point to the existence of remarkably diverse and personal phage families that are stable in time and apparently present in people around the world.", "doi": "10.1101/516864", "publication_date": "2019-01-10" }, { "id": "authors:r5nqq-m9640", "collection": "authors", "collection_id": "r5nqq-m9640", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20181128-093526476", "type": "article", "title": "Measuring cis-regulatory energetics in living cells using allelic manifolds", "author": [ { "family_name": "Forcier", "given_name": "Talitha L.", "clpid": "Forcier-T-L" }, { "family_name": "Ayaz", "given_name": "Andalus", "clpid": "Ayaz-A" }, { "family_name": "Gill", "given_name": "Manraj S.", "clpid": "Gill-M-S" }, { "family_name": "Jones", "given_name": "Daniel", "orcid": "0000-0002-7902-996X", "clpid": "Jones-D-L" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Kinney", "given_name": "Justin B.", "orcid": "0000-0003-1897-3778", "clpid": "Kinney-J-B" } ], "abstract": "Gene expression in all organisms is controlled by cooperative interactions between DNA-bound transcription factors (TFs), but quantitatively measuring TF-DNA and TF-TF interactions remains difficult. Here we introduce a strategy for precisely measuring the Gibbs free energy of such interactions in living cells. This strategy centers on the measurement and modeling of 'allelic manifolds', a multidimensional generalization of the classical genetics concept of allelic series. Allelic manifolds are measured using reporter assays performed on strategically designed cis-regulatory sequences. Quantitative biophysical models are then fit to the resulting data. We used this strategy to study regulation by two Escherichia coliTFs, CRP and \u03c3^(70) RNA polymerase. Doing so, we consistently obtained energetic measurements precise to \u223c0.1 kcal/mol. We also obtained multiple results that deviate from the prior literature. Our strategy is compatible with massively parallel reporter assays in both prokaryotes and eukaryotes, and should therefore be highly scalable and broadly applicable.", "doi": "10.7554/eLife.40618", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2018-12-20", "volume": "7", "pages": "Art. No. e40618" }, { "id": "authors:ndy38-a2a63", "collection": "authors", "collection_id": "ndy38-a2a63", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20181128-093526917", "type": "monograph", "title": "Modeling Entropic and Energetic Effects of Linker Length and Rigidity within Synthetic HIV-1 Antibodies designed to Bind Bivalently to Env Spikes", "author": [ { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-T" }, { "family_name": "Gnanapragasam", "given_name": "Priyanthi N. P.", "clpid": "Gnanapragasam-P-N-P" }, { "family_name": "Yazdi", "given_name": "Shahrzad", "clpid": "Yazdi-S" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "abstract": "Due to the low density of envelope (Env) spikes on the surface of HIV-1, neutralizing IgG antibodies rarely bind bivalently using both antigen-binding arms (Fabs) to crosslink between spikes (inter-spike crosslinking), instead resorting to weaker monovalent binding that is more sensitive to Env mutations. Antibodies capable of bivalently binding within single Env trimers (intra-spike crosslinking) should exhibit increased neutralization potencies. Here we consider synthetic diFabs joined by varying lengths of rigid double-stranded DNA (dsDNA). We asked whether linkers with different rigidities could enhance diFab potency by modeling DNA-Fabs containing different combinations of rigid dsDNA and flexible single-stranded DNA (ssDNA) and created a model that predicts their neutralization potencies. Model predictions showed that although a long flexible polymer may be capable of bivalent binding, it exhibits weak neutralization due to the large loss in entropic degrees of freedom during bivalent binding. In contrast, the strongest neutralization potencies were predicted to require a rigid linker that optimally spanned the distance between two Fab binding sites on an Env trimer. These results inform the design of bivalent anti-HIV-1 therapeutics that utilize avidity effects to remain potent against HIV-1 in the face of the rapid mutation of Env spikes.", "doi": "10.1101/406454", "publication_date": "2018-11-12" }, { "id": "authors:0kefq-g9a55", "collection": "authors", "collection_id": "0kefq-g9a55", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180917-125104010", "type": "article", "title": "Connecting the dots between mechanosensitive channel abundance, osmotic shock, and survival at single-cell resolution", "author": [ { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-G" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Rasmussen", "given_name": "Akiko", "clpid": "Rasmussen-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Rapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks, Escherichia coli and other microbes express several types of transmembrane channels that open and close in response to changes in membrane tension. In E. coli, one of the most abundant channels is the mechanosensitive channel of large conductance (MscL). While this channel has been heavily characterized through structural methods, electrophysiology, and theoretical modeling, our understanding of its physiological role in preventing cell death by alleviating high membrane tension remains tenuous. In this work, we examine the contribution of MscL alone to cell survival after osmotic shock at single-cell resolution using quantitative fluorescence microscopy. We conducted these experiments in an E. coli strain which is lacking all mechanosensitive channel genes save for MscL, whose expression was tuned across 3 orders of magnitude through modifications of the Shine-Dalgarno sequence. While theoretical models suggest that only a few MscL channels would be needed to alleviate even large changes in osmotic pressure, we find that between 500 and 700 channels per cell are needed to convey upwards of 80% survival. This number agrees with the average MscL copy number measured in wild-type E. coli cells through proteomic studies and quantitative Western blotting. Furthermore, we observed zero survival events in cells with fewer than \u223c100 channels per cell. This work opens new questions concerning the contribution of other mechanosensitive channels to survival, as well as regulation of their activity.", "doi": "10.1128/jb.00460-18", "pmcid": "PMC6222198", "issn": "0021-9193", "publisher": "American Society for Microbiology", "publication": "Journal of Bacteriology", "publication_date": "2018-11", "series_number": "23", "volume": "200", "issue": "23", "pages": "Art. No. e00460-18" }, { "id": "authors:8apf9-6sm71", "collection": "authors", "collection_id": "8apf9-6sm71", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20181009-105623284", "type": "article", "title": "Theoretical analysis of inducer and operator binding for cyclic-AMP receptor protein mutants", "author": [ { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-T" }, { "family_name": "Duque", "given_name": "Julia", "clpid": "Duque-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Allosteric transcription factors undergo binding events at inducer binding sites as well as at distinct DNA binding domains, and it is difficult to disentangle the structural and functional consequences of these two classes of interactions. We compare the ability of two statistical mechanical models\u2014the Monod-Wyman-Changeux (MWC) and the Koshland-N\u00e9methy-Filmer (KNF) models of protein conformational change\u2014to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. We use these models to examine a beautiful recent experiment that created a single-chain version of the CRP homodimer, creating six mutants using all possible combinations of the wild type, D53H, and S62F subunits. We demonstrate that the MWC model can explain the behavior of all six mutants using a small, self-consistent set of parameters whose complexity scales with the number of subunits, providing a significant benefit over previous models. In comparison, the KNF model not only leads to a poorer characterization of the available data but also fails to generate parameter values in line with the available structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.", "doi": "10.1371/journal.pone.0204275", "pmcid": "PMC6157895", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLoS ONE", "publication_date": "2018-09-26", "series_number": "9", "volume": "13", "issue": "9", "pages": "Art. No. e0204275" }, { "id": "authors:n5gev-b2594", "collection": "authors", "collection_id": "n5gev-b2594", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200117-131810813", "type": "monograph", "title": "Harnessing Avidity: Quantifying Entropic and Energetic Effects of Linker Length and Rigidity Required for Multivalent Binding of Antibodies to HIV-1 Spikes", "author": [ { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-T" }, { "family_name": "Yazdi", "given_name": "Shahrzad", "clpid": "Yazdi-S" }, { "family_name": "Coey", "given_name": "Aaron", "clpid": "Coey-A" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Due to the low density of envelope (Env) spikes on the surface of HIV-1, neutralizing IgG antibodies rarely bind bivalently using both antigen-binding arms (Fabs) to crosslink between spikes (inter-spike crosslinking), instead resorting to weaker monovalent binding that is more sensitive to Env mutations. Synthetic antibodies designed to bivalently bind a single Env trimer (intra-spike crosslinking) were previously shown to exhibit increased neutralization potencies. In initial work, diFabs joined by varying lengths of rigid double-stranded DNA (dsDNA) were considered. Anticipating future experiments to improve synthetic antibodies, we investigate whether linkers with different rigidities could enhance diFab potency by modeling DNA-Fabs containing different combinations of rigid dsDNA and flexible single-stranded DNA (ssDNA) and characterizing their neutralization potential. Model predictions suggest that while a long flexible polymer may be capable of bivalent binding, it exhibits weak neutralization due to the large loss in entropic degrees of freedom when both Fabs are bound. In contrast, the strongest neutralization potencies are predicted to require a rigid linker that optimally spans the distance between two Fab binding sites on an Env trimer, and avidity can be further boosted by incorporating more Fabs into these constructs. These results inform the design of multivalent anti-HIV-1 therapeutics that utilize avidity effects to remain potent against HIV-1 in the face of the rapid mutation of Env spikes.", "doi": "10.1101/406454", "publication_date": "2018-09-03" }, { "id": "authors:cqapn-bg741", "collection": "authors", "collection_id": "cqapn-bg741", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180927-114224268", "type": "monograph", "title": "Quantifying the Entropic and Energetic Effects of Linker Length and Rigidity within Synthetic HIV-1 Antibodies designed to Bind Bivalently to Env Spikes", "author": [ { "family_name": "Galimidi", "given_name": "Rachel P.", "clpid": "Galimidi-Rachel-P" }, { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-T" }, { "family_name": "Gnanapragasam", "given_name": "Priyanthi N. P.", "clpid": "Gnanapragasam-Priyanthi-N-P" }, { "family_name": "Joshi", "given_name": "Devashish S.", "clpid": "Joshi-D-S" }, { "family_name": "Lynch", "given_name": "Anne M.", "clpid": "Lynch-A-M" }, { "family_name": "Yazdi", "given_name": "Shahrzad", "clpid": "Yazdi-S" }, { "family_name": "West", "given_name": "Anthony P., Jr.", "clpid": "West-A-P-Jr" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "abstract": "Due to the low density of envelope (Env) spikes on the surface of HIV-1, neutralizing IgG antibodies rarely bind bivalently using both antigen-binding arms (Fabs) to crosslink between spikes, instead resorting to weaker monovalent binding that is more sensitive to Env mutations. Synthetic antibodies capable of bivalently binding within single Env trimers (intra-spike crosslinking) were previously shown to exhibit increased neutralization potencies. In the initial work, diFabs joined by varying lengths of rigid double-stranded DNA (dsDNA) were considered. Here we investigated whether linkers with different rigidities could enhance diFab potency by synthesizing DNA-Fabs containing different combinations of rigid dsDNA and flexible single-stranded DNA (ssDNA) and created a model that predicts their neutralization potencies. Model predictions, verified by experimental data, show that although a long flexible polymer may be capable of bivalent binding, it exhibits weak neutralization due to the large loss in entropic degrees of freedom during bivalent binding. In contrast, the strongest neutralization potencies require a rigid linker that optimally spans the distance between two Fab binding sites on an Env trimer. These results inform the design of bivalent anti-HIV-1 therapeutics that utilize avidity effects to remain potent against HIV-1 in the face of the rapid mutation of Env spikes.", "doi": "10.1101/406454", "publication_date": "2018-09-03" }, { "id": "authors:djxd4-dcc69", "collection": "authors", "collection_id": "djxd4-dcc69", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180927-114222775", "type": "monograph", "title": "Precision measurement of cis-regulatory energetics in living cells", "author": [ { "family_name": "Forcier", "given_name": "Talitha", "clpid": "Forcier-T" }, { "family_name": "Ayaz", "given_name": "Andalus", "clpid": "Ayaz-A" }, { "family_name": "Gill", "given_name": "Manraj S.", "clpid": "Gill-M-S" }, { "family_name": "Jones", "given_name": "Daniel", "orcid": "0000-0002-7902-996X", "clpid": "Jones-D-L" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Kinney", "given_name": "Justin B.", "orcid": "0000-0003-1897-3778", "clpid": "Kinney-J-B" } ], "abstract": "Gene expression in all organisms is controlled by cooperative interactions between DNA-bound transcription factors (TFs). However, measuring TF-TF interactions that occur at individual cis-regulatory sequences remains difficult. Here we introduce a strategy for precisely measuring the Gibbs free energy of such interactions in living cells. Our strategy uses reporter assays performed on strategically designed cis-regulatory sequences, together with a biophysical modeling approach we call \"expression manifolds\". We applied this strategy in Escherichia coli to interactions between two paradigmatic TFs: CRP and RNA polymerase (RNAP). Doing so, we consistently obtain measurements precise to ~0.1 kcal/mol. Unexpectedly, CRP-RNAP interactions are seen to deviate in multiple ways from the prior literature. Moreover, the well-known RNAP binding motif is found to be a surprisingly unreliable predictor of RNAP-DNA binding energy. Our strategy is compatible with massively parallel reporter assays in both prokaryotes and eukaryotes, and should thus be highly scalable and broadly applicable.", "doi": "10.1101/380972", "publication_date": "2018-07-31" }, { "id": "authors:cgg81-yrq25", "collection": "authors", "collection_id": "cgg81-yrq25", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180521-155526693", "type": "article", "title": "The biomass distribution on Earth", "author": [ { "family_name": "Bar-On", "given_name": "Yinon M.", "orcid": "0000-0001-8477-609X", "clpid": "Bar-On-Yinon-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" } ], "abstract": "A census of the biomass on Earth is key for understanding the structure and dynamics of the biosphere. However, a global, quantitative view of how the biomass of different taxa compare with one another is still lacking. Here, we assemble the overall biomass composition of the biosphere, establishing a census of the \u2248550 gigatons of carbon (Gt C) of biomass distributed among all of the kingdoms of life. We find that the kingdoms of life concentrate at different locations on the planet; plants (\u2248450 Gt C, the dominant kingdom) are primarily terrestrial, whereas animals (\u22482 Gt C) are mainly marine, and bacteria (\u224870 Gt C) and archaea (\u22487 Gt C) are predominantly located in deep subsurface environments. We show that terrestrial biomass is about two orders of magnitude higher than marine biomass and estimate a total of \u22486 Gt C of marine biota, doubling the previous estimated quantity. Our analysis reveals that the global marine biomass pyramid contains more consumers than producers, thus increasing the scope of previous observations on inverse food pyramids. Finally, we highlight that the mass of humans is an order of magnitude higher than that of all wild mammals combined and report the historical impact of humanity on the global biomass of prominent taxa, including mammals, fish, and plants.", "doi": "10.1073/pnas.1711842115", "pmcid": "PMC6016768", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2018-06-19", "series_number": "25", "volume": "115", "issue": "25", "pages": "6506-6511" }, { "id": "authors:xdtgs-vwm72", "collection": "authors", "collection_id": "xdtgs-vwm72", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180507-083945479", "type": "article", "title": "Systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria", "author": [ { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-Nathan-M" }, { "family_name": "Barnes", "given_name": "Stephanie L.", "orcid": "0000-0002-5237-603X", "clpid": "Barnes-Stephanie-L" }, { "family_name": "Ireland", "given_name": "William T.", "orcid": "0000-0003-0971-2904", "clpid": "Ireland-William-T" }, { "family_name": "Jones", "given_name": "Daniel L.", "orcid": "0000-0002-7902-996X", "clpid": "Jones-Daniel-L" }, { "family_name": "Sweredoski", "given_name": "Michael J.", "orcid": "0000-0003-0878-3831", "clpid": "Sweredoski-Michael-J" }, { "family_name": "Moradian", "given_name": "Annie", "orcid": "0000-0002-0407-2031", "clpid": "Moradian-Annie" }, { "family_name": "Hess", "given_name": "Sonja", "orcid": "0000-0002-5904-9816", "clpid": "Hess-Sonja" }, { "family_name": "Kinney", "given_name": "Justin B.", "orcid": "0000-0003-1897-3778", "clpid": "Kinney-Justin-B" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Gene regulation is one of the most ubiquitous processes in biology. However, while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. At present, characterizing the molecular mechanisms by which individual regulatory sequences operate requires focused efforts using low-throughput methods. Here, we take a first step toward multipromoter dissection and show how a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect multiple bacterial promoters in a systematic way. We show this approach on both well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all cases, we recover nucleotide-resolution models of promoter mechanism. For some promoters, including previously unannotated ones, the approach allowed us to further extract quantitative biophysical models describing input\u2013output relationships. Given the generality of the approach presented here, it opens up the possibility of quantitatively dissecting the mechanisms of promoter function in E. coli and a wide range of other bacteria.", "doi": "10.1073/pnas.1722055115", "pmcid": "PMC6003448", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2018-05-22", "series_number": "21", "volume": "115", "issue": "21", "pages": "E4796-E4805" }, { "id": "authors:w6zry-9qr04", "collection": "authors", "collection_id": "w6zry-9qr04", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171204-090149985", "type": "article", "title": "Tuning transcriptional regulation through signaling: A predictive theory of allosteric induction", "author": [ { "family_name": "Razo-Mejia", "given_name": "Manuel", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-Manuel" }, { "family_name": "Barnes", "given_name": "Stephanie L.", "orcid": "0000-0002-5237-603X", "clpid": "Barnes-Stephanie-L" }, { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-Nathan-M" }, { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-Griffin-D" }, { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-Tal" }, { "family_name": "Lewis", "given_name": "Mitchell", "clpid": "Lewis-Mitchell" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Allosteric regulation is found across all domains of life, yet we still lack simple, predictive theories that directly link the experimentally tunable parameters of a system to its input-output response. To that end, we present a general theory of allosteric transcriptional regulation using the Monod-Wyman-Changeux model. We rigorously test this model using the ubiquitous simple repression motif in bacteria by first predicting the behavior of strains that span a large range of repressor copy numbers and DNA binding strengths and then constructing and measuring their response. Our model not only accurately captures the induction profiles of these strains, but also enables us to derive analytic expressions for key properties such as the dynamic range and [EC_(50)]. Finally, we derive an expression for the free energy of allosteric repressors that enables us to collapse our experimental data onto a single master curve that captures the diverse phenomenology of the induction profiles.", "doi": "10.1016/j.cels.2018.02.004", "pmcid": "PMC5991102", "issn": "2405-4712", "publisher": "Cell Press", "publication": "Cell Systems", "publication_date": "2018-04-25", "series_number": "4", "volume": "6", "issue": "4", "pages": "456-469" }, { "id": "authors:nn1ws-mbd79", "collection": "authors", "collection_id": "nn1ws-mbd79", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180409-140949316", "type": "article", "title": "Research: A comprehensive and quantitative exploration of thousands of viral genomes", "author": [ { "family_name": "Mahmoudabadi", "given_name": "Gita", "orcid": "0000-0002-8812-7246", "clpid": "Mahmoudabadi-Gita" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The complete assembly of viral genomes from metagenomic datasets (short genomic sequences gathered from environmental samples) has proven to be challenging, so there are significant blind spots when we view viral genomes through the lens of metagenomics. One approach to overcoming this problem is to leverage the thousands of complete viral genomes that are publicly available. Here we describe our efforts to assemble a comprehensive resource that provides a quantitative snapshot of viral genomic trends \u2013 such as gene density, noncoding percentage, and abundances of functional gene categories \u2013 across thousands of viral genomes. We have also developed a coarse-grained method for visualizing viral genome organization for hundreds of genomes at once, and have explored the extent of the overlap between bacterial and bacteriophage gene pools. Existing viral classification systems were developed prior to the sequencing era, so we present our analysis in a way that allows us to assess the utility of the different classification systems for capturing genomic trends.", "doi": "10.7554/eLife.31955", "pmcid": "PMC5908442", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2018-04-06", "volume": "7", "pages": "Art. No. e31955" }, { "id": "authors:ckvn6-1qd95", "collection": "authors", "collection_id": "ckvn6-1qd95", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180501-112937401", "type": "article", "title": "Two-Stage Dynamics of In Vivo Bacteriophage Genome Ejection", "author": [ { "family_name": "Chen", "given_name": "Yi-Ju", "clpid": "Chen-Yi-Ju" }, { "family_name": "Wu", "given_name": "David", "clpid": "Wu-David" }, { "family_name": "Gelbart", "given_name": "William", "clpid": "Gelbart-W-M" }, { "family_name": "Knobler", "given_name": "Charles M.", "clpid": "Knobler-C-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Kegel", "given_name": "Willem K.", "clpid": "Kegel-W-K" } ], "abstract": "Biopolymer translocation is a key step in viral infection processes. The transfer of information-encoding genomes allows viruses to reprogram the cell fate of their hosts. Constituting 96% of all known bacterial viruses [A. Fokine and M.\u2009G. Rossmann, Molecular architecture of tailed double-stranded DNA phages, Bacteriophage 4, e28281 (2014)], the tailed bacteriophages deliver their DNA into host cells via an \"ejection\" process, leaving their protein shells outside of the bacteria; a similar scenario occurs for mammalian viruses like herpes, where the DNA genome is ejected into the nucleus of host cells, while the viral capsid remains bound outside to a nuclear-pore complex. In light of previous experimental measurements of in vivo bacteriophage \u03bb ejection, we analyze here the physical processes that give rise to the observed dynamics. We propose that, after an initial phase driven by self-repulsion of DNA in the capsid, the ejection is driven by anomalous diffusion of phage DNA in the crowded bacterial cytoplasm. We expect that this two-step mechanism is general for phages that operate by pressure-driven ejection, and we discuss predictions of our theory to be tested in future experiments.", "doi": "10.1103/PhysRevX.8.021029", "issn": "2160-3308", "publisher": "American Physical Society", "publication": "Physical Review X", "publication_date": "2018-04", "series_number": "2", "volume": "8", "issue": "2", "pages": "Art. No. 021029" }, { "id": "authors:b1jg4-4v262", "collection": "authors", "collection_id": "b1jg4-4v262", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180510-124207785", "type": "article", "title": "Skewing the Playing Field: A Single-Molecule Study on how RSS Sequence Influences Gene Segment Selection", "author": [ { "family_name": "Hirokawa", "given_name": "Soichi", "orcid": "0000-0001-5584-2676", "clpid": "Hirokawa-Soichi" }, { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-N-M" }, { "family_name": "Lovely", "given_name": "Geoffrey A.", "clpid": "Lovely-G-A" }, { "family_name": "Anaya", "given_name": "Michael", "orcid": "0000-0002-6944-3614", "clpid": "Anaya-Michael-A" }, { "family_name": "Schatz", "given_name": "David G.", "clpid": "Schatz-David-G" }, { "family_name": "Baltimore", "given_name": "David", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "V(D)J recombination, the cut-and-paste process that combines various antibody-encoding gene segments, provides jawed vertebrates with a combinatorically diverse arsenal of unique antibodies that allows the organism to identify virtually any invading bacterium or infected cell. To initiate this process, the RAG enzymatic complex binds two recognizable recombination signal sequences (RSSs) neighboring the gene segments and subsequently cleaves the DNA to expose the segments before additional enzymes join the two ends to create a continuous antibody-encoding gene. While the general process is well understood, the root causes for the non-uniform distribution of gene segment selection have not been clearly parsed out. In this project, we examine how the sequence diversity of RSSs affect RAG's affinity for forming paired complexes and its propensity for cleaving the DNA, the two key steps for passing two gene segments onward to generate an antibody-encoding gene. Using a single-molecule method known as tethered particle motion (TPM), we systemically study a range of RSS sequences, from single mutation deviations away from the consensus sequence to endogenous RSSs, to determine that RSS sequence is a significant determinant for whether a given gene segment in any antibody gene loci will be selected to produce an antibody. We expect that this work will set us on an initial path to better understanding how the dynamic nature of the genome influences the likelihood that a lymphocyte will produce a particular antibody.", "doi": "10.1016/j.bpj.2017.11.511", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2018-02-02", "series_number": "3", "volume": "114", "issue": "3", "pages": "86a" }, { "id": "authors:st2zx-zc567", "collection": "authors", "collection_id": "st2zx-zc567", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180523-152301882", "type": "article", "title": "Theoretical Analysis of Allosteric and Operator Binding for Cyclic-AMP Receptor Protein Mutants", "author": [ { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-T" }, { "family_name": "Duque", "given_name": "Julia", "clpid": "Duque-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Allosteric transcription factors undergo binding events both at their operator binding sites as well as at distinct allosteric sites, and it is often difficult to disentangle the structural and functional consequences of the two types of binding. In this work, we compare the ability of two statistical mechanical models \u2013 the Monod-Wyman-Changeux (MWC) and the Koshland-N\u00e9methy-Filmer (KNF) models of allostery \u2013 to characterize the multi-step activation mechanism of the cyclic-AMP receptor protein (CRP). We first analyze the allosteric transition, where cyclic-AMP binds to CRP, then model how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. We examine data from a recent beautiful experiment that created a single-chain version of the CRP homodimer, thereby enabling each subunit to be mutated separately. Using this construct, six mutants were created using every possible combination of the wild type subunit, a D53H mutant subunit, and an S62F mutant subunit. We show that both the MWC and KNF models can simultaneously characterize the cyclic-AMP and DNA binding of all six CRP constructs based solely on their subunit compositions, thereby tying together the behavior of the mutants to a small, self-consistent set of parameters.", "doi": "10.1016/j.bpj.2017.11.2330", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2018-02-02", "series_number": "3", "volume": "114", "issue": "3", "pages": "420A-421A" }, { "id": "authors:4964v-yks16", "collection": "authors", "collection_id": "4964v-yks16", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180510-092357051", "type": "article", "title": "Membrane Tension and the Charge State of Cells", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Many of the ways that cells talk and listen to the external world center on the presence of proteins on the cell surface. Indeed, the cell membrane is an amazingly diverse lipid environment, riddled in turn with a host of different proteins that perform tasks ranging from sensing and measuring chemical signals to the transport of sugars needed for cell division to the detection of potentially lethal osmotic pressures. This talk will focus on recent progress in the dissection of the mechanisms of mechanosensation in bacteria with special reference to the rich interplay between certain classes of ion channels and the surrounding lipids and how that interplay sculpts the response of bacteria to osmotic insults.", "doi": "10.1016/j.bpj.2017.11.203", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2018-02-02", "series_number": "3", "volume": "114", "issue": "3", "pages": "29a" }, { "id": "authors:nvs56-pp489", "collection": "authors", "collection_id": "nvs56-pp489", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180523-143351877", "type": "article", "title": "A Systematic and Scalable Approach for Dissecting the Molecular Mechanisms of Transcriptional Regulation in Bacteria", "author": [ { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-N-M" }, { "family_name": "Barnes", "given_name": "Stephanie L.", "orcid": "0000-0002-5237-603X", "clpid": "Barnes-S-L" }, { "family_name": "Ireland", "given_name": "William T.", "orcid": "0000-0003-0971-2904", "clpid": "Ireland-W-T" }, { "family_name": "Beeler", "given_name": "Suzannah M.", "orcid": "0000-0002-1930-4827", "clpid": "Beeler-S-M" }, { "family_name": "Kinney", "given_name": "Justin B.", "orcid": "0000-0003-1897-3778", "clpid": "Kinney-J-B" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Organisms across all domains of life must make regulatory decisions in response to changing environments. The decision about when and where to turn on transcription in bacteria is mainly controlled through the binding of transcription factors to promoter regions of the DNA. However, while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. Even for the organism Escherichia coli, whose regulation is arguably best understood, we still have no indication if or how more than half of the genes are regulated. Here we show how a combination of massively-parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect bacterial promoters. We use the approach to recover nucleotide-resolution models of promoter mechanism for a variety of well-characterized and previously unannotated promoters in E. coli. We believe this approach opens up the possibility of quantitatively dissecting the mechanisms of promoter function across the genome of E. coli and other bacteria as well.", "doi": "10.1016/j.bpj.2017.11.849", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2018-02-02", "series_number": "3", "volume": "114", "issue": "3", "pages": "151A" }, { "id": "authors:09b86-mx316", "collection": "authors", "collection_id": "09b86-mx316", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171128-123653098", "type": "article", "title": "The role of DNA sequence in nucleosome breathing", "author": [ { "family_name": "Culkin", "given_name": "Jamie", "clpid": "Culkin-Jamie" }, { "family_name": "de Bruin", "given_name": "Lennart", "clpid": "de-Bruin-Lennart" }, { "family_name": "Tompitak", "given_name": "Marco", "orcid": "0000-0003-4275-2262", "clpid": "Tompitak-Marco" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Schiessel", "given_name": "Helmut", "orcid": "0000-0001-5040-6488", "clpid": "Schiessel-Helmut" } ], "abstract": "Roughly 3/4 of human genomes are sequestered by nucleosomes, DNA spools with a protein core, dictating a broad range of biological processes, ranging from gene regulation, recombination, and replication, to chromosome condensation. Nucleosomes are dynamical structures and temporarily expose wrapped DNA through spontaneous unspooling from either end, a process called site exposure or nucleosome breathing. Here we ask how this process is influenced by the mechanical properties of the wrapped DNA, which is known to depend on the underlying base pair sequence. Using a coarse-grained nucleosome model we calculate the accessibility profiles for site exposure. We find that the process is very sensitive to sequence effects, so that evolution could potentially tune the accessibility of nucleosomal DNA and would only need a small number of mutations to do so.", "doi": "10.1140/epje/i2017-11596-2", "pmcid": "PMC7001874", "issn": "1292-8941", "publisher": "Springer", "publication": "European Physical Journal E", "publication_date": "2017-11", "series_number": "11", "volume": "40", "issue": "11", "pages": "Art. No. 106" }, { "id": "authors:g8dca-m8d69", "collection": "authors", "collection_id": "g8dca-m8d69", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171009-100434563", "type": "article", "title": "Musings on mechanism: quest for a quark theory of proteins?", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The give and take between biology and physics is an important part of the history of modern science, with this partnership perhaps now more intimate than ever. In this essay, I explore the ways in which these different fields can strengthen each other through their distinct outlooks on some of the most important questions being asked about the living world. In particular, I highlight three areas where the perspective from physics might lead to deeper insights into the workings of cells. First, I consider what it means for two ostensibly unrelated problems to be the same, and how such sameness can provide unexpected insights into apparently unrelated phenomena. Second, I consider different conceptions of what constitutes a mechanistic understanding of a given phenomenon with an emphasis on nonmolecular notions of mechanism. The third idea is the importance of \"toy problems\" as a way of providing foundational insights into the real problems. Though my focus here is primarily on ways in which physics approaches might prove interesting in biology, I close with an example of how biology might substantially alter physics by providing a forum and the tools to uncover a fundamental understanding of nonequilibrium phenomena.\u2014Phillips, R. Musings on mechanism: quest for a quark theory of proteins?", "doi": "10.1096/fj.201700594", "issn": "0892-6638", "publisher": "Federation of American Societies for Experimental Biology", "publication": "FASEB Journal", "publication_date": "2017-10", "series_number": "10", "volume": "31", "issue": "10", "pages": "4207-4215" }, { "id": "authors:ngdk4-1a462", "collection": "authors", "collection_id": "ngdk4-1a462", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170717-090138223", "type": "article", "title": "Self-consistent theory of transcriptional control in complex regulatory architectures", "author": [ { "family_name": "Landman", "given_name": "Jasper", "clpid": "Landman-J" }, { "family_name": "Brewster", "given_name": "Robert C.", "clpid": "Brewster-R-C" }, { "family_name": "Weinert", "given_name": "Franz M.", "clpid": "Weinert-F-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Kegel", "given_name": "Willem K.", "clpid": "Kegel-W-K" } ], "abstract": "Individual regulatory proteins are typically charged with the simultaneous regulation of a battery of different genes. As a result, when one of these proteins is limiting, competitive effects have a significant impact on the transcriptional response of the regulated genes. Here we present a general framework for the analysis of any generic regulatory architecture that accounts for the competitive effects of the regulatory environment by isolating these effects into an effective concentration parameter. These predictions are formulated using the grand-canonical ensemble of statistical mechanics and the fold-change in gene expression is predicted as a function of the number of transcription factors, the strength of interactions between the transcription factors and their DNA binding sites, and the effective concentration of the transcription factor. The effective concentration is set by the transcription factor interactions with competing binding sites within the cell and is determined self-consistently. Using this approach, we analyze regulatory architectures in the grand-canonical ensemble ranging from simple repression and simple activation to scenarios that include repression mediated by DNA looping of distal regulatory sites. It is demonstrated that all the canonical expressions previously derived in the case of an isolated, non-competing gene, can be generalised by a simple substitution to their grand canonical counterpart, which allows for simple intuitive incorporation of the influence of multiple competing transcription factor binding sites. As an example of the strength of this approach, we build on these results to present an analytical description of transcriptional regulation of the lac operon.", "doi": "10.1371/journal.pone.0179235", "pmcid": "PMC5501422", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLOS ONE", "publication_date": "2017-07", "series_number": "7", "volume": "12", "issue": "7", "pages": "Art. No. e0179235" }, { "id": "authors:wv62b-avv82", "collection": "authors", "collection_id": "wv62b-avv82", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170516-095417507", "type": "article", "title": "Energetic cost of building a virus", "author": [ { "family_name": "Mahmoudabadi", "given_name": "Gita", "orcid": "0000-0002-8812-7246", "clpid": "Mahmoudabadi-Gita" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Viruses are incapable of autonomous energy production. Although many experimental studies make it clear that viruses are parasitic entities that hijack the molecular resources of the host, a detailed estimate for the energetic cost of viral synthesis is largely lacking. To quantify the energetic cost of viruses to their hosts, we enumerated the costs associated with two very distinct but representative DNA and RNA viruses, namely, T4 and influenza. We found that, for these viruses, translation of viral proteins is the most energetically expensive process. Interestingly, the costs of building a T4 phage and a single influenza virus are nearly the same. Due to influenza's higher burst size, however, the overall cost of a T4 phage infection is only 2\u20133% of the cost of an influenza infection. The costs of these infections relative to their host's estimated energy budget during the infection reveal that a T4 infection consumes about a third of its host's energy budget, whereas an influenza infection consumes only \u2248 1%. Building on our estimates for T4, we show how the energetic costs of double-stranded DNA phages scale with the capsid size, revealing that the dominant cost of building a virus can switch from translation to genome replication above a critical size. Last, using our predictions for the energetic cost of viruses, we provide estimates for the strengths of selection and genetic drift acting on newly incorporated genetic elements in viral genomes, under conditions of energy limitation.", "doi": "10.1073/pnas.1701670114", "pmcid": "PMC5465929", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2017-05-30", "series_number": "22", "volume": "114", "issue": "22", "pages": "E4324-E4333" }, { "id": "authors:f36gb-far86", "collection": "authors", "collection_id": "f36gb-far86", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170131-092637914", "type": "article", "title": "Monod-Wyman-Changeux Analysis of Ligand-Gated Ion Channel Mutants", "author": [ { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-Tal" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "We present a framework for computing the gating properties of ligand-gated ion channel mutants using the Monod-Wyman-Changeux (MWC) model of allostery. We derive simple analytic formulas for key functional properties such as the leakiness, dynamic range, half-maximal effective concentration ([EC_(50)]), and effective Hill coefficient, and explore the full spectrum of phenotypes that are accessible through mutations. Specifically, we consider mutations in the channel pore of nicotinic acetylcholine receptor (nAChR) and the ligand binding domain of a cyclic nucleotide-gated (CNG) ion channel, demonstrating how each mutation can be characterized as only affecting a subset of the biophysical parameters. In addition, we show how the unifying perspective offered by the MWC model allows us, perhaps surprisingly, to collapse the plethora of dose-response data from different classes of ion channels into a universal family of curves.", "doi": "10.1021/acs.jpcb.6b12672", "pmcid": "PMC5551692", "issn": "1520-6106", "publisher": "American Chemical Society", "publication": "Journal of Physical Chemistry B", "publication_date": "2017-04-20", "series_number": "15", "volume": "121", "issue": "15", "pages": "3813-3824" }, { "id": "authors:b03gp-07610", "collection": "authors", "collection_id": "b03gp-07610", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180730-091900653", "type": "monograph", "title": "Membranes by the Numbers", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Many of the most important processes in cells take place on and across membranes. With the rise of an impressive array of powerful quantitative methods for characterizing these membranes, it is an opportune time to reflect on the structure and function of membranes from the point of view of biological numeracy. To that end, in this article, I review the quantitative parameters that characterize the mechanical, electrical and transport properties of membranes and carry out a number of corresponding order of magnitude estimates that help us understand the values of those parameters.", "doi": "10.48550/arXiv.1703.02066", "publication_date": "2017-03-06" }, { "id": "authors:yr6t3-8cg20", "collection": "authors", "collection_id": "yr6t3-8cg20", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170619-150117855", "type": "monograph", "title": "An allosteric theory of transcription factor induction", "author": [ { "family_name": "Razo-Mejia", "given_name": "Manuel", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-M" }, { "family_name": "Barnes", "given_name": "Stephanie L.", "orcid": "0000-0002-5237-603X", "clpid": "Barnes-S-L" }, { "family_name": "Belliveau", "given_name": "Nathan M.", "orcid": "0000-0002-1536-1963", "clpid": "Belliveau-N-M" }, { "family_name": "Chure", "given_name": "Griffin", "orcid": "0000-0002-2216-2057", "clpid": "Chure-G" }, { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-T" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Allosteric molecules serve as regulators of cellular activity across all domains of life. We present a general theory of allosteric transcriptional regulation that permits quantitative predictions for how physiological responses are tuned to environmental stimuli. To test the model's predictive power, we apply it to the specific case of the ubiquitous simple repression motif in bacteria. We measure the fold-change in gene expression at different inducer concentrations in a collection of strains that span a range of repressor copy numbers and operator binding strengths. After inferring the inducer dissociation constants using data from one of these strains, we show the broad reach of the model by predicting the induction profiles of all other strains. Finally, we derive an expression for the free energy of allosteric transcription factors which enables us to collapse the data from all of our experiments onto a single master curve, capturing the diverse phenomenology of the induction profiles.", "doi": "10.1101/111013", "publication_date": "2017-02-22" }, { "id": "authors:yd05z-nh425", "collection": "authors", "collection_id": "yd05z-nh425", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170331-134057165", "type": "article", "title": "The great ideas of biology: Exploration through experimentation in an undergraduate lab course", "author": [ { "family_name": "Finch", "given_name": "L.", "clpid": "Finch-L" }, { "family_name": "Horii", "given_name": "C. V.", "orcid": "0000-0001-8609-1430", "clpid": "Horii-Cassandra-Volpe" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Bois", "given_name": "J. S.", "orcid": "0000-0001-7137-8746", "clpid": "Bois-J-S" } ], "abstract": "We developed an introductory laboratory course to provide a visceral experience that aims at getting students truly excited about scientific study of the living world. Our vehicle to do that was to focus on what Paul Nurse dubbed \"the great ideas of biology\" rather than an approach to biology that celebrates specific factual knowledge. To that end, we developed eight diverse experimental \nmodules, each of which highlights a key biological concept and gives an opportunity to use theory to generate testable hypotheses, to perform high quality measurements to test those hypotheses (some of which are clearly wrong), and to perform sophisticated computational data analysis. Some \nmodules incorporate modern microscopy and computational techniques in classic experiments, such as bacterial growth and the Luria\u2010Delbr\u00fcck experiment, while others address current research questions using methods like optogenetics and single molecule measurements. We have offered the course eight times, and in the most recent edition of the course, we conducted pre/post\u2010course interviews and \nattitude surveys. The students, both bio and non\u2010bio majors alike, reported being captivated by seeing life occur across the broad range of experiments and model organisms. We observed demonstrable development of their curiosity and enthusiasm for biology. Additionally, we found that \nprior to the course, students had only vague notions about what it means to make quantitative biological measurements and interpret them. They completed the course with a clearer understanding of scientific inquiry in biology and the skills and confidence to actually perform and interpret measurements in living systems.", "issn": "1059-1524", "publisher": "American Society for Cell Biology", "publication": "Molecular Biology of the Cell", "publication_date": "2016-12", "volume": "27", "pages": "Art. No. P830" }, { "id": "authors:ps3y5-mg111", "collection": "authors", "collection_id": "ps3y5-mg111", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160502-112340784", "type": "article", "title": "Statistical Mechanics of Allosteric Enzymes", "author": [ { "family_name": "Einav", "given_name": "Tal", "orcid": "0000-0003-0777-1193", "clpid": "Einav-Tal" }, { "family_name": "Mazutis", "given_name": "Linas", "orcid": "0000-0002-5552-6427", "clpid": "Mazutis-Linas" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The concept of allostery in which macromolecules switch between two different conformations is a central theme in biological processes ranging from gene regulation to cell signaling to enzymology. Allosteric enzymes pervade metabolic processes, yet a simple and unified treatment of the effects of allostery in enzymes has been lacking. In this work, we take a step toward this goal by modeling allosteric enzymes and their interaction with two key molecular players\u2014allosteric regulators and competitive inhibitors. We then apply this model to characterize existing data on enzyme activity, comment on how enzyme parameters (such as substrate binding affinity) can be experimentally tuned, and make novel predictions on how to control phenomena such as substrate inhibition.", "doi": "10.1021/acs.jpcb.6b01911", "pmcid": "PMC5452729", "issn": "1520-6106", "publisher": "American Chemical Society", "publication": "Journal of Physical Chemistry B", "publication_date": "2016-07-07", "series_number": "26", "volume": "120", "issue": "26", "pages": "6021-6037" }, { "id": "authors:21me8-m0c96", "collection": "authors", "collection_id": "21me8-m0c96", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160509-153942313", "type": "article", "title": "Design Principles of Length Control of Cytoskeletal Structures", "author": [ { "family_name": "Mohapatra", "given_name": "Lishibanya", "orcid": "0000-0003-4332-1675", "clpid": "Mohapatra-Lishibanya" }, { "family_name": "Goode", "given_name": "Bruce L.", "orcid": "0000-0002-6443-5893", "clpid": "Goode-Bruce-L" }, { "family_name": "Jelenkovic", "given_name": "Predrag", "clpid": "Jelenkovic-Predrag" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Kondev", "given_name": "Jane", "orcid": "0000-0001-7522-7144", "clpid": "Kondev-Jane" } ], "abstract": "Cells contain elaborate and interconnected networks of protein polymers, which make up the cytoskeleton. The cytoskeleton governs the internal positioning and movement of vesicles and organelles and controls dynamic changes in cell polarity, shape, and movement. Many of these processes require tight control of the size and shape of cytoskeletal structures, which is achieved despite rapid turnover of their molecular components. Here we review mechanisms by which cells control the size of filamentous cytoskeletal structures, from the point of view of simple quantitative models that take into account stochastic dynamics of their assembly and disassembly. Significantly, these models make experimentally testable predictions that distinguish different mechanisms of length control. Although the primary focus of this review is on cytoskeletal structures, we believe that the broader principles and mechanisms discussed herein will apply to a range of other subcellular structures whose sizes are tightly controlled and are linked to their functions.", "doi": "10.1146/annurev-biophys-070915-094206", "pmcid": "PMC5466818", "issn": "1936-122X", "publisher": "Annual Reviews", "publication": "Annual Review of Biophysics", "publication_date": "2016-07", "volume": "45", "pages": "85-116" }, { "id": "authors:h0jy4-95736", "collection": "authors", "collection_id": "h0jy4-95736", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160311-132630114", "type": "article", "title": "Using synthetic biology to make cells tomorrow's test tubes", "author": [ { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Brewster", "given_name": "Robert C.", "clpid": "Brewster-R-C" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The main tenet of physical biology is that biological phenomena can be subject to the same quantitative and predictive understanding that physics has afforded in the context of inanimate matter. However, the inherent complexity of many of these biological processes often leads to the derivation of complex theoretical descriptions containing a plethora of unknown parameters. Such complex descriptions pose a conceptual challenge to the establishment of a solid basis for predictive biology. In this article, we present various exciting examples of how synthetic biology can be used to simplify biological systems and distill these phenomena down to their essential features as a means to enable their theoretical description. Here, synthetic biology goes beyond previous efforts to engineer nature and becomes a tool to bend nature to understand it. We discuss various recent and classic experiments featuring applications of this synthetic approach to the elucidation of problems ranging from bacteriophage infection, to transcriptional regulation in bacteria and in developing embryos, to evolution. In all of these examples, synthetic biology provides the opportunity to turn cells into the equivalent of a test tube, where biological phenomena can be reconstituted and our theoretical understanding put to test with the same ease that these same phenomena can be studied in the in vitro setting.", "doi": "10.1039/C6IB00006A", "pmcid": "PMC4837077", "issn": "1757-9694", "publisher": "Royal Society of Chemistry", "publication": "Integrative Biology", "publication_date": "2016-04", "series_number": "4", "volume": "8", "issue": "4", "pages": "431-450" }, { "id": "authors:yz49a-pec73", "collection": "authors", "collection_id": "yz49a-pec73", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160321-083721510", "type": "article", "title": "SnapShot: Timescales in Cell Biology", "author": [ { "family_name": "Shamir", "given_name": "Maya", "clpid": "Shamir-Maya" }, { "family_name": "Bar-On", "given_name": "Yinon", "orcid": "0000-0001-8477-609X", "clpid": "Bar-On-Y-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" } ], "abstract": "Knowing key timescales enables us to quickly gain intuition, perform sanity checks and serves as a sixth sense in understanding how cells grow and communicate. This SnapShot briefly communicates some of the insights we collected about rates and durations of these processes from thinking about cell biology by the numbers.", "doi": "10.1016/j.cell.2016.02.058", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "2016-03-10", "series_number": "6", "volume": "164", "issue": "6", "pages": "1302-1302.e1" }, { "id": "authors:e6etk-g5t77", "collection": "authors", "collection_id": "e6etk-g5t77", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160106-065143620", "type": "article", "title": "Predicting the impact of promoter variability on regulatory outputs", "author": [ { "family_name": "Kreamer", "given_name": "Naomi N.", "clpid": "Kreamer-Naomi-N-K" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Boedicker", "given_name": "James Q.", "orcid": "0000-0003-4107-3719", "clpid": "Boedicker-J-Q" } ], "abstract": "The increased availability of whole genome sequences calls for quantitative models of global gene expression, yet predicting gene expression patterns directly from genome sequence remains a challenge. We examine the contributions of an individual regulator, the ferrous iron-responsive regulatory element, BqsR, on global patterns of gene expression in Pseudomonas aeruginosa. The position weight matrix (PWM) derived for BqsR uncovered hundreds of likely binding sites throughout the genome. Only a subset of these potential binding sites had a regulatory consequence, suggesting that BqsR/DNA interactions were not captured within the PWM or that the broader regulatory context at each promoter played a greater role in setting promoter outputs. The architecture of the BqsR operator was systematically varied to understand how binding site parameters influence expression. We found that BqsR operator affinity was predicted by the PWM well. At many promoters the surrounding regulatory context, including overlapping operators of BqsR or the presence of RhlR binding sites, were influential in setting promoter outputs. These results indicate more comprehensive models that include local regulatory contexts are needed to develop a predictive understanding of global regulatory outputs.", "doi": "10.1038/srep18238", "pmcid": "PMC4682146", "issn": "2045-2322", "publisher": "Nature Publishing Group", "publication": "Scientific Reports", "publication_date": "2015-12-17", "volume": "5", "pages": "Art. No. 18238" }, { "id": "authors:cdrkf-jd020", "collection": "authors", "collection_id": "cdrkf-jd020", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20151130-110111581", "type": "article", "title": "Theory in Biology: Figure 1 or Figure 7?", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The pace of modern science is staggering. The quantities of data now flowing from DNA sequencers, fluorescence and electron microscopes, mass spectrometers, and other mind-blowing instruments leave us faced with information overload. This explosion in data has brought on its heels a concomitant need for efforts at the kinds of synthesis and unification we see in theoretical physics. Often in cell biology, when theoretical modeling takes place, it is as a figure 7 reflection on experiments that have already been done, with data fitting providing a metric of success. Figure 1 theory, by way of contrast, is about living dangerously by turning our thinking into formal mathematical predictions and confronting that math with experiments that have not yet been done.", "doi": "10.1016/j.tcb.2015.10.007", "pmcid": "PMC4666001", "issn": "0962-8924", "publisher": "Elsevier", "publication": "Trends in Cell Biology", "publication_date": "2015-12", "series_number": "12", "volume": "25", "issue": "12", "pages": "723-729" }, { "id": "authors:h1kda-88177", "collection": "authors", "collection_id": "h1kda-88177", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150810-075722055", "type": "article", "title": "Interplay of Protein Binding Interactions, DNA Mechanics, and Entropy in DNA Looping Kinetics", "author": [ { "family_name": "Mulligan", "given_name": "Peter J.", "clpid": "Mulligan-P-J" }, { "family_name": "Chen", "given_name": "Yi-Ju", "clpid": "Chen-Yi-Ju" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Spakowitz", "given_name": "Andrew J.", "orcid": "0000-0002-0585-1942", "clpid": "Spakowitz-A-J" } ], "abstract": "DNA looping plays a key role in many fundamental biological processes, including gene regulation, recombination, and chromosomal organization. The looping of DNA is often mediated by proteins whose structural features and physical interactions can alter the length scale at which the looping occurs. Looping and unlooping processes are controlled by thermodynamic contributions associated with mechanical deformation of the DNA strand and entropy arising from thermal fluctuations of the conformation. To determine how these confounding effects influence DNA looping and unlooping kinetics, we present a theoretical model that incorporates the role of the protein interactions, DNA mechanics, and conformational entropy. We show that for shorter DNA strands the interaction distance affects the transition state, resulting in a complex relationship between the looped and unlooped state lifetimes and the physical properties of the looped DNA. We explore the range of behaviors that arise with varying interaction distance and DNA length. These results demonstrate how DNA deformation and entropy dictate the scaling of the looping and unlooping kinetics versus the J-factor, establishing the connection between kinetic and equilibrium behaviors. Our results show how the twist-and-bend elasticity of the DNA chain modulates the kinetics and how the influence of the interaction distance fades away at intermediate to longer chain lengths, in agreement with previous scaling predictions.", "doi": "10.1016/j.bpj.2015.06.054", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2015-08-04", "series_number": "3", "volume": "109", "issue": "3", "pages": "618-629" }, { "id": "authors:1jbdr-6e168", "collection": "authors", "collection_id": "1jbdr-6e168", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150324-125537305", "type": "article", "title": "Single-molecule analysis of RAG-mediated V(D)J DNA cleavage", "author": [ { "family_name": "Lovely", "given_name": "Geoffrey A.", "clpid": "Lovely-Geoffrey-A" }, { "family_name": "Brewster", "given_name": "Robert C.", "clpid": "Brewster-Robert-C" }, { "family_name": "Schatz", "given_name": "David G.", "clpid": "Schatz-David-G" }, { "family_name": "Baltimore", "given_name": "David", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed single-molecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only \u223c40% of observed synapses resulting in cleavage at consensus RSS binding sites.", "doi": "10.1073/pnas.1503477112", "pmcid": "PMC4394307", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2015-04-07", "series_number": "14", "volume": "112", "issue": "14", "pages": "E1715-E1723" }, { "id": "authors:nfv7t-72584", "collection": "authors", "collection_id": "nfv7t-72584", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150409-132742605", "type": "article", "title": "Napoleon Is in Equilibrium", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "It has been said that the cell is the test tube of the twenty-first century. If so, the theoretical tools needed to quantitatively and predictively describe what goes on in such test tubes lag sorely behind the stunning experimental advances in biology seen in the decades since the molecular biology revolution began. Perhaps surprisingly, one of the theoretical tools that has been used with great success on problems ranging from how cells communicate with their environment and each other to the nature of the organization of proteins and lipids within the cell membrane is statistical mechanics. A knee-jerk reaction to the use of statistical mechanics in the description of cellular processes is that living organisms are so far from equilibrium that one has no business even thinking about it. But such reactions are probably too hasty given that there are many regimes in which, because of a separation of timescales, for example, such an approach can be a useful first step. In this article, we explore the power of statistical mechanical thinking in the biological setting, with special emphasis on cell signaling and regulation. We show how such models are used to make predictions and describe some recent experiments designed to test them. We also consider the limits of such models based on the relative timescales of the processes of interest.", "doi": "10.1146/annurev-conmatphys-031214-014558", "pmcid": "PMC4946674", "issn": "1947-5454", "publisher": "Annual Reviews", "publication": "Annual Review of Condensed Matter Physics", "publication_date": "2015-03", "volume": "6", "pages": "85-111" }, { "id": "authors:4cy8x-k8v15", "collection": "authors", "collection_id": "4cy8x-k8v15", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170616-133655981", "type": "book_section", "title": "Host-Virus Interaction: From Metagenomics to Single-Cell Genomics", "book_title": "Encyclopedia of Metagenomics", "author": [ { "family_name": "Tadmor", "given_name": "Arbel D.", "clpid": "Tadmor-A-D" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "contributor": [ { "family_name": "Nelson", "given_name": "Karen E.", "clpid": "Nelson-K-E" } ], "abstract": "It is widely appreciated today that viruses are a dominant and critical part of Earth's biosphere. Yet despite the major advances in the study of environmental viruses in most cases, our knowledge of which viruses go with which hosts is meager. In the classic phage isolation technique, known as the plaque assay, a confluent layer of host cells is infected with a low density of viral particles. When a virus infects a cell within this \"lawn\" of host cells, the cell lyses, and new viral particles infect adjacent cells thereby creating a clearing, or plaque, in the lawn. This technique for isolating viruses requires that the host of the virus be culturable. However, given that >99% of microbes on Earth cannot be cultured at this time, the vast majority of phage-host systems cannot be investigated in the laboratory using these classical phage enrichment techniques. Consequently, little is known about the biology of most viruses and their host specificity in the wild.", "doi": "10.1007/978-1-4899-7478-5_792", "isbn": "978-1-4899-7477-8", "publisher": "Springer", "place_of_publication": "New York, NY", "publication_date": "2015-01-04", "pages": "257-265" }, { "id": "authors:d4sap-kt662", "collection": "authors", "collection_id": "d4sap-kt662", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150108-145321538", "type": "article", "title": "The Rate of Osmotic Downshock Determines the Survival Probability of Bacterial Mechanosensitive Channel Mutants", "author": [ { "family_name": "Bialecka-Fornal", "given_name": "Maja", "clpid": "Bialecka-Fornal-M" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Mechanosensitive (MS) channels allow cells to sense and respond to environmental changes. In bacteria, these channels are believed to protect against an osmotic shock. The physiological function of these channels has been characterized primarily by a standardized assay, where aliquots of batch-cultured cells are rapidly pipetted into a hypotonic medium. Under this method, it has been inferred many types of MS channels (MscS homologs in Escherichia coli) demonstrate limited effectiveness against shock, typically rescuing less than 10% of the cells when expressed at native levels. We introduce a single-cell-based assay which allows us to control how fast the osmolarity changes, over time scales ranging from a fraction of a second to several minutes. We find that the protection provided by MS channels depends strongly on the rate of osmotic change, revealing that, under a slow enough osmotic drop, MscS homologs can lead to survival rates comparable to those found in wild-type strains. Further, after the osmotic downshift, we observe multiple death phenotypes, which are inconsistent with the prevailing paradigm of how cells lyse. Both of these findings require a reevaluation of our basic understanding of the physiology of MS channels.", "doi": "10.1128/JB.02175-14", "pmcid": "PMC4288683", "issn": "0021-9193", "publisher": "American Society for Microbiology", "publication": "Journal of Bacteriology", "publication_date": "2015-01", "series_number": "1", "volume": "197", "issue": "1", "pages": "231-237" }, { "id": "authors:6bh3h-mcw62", "collection": "authors", "collection_id": "6bh3h-mcw62", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150129-091025277", "type": "article", "title": "The Influence of Promoter Architectures and Regulatory Motifs on Gene Expression in Escherichia coli", "author": [ { "family_name": "Rydenfelt", "given_name": "Mattias", "clpid": "Rydenfelt-M" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Cox", "given_name": "Robert Sidney, III", "clpid": "Cox-R-S-III" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The ability to regulate gene expression is of central importance for the adaptability of living organisms to changes in their external and internal environment. At the transcriptional level, binding of transcription factors (TFs) in the promoter region can modulate the transcription rate, hence making TFs central players in gene regulation. For some model organisms, information about the locations and identities of discovered TF binding sites have been collected in continually updated databases, such as RegulonDB for the well-studied case of E. coli. In order to reveal the general principles behind the binding-site arrangement and function of these regulatory architectures we propose a random promoter architecture model that preserves the overall abundance of binding sites to identify overrepresented binding site configurations. This model is analogous to the random network model used in the study of genetic network motifs, where regulatory motifs are identified through their overrepresentation with respect to a \"randomly connected\" genetic network. Using our model we identify TF pairs which coregulate operons in an overrepresented fashion, or individual TFs which act at multiple binding sites per promoter by, for example, cooperative binding, DNA looping, or through multiple binding domains. We furthermore explore the relationship between promoter architecture and gene expression, using three different genome-wide protein copy number censuses. Perhaps surprisingly, we find no systematic correlation between the number of activator and repressor binding sites regulating a gene and the level of gene expression. A position-weight-matrix model used to estimate the binding affinity of RNA polymerase (RNAP) to the promoters of activated and repressed genes suggests that this lack of correlation might in part be due to differences in basal transcription levels, with repressed genes having a higher basal activity level. This quantitative catalogue relating promoter architecture and function provides a first step towards genome-wide predictive models of regulatory function.", "doi": "10.1371/journal.pone.0114347", "pmcid": "PMC4280137", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLoS ONE", "publication_date": "2014-12-30", "series_number": "12", "volume": "9", "issue": "12", "pages": "Art. No. e114347" }, { "id": "authors:cm7v0-rkb85", "collection": "authors", "collection_id": "cm7v0-rkb85", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20141223-110116617", "type": "article", "title": "Promoter architecture dictates cell-to-cell variability in gene expression", "author": [ { "family_name": "Jones", "given_name": "Daniel L.", "orcid": "0000-0002-7902-996X", "clpid": "Jones-D-L" }, { "family_name": "Brewster", "given_name": "Robert C.", "clpid": "Brewster-R-C" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Variability in gene expression among genetically identical cells has emerged as a central\npreoccupation in the study of gene regulation; however, a divide exists between the\npredictions of molecular models of prokaryotic transcriptional regulation and genome-wide\nexperimental studies suggesting that this variability is indifferent to the underlying regulatory\narchitecture. We constructed a set of promoters in Escherichia coli in which promoter\nstrength, transcription factor binding strength, and transcription factor copy numbers are\nsystematically varied, and used messenger RNA (mRNA) fluorescence in situ hybridization to\nobserve how these changes affected variability in gene expression. Our parameter-free models\npredicted the observed variability; hence, the molecular details of transcription dictate\nvariability in mRNA expression, and transcriptional noise is specifically tunable and thus\nrepresents an evolutionarily accessible phenotypic parameter.", "doi": "10.1126/science.1255301", "pmcid": "PMC4388425", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2014-12-19", "series_number": "6216", "volume": "346", "issue": "6216", "pages": "1533-1536" }, { "id": "authors:jkmty-y9t04", "collection": "authors", "collection_id": "jkmty-y9t04", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150219-110954794", "type": "article", "title": "Scaling of Gene Expression with Transcription-Factor Fugacity", "author": [ { "family_name": "Weinert", "given_name": "Franz M.", "clpid": "Weinert-F-M" }, { "family_name": "Brewster", "given_name": "Robert C.", "clpid": "Brewster-R-C" }, { "family_name": "Rydenfelt", "given_name": "Mattias", "clpid": "Rydenfelt-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Kegel", "given_name": "Willem K.", "clpid": "Kegel-W-K" } ], "abstract": "The proteins associated with gene regulation are often shared between multiple pathways simultaneously. By way of contrast, models in regulatory biology often assume these pathways act independently. We demonstrate a framework for calculating the change in gene expression for the interacting case by decoupling repressor occupancy across the cell from the gene of interest by way of a chemical potential. The details of the interacting regulatory architecture are encompassed in an effective concentration, and thus, a single scaling function describes a collection of gene expression data from diverse regulatory situations and collapses it onto a single master curve.", "doi": "10.1103/PhysRevLett.113.258101", "pmcid": "PMC4386862", "issn": "0031-9007", "publisher": "American Physical Society", "publication": "Physical Review Letters", "publication_date": "2014-12-19", "series_number": "25", "volume": "113", "issue": "25", "pages": "Art. No. 258101" }, { "id": "authors:nrxc8-ce464", "collection": "authors", "collection_id": "nrxc8-ce464", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20141209-161207781", "type": "article", "title": "Modulation of DNA loop lifetimes by the free energy of loop formation", "author": [ { "family_name": "Chen", "given_name": "Yi-Ju", "clpid": "Chen-Yi-Ju" }, { "family_name": "Johnson", "given_name": "Stephanie", "orcid": "0000-0001-9487-8583", "clpid": "Johnson-S" }, { "family_name": "Mulligan", "given_name": "Peter", "clpid": "Mulligan-P" }, { "family_name": "Spakowitz", "given_name": "Andrew J.", "orcid": "0000-0002-0585-1942", "clpid": "Spakowitz-A-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Storage and retrieval of the genetic information in cells is a dynamic process that requires the DNA to undergo dramatic structural rearrangements. DNA looping is a prominent example of such a structural rearrangement that is essential for transcriptional regulation in both prokaryotes and eukaryotes, and the speed of such regulations affects the fitness of individuals. Here, we examine the in vitro looping dynamics of the classic Lac repressor gene-regulatory motif. We show that both loop association and loop dissociation at the DNA-repressor junctions depend on the elastic deformation of the DNA and protein, and that both looping and unlooping rates approximately scale with the looping J factor, which reflects the system's deformation free energy. We explain this observation by transition state theory and model the DNA\u2013protein complex as an effective worm-like chain with twist. We introduce a finite protein\u2013DNA binding interaction length, in competition with the characteristic DNA deformation length scale, as the physical origin of the previously unidentified loop dissociation dynamics observed here, and discuss the robustness of this behavior to perturbations in several polymer parameters.", "doi": "10.1073/pnas.1415685111", "pmcid": "PMC4267329", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2014-12-09", "series_number": "49", "volume": "111", "issue": "49", "pages": "17396-17401" }, { "id": "authors:n9svj-ja346", "collection": "authors", "collection_id": "n9svj-ja346", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20141204-100345751", "type": "article", "title": "The quantified cell", "author": [ { "family_name": "Flamholz", "given_name": "Avi", "orcid": "0000-0002-9278-5479", "clpid": "Flamholz-Avi" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" } ], "abstract": "The microscopic world of a cell can be as alien to our human-centered intuition as the confinement of quarks within protons or the event horizon of a black hole. We are prone to thinking by analogy\u2014Golgi cisternae stack like pancakes, red blood cells look like donuts\u2014but very little in our human experience is truly comparable to the immensely crowded, membrane-subdivided interior of a eukaryotic cell or the intricately layered structures of a mammalian tissue. So in our daily efforts to understand how cells work, we are faced with a challenge: how do we develop intuition that works at the microscopic scale?", "doi": "10.1091/mbc.E14-09-1347", "pmcid": "PMC4230611", "issn": "1059-1524", "publisher": "American Society for Cell Biology", "publication": "Molecular Biology of the Cell", "publication_date": "2014-11-05", "series_number": "22", "volume": "25", "issue": "22", "pages": "3497-3500" }, { "id": "authors:gkzt6-wcf38", "collection": "authors", "collection_id": "gkzt6-wcf38", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140224-115855856", "type": "article", "title": "Multiple LacI-mediated loops revealed by Bayesian statistics and tethered particle motion", "author": [ { "family_name": "Johnson", "given_name": "Stephanie", "orcid": "0000-0001-9487-8583", "clpid": "Johnson-S" }, { "family_name": "van de Meent", "given_name": "Jan-Willem", "clpid": "van-de-Meent-J-W" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Wiggins", "given_name": "Chris H.", "clpid": "Wiggins-C-H" }, { "family_name": "Lind\u00e9n", "given_name": "Martin", "clpid": "Lind\u00e9n-M" } ], "abstract": "The bacterial transcription factor LacI loops DNA by binding to two separate locations on the DNA simultaneously. Despite being one of the best-studied model systems for transcriptional regulation, the number and conformations of loop structures accessible to LacI remain unclear, though the importance of multiple coexisting loops has been implicated in interactions between LacI and other cellular regulators of gene expression. To probe this issue, we have developed a new analysis method for tethered particle motion, a versatile and commonly used in vitro single-molecule technique. Our method, vbTPM, performs variational Bayesian inference in hidden Markov models. It learns the number of distinct states (i.e. DNA\u2013protein conformations) directly from tethered particle motion data with better resolution than existing methods, while easily correcting for common experimental artifacts. Studying short (roughly 100 bp) LacI-mediated loops, we provide evidence for three distinct loop structures, more than previously reported in single-molecule studies. Moreover, our results confirm that changes in LacI conformation and DNA-binding topology both contribute to the repertoire of LacI-mediated loops formed in vitro, and provide qualitatively new input for models of looping and transcriptional regulation. We expect vbTPM to be broadly useful for probing complex protein\u2013nucleic acid interactions.", "doi": "10.1093/nar/gku563", "pmcid": "PMC4176382", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "2014-09-15", "series_number": "16", "volume": "42", "issue": "16", "pages": "10265-10277" }, { "id": "authors:s1914-vgw29", "collection": "authors", "collection_id": "s1914-vgw29", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140509-111341446", "type": "article", "title": "Comparison of the theoretical and real-world evolutionary potential of a genetic circuit", "author": [ { "family_name": "Razo-Mejia", "given_name": "M.", "orcid": "0000-0002-9510-0527", "clpid": "Razo-Mejia-M" }, { "family_name": "Boedicker", "given_name": "J. Q.", "clpid": "Boedicker-J-Q" }, { "family_name": "Jones", "given_name": "D.", "orcid": "0000-0002-7902-996X", "clpid": "Jones-D-L" }, { "family_name": "DeLuna", "given_name": "A.", "clpid": "DeLuna-A" }, { "family_name": "Kinney", "given_name": "J. B.", "orcid": "0000-0003-1897-3778", "clpid": "Kinney-J-B" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "With the development of next-generation sequencing technologies, many large scale experimental efforts aim to map genotypic variability among individuals. This natural variability in populations fuels many fundamental biological processes, ranging from evolutionary adaptation and speciation to the spread of genetic diseases and drug resistance. An interesting and important component of this variability is present within the regulatory regions of genes. As these regions evolve, accumulated mutations lead to modulation of gene expression, which may have consequences for the phenotype. A simple model system where the link between genetic variability, gene regulation and function can be studied in detail is missing. In this article we develop a model to explore how the sequence of the wild-type lac promoter dictates the fold-change in gene expression. The model combines single-base pair resolution maps of transcription factor and RNA polymerase binding energies with a comprehensive thermodynamic model of gene regulation. The model was validated by predicting and then measuring the variability of lac operon regulation in a collection of natural isolates. We then implement the model to analyze the sensitivity of the promoter sequence to the regulatory output, and predict the potential for regulation to evolve due to point mutations in the promoter region.", "doi": "10.1088/1478-3975/11/2/026005", "pmcid": "PMC4051709", "issn": "1478-3967", "publisher": "IOP", "publication": "Physical Biology", "publication_date": "2014-04", "series_number": "2", "volume": "11", "issue": "2", "pages": "Art. No. 026005" }, { "id": "authors:cb2wh-f8416", "collection": "authors", "collection_id": "cb2wh-f8416", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140320-130336013", "type": "article", "title": "The Transcription Factor Titration Effect Dictates Level of Gene Expression", "author": [ { "family_name": "Brewster", "given_name": "Robert C.", "clpid": "Brewster-R-C" }, { "family_name": "Weinert", "given_name": "Franz M.", "clpid": "Weinert-F-M" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Song", "given_name": "Dan", "clpid": "Song-D" }, { "family_name": "Rydenfelt", "given_name": "Mattias", "clpid": "Rydenfelt-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number\u2014in multiple identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally, we use these experiments to dynamically measure plasmid copy number through the cell cycle.", "doi": "10.1016/j.cell.2014.02.022", "pmcid": "PMC4080642", "issn": "0092-8674", "publisher": "Elsevier", "publication": "Cell", "publication_date": "2014-03-13", "series_number": "6", "volume": "156", "issue": "6", "pages": "1312-1323" }, { "id": "authors:gcq33-c1360", "collection": "authors", "collection_id": "gcq33-c1360", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140710-094352199", "type": "article", "title": "The Rate of Osmotic Shock Determines Bacterial Survival", "author": [ { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Bialecka-Fornal", "given_name": "Maja", "clpid": "Bialecka-Fornal-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Mechanosensitive (MS) channels allow cells to sense and respond to environmental changes. In bacteria, these channels are believed to protect against an osmotic shock. The physiological function of these channels has been primarily characterized by a standardized assay, where aliquots of batch cultured cells are rapidly pipetted into a hypotonic medium. Under this method, it has been inferred many types of MS channels (MscS homologs in E. coli) demonstrate questionable effectiveness against shock. We introduce a single-cell based assay which allows us to control how fast the osmolarity changes, over time scales ranging from a fraction of second to several minutes. We find that the protection provided by MS channels depends strongly on the rate of osmotic change, revealing that, under a slow enough osmotic drop, even \"ineffective\" MscS homologs can lead to survival rates comparable to those found in wild-type strains. Further, after the osmotic downshift, we observe multiple death phenotypes, which are inconsistent with the prevailing paradigm of how cells lyse. Both of these findings require a re-evaluation of our basic understanding of the physiology of MS channels.", "doi": "10.1016/j.bpj.2013.11.3084", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2014-01-28", "series_number": "2", "volume": "106", "issue": "2", "pages": "554A" }, { "id": "authors:mv0dn-nf254", "collection": "authors", "collection_id": "mv0dn-nf254", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140710-081414989", "type": "article", "title": "Dissecting the Role of Ferrous Iron in Pseudomonas Aeruginosa Gene Regulation", "author": [ { "family_name": "Boedicker", "given_name": "James", "clpid": "Boedicker-J-Q" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "In our quest towards a predictive understanding of biological systems, we have combined quantitative models of gene regulation with careful experimental tests of model predictions in order to determine how promoter architecture dictates the output level of gene expression. We have applied this quantitative framework to understand how cells sense and respond to extracellular cues, using ferrous iron-mediated gene regulation in Pseudomonas aeruginosa as an important case study.\n\nUpon recent discovery of a two-component system that specifically responds to ferrous iron, there is an emerging picture of the critical role of Fe(II) availability in the regulatory outputs of Pseudomonas aeruginosa. On the bench top Fe(II) is readily oxidized to Fe(III), however in infections such as in the lungs of cystic fibrosis patients, cells can encounter reducing environments containing Fe(II). Ferrous iron regulates genes related to both pathogenicity and biofilm formation. These pathways are also controlled by the quorum sensing systems and the availability of ferric iron. It is not yet clear why multiple pathways are used to sense extracellular iron or how the cell integrates information from these different pathways to effectively respond to changes in the availability and oxidation state of iron.\n\nUsing statistical mechanical models of gene regulation and the tools of synthetic biology, the rules of the ferrous iron regulon are beginning to emerge. We quantified how promoter architecture dictates the regulatory response to Fe(II). A quantitative understanding of how promoters encode a ferrous iron response enabled us to predict how Fe(II) influences global gene expression patterns. Some genes respond to both Fe(II) and additional regulatory inputs, such as other transcription factors or two-component systems. We dissected these multi-input promoters to understand how cells combine ferrous iron availability with other regulatory factors to make transcriptional decisions.", "doi": "10.1016/j.bpj.2013.11.2115", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2014-01-28", "series_number": "2", "volume": "106", "issue": "2", "pages": "374A" }, { "id": "authors:kjf1p-e4r98", "collection": "authors", "collection_id": "kjf1p-e4r98", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140710-100757843", "type": "article", "title": "Multiple Lac-Mediated Loops Revealed by Bayesian Statistics and Tethered Particle Motion", "author": [ { "family_name": "Lind\u00e9n", "given_name": "Martin", "clpid": "Lind\u00e9n-M" }, { "family_name": "Johnson", "given_name": "Stephanie", "orcid": "0000-0001-9487-8583", "clpid": "Johnson-S" }, { "family_name": "van de Meent", "given_name": "Jan-Willem", "clpid": "van-de-Meent-J-W" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Wiggins", "given_name": "Chris", "clpid": "Wiggins-C-H" } ], "abstract": "The bacterial transcription factor LacI loops DNA by binding to two distinct binding sites on DNA. Despite being one of the most well-studied model systems for transcriptional regulation, the number of possible loops, and their structures, are unclear. To clarify this question, we have developed a new analysis method for tethered particle motion (TPM), a versatile single-molecule technique commonly used to study Lac-mediated looping and other DNA-protein interactions in vitro. The method, called vbTPM, is based on a variational Bayes treatment of hidden Markov models. It learns the number of distinct states (e.g., DNA-protein conformations) in a TPM trajectory directly from the data with better resolution than existing methods, and also corrects for common experimental artifacts. Studying short (about 100 bp) LacI-mediated loops, we are able to resolve states and interconversion patterns that are best explained in terms of at least three distinct loop structures. These results indicate that changes in Lac conformation and DNA binding orientation both contribute to the repertoire of LacI-mediated loops, and provides new input for quantitative models of looping and transcriptional regulation.", "doi": "10.1016/j.bpj.2013.11.174", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2014-01-28", "series_number": "2", "volume": "106", "issue": "2", "pages": "22A" }, { "id": "authors:nj0xj-bsc91", "collection": "authors", "collection_id": "nj0xj-bsc91", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140710-102048609", "type": "article", "title": "Single Molecule Dynamics Governing the Initiation of V(D)J Recombination", "author": [ { "family_name": "Lovely", "given_name": "Geoffrey", "clpid": "Lovely-Geoffrey-A" }, { "family_name": "Lind\u00e9n", "given_name": "Martin", "clpid": "Lind\u00e9n-Martin" }, { "family_name": "Ramesh", "given_name": "Pradeep", "clpid": "Ramesh-P" }, { "family_name": "Schatz", "given_name": "David", "clpid": "Schatz-David-G" }, { "family_name": "Baltimore", "given_name": "David", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The recombination activating genes (RAG)1 and RAG2 perform V(D)J recombination by rearranging conserved recombination signal sequences (RSSs) to generate antigen-receptors during lymphopoiesis. However the orchestration of V(D)J recombination on biologically relevant (long) length scales has resisted experimental investigation. Here we develop single-molecule assays to watch in real time as RAG1/2 and its co-factor HMGB1 carry out V(D)J recombination from start (RSS binding) to finish (hairpin formation) on long DNA molecules. We capture various intermediate states preceding hairpin formation, show how RAG1/2 and HMGB1 form bends on the DNA, demonstrate how the identity of the recombination signal sequence modulates bending with single bp resolution and show HMGB1 must compact DNA flanking RSSs to form hairpins. Our results provide single-molecule mechanistic insight into the orchestration of V(D)J recombination.", "doi": "10.1016/j.bpj.2013.11.3826", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2014-01-28", "series_number": "2", "volume": "106", "issue": "2", "pages": "692A" }, { "id": "authors:8sbt9-ew193", "collection": "authors", "collection_id": "8sbt9-ew193", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140710-091408000", "type": "article", "title": "Time-Resolved Plasmid Counting by Way of Transcription Factor Sequestration", "author": [ { "family_name": "Brewster", "given_name": "Robert", "clpid": "Brewster-R-C" }, { "family_name": "Weinert", "given_name": "Franz", "clpid": "Weinert-F-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "In contrast to the chromosome, genes expressed from plasmids exist in high copy number which changes continuously during the cell cycle. There are many models for how the control mechanisms associated with plasmid copy number dictate the cell-cycle dependent plasmid copy number, which have unique predictions for the dynamics of replication. We demonstrate a non-invasive method for dynamical quantification of plasmid copy number over the course of the cell cycle using the level of transcription from a reporter gene which shares a transcription factor with the plasmid. Using this method, we explore the cell-cycle dependent nature of plasmid replication for different origins of replication. Our method allows for discrimination of single-cell characteristics such as the single-cell division time and morphology and may permit discrimination of different replication mechanisms.", "doi": "10.1016/j.bpj.2013.11.1606", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2014-01-28", "series_number": "2", "volume": "106", "issue": "2", "pages": "274A" }, { "id": "authors:jap1m-sdh09", "collection": "authors", "collection_id": "jap1m-sdh09", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140710-084044769", "type": "article", "title": "Dynamic Gene Expression and Design Principles of Viral Infection Pathway", "author": [ { "family_name": "Chen", "given_name": "Yi-Ju", "clpid": "Chen-Yi-Ju" }, { "family_name": "Zhiyentayev", "given_name": "Timur", "clpid": "Zhiyentayev-T" }, { "family_name": "Wu", "given_name": "David", "clpid": "Wu-David" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The study of gene expression is often couched in the language of steady-states. On the other hand, non-steady-state conditions are clearly of critical importance. One example that opens a unique window to studying non-steady-state gene expression patterns is viral infection. The lytic pathway of most bacteriophages leads to production of the machinery of the virus and eventually the infected cells burst. Here we use phage lambda and E. coli as a model system to study the intracellular dynamics of viral products from the nucleic acid to protein level.\n\nWe use single-molecule fluorescence in situ hybridization and fluorescence fusion proteins to quantify the copy number of viral nucleic acid and protein products in single cells during the lytic pathway as a function of time. We ask how the growth curves of the viral products can be explained by using simple master equations describing the replication/transcription/translation of the well-characterized phage lambda genetic regulatory circuit. We also examine the molecular balance in the viral production process, as balance in sub-components is a recurring principle in the biosynthesis of ribosome, protein complexes and metabolic pathways. In particular, how wasteful is the production compared to the burst size (the average number of viral particles released per cell as measured in titer experiments), and whether the viral components (for example the capsid protein to genome ratio) are produced to the correct stoichiometry that is well-defined from structural studies for mature viral particles.", "doi": "10.1016/j.bpj.2013.11.2111", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2014-01-28", "series_number": "2", "volume": "106", "issue": "2", "pages": "373A" }, { "id": "authors:9qtzw-wa037", "collection": "authors", "collection_id": "9qtzw-wa037", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140710-094648257", "type": "article", "title": "The Transcription Factor Titration Effect Dictates Level of Gene Expression", "author": [ { "family_name": "Weinert", "given_name": "Franz M.", "clpid": "Weinert-F-M" }, { "family_name": "Brewster", "given_name": "Robert C.", "clpid": "Brewster-R-C" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Song", "given_name": "Linda", "clpid": "Song-Linda" }, { "family_name": "Rydenfelt", "given_name": "Mattias", "clpid": "Rydenfelt-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Models of transcription are often built around a picture of RNA polymerase and transcription factors acting on a single copy of a promoter. However, many transcription factors are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number; in multiple, identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between transcription factor copy number and the demand for that transcription factor. Using video microscopy, we measure this effect and demonstrate the parameter-free predictive power of the thermodynamic model as a function of the copy number of the transcription factor and the number and affinities of the available specific binding sites. Understanding how to account for the effects of competing binding sites is an important facet of predictive control of transcription and gene circuit design, where transcription factors regularly navigate complex DNA binding landscapes.", "doi": "10.1016/j.bpj.2013.11.4483", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2014-01-28", "series_number": "2", "volume": "106", "issue": "2", "pages": "489A" }, { "id": "authors:cenkr-0yv40", "collection": "authors", "collection_id": "cenkr-0yv40", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140710-080835668", "type": "article", "title": "Promoter Architecture Dictates Variability in Gene Expression", "author": [ { "family_name": "Jones", "given_name": "Daniel L.", "orcid": "0000-0002-7902-996X", "clpid": "Jones-D-L" }, { "family_name": "Brewster", "given_name": "Robert", "clpid": "Brewster-R-C" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Recent experimental studies have reported that cell-to-cell variability in gene expression is a universal function of mean gene expression levels, implying that knowledge of mean levels of gene expression alone is sufficient to determine the level of variability. This result seems at odds with the physical intuition underlying efforts in the literature to predict the level of variability for different promoter architectures (where we take \"promoter architecture\" to mean the numbers, positions, and strengths of transcription factor and RNA polymerase binding sites). For instance, we would expect slow TF binding/unbinding kinetics to yield a relatively higher level of variability at a given mean than fast kinetics, as the switching between distinct transcriptional states will yield a broad distribution in mRNA production.\n\nTo address this apparent conundrum, we constructed a set of 18 constitutive (unregulated) promoters with expression levels spanning a range of 2.5 orders of magnitude, and found that the observed mRNA distributions were consistent with Poissonian production of mRNA. We then placed one of these promoters under regulation by the repressor transcription factor LacI, and measured the resulting mRNA distributions at a range of LacI concentrations. We found that, for the regulated promoter, the level of variability as a function of mean expression followed a distinct, promoter architecture dependent curve as compared with the unregulated promoter. These results demonstrate that the level of variability in gene expression depends on the specific promoter architecture in play.", "doi": "10.1016/j.bpj.2013.11.4481", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2014-01-28", "series_number": "2", "volume": "106", "issue": "2", "pages": "489A" }, { "id": "authors:5w1st-5mb53", "collection": "authors", "collection_id": "5w1st-5mb53", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140710-104425641", "type": "article", "title": "Identification and Analysis of the Transcriptional Regulatory Networks Governing Mechanosensitive Channels in E. coli", "author": [ { "family_name": "Barnes", "given_name": "Stephanie", "clpid": "Barnes-S" }, { "family_name": "Jones", "given_name": "Daniel", "orcid": "0000-0002-7902-996X", "clpid": "Jones-D-L" }, { "family_name": "Belliveau", "given_name": "Nathan", "clpid": "Belliveau-N" }, { "family_name": "Sundaresh", "given_name": "Sushant", "clpid": "Sundaresh-S" }, { "family_name": "Kinney", "given_name": "Justin", "clpid": "Kinney-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Mechanosensitive (MS) channels are membrane proteins which are gated by mechanical stress in the cell membrane. Channel opening in response to mechanical stresses makes it possible for molecules to pass through the membrane, allowing an organism to alleviate solute imbalances which place osmotic stress on the membrane and can cause cell lysis and death. Two of the seven known MS channels in E. coli, MscL and MscS, are individually capable of rescuing these bacteria from sudden changes in osmolarity (osmotic shock). If MscL and MscS are both sufficient for ensuring survival under osmotic shock conditions, what is the purpose of the other five MS channels? In the interest of exploring the physiological roles of the various MS channels, we hypothesize that understanding how they are regulated will shed light on their function. Specifically, we investigate the regulatory context of the MS genes by identifying the regulatory architecture and transcription factors responsible for controlling MS gene output. We use a method known as \"Sort-Seq\" to locate transcription factor binding sites with base pair resolution, and DNA affinity chromatography to determine transcription factor identity. Understanding how the MS genes are regulated will give us clues as to the specific stressors which they defend the cell against. This work provides a specific case study of a method which we think will have much broader reach in mapping out regulatory networks genome-wide.", "doi": "10.1016/j.bpj.2013.11.4472", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2014-01-28", "series_number": "2", "volume": "106", "issue": "2", "pages": "487A" }, { "id": "authors:z0a7b-t1w70", "collection": "authors", "collection_id": "z0a7b-t1w70", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140404-102949606", "type": "article", "title": "Statistical mechanical model of coupled transcription from multiple promoters due to transcription factor titration", "author": [ { "family_name": "Rydenfelt", "given_name": "Mattias", "clpid": "Rydenfelt-M" }, { "family_name": "Cox", "given_name": "Robert Sidney, III", "clpid": "Cox-R-S-III" }, { "family_name": "Garcia", "given_name": "Hernan", "clpid": "Garcia-H" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Transcription factors (TFs) with regulatory action at multiple promoter targets is the rule rather than the exception, with examples ranging from the cAMP receptor protein (CRP) in E. coli that regulates hundreds of different genes simultaneously to situations involving multiple copies of the same gene, such as plasmids, retrotransposons, or highly replicated viral DNA. When the number of TFs heavily exceeds the number of binding sites, TF binding to each promoter can be regarded as independent. However, when the number of TF molecules is comparable to the number of binding sites, TF titration will result in correlation (\"promoter entanglement\") between transcription of different genes. We develop a statistical mechanical model which takes the TF titration effect into account and use it to predict both the level of gene expression for a general set of promoters and the resulting correlation in transcription rates of different genes. Our results show that the TF titration effect could be important for understanding gene expression in many regulatory settings.", "doi": "10.1103/PhysRevE.89.012702", "pmcid": "PMC4043999", "issn": "1539-3755", "publisher": "American Physical Society", "publication": "Physical Review E", "publication_date": "2014-01-06", "series_number": "1", "volume": "89", "issue": "1", "pages": "Art. No. 012702" }, { "id": "authors:91kpj-znn93", "collection": "authors", "collection_id": "91kpj-znn93", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140326-113402727", "type": "article", "title": "Distinct structural features of TFAM drive mitochondrial DNA packaging versus transcriptional activation", "author": [ { "family_name": "Ngo", "given_name": "Huu B.", "clpid": "Ngo-Huu-B" }, { "family_name": "Lovely", "given_name": "Geoffrey A.", "clpid": "Lovely-G-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" } ], "abstract": "TFAM (transcription factor A, mitochondrial) is a DNA-binding protein that activates transcription at the two major promoters of mitochondrial DNA (mtDNA)\u2014the light strand promoter (LSP) and the heavy strand promoter 1 (HSP1). Equally important, it coats and packages the mitochondrial genome. TFAM has been shown to impose a U-turn on LSP DNA; however, whether this distortion is relevant at other sites is unknown. Here we present crystal structures of TFAM bound to HSP1 and to nonspecific DNA. In both, TFAM similarly distorts the DNA into a U-turn. Yet, TFAM binds to HSP1 in the opposite orientation from LSP explaining why transcription from LSP requires DNA bending, whereas transcription at HSP1 does not. Moreover, the crystal structures reveal dimerization of DNA-bound TFAM. This dimerization is dispensable for DNA bending and transcriptional activation but is important in DNA compaction. We propose that TFAM dimerization enhances mitochondrial DNA compaction by promoting looping of the DNA.", "doi": "10.1038/ncomms4077", "pmcid": "PMC3936014", "issn": "2041-1723", "publisher": "Nature Publishing Group", "publication": "Nature Communications", "publication_date": "2014-01", "series_number": "1", "volume": "5", "issue": "1", "pages": "Art. No. 3077" }, { "id": "authors:790gr-ppf60", "collection": "authors", "collection_id": "790gr-ppf60", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140103-091544508", "type": "article", "title": "The Feynman Lectures on Physics", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Over the past three decades, I have asked hundreds of people to name the five or ten books that have meant the most to them. Although Jane Austen's Pride and Prejudice tops the list, The Feynman Lectures on Physics is the science title most often cited. That may say something about the kind of readers I talk to, but it is an accurate reflection of the broad reach of this half-century-old scientific classic.", "doi": "10.1038/504030a", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2013-12-05", "series_number": "7478", "volume": "504", "issue": "7478", "pages": "30-31" }, { "id": "authors:5vacd-kkj67", "collection": "authors", "collection_id": "5vacd-kkj67", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20131204-074505690", "type": "article", "title": "DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation", "author": [ { "family_name": "Boedicker", "given_name": "James Q.", "clpid": "Boedicker-J-Q" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Johnson", "given_name": "Stephanie", "orcid": "0000-0001-9487-8583", "clpid": "Johnson-S" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution.", "doi": "10.1088/1478-3975/10/6/066005", "pmcid": "PMC3915735", "issn": "1478-3967", "publisher": "IOP", "publication": "Physical Biology", "publication_date": "2013-12", "series_number": "6", "volume": "10", "issue": "6", "pages": "Art. No. 066005" }, { "id": "authors:d1s86-cx492", "collection": "authors", "collection_id": "d1s86-cx492", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20131205-092633341", "type": "article", "title": "Poly(dA:dT)-Rich DNAs Are Highly Flexible in the Context\n of DNA Looping", "author": [ { "family_name": "Johnson", "given_name": "Stephanie", "clpid": "Johnson-Stephanie" }, { "family_name": "Chen", "given_name": "Yi-Ju", "clpid": "Chen-Yi-Ju" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Large-scale DNA deformation is ubiquitous in transcriptional regulation in prokaryotes and eukaryotes alike. Though much is known about how transcription factors and constellations of binding sites dictate where and how gene regulation will occur, less is known about the role played by the intervening DNA. In this work we explore the effect of sequence flexibility on transcription factor-mediated DNA looping, by drawing on sequences identified in nucleosome formation and ligase-mediated cyclization assays as being especially favorable for or resistant to large deformations. We examine a poly(dA:dT)-rich, nucleosome-repelling sequence that is often thought to belong to a class of highly inflexible DNAs; two strong nucleosome positioning sequences that share a set of particular sequence features common to nucleosome-preferring DNAs; and a CG-rich sequence representative of high G+C-content genomic regions that correlate with high nucleosome occupancy in vivo. To measure the flexibility of these sequences in the context of DNA looping, we combine the in vitro single-molecule tethered particle motion assay, a canonical looping protein, and a statistical mechanical model that allows us to quantitatively relate the looping probability to the looping free energy. We show that, in contrast to the case of nucleosome occupancy, G+C content does not positively correlate with looping probability, and that despite sharing sequence features that are thought to determine nucleosome affinity, the two strong nucleosome positioning sequences behave markedly dissimilarly in the context of looping. Most surprisingly, the poly(dA:dT)-rich DNA that is often characterized as highly inflexible in fact exhibits one of the highest propensities for looping that we have measured. These results argue for a need to revisit our understanding of the mechanical properties of DNA in a way that will provide a basis for understanding DNA deformation over the entire range of biologically relevant scenarios that are impacted by DNA deformability.", "doi": "10.1371/journal.pone.0075799", "pmcid": "PMC3795714", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLOS ONE", "publication_date": "2013-10-11", "series_number": "10", "volume": "8", "issue": "10", "pages": "Art. No. e75799" }, { "id": "authors:prcar-1rf35", "collection": "authors", "collection_id": "prcar-1rf35", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130614-144031370", "type": "article", "title": "Statistical Mechanics of Monod\u2013Wyman\u2013Changeux (MWC) Models", "author": [ { "family_name": "Marzen", "given_name": "Sarah", "clpid": "Marzen-S" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The 50th anniversary of the classic Monod\u2013Wyman\u2013Changeux (MWC) model provides an opportunity to survey the broader conceptual and quantitative implications of this quintessential biophysical model. With the use of statistical mechanics, the mathematical implementation of the MWC concept links problems that seem otherwise to have no ostensible biological connection including ligand\u2013receptor binding, ligand-gated ion channels, chemotaxis, chromatin structure and gene regulation. Hence, a thorough mathematical analysis of the MWC model can illuminate the performance limits of a number of unrelated biological systems in one stroke. The goal of our review is twofold. First, we describe in detail the general physical principles that are used to derive the activity of MWC molecules as a function of their regulatory ligands. Second, we illustrate the power of ideas from information theory and dynamical systems for quantifying how well the output of MWC molecules tracks their sensory input, giving a sense of the \"design\" constraints faced by these receptors.", "doi": "10.1016/j.jmb.2013.03.013", "pmcid": "PMC3786005", "issn": "0022-2836", "publisher": "Elsevier", "publication": "Journal of Molecular Biology", "publication_date": "2013-05-13", "series_number": "9", "volume": "425", "issue": "9", "pages": "1433-1460" }, { "id": "authors:s04ah-pca43", "collection": "authors", "collection_id": "s04ah-pca43", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130718-104555723", "type": "article", "title": "Connection between Oligomeric State and Gating Characteristics of Mechanosensitive Ion Channels", "author": [ { "family_name": "Haselwandter", "given_name": "Christoph A.", "clpid": "Haselwandter-C-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The mechanosensitive channel of large conductance (MscL) is capable of transducing mechanical stimuli such as membrane tension into an electrochemical response. MscL provides a widely-studied model system for mechanotransduction and, more generally, for how bilayer mechanical properties regulate protein conformational changes. Much effort has been expended on the detailed experimental characterization of the molecular structure and biological function of MscL. However, despite its central significance, even basic issues such as the physiologically relevant oligomeric states and molecular structures of MscL remain a matter of debate. In particular, tetrameric, pentameric, and hexameric oligomeric states of MscL have been proposed, together with a range of detailed molecular structures of MscL in the closed and open channel states. Previous theoretical work has shown that the basic phenomenology of MscL gating can be understood using an elastic model describing the energetic cost of the thickness deformations induced by MscL in the surrounding lipid bilayer. Here, we generalize this elastic model to account for the proposed oligomeric states and hydrophobic shapes of MscL. We find that the oligomeric state and hydrophobic shape of MscL are reflected in the energetic cost of lipid bilayer deformations. We make quantitative predictions pertaining to the gating characteristics associated with various structural models of MscL and, in particular, show that different oligomeric states and hydrophobic shapes of MscL yield distinct membrane contributions to the gating energy and gating tension. Thus, the functional properties of MscL provide a signature of the oligomeric state and hydrophobic shape of MscL. Our results suggest that, in addition to the hydrophobic mismatch between membrane proteins and the surrounding lipid bilayer, the symmetry and shape of the hydrophobic surfaces of membrane proteins play an important role in the regulation of protein function by bilayer membranes.", "doi": "10.1371/journal.pcbi.1003055", "pmcid": "PMC3656111", "issn": "1553-734X", "publisher": "Public Library of Science", "publication": "PLoS Computational Biology", "publication_date": "2013-05", "series_number": "5", "volume": "9", "issue": "5", "pages": "Art. No. e1003055" }, { "id": "authors:yss2d-79s72", "collection": "authors", "collection_id": "yss2d-79s72", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130523-084817493", "type": "article", "title": "Directional interactions and cooperativity between mechanosensitive membrane proteins", "author": [ { "family_name": "Haselwandter", "given_name": "Christoph A.", "clpid": "Haselwandter-C-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "While modern structural biology has provided us with a rich and diverse picture of membrane proteins, the biological function of membrane proteins is often influenced by the mechanical properties of the surrounding lipid bilayer. Here we explore the relation between the shape of membrane proteins and the cooperative function of membrane proteins induced by membrane-mediated elastic interactions. For the experimental model system of mechanosensitive ion channels we find that the sign and strength of elastic interactions depend on the protein shape, yielding distinct cooperative gating curves for distinct protein orientations. Our approach predicts how directional elastic interactions affect the molecular structure, organization, and biological function of proteins in crowded membranes.", "doi": "10.1209/0295-5075/101/68002", "pmcid": "PMC4193682", "issn": "0295-5075", "publisher": "IOP", "publication": "Europhysics Letters", "publication_date": "2013-03", "series_number": "6", "volume": "101", "issue": "6", "pages": "Art. No. 68002" }, { "id": "authors:e6ncb-ggp59", "collection": "authors", "collection_id": "e6ncb-ggp59", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130425-140427051", "type": "article", "title": "Bacteria are Stressed Out Too: The Physics of Mechanosensation", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Many of the ways that cells talk and listen to the external world center on the presence of proteins on the cell surface. Indeed, the cell membrane is an amazingly diverse lipid environment, riddled in turn with a host of different proteins that perform tasks ranging from sensing and measuring chemical signals to the transport of sugars needed for cell division to the detection of potentially lethal osmotic pressures. This talk will focus on recent progress in the dissection of the mechanisms of mechanosensation in bacteria with special reference to the rich interplay between certain classes of ion channels and the surrounding lipids. Using simple arguments from elasticity theory, I will describe the membrane deformation footprint surrounding ion channels and how this deformation footprint contributes to the free energy of channel gating. In turn, I will show how these ideas can be parlayed into an experimental strategy for better understanding mechanosensation by watching individual cells as they are subjected to controlled levels of osmotic shock.", "doi": "10.1016/j.bpj.2012.11.072", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2013-01-29", "series_number": "2", "volume": "104", "issue": "2", "pages": "8A-8A" }, { "id": "authors:2nah1-sx468", "collection": "authors", "collection_id": "2nah1-sx468", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130429-081637377", "type": "article", "title": "Analysis of DNA Looping Kinetics in Tethered Particle Motion Experiments using Hidden Markov Models", "author": [ { "family_name": "Lind\u00e9n", "given_name": "Martin", "clpid": "Lind\u00e9n-M" }, { "family_name": "Johnson", "given_name": "Stephanie", "orcid": "0000-0001-9487-8583", "clpid": "Johnson-S" }, { "family_name": "van de Meent", "given_name": "Jan-Willem", "clpid": "van-de-Meent-J-W" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Wiggins", "given_name": "Chris H.", "clpid": "Wiggins-C-H" } ], "abstract": "Tethered particle motion (TPM) is a powerful method for measuring DNA-protein interactions at the single molecule level. TPM experiments monitor the Brownian motion of beads tethered to a microscope cover slip. The Brownian motion changes when a protein binds to and deforms the DNA 'leash', for example in the formation of a DNA loop. A complicating factor in the interpretation of TPM data is that the number of observable states, corresponding to different conformations of the DNA-protein complex, is often not known in advance. Moreover, conformational transitions that occur on time scales comparable to the diffusive motion of the bead are difficult to extract from the data.\n\nWe present an analysis method for TPM data that overcomes these limitations in existing approaches. Our method relies on variational Bayesian inference on a variant of the Hidden Markov model. This variational approach allows us to determine the number of states directly from the data in a statistically principled manner.\n\nMoreover, by operating directly on the position data, we achieve significantly better time resolution compared to methods based on running averages of the bead root-mean-square distance from the tethering point. Finally, we show that hierarchical techniques developed in the context of single molecule FRET experiments can be adapted to our TPM methods to perform pooled analysis on many trajectories. This increases the accuracy of the method despite considerable bead-to-bead variability, and allows a more precise characterization of rare events.\n\nWe apply our method to Lac-mediated loop formation on a short (107 bp) construct, and demonstrate direct interconversion between two different looped states, with implications for structual models of the looped Lac-DNA complex.", "doi": "10.1016/j.bpj.2012.11.2329", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2013-01-29", "series_number": "2", "volume": "104", "issue": "2", "pages": "418A-418A" }, { "id": "authors:727rt-c0462", "collection": "authors", "collection_id": "727rt-c0462", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130205-103824324", "type": "article", "title": "Theoretical and Experimental Dissection of DNA Loop-Mediated Repression", "author": [ { "family_name": "Boedicker", "given_name": "James Q.", "clpid": "Boedicker-J-Q" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Transcriptional networks across all domains of life feature a wide range of regulatory architectures. Theoretical models now make clear predictions about how key parameters describing those architectures modulate gene expression, and the ability to construct genetic circuits with tunable parameters enables precise tests of such models. We dissect gene regulation through DNA looping by tuning network parameters such as repressor copy number, DNA binding strengths, and loop length in both thermodynamic models and experiments. Our results help clarify the short-length mechanical properties of DNA.", "doi": "10.1103/PhysRevLett.110.018101", "pmcid": "PMC3716456", "issn": "0031-9007", "publisher": "American Physical Society", "publication": "Physical Review Letters", "publication_date": "2013-01-04", "series_number": "1", "volume": "110", "issue": "1", "pages": "Art. No. 018101" }, { "id": "authors:4ttp3-w9h81", "collection": "authors", "collection_id": "4ttp3-w9h81", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130118-102317460", "type": "article", "title": "Interdisciplinary Graduate Training in Teaching Labs", "author": [ { "family_name": "Vale", "given_name": "Ronald D.", "clpid": "Vale-R-D" }, { "family_name": "DeRisi", "given_name": "Joseph", "clpid": "DeRisi-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Mullins", "given_name": "R. Dyche", "clpid": "Mullins-R-D" }, { "family_name": "Waterman", "given_name": "Clare", "clpid": "Waterman-C" }, { "family_name": "Mitchison", "given_name": "Timothy J.", "clpid": "Mitchison-T-J" } ], "abstract": "Modern research and training in the life sciences require cross-disciplinary programs, integrating concepts and methods from biology, physics, chemistry, and mathematics. We describe the structure and outcomes from an example of one such approach, the Physiology Course at the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts, and discuss how similar intensive, team-building research courses are also being applied to improve graduate education in universities. These courses are based on teaching laboratories that have students address contemporary research questions by combining ideas and approaches from biology, computation, and physics.", "doi": "10.1126/science.1216570", "pmcid": "PMC3810400", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2012-12-21", "series_number": "6114", "volume": "338", "issue": "6114", "pages": "1542-1543" }, { "id": "authors:7154e-yc170", "collection": "authors", "collection_id": "7154e-yc170", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130211-115816554", "type": "article", "title": "Tuning Promoter Strength through RNA Polymerase Binding Site Design in Escherichia coli", "author": [ { "family_name": "Brewster", "given_name": "Robert C.", "clpid": "Brewster-R-C" }, { "family_name": "Jones", "given_name": "Daniel L.", "orcid": "0000-0002-7902-996X", "clpid": "Jones-D-L" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "One of the paramount goals of synthetic biology is to have the ability to tune transcriptional networks to targeted levels of expression at will. As a step in that direction, we have constructed a set of 18 unique binding sites for E. coli RNA Polymerase (RNAP) \u03c3^(70) holoenzyme, designed using a model of sequence-dependent binding energy combined with a thermodynamic model of transcription to produce a targeted level of gene expression. This promoter set allows us to determine the correspondence between the absolute numbers of mRNA molecules or protein products and the predicted promoter binding energies measured in K_(B)T energy units. These binding sites adhere on average to the predicted level of gene expression over orders of magnitude in constitutive gene expression, to within a factor of in both protein and mRNA copy number. With these promoters in hand, we then place them under the regulatory control of a bacterial repressor and show that again there is a strict correspondence between the measured and predicted levels of expression, demonstrating the transferability of the promoters to an alternate regulatory context. In particular, our thermodynamic model predicts the expression from our promoters under a range of repressor concentrations between several per cell up to over 100 per cell. After correcting the predicted polymerase binding strength using the data from the unregulated promoter, the thermodynamic model accurately predicts the expression for the simple repression strains to within 30%. Demonstration of modular promoter design, where parts of the circuit (such as RNAP/TF binding strength and transcription factor copy number) can be independently chosen from a stock list and combined to give a predictable result, has important implications as an engineering tool for use in synthetic biology.", "doi": "10.1371/journal.pcbi.1002811", "pmcid": "PMC3521663", "issn": "1553-734X", "publisher": "Public Library of Science", "publication": "PLoS Computational Biology", "publication_date": "2012-12", "series_number": "12", "volume": "8", "issue": "12", "pages": "Art. No. e1002811" }, { "id": "authors:ndj0h-tq609", "collection": "authors", "collection_id": "ndj0h-tq609", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120709-133545138", "type": "article", "title": "Sequence Dependence of Transcription Factor-Mediated DNA Looping", "author": [ { "family_name": "Johnson", "given_name": "Stephanie", "orcid": "0000-0001-9487-8583", "clpid": "Johnson-S" }, { "family_name": "Lind\u00e9n", "given_name": "Martin", "clpid": "Lind\u00e9n-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "DNA is subject to large deformations in a wide range of biological processes. Two key examples illustrate how such deformations influence the readout of the genetic information: the sequestering of eukaryotic genes by nucleosomes, and DNA looping in transcriptional regulation in both prokaryotes and eukaryotes. These kinds of regulatory problems are now becoming amenable to systematic quantitative dissection with a powerful dialogue between theory and experiment. Here we use a single-molecule experiment in conjunction with a statistical mechanical model to test quantitative predictions for the behavior of DNA looping at short length scales, and to determine how DNA sequence affects looping at these lengths. We calculate and measure how such looping depends upon four key biological parameters: the strength of the transcription factor binding sites, the concentration of the transcription factor, and the length and sequence of the DNA loop. Our studies lead to the surprising insight that sequences that are thought to be especially favorable for nucleosome formation because of high flexibility lead to no systematically detectable effect of sequence on looping, and begin to provide a picture of the distinctions between the short length scale mechanics of nucleosome formation and looping.", "doi": "10.1093/nar/gks473", "pmcid": "PMC3439888", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "2012-09", "series_number": "16", "volume": "40", "issue": "16", "pages": "7728-7738" }, { "id": "authors:3cykd-9b915", "collection": "authors", "collection_id": "3cykd-9b915", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120827-142257698", "type": "article", "title": "A single-molecule view of protein action at a distance", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Decision making as a result of changing\nenvironmental conditions is a signature feature of\nall living organisms. One of the most common ways\nin which cells implement these decisions is through\nproteins regulating the process of transcription. An\nimpressive array of quantitative measurements now\nmake it possible to query the transcription process\nin exquisite detail and as a result have raised the bar\non what it means to understand transcription. After\ngiving an overview of the \"by the numbers\" approach\nto transcription, I will then highlight a three-pronged\nstrategy for the study of this crucial part of the central\ndogma based upon theoretical modeling using the tools\nof statistical mechanics, single-molecule approaches\nwhich allow us to watch transcription factors one at a\ntime and single-cell approaches which permit systematic\nmeasurements of the level of gene expression for a\nnumber of different regulatory architectures.", "issn": "0961-8368", "publisher": "Wiley", "publication": "Protein Science", "publication_date": "2012-08", "series_number": "S1", "volume": "21", "issue": "S1", "pages": "65" }, { "id": "authors:6ekzz-h6011", "collection": "authors", "collection_id": "6ekzz-h6011", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120814-102205172", "type": "article", "title": "Operator Sequence Alters Gene Expression Independently of Transcription Factor Occupancy in Bacteria", "author": [ { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Sanchez", "given_name": "Alvaro", "clpid": "Sanchez-A" }, { "family_name": "Boedicker", "given_name": "James Q.", "clpid": "Boedicker-J-Q" }, { "family_name": "Osborne", "given_name": "Melissa", "clpid": "Osborne-M" }, { "family_name": "Gelles", "given_name": "Jeff", "clpid": "Gelles-J" }, { "family_name": "Kondev", "given_name": "Jane", "clpid": "Kondev-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "A canonical quantitative view of transcriptional regulation holds that the only role of operator sequence is to set the probability of transcription factor binding, with operator occupancy determining the level of gene expression. In this work, we test this idea by characterizing repression in vivo and the binding of RNA polymerase in vitro in experiments where operators of various sequences were placed either upstream or downstream from the promoter in Escherichia coli. Surprisingly, we find that operators with a weaker binding affinity can yield higher repression levels than stronger operators. Repressor bound to upstream operators modulates promoter escape, and the magnitude of this modulation is not correlated with the repressor-operator binding affinity. This suggests that operator sequences may modulate transcription by altering the nature of the interaction of the bound transcription factor with the transcriptional machinery, implying a new layer of sequence dependence that must be confronted in the quantitative understanding of gene expression.", "doi": "10.1016/j.celrep.2012.06.004", "pmcid": "PMC3616187", "issn": "2211-1247", "publisher": "Elsevier", "publication": "Cell Reports", "publication_date": "2012-07-26", "series_number": "1", "volume": "2", "issue": "1", "pages": "150-161" }, { "id": "authors:nbt4d-ajs72", "collection": "authors", "collection_id": "nbt4d-ajs72", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120716-083157294", "type": "article", "title": "A Single-Molecule Hershey-Chase Experiment", "author": [ { "family_name": "Van Valen", "given_name": "David", "orcid": "0000-0001-7534-7621", "clpid": "Van-Valen-D" }, { "family_name": "Wu", "given_name": "David", "clpid": "Wu-David" }, { "family_name": "Chen", "given_name": "Yi-Ju", "clpid": "Chen-Yi-Ju" }, { "family_name": "Tuson", "given_name": "Hannah", "clpid": "Tuson-H" }, { "family_name": "Wiggins", "given_name": "Paul", "clpid": "Wiggins-P" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Ever since Hershey and Chase used phages to establish DNA as the carrier of genetic information in 1952, the precise mechanisms of phage DNA translocation have been a mystery. While bulk measurements have set a time scale for in vivo DNA translocation during bacteriophage infection, measurements of DNA ejection by single bacteriophages have only been made in vitro. Here, we present direct visualization of single bacteriophages infecting individual Escherichia coli cells. For bacteriophage lambda, we establish a mean ejection time of roughly 5 minutes with significant cell-to-cell variability, including pausing events. In contrast, corresponding in vitro single-molecule ejections take only 10 seconds to reach completion and do not exhibit significant variability. Our data reveal that the velocity of ejection for two different genome lengths collapses onto a single curve. This suggests that in vivo ejections are controlled by the amount of DNA ejected, in contrast with in vitro DNA ejections, which are governed by the amount of DNA left inside the capsid. This analysis provides evidence against a purely intrastrand repulsion based mechanism, and suggests that cell-internal processes dominate. This provides a picture of the early stages of phage infection and sheds light on the problem of polymer translocation.", "doi": "10.1016/j.cub.2012.05.023", "pmcid": "PMC3462812", "issn": "0960-9822", "publisher": "Cell Press", "publication": "Current Biology", "publication_date": "2012-07-24", "series_number": "14", "volume": "22", "issue": "14", "pages": "1339-1343" }, { "id": "authors:p9s9r-wfc55", "collection": "authors", "collection_id": "p9s9r-wfc55", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120706-080343895", "type": "article", "title": "Focus on the physics of the cell membrane", "author": [ { "family_name": "Bassereau", "given_name": "Patricia", "clpid": "Bassereau-P" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Schwille", "given_name": "Petra", "clpid": "Schwille-P" } ], "abstract": "This focus issue on membrane biophysics presents a collection of papers illustrating new developments in modern biophysical research on cell membranes. The work described here addresses questions from a broad range of areas, including cell adhesion, membrane trafficking and activation of cells of the immune system. It also presents recent views on membrane mechanics, the effect of electric fields, as well as on the interplay of mechanics and chemistry and organization at many different scales.", "doi": "10.1088/1367-2630/14/5/055021", "issn": "1367-2630", "publisher": "IOP", "publication": "New Journal of Physics", "publication_date": "2012-05-21", "volume": "14", "pages": "Art. No. 055021" }, { "id": "authors:mems0-81b42", "collection": "authors", "collection_id": "mems0-81b42", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120523-100035590", "type": "article", "title": "Single-Cell Census of Mechanosensitive Channels in Living Bacteria", "author": [ { "family_name": "Bialecka-Fornal", "given_name": "Maja", "clpid": "Bialecka-Fornal-M" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "DeBerg", "given_name": "Hannah A.", "clpid": "DeBerg-H-A" }, { "family_name": "Gandhi", "given_name": "Chris S.", "clpid": "Gandhi-C-S" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Bacteria are subjected to a host of different environmental stresses. One such insult occurs when cells encounter changes in the osmolarity of the surrounding media resulting in an osmotic shock. In recent years, a great deal has been learned about mechanosensitive (MS) channels which are thought to provide osmoprotection in these circumstances by opening emergency release valves in response to membrane tension. However, even the most elementary physiological parameters such as the number of MS channels per cell, how MS channel expression levels influence the physiological response of the cells, and how this mean number of channels varies from cell to cell remain unanswered. In this paper, we make a detailed quantitative study of the expression of the mechanosensitive channel of large conductance (MscL) in different media and at various stages in the growth history of bacterial cultures. Using both quantitative fluorescence microscopy and quantitative Western blots our study complements earlier electrophysiology-based estimates and results in the following key insights: i) the mean number of channels per cell is much higher than previously estimated, ii) measurement of the single-cell distributions of such channels reveals marked variability from cell to cell and iii) the mean number of channels varies under different environmental conditions. The regulation of MscL expression displays rich behaviors that depend strongly on culturing conditions and stress factors, which may give clues to the physiological role of MscL. The number of stress-induced MscL channels and the associated variability have far reaching implications for the in vivo response of the channels and for modeling of this response. As shown by numerous biophysical models, both the number of such channels and their variability can impact many physiological processes including osmoprotection, channel gating probability, and channel clustering.", "doi": "10.1371/journal.pone.0033077", "pmcid": "PMC3302805", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLoS ONE", "publication_date": "2012-03-13", "series_number": "3", "volume": "7", "issue": "3", "pages": "Art. No. e33077" }, { "id": "authors:zemhp-b3a92", "collection": "authors", "collection_id": "zemhp-b3a92", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120501-131827231", "type": "article", "title": "Entropic Tension in Crowded Membranes", "author": [ { "family_name": "Lind\u00e9n", "given_name": "Martin", "clpid": "Lind\u00e9n-M" }, { "family_name": "Sens", "given_name": "Pierre", "clpid": "Sens-P" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can alter the critical membrane tension at which they undergo a transition from a closed to an open state, and therefore influence protein function in vivo. Despite their obvious importance, crowding phenomena in membranes are much less well studied\nthan in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than 2 k_(B)T in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.", "doi": "10.1371/journal.pcbi.1002431", "pmcid": "PMC3305330", "issn": "1553-734X", "publisher": "Public Library of Science", "publication": "PLoS Computational Biology", "publication_date": "2012-03", "series_number": "3", "volume": "8", "issue": "3", "pages": "Art. no. e1002431" }, { "id": "authors:hem0e-q7e74", "collection": "authors", "collection_id": "hem0e-q7e74", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20111111-151109702", "type": "article", "title": "Lipid Bilayer Mechanics in a Pipette with Glass-Bilayer Adhesion", "author": [ { "family_name": "Ursell", "given_name": "Tristan", "clpid": "Ursell-T" }, { "family_name": "Agrawal", "given_name": "Ashutosh", "clpid": "Agrawal-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Electrophysiology is a central tool for measuring how different driving forces (e.g., ligand concentration, transmembrane voltage, or lateral tension) cause a channel protein to gate. Upon formation of the high resistance seal between a lipid bilayer and a glass pipette, the so-called \"giga-seal\", channel activity can be recorded electrically. In this article, we explore the implications of giga-seal formation on the mechanical state of a lipid bilayer patch. We use a mechanical model for the free energy of bilayer geometry in the presence of glass-bilayer adhesion to draw three potentially important conclusions. First, we use our adhesion model to derive an explicit relationship between applied pressure and patch shape that is consistent with the Laplace-Young Law, giving an alternative method of calculating patch tension under pressure. With knowledge of the adhesion constant, which we find to be in the range ~0.4\u20134 mN/m, and the pipette size, one can precisely calculate the patch tension as a function of pressure, without the difficultly of obtaining an optical measurement of the bilayer radius of curvature. Second, we use data from previous electrophysiological experiments to show that over a wide range of lipids, the resting tension on a electrophysiological patch is highly variable and can be 10\u2013100 times higher than estimates of the tension in a typical cell membrane. This suggests that electrophysiological experiments may be systematically altering channel-gating characteristics and querying the channels under conditions that are not the same as their physiological counterparts. Third, we show that reversible adhesion leads to a predictable change in the population response of gating channels in a bilayer patch.", "doi": "10.1016/j.bpj.2011.08.057", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2011-10-19", "series_number": "8", "volume": "101", "issue": "8", "pages": "1913-1920" }, { "id": "authors:jcmjv-1gb94", "collection": "authors", "collection_id": "jcmjv-1gb94", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20111114-111309696", "type": "article", "title": "Mechanosensitive Channels: What Can They Do and How Do They Do It?", "author": [ { "family_name": "Haswell", "given_name": "Elizabeth S.", "orcid": "0000-0002-4246-065X", "clpid": "Haswell-E-S" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "abstract": "While mechanobiological processes employ diverse mechanisms, at their heart are force-induced perturbations in the structure and dynamics of molecules capable of triggering subsequent events. Among the best characterized force-sensing systems are bacterial mechanosensitive channels. These channels reflect an intimate coupling of protein conformation with the mechanics of the surrounding membrane; the membrane serves as an adaptable sensor that responds to an input of applied force and converts it into an output signal, interpreted for the cell by mechanosensitive channels. The cell can exploit this information in a number of ways: ensuring cellular viability in the presence of osmotic stress and perhaps also serving as a signal transducer for membrane tension or other functions. This review focuses on the bacterial mechanosensitive channels of large (MscL) and small (MscS) conductance and their eukaryotic homologs, with an emphasis on the outstanding issues surrounding the function and mechanism of this fascinating class of molecules.", "doi": "10.1016/j.str.2011.09.005", "pmcid": "PMC3203646", "issn": "0969-2126", "publisher": "Cell Press", "publication": "Structure", "publication_date": "2011-10-12", "series_number": "10", "volume": "19", "issue": "10", "pages": "1356-1369" }, { "id": "authors:tx4br-6w882", "collection": "authors", "collection_id": "tx4br-6w882", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110909-100811989", "type": "article", "title": "Jonathan Widom (1955\u20132011)", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "One of the fundamental paradoxes of DNA, life's most iconic molecule, is that nearly a metre of this polymer must be packed into the microscopic nucleus of every human cell while still granting access for the many biochemical and biophysical transactions needed to exploit its genetic information. Jonathan Widom rigorously and creatively applied physical and chemical principles to unravel the mystery of how genomes can at once be tightly packed and yet available for read-out. He did this over a 30-year career that ended unexpectedly and prematurely on 18 July 2011. He was 55.", "doi": "10.1038/476400a", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2011-08-25", "series_number": "7361", "volume": "476", "issue": "7361", "pages": "400-400" }, { "id": "authors:yfy5g-zhf66", "collection": "authors", "collection_id": "yfy5g-zhf66", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110929-140259410", "type": "article", "title": "Comparison and Calibration of Different Reporters for Quantitative Analysis of Gene Expression", "author": [ { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Boedicker", "given_name": "James Q.", "clpid": "Boedicker-J-Q" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Absolute levels of gene expression in bacteria are observed to vary over as much as six orders of magnitude. Thermodynamic models have been proposed as a tool to describe the expression levels of a given transcriptional circuit. In this context, it is essential to understand both the limitations and linear range of the different methods for measuring gene expression and to determine to what extent measurements from different reporters can be directly compared with one aim being the stringent testing of theoretical descriptions of gene expression. In this article, we compare two protein reporters by measuring both the absolute level of expression and fold-change in expression using the fluorescent protein EYFP and the enzymatic reporter \u03b2-galactosidase. We determine their dynamic and linear range and show that they are interchangeable for measuring mean levels of expression over four orders of magnitude. By calibrating these reporters such that they can be interpreted in terms of absolute molecular counts, we establish limits for their applicability: autofluorescence on the lower end of expression for EYFP (at \u223c10 molecules per cell) and interference with cellular growth on the high end for \u03b2-galactosidase (at \u223c20,000 molecules per cell). These qualities make the reporters complementary and necessary when trying to experimentally verify the predictions from the theoretical models.", "doi": "10.1016/j.bpj.2011.06.026", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2011-08-03", "series_number": "3", "volume": "101", "issue": "3", "pages": "535-544" }, { "id": "authors:71drw-0bb08", "collection": "authors", "collection_id": "71drw-0bb08", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110801-102535300", "type": "article", "title": "Quantitative dissection of the simple repression input\u2013output function", "author": [ { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "We present a quantitative case study of transcriptional regulation in which we carry out a systematic dialogue between theory and measurement for an important and ubiquitous regulatory motif in bacteria, namely, that of simple repression. This architecture is realized by a single repressor binding site overlapping the promoter. From the theory point of view, this motif is described by a single gene regulation function based upon only a few parameters that are convenient theoretically and accessible experimentally. The usual approach is turned on its side by using the mathematical description of these regulatory motifs as a predictive tool to determine the number of repressors in a collection of strains with a large variation in repressor copy number. The predictions and corresponding measurements are carried out over a large dynamic range in both expression fold change (spanning nearly four orders of magnitude) and repressor copy number (spanning about two orders of magnitude). The predictions are tested by measuring the resulting level of gene expression and are then validated by using quantitative immunoblots. The key outcomes of this study include a systematic quantitative analysis of the limits and validity of the input\u2013output relation for simple repression, a precise determination of the in vivo binding energies for DNA\u2013repressor interactions for several distinct repressor binding sites, and a repressor census for Lac repressor in Escherichia coli.", "doi": "10.1073/pnas.1015616108", "pmcid": "PMC3141941", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2011-07-19", "series_number": "29", "volume": "108", "issue": "29", "pages": "12173-12178" }, { "id": "authors:tgp6k-cnr76", "collection": "authors", "collection_id": "tgp6k-cnr76", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110725-075342594", "type": "article", "title": "Building Enhancers from the Ground Up: A Synthetic Biology Approach", "author": [ { "family_name": "Amit", "given_name": "Roee", "clpid": "Amit-R" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" } ], "abstract": "A challenge of the synthetic biology approach is to use our understanding of a system to recreate a biological function with specific properties. We have applied this framework to bacterial enhancers, combining a driver, transcription factor binding sites, and a poised polymerase to create synthetic modular enhancers. Our findings suggest that enhancer-based transcriptional control depends critically and quantitatively on DNA looping, leading to complex regulatory effects when the enhancer cassettes contain additional transcription factor binding sites for TetR, a bacterial transcription factor. We show through a systematic interplay of experiment and thermodynamic modeling that the level of gene expression can be modulated to convert a variable inducer concentration input into discrete or step-like output expression levels. Finally, using a different DNA-binding protein (TraR), we show that the regulatory output is not a particular feature of the specific DNA-binding protein used for the enhancer but a general property of synthetic bacterial enhancers.", "doi": "10.1016/j.cell.2011.06.024", "pmcid": "PMC3155781", "issn": "0092-8674", "publisher": "Elsevier", "publication": "Cell", "publication_date": "2011-07-08", "series_number": "1", "volume": "146", "issue": "1", "pages": "105-118" }, { "id": "authors:0pbze-dgk14", "collection": "authors", "collection_id": "0pbze-dgk14", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110706-095349883", "type": "article", "title": "Probing Individual Environmental Bacteria for Viruses by Using Microfluidic Digital PCR", "author": [ { "family_name": "Tadmor", "given_name": "Arbel D.", "clpid": "Tadmor-A-D" }, { "family_name": "Ottesen", "given_name": "Elizabeth A.", "clpid": "Ottesen-E-A" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Viruses may very well be the most abundant biological entities on the planet. Yet neither metagenomic studies nor classical phage isolation techniques have shed much light on the identity of the hosts of most viruses. We used a microfluidic digital polymerase chain reaction (PCR) approach to physically link single bacterial cells harvested from a natural environment with a viral marker gene. When we implemented this technique on the microbial community residing in the termite hindgut, we found genus-wide infection patterns displaying remarkable intragenus selectivity. Viral marker allelic diversity revealed restricted mixing of alleles between hosts, indicating limited lateral gene transfer of these alleles despite host proximity. Our approach does not require culturing hosts or viruses and provides a method for examining virus-bacterium interactions in many environments.", "doi": "10.1126/science.1200758", "pmcid": "PMC3261838", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2011-07-01", "series_number": "6038", "volume": "333", "issue": "6038", "pages": "58-62" }, { "id": "authors:d0z4x-vkt05", "collection": "authors", "collection_id": "d0z4x-vkt05", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110621-092111171", "type": "article", "title": "Elastic energy of polyhedral bilayer vesicles", "author": [ { "family_name": "Haselwandter", "given_name": "Christoph A.", "clpid": "Haselwandter-C-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "In recent experiments [M. Dubois, B. Dem\u00e9, T. Gulik-Krzywicki, J.-C. Dedieu, C. Vautrin, S. D\u00e9sert, E. Perez, and T. Zemb, Nature (London) 411, 672 (2001)] the spontaneous formation of hollow bilayer vesicles with polyhedral symmetry has been observed. On the basis of the experimental phenomenology it was suggested [M. Dubois, V. Lizunov, A. Meister, T. Gulik-Krzywicki, J. M. Verbavatz, E. Perez, J. Zimmerberg, and T. Zemb, Proc. Natl. Acad. Sci. USA 101, 15082 (2004)] that the mechanism for the formation of bilayer polyhedra is minimization of elastic bending energy. Motivated by these experiments, we study the elastic bending energy of polyhedral bilayer vesicles. In agreement with experiments, and provided that excess amphiphiles exhibiting spontaneous curvature are present in sufficient quantity, we find that polyhedral bilayer vesicles can indeed be energetically favorable compared to spherical bilayer vesicles. Consistent with experimental observations we also find that the bending energy associated with the vertices of bilayer polyhedra can be locally reduced through the formation of pores. However, the stabilization of polyhedral bilayer vesicles over spherical bilayer vesicles relies crucially on molecular segregation of excess amphiphiles along the ridges rather than the vertices of bilayer polyhedra. Furthermore, our analysis implies that, contrary to what has been suggested on the basis of experiments, the icosahedron does not minimize elastic bending energy among arbitrary polyhedral shapes and sizes. Instead, we find that, for large polyhedron sizes, the snub dodecahedron and the snub cube both have lower total bending energies than the icosahedron.", "doi": "10.1103/PhysRevE.83.061901", "pmcid": "PMC3236088", "issn": "1539-3755", "publisher": "American Physical Society", "publication": "Physical Review E", "publication_date": "2011-06-01", "series_number": "6", "volume": "83", "issue": "6", "pages": "Art. No. 061901" }, { "id": "authors:1d78d-gtf34", "collection": "authors", "collection_id": "1d78d-gtf34", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110422-111812818", "type": "article", "title": "Effect of Promoter Architecture on the Cell-to-Cell Variability in Gene Expression", "author": [ { "family_name": "Sanchez", "given_name": "Alvaro", "clpid": "Sanchez-A" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Jones", "given_name": "Daniel", "orcid": "0000-0002-7902-996X", "clpid": "Jones-D-L" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Kondev", "given_name": "Jan\u00e9", "clpid": "Kondev-J" } ], "abstract": "According to recent experimental evidence, promoter architecture, defined by the number, strength and regulatory role of the operators that control transcription, plays a major role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effort that addresses the question of how changes in promoter architecture affect variability in gene expression in a systematic rather than case-by-case fashion. In this article we make such a systematic investigation, based on a microscopic model of gene regulation that incorporates stochastic effects. In particular, we show how operator strength and operator multiplicity affect this variability. We examine different modes of transcription factor binding to complex promoters (cooperative, independent, simultaneous) and how each of these affects the level of variability in transcriptional output from cell-to-cell. We propose that direct comparison between in vivo single-cell experiments and theoretical predictions for the moments of the probability distribution of mRNA number per cell can be used to test kinetic models of gene regulation. The emphasis of the discussion is on prokaryotic gene regulation, but our analysis can be extended to eukaryotic cells as well.", "doi": "10.1371/journal.pcbi.1001100", "pmcid": "PMC3048382", "issn": "1553-734X", "publisher": "Public Library of Science", "publication": "PLoS Computational Biology", "publication_date": "2011-03", "series_number": "3", "volume": "7", "issue": "3", "pages": "Art. No. e1001100" }, { "id": "authors:4y55t-1qq45", "collection": "authors", "collection_id": "4y55t-1qq45", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110324-162735185", "type": "article", "title": "Morphological Phase Diagram for Lipid Membrane Domains with Entropic Tension", "author": [ { "family_name": "Rim", "given_name": "J. E.", "clpid": "Rim-J-E" }, { "family_name": "Ursell", "given_name": "T. S.", "clpid": "Ursell-T-S" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Klug", "given_name": "W. S.", "clpid": "Klug-W-S" } ], "abstract": "Circular domains in phase-separated lipid vesicles with symmetric leaflet composition commonly exhibit three stable morphologies: flat, dimpled, and budded. However, stable dimples (i.e., partially budded domains) present a puzzle since simple elastic theories of domain shape predict that only flat and spherical budded domains are mechanically stable in the absence of spontaneous curvature. We argue that this inconsistency arises from the failure of the constant surface tension ensemble to properly account for the effect of entropic bending fluctuations. Formulating membrane elasticity within an entropic tension ensemble, wherein tension represents the free energy cost of extracting membrane area from thermal bending of the membrane, we calculate a morphological phase diagram that contains regions of mechanical stability for each of the flat, dimpled, and budded domain morphologies.", "doi": "10.1103/PhysRevLett.106.057801", "issn": "0031-9007", "publisher": "American Physical Society", "publication": "Physical Review Letters", "publication_date": "2011-02-03", "series_number": "5", "volume": "106", "issue": "5", "pages": "057801" }, { "id": "authors:v3def-aaj52", "collection": "authors", "collection_id": "v3def-aaj52", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110531-101647091", "type": "book_section", "title": "Thermodynamics of Biological Processes", "book_title": "Biothermodynamics. Part D", "author": [ { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Kondev", "given_name": "Jane", "clpid": "Kondev-J" }, { "family_name": "Orme", "given_name": "Nigel", "clpid": "Orme-N" }, { "family_name": "Theriot", "given_name": "Julie A.", "clpid": "Theriot-J-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "There is a long and rich tradition of using ideas from both equilibrium thermodynamics and its microscopic partner theory of equilibrium statistical mechanics. In this chapter, we provide some background on the origins of the seemingly unreasonable effectiveness of ideas from both thermodynamics and statistical mechanics in biology. After making a description of these foundational issues, we turn to a series of case studies primarily focused on binding that are intended to illustrate the broad biological reach of equilibrium thinking in biology. These case studies include ligand-gated ion channels, thermodynamic models of transcription, and recent applications to the problem of bacterial chemotaxis. As part of the description of these case studies, we explore a number of different uses of the famed Monod\u2013Wyman\u2013Changeux (MWC) model as a generic tool for providing a mathematical characterization of two-state systems. These case studies should provide a template for tailoring equilibrium ideas to other problems of biological interest.", "doi": "10.1016/B978-0-12-381268-1.00014-8", "isbn": "9780123860033", "publisher": "Elsevier", "place_of_publication": "Amsterdam", "publication_date": "2011", "pages": "27-59" }, { "id": "authors:3qcyf-yah54", "collection": "authors", "collection_id": "3qcyf-yah54", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110207-083519894", "type": "article", "title": "Transcription by the numbers redux: experiments and calculations that surprise", "author": [ { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Sanchez", "given_name": "Alvaro", "clpid": "Sanchez-A" }, { "family_name": "Kuhlman", "given_name": "Thomas", "clpid": "Kuhlman-T" }, { "family_name": "Kondev", "given_name": "Jane", "clpid": "Kondev-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The study of transcription has witnessed an explosion of quantitative effort both experimentally and theoretically. In this article we highlight some of the exciting recent experimental efforts in the study of transcription with an eye to the demands that such experiments put on theoretical models of transcription. From a modeling perspective, we focus on two broad classes of models: the so-called thermodynamic models that use statistical mechanics to reckon the level of gene expression as probabilities of promoter occupancy, and rate-equation treatments that focus on the temporal evolution of the activity of a given promoter and that make it possible to compute the distributions of messenger RNA and proteins. We consider several appealing case studies to illustrate how quantitative models have been used to dissect transcriptional regulation.", "doi": "10.1016/j.tcb.2010.07.002", "pmcid": "PMC3174145", "issn": "0962-8924", "publisher": "Elsevier", "publication": "Trends in Cell Biology", "publication_date": "2010-12", "series_number": "12", "volume": "20", "issue": "12", "pages": "723-733" }, { "id": "authors:w53vt-bzx13", "collection": "authors", "collection_id": "w53vt-bzx13", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110302-102608259", "type": "article", "title": "Minimal Bending Energies of Bilayer Polyhedra", "author": [ { "family_name": "Haselwandter", "given_name": "Christoph A.", "clpid": "Haselwandter-C-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Motivated by recent experiments on bilayer polyhedra composed of amphiphilic molecules, we study the elastic bending energies of bilayer vesicles forming polyhedral shapes. Allowing for segregation of excess amphiphiles along the ridges of polyhedra, we find that bilayer polyhedra can indeed have lower bending energies than spherical bilayer vesicles. However, our analysis also implies that, contrary to what has been suggested on the basis of experiments, the snub dodecahedron, rather than the icosahedron, generally represents the energetically favorable shape of bilayer polyhedra.", "doi": "10.1103/PhysRevLett.105.228101", "pmcid": "PMC3169433", "issn": "0031-9007", "publisher": "American Physical Society", "publication": "Physical Review Letters", "publication_date": "2010-11-24", "series_number": "22", "volume": "105", "issue": "22", "pages": "Art. No. 228101" }, { "id": "authors:ee1g1-pa436", "collection": "authors", "collection_id": "ee1g1-pa436", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20101004-094321543", "type": "article", "title": "Dynamical fluctuations in biochemical reactions and cycles", "author": [ { "family_name": "Press\u00e9", "given_name": "S.", "clpid": "Press\u00e9-S" }, { "family_name": "Ghosh", "given_name": "K.", "clpid": "Ghosh-K" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Dill", "given_name": "K. A.", "clpid": "Dill-K-A" } ], "abstract": "We develop theory for the dynamics and fluctuations in some cyclic and linear biochemical reactions. We use the approach of maximum caliber, which computes the ensemble of paths taken by the system, given a few experimental observables. This approach may be useful for interpreting single-molecule or few-particle experiments on molecular motors, enzyme reactions, ion-channels, and phosphorylation-driven biological clocks. We consider cycles where all biochemical states are observable. Our method shows how: (1) the noise in cycles increases with cycle size and decreases with the driving force that spins the cycle and (2) provides a recipe for estimating small-number features, such as probability of backward spin in small cycles, from experimental data. The back-spin probability diminishes exponentially with the deviation from equilibrium. We believe this method may also be useful for other few-particle nonequilibrium biochemical reaction systems.", "doi": "10.1103/PhysRevE.82.031905", "issn": "1539-3755", "publisher": "American Physical Society", "publication": "Physical Review E", "publication_date": "2010-09-15", "series_number": "3", "volume": "82", "issue": "3", "pages": "Art. No. 031905" }, { "id": "authors:qe7vg-w2207", "collection": "authors", "collection_id": "qe7vg-w2207", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20100907-101147251", "type": "article", "title": "Ion-Dependent Dynamics of DNA Ejections for Bacteriophage \u03bb", "author": [ { "family_name": "Wu", "given_name": "David", "clpid": "Wu-David" }, { "family_name": "Van Valen", "given_name": "David", "orcid": "0000-0001-7534-7621", "clpid": "Van-Valen-D" }, { "family_name": "Hu", "given_name": "Qicong", "clpid": "Hu-Qicong" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "We studied the control parameters that govern the dynamics of in vitro DNA ejection in bacteriophage \u03bb. Previous work demonstrated that bacteriophage DNA is highly pressurized, and this pressure has been hypothesized to help drive DNA ejection. Ions influence this process by screening charges on DNA; however, a systematic variation of salt concentrations to explore these effects has not been undertaken. To study the nature of the forces driving DNA ejection, we performed in vitro measurements of DNA ejection in bulk and at the single-phage level. We present measurements on the dynamics of ejection and on the self-repulsion force driving ejection. We examine the role of ion concentration and identity in both measurements, and show that the charge of counterions is an important control parameter. These measurements show that the mobility of ejecting DNA is independent of ionic concentrations for a given amount of DNA in the capsid. We also present evidence that phage DNA forms loops during ejection, and confirm that this effect occurs using optical tweezers.", "doi": "10.1016/j.bpj.2010.06.024", "pmcid": "PMC2920739", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2010-08-18", "series_number": "4", "volume": "99", "issue": "4", "pages": "1101-1109" }, { "id": "authors:qvmmn-sgd49", "collection": "authors", "collection_id": "qvmmn-sgd49", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20100119-145049392", "type": "article", "title": "A feeling for the numbers in biology", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Milo", "given_name": "Ron", "orcid": "0000-0003-1641-2299", "clpid": "Milo-Ron" } ], "abstract": "Although the quantitative description of biological systems has been going on for centuries, recent advances in the measurement of phenomena ranging from metabolism to gene expression to signal transduction have resulted in a new emphasis on biological numeracy. This article describes the confluence of two different approaches to biological numbers. First, an impressive array of quantitative measurements make it possible to develop intuition about biological numbers ranging from how many gigatons of atmospheric carbon are fixed every year in the process of photosynthesis to the number of membrane transporters needed to provide sugars to rapidly dividing Escherichia coli cells. As a result of the vast array of such quantitative data, the BioNumbers web site has recently been developed as a repository for biology by the numbers. Second, a complementary and powerful tradition of numerical estimates familiar from the physical sciences and canonized in the so-called \"Fermi problems\" calls for efforts to estimate key biological quantities on the basis of a few foundational facts and simple ideas from physics and chemistry. In this article, we describe these two approaches and illustrate their synergism in several particularly appealing case studies. These case studies reveal the impact that an emphasis on numbers can have on important biological questions.", "doi": "10.1073/pnas.0907732106", "pmcid": "PMC2799844", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2009-12-22", "series_number": "51", "volume": "106", "issue": "51", "pages": "21465-21471" }, { "id": "authors:fc5p7-7d721", "collection": "authors", "collection_id": "fc5p7-7d721", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170409-072401658", "type": "article", "title": "RNA-protein binding kinetics in an automated microfluidic reactor", "author": [ { "family_name": "Ridgeway", "given_name": "William K.", "clpid": "Ridgeway-W-K" }, { "family_name": "Seitaridou", "given_name": "Effrosyni", "clpid": "Seitaridou-E" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Williamson", "given_name": "James R.", "clpid": "Williamson-J-R" } ], "abstract": "Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA\u2013protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic 'Riboreactor' has been designed and constructed to facilitate the study of kinetics of RNA\u2013protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA\u2013protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.", "doi": "10.1093/nar/gkp733", "pmcid": "PMC2790880", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "2009-11", "series_number": "21", "volume": "37", "issue": "21", "pages": "e142-e142" }, { "id": "authors:qejar-8a961", "collection": "authors", "collection_id": "qejar-8a961", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20100114-101620211", "type": "article", "title": "RNA\u2013protein binding kinetics in an automated microfluidic reactor", "author": [ { "family_name": "Ridgeway", "given_name": "William K.", "clpid": "Ridgeway-W-K" }, { "family_name": "Seitaridou", "given_name": "Effrosyni", "clpid": "Seitaridou-E" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Williamson", "given_name": "James R.", "clpid": "Williamson-J-R" } ], "abstract": "Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA\u2013protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic 'Riboreactor' has been designed and constructed to facilitate the study of kinetics of RNA\u2013protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA\u2013protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.", "doi": "10.1093/nar/gkp733", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "2009-09-16", "series_number": "21", "volume": "37", "issue": "21", "pages": "Art. e142" }, { "id": "authors:tphnr-vqz10", "collection": "authors", "collection_id": "tphnr-vqz10", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20090828-231031757", "type": "article", "title": "Morphology and interaction between lipid domains", "author": [ { "family_name": "Ursell", "given_name": "Tristan S.", "clpid": "Ursell-T-S" }, { "family_name": "Klug", "given_name": "William S.", "clpid": "Klug-W-S" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Cellular membranes are a heterogeneous mix of lipids, proteins and small molecules. Special groupings enriched in saturated lipids and cholesterol form liquid-ordered domains, known as \"lipid rafts,\" thought to serve as platforms for signaling, trafficking and material transport throughout the secretory pathway. Questions remain as to how the cell maintains small fluid lipid domains, through time, on a length scale consistent with the fact that no large-scale phase separation is observed. Motivated by these examples, we have utilized a combination of mechanical modeling and in vitro experiments to show that membrane morphology plays a key role in maintaining small domain sizes and organizing domains in a model membrane. We demonstrate that lipid domains can adopt a flat or dimpled morphology, where the latter facilitates a repulsive interaction that slows coalescence and helps regulate domain size and tends to laterally organize domains in the membrane.", "doi": "10.1073/pnas.0903825106", "pmcid": "PMC2726347", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2009-08-11", "series_number": "32", "volume": "106", "issue": "32", "pages": "13301-13306" }, { "id": "authors:aqkmt-jr394", "collection": "authors", "collection_id": "aqkmt-jr394", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20090817-144809908", "type": "article", "title": "Trajectory Approach to Two-State Kinetics of Single Particles on Sculpted Energy Landscapes", "author": [ { "family_name": "Wu", "given_name": "David", "orcid": "0000-0003-4587-0280", "clpid": "Wu-David" }, { "family_name": "Ghosh", "given_name": "Kingshuk", "orcid": "0000-0003-4976-0986", "clpid": "Ghosh-Kingshuk" }, { "family_name": "Inamdar", "given_name": "Mandar", "orcid": "0000-0001-8549-8490", "clpid": "Inamdar-Mandar-M" }, { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Fraser", "given_name": "Scott", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Dill", "given_name": "Ken", "orcid": "0000-0002-2390-2002", "clpid": "Dill-Ken" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "We study the trajectories of a single colloidal particle as it hops between two energy wells which are sculpted using optical traps. Whereas the dynamical behaviors of such systems are often treated by master-equation methods that focus on particles as actors, we analyze them instead using a trajectory-based variational method called maximum caliber (MaxCal). We show that the MaxCal strategy accurately predicts the full dynamics that we observe in the experiments: From the observed averages, it predicts second and third moments and covariances, with no free parameters. The covariances are the dynamical equivalents of Maxwell-like equilibrium reciprocal relations and Onsager-like dynamical relations.", "doi": "10.1103/PhysRevLett.103.050603", "pmcid": "PMC3273425", "issn": "0031-9007", "publisher": "American Physical Society", "publication": "Physical Review Letters", "publication_date": "2009-07-31", "series_number": "5", "volume": "103", "issue": "5", "pages": "Art. No. 050603" }, { "id": "authors:bptnn-jrw02", "collection": "authors", "collection_id": "bptnn-jrw02", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20090727-143233526", "type": "article", "title": "Reduced amino acid alphabets exhibit an improved sensitivity and selectivity in fold assignment", "author": [ { "family_name": "Peterson", "given_name": "Eric L.", "clpid": "Peterson-E-L" }, { "family_name": "Kondev", "given_name": "Jan\u00e9", "clpid": "Kondev-J" }, { "family_name": "Theriot", "given_name": "Julie A.", "clpid": "Theriot-J-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Motivation: Many proteins with vastly dissimilar sequences are found to share a common fold, as evidenced in the wealth of structures now available in the Protein Data Bank. One idea that has found success in various applications is the concept of a reduced amino acid alphabet, wherein similar amino acids are clustered together. Given the structural similarity exhibited by many apparently dissimilar sequences, we undertook this study looking for improvements in fold recognition by comparing protein sequences written in a reduced alphabet. \n\nResults: We tested over 150 of the amino acid clustering schemes proposed in the literature with all-versus-all pairwise sequence alignments of sequences in the Distance matrix alignment (DALI) database. We combined several metrics from information retrieval popular in the literature: mean precision, area under the Receiver Operating Characteristic curve and recall at a fixed error rate and found that, in contrast to previous work, reduced alphabets in many cases outperform full alphabets. We find that reduced alphabets can perform at a level comparable to full alphabets in correct pairwise alignment of sequences and can show increased sensitivity to pairs of sequences with structural similarity but low-sequence identity. Based on these results, we hypothesize that reduced alphabets may also show performance gains with more sophisticated methods such as profile and pattern searches.", "doi": "10.1093/bioinformatics/btp164", "pmcid": "PMC2732308", "issn": "1367-4803", "publisher": "Oxford University Press", "publication": "Bioinformatics", "publication_date": "2009-06-01", "series_number": "11", "volume": "25", "issue": "11", "pages": "1356-1362" }, { "id": "authors:j28ym-kd539", "collection": "authors", "collection_id": "j28ym-kd539", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20090914-090009819", "type": "article", "title": "First-principles calculation of DNA looping in tethered particle experiments", "author": [ { "family_name": "Towles", "given_name": "Kevin B.", "clpid": "Towles-K-B" }, { "family_name": "Beausang", "given_name": "John F", "clpid": "Beausang-J-F" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Nelson", "given_name": "Philip C.", "clpid": "Nelson-P-C" } ], "abstract": "We calculate the probability of DNA loop formation mediated by regulatory proteins such as Lac repressor (LacI), using a mathematical model of DNA elasticity. Our model is adapted to calculating quantities directly observable in tethered particle motion (TPM) experiments, and it accounts for all the entropic forces present in such experiments. Our model has no free parameters; it characterizes DNA elasticity using information obtained in other kinds of experiments. It assumes a harmonic elastic energy function (or wormlike chain type elasticity), but our Monte Carlo calculation scheme is flexible enough to accommodate arbitrary elastic energy functions. We show how to compute both the 'looping J factor' (or equivalently, the looping free energy) for various DNA construct geometries and LacI concentrations, as well as the detailed probability density function of bead excursions. We also show how to extract the same quantities from recent experimental data on TPM, and then compare to our model's predictions. In particular, we present a new method to correct observed data for finite camera shutter time and other experimental effects. Although the currently available experimental data give large uncertainties, our first-principles predictions for the looping free energy change are confirmed to within about 1 k_BT, for loops of length around 300 basepairs. More significantly, our model successfully reproduces the detailed distributions of bead excursion, including their surprising three-peak structure, without any fit parameters and without invoking any alternative conformation of the LacI tetramer. Indeed, the model qualitatively reproduces the observed dependence of these distributions on tether length (e.g., phasing) and on LacI concentration (titration). However, for short DNA loops (around 95 basepairs) the experiments show more looping than is predicted by the harmonic-elasticity model, echoing other recent experimental results. Because the experiments we study are done in vitro, this anomalously high looping cannot be rationalized as resulting from the presence of DNA-bending proteins or other cellular machinery. We also show that it is unlikely to be the result of a hypothetical 'open' conformation of the LacI tetramer.", "doi": "10.1088/1478-3975/6/2/025001", "issn": "1478-3967", "publisher": "IOP", "publication": "Physical Biology", "publication_date": "2009-06", "series_number": "2", "volume": "6", "issue": "2", "pages": "Art. No. 025001" }, { "id": "authors:kj1g3-rw007", "collection": "authors", "collection_id": "kj1g3-rw007", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20100120-154513518", "type": "article", "title": "Concentration and Length Dependence of DNA Looping in Transcriptional Regulation", "author": [ { "family_name": "Han", "given_name": "Lin", "clpid": "Han-Lin" }, { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Blumberg", "given_name": "Seth", "clpid": "Blumberg-S" }, { "family_name": "Towles", "given_name": "Kevin B.", "clpid": "Towles-K-B" }, { "family_name": "Beausang", "given_name": "John F.", "clpid": "Beausang-J-F" }, { "family_name": "Nelson", "given_name": "Philip C.", "clpid": "Nelson-P-C" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage), to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least) two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the J-factor for looping.", "doi": "10.1371/journal.pone.0005621", "pmcid": "PMC2682762", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLoS ONE", "publication_date": "2009-05-25", "series_number": "5", "volume": "4", "issue": "5", "pages": "Art. No. e5621" }, { "id": "authors:99b2g-6e507", "collection": "authors", "collection_id": "99b2g-6e507", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20090814-082130740", "type": "article", "title": "Emerging roles for lipids in shaping membrane-protein function", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Ursell", "given_name": "Tristan", "clpid": "Ursell-T" }, { "family_name": "Wiggins", "given_name": "Paul", "clpid": "Wiggins-P-A" }, { "family_name": "Sens", "given_name": "Pierre", "clpid": "Sens-P" } ], "abstract": "Studies of membrane proteins have revealed a direct link between the lipid environment and the structure and function of some of these proteins. Although some of these effects involve specific chemical interactions between lipids and protein residues, many can be understood in terms of protein-induced perturbations to the membrane shape. The free-energy cost of such perturbations can be estimated quantitatively, and measurements of channel gating in model systems of membrane proteins with their lipid partners are now confirming predictions of simple models.", "doi": "10.1038/nature08147", "pmcid": "PMC3169427", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2009-05-21", "series_number": "7245", "volume": "459", "issue": "7245", "pages": "379-385" }, { "id": "authors:ejfb3-65p30", "collection": "authors", "collection_id": "ejfb3-65p30", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20090825-145726010", "type": "article", "title": "Biochemistry on a Leash: The Roles of Tether Length and Geometry in Signal Integration Proteins", "author": [ { "family_name": "Van Valen", "given_name": "David", "orcid": "0000-0001-7534-7621", "clpid": "Van-Valen-D" }, { "family_name": "Haataja", "given_name": "Mikko", "orcid": "0000-0001-8459-7331", "clpid": "Haataja-Mikko" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "We use statistical mechanics and simple ideas from polymer physics to develop a quantitative model of proteins whose activity is controlled by flexibly tethered ligands and receptors. We predict how the properties of tethers influence the function of these proteins and demonstrate how their tether length dependence can be exploited to construct proteins whose integration of multiple signals can be tuned. One case study to which we apply these ideas is that of the Wiskott-Aldrich Syndrome Proteins as activators of actin polymerization. More generally, tethered ligands competing with those free in solution are common phenomena in biology, making this an important specific example of a widespread biological idea.", "doi": "10.1016/j.bpj.2008.10.052", "pmcid": "PMC2717227", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2009-02-18", "series_number": "4", "volume": "96", "issue": "4", "pages": "1275-1292" }, { "id": "authors:qnfz1-ybs36", "collection": "authors", "collection_id": "qnfz1-ybs36", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20100817-074128416", "type": "book_section", "title": "Calibration of Tethered Particle Motion Experiments", "book_title": "Mathematics of DNA structure, function and interactions", "author": [ { "family_name": "Han", "given_name": "Lin", "clpid": "Han-Lin" }, { "family_name": "Lui", "given_name": "Bertrand H.", "clpid": "Lui-Bertrand-H" }, { "family_name": "Blumberg", "given_name": "Seth", "clpid": "Blumberg-S" }, { "family_name": "Beausang", "given_name": "John F.", "clpid": "Beausang-J-F" }, { "family_name": "Nelson", "given_name": "Philip C.", "clpid": "Nelson-P-C" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "contributor": [ { "family_name": "Benham", "given_name": "Craig John", "clpid": "Benham-C-J" } ], "abstract": "The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA loping and transcription. TPM experiments exploit the Brownian motion of a DNA-tethered bead to probe biologically relevant conformational changes of the tether. In these experiments, a change in the extent of the bead's random motion is used as a reporter of the underlying macromolecular dynamics and is often deemed sufficient for TPM analysis. However, a complete understanding of how the motion depends on the physical properties of the tethered particle complex would permit more quantitative and accurate evaluation of TPM data. For instance, such understanding can help extract details about a looped complex geometry (or multiple coexisting geometries) from TPM data. To better characterize the measurement capabilities of TPM experiments involving DNA tethers, we have carried out a detailed calibration of TPM magnitude as a function of DNA length and particle size. We also explore how experimental parameters such as acquisition time and exposure time affect the apparent motion of the tethered particle. We vary the DNA length from 200 bp to 2.6 kbp and consider particle diameters of 200, 490 and 970 nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation.", "doi": "10.1007/978-1-4419-0670-0_6", "isbn": "978-1-4419-0670-0", "publisher": "Springer", "place_of_publication": "New York", "publication_date": "2009", "pages": "123-138" }, { "id": "authors:53aty-csq63", "collection": "authors", "collection_id": "53aty-csq63", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:LEEpnas08", "type": "article", "title": "Membrane shape as a reporter for applied forces", "author": [ { "family_name": "Lee", "given_name": "Heun Jin", "clpid": "Lee-Heun-Jin" }, { "family_name": "Peterson", "given_name": "Eric-L", "clpid": "Peterson-E-L" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Klug", "given_name": "William S.", "clpid": "Klug-W-S" }, { "family_name": "Wiggins", "given_name": "Paul A.", "clpid": "Wiggins-P-A" } ], "abstract": "Recent advances have enabled 3-dimensional reconstructions of biological structures in vivo, ranging in size and complexity from single proteins to multicellular structures. In particular, tomography and confocal microscopy have been exploited to capture detailed 3-dimensional conformations of membranes in cellular processes ranging from viral budding and organelle maintenance to phagocytosis. Despite the wealth of membrane structures available, there is as yet no generic, quantitative method for their interpretation. We propose that by modeling these observed biomembrane shapes as fluid lipid bilayers in mechanical equilibrium, the externally applied forces as well as the pressure, tension, and spontaneous curvature can be computed directly from the shape alone. To illustrate the potential power of this technique, we apply an axial force with optical tweezers to vesicles and explicitly demonstrate that the applied force is equal to the force computed from the membrane conformation.", "doi": "10.1073/pnas.0806814105", "pmcid": "PMC2614748", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2008-12-09", "series_number": "49", "volume": "105", "issue": "49", "pages": "19252-19256" }, { "id": "authors:57wdc-rmx77", "collection": "authors", "collection_id": "57wdc-rmx77", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:REEpre08", "type": "article", "title": "Membrane mechanics as a probe of ion-channel gating mechanisms", "author": [ { "family_name": "Reeves", "given_name": "Daniel", "clpid": "Reeves-D" }, { "family_name": "Ursell", "given_name": "Tristan", "clpid": "Ursell-T" }, { "family_name": "Sens", "given_name": "Pierre", "clpid": "Sens-P" }, { "family_name": "Kondev", "given_name": "Jane", "clpid": "Kondev-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The details of conformational changes undergone by transmembrane ion channels in response to stimuli, such as electric fields and membrane tension, remain controversial. We approach this problem by considering how the conformational changes impose deformations in the lipid bilayer. We focus on the role of bilayer deformations in the context of voltage-gated channels because we hypothesize that such deformations are relevant in this case as well as for channels that are explicitly mechanosensitive. As a result of protein conformational changes, we predict that the lipid bilayer suffers deformations with a characteristic free-energy scale of 10kBT. This free energy is comparable to the voltage-dependent part of the total gating energy, and we argue that these deformations could play an important role in the overall free-energy budget of gating. As a result, channel activity will depend upon mechanical membrane parameters such as tension and leaflet thickness. We further argue that the membrane deformation around any channel can be divided into three generic classes of deformation that exhibit different mechanosensitive properties. Finally, we provide the theoretical framework that relates conformational changes during gating to tension and leaflet thickness dependence in the critical gating voltage. This line of investigation suggests experiments that could discern the dominant deformation imposed upon the membrane as a result of channel gating, thus providing clues as to the channel deformation induced by the stimulus.", "doi": "10.1103/PhysRevE.78.041901", "issn": "1539-3755", "publisher": "American Physical Society", "publication": "Physical Review E", "publication_date": "2008-10", "series_number": "4", "volume": "78", "issue": "4", "pages": "Art. No. 041901" }, { "id": "authors:kjnfq-jfp81", "collection": "authors", "collection_id": "kjnfq-jfp81", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:GRApnas07", "type": "article", "title": "Real-time observations of single bacteriophage \u03bb DNA ejections in vitro", "author": [ { "family_name": "Grayson", "given_name": "Paul", "clpid": "Grayson-P" }, { "family_name": "Han", "given_name": "Lin", "clpid": "Han-Lin" }, { "family_name": "Winther", "given_name": "Tabita", "clpid": "Winther-T" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "The physical, chemical, and structural features of bacteriophage genome release have been the subject of much recent attention. Many theoretical and experimental studies have centered on the internal forces driving the ejection process. Recently, Mangenot et al. [Mangenot S, Hochrein M, R\u00e4dler J, Letellier L (2005) Curr Biol 15:430\u2013435.] reported fluorescence microscopy of phage T5 ejections, which proceeded stepwise between DNA nicks, reaching a translocation speed of 75 kbp/s or higher. It is still unknown how high the speed actually is. This paper reports real-time measurements of ejection from phage {lambda}, revealing how the speed depends on key physical parameters such as genome length and ionic state of the buffer. Except for a pause before DNA is finally released, the entire 48.5-kbp genome is translocated in {approx}1.5 s without interruption, reaching a speed of 60 kbp/s. The process gives insights particularly into the effects of two parameters: a shorter genome length results in lower speed but a shorter total time, and the presence of divalent magnesium ions (replacing sodium) reduces the pressure, increasing ejection time to 8\u201311 s. Pressure caused by DNA\u2013DNA interactions within the head affects the initiation of ejection, but the close packing is also the dominant source of friction: more tightly packed phages initiate ejection earlier, but with a lower initial speed. The details of ejection revealed in this study are probably generic features of DNA translocation in bacteriophages and have implications for the dynamics of DNA in other biological systems.", "doi": "10.1073/pnas.0703274104", "pmcid": "PMC1976217", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2007-09-11", "series_number": "37", "volume": "104", "issue": "37", "pages": "14652-14657" }, { "id": "authors:zthg5-hem02", "collection": "authors", "collection_id": "zthg5-hem02", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:URSploscb07", "type": "article", "title": "Cooperative Gating and Spatial Organization of Membrane Proteins through Elastic Interactions", "author": [ { "family_name": "Ursell", "given_name": "Tristan", "clpid": "Ursell-T" }, { "family_name": "Huang", "given_name": "Kerwin Casey", "clpid": "Huang-Karwin-Casey" }, { "family_name": "Peterson", "given_name": "Eric", "clpid": "Peterson-E-L" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Biological membranes are elastic media in which the presence of a transmembrane protein leads to local bilayer deformation. The energetics of deformation allow two membrane proteins in close proximity to influence each other's equilibrium conformation via their local deformations, and spatially organize the proteins based on their geometry. We use the mechanosensitive channel of large conductance (MscL) as a case study to examine the implications of bilayer-mediated elastic interactions on protein conformational statistics and clustering. The deformations around MscL cost energy on the order of 10 kBT and extend \u223c3 nm from the protein edge, as such elastic forces induce cooperative gating, and we propose experiments to measure these effects. Additionally, since elastic interactions are coupled to protein conformation, we find that conformational changes can severely alter the average separation between two proteins. This has important implications for how conformational changes organize membrane proteins into functional groups within membranes.", "doi": "10.1371/journal.pcbi.0030081", "pmcid": "PMC1864995", "issn": "1553-7358", "publisher": "Public Library of Science", "publication": "PLOS Computational Biology", "publication_date": "2007-05", "series_number": "5", "volume": "3", "issue": "5", "pages": "803-812" }, { "id": "authors:0qf0e-dsk21", "collection": "authors", "collection_id": "0qf0e-dsk21", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170526-112238161", "type": "article", "title": "Measuring Flux Distributions for Diffusion in the Small-Numbers Limit", "author": [ { "family_name": "Seitaridou", "given_name": "Effrosyni", "clpid": "Seitaridou-E" }, { "family_name": "Inamdar", "given_name": "Mandar M.", "clpid": "Inamdar-M-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Ghosh", "given_name": "Kingshuk", "clpid": "Ghosh-K" }, { "family_name": "Dill", "given_name": "Ken", "clpid": "Dill-K" } ], "abstract": "For the classical diffusion of independent particles, Fick's law gives a well-known relationship between the average flux and the average concentration gradient. What has not yet been explored experimentally, however, is the dynamical distribution of diffusion rates in the limit of small particle numbers. Here, we measure the distribution of diffusional fluxes using a microfluidics device filled with a colloidal suspension of a small number of microspheres. Our experiments show that (1) the flux distribution is accurately described by a Gaussian function; (2) Fick's law, that the average flux is proportional to the particle gradient, holds even for particle gradients down to a single particle difference; (3) the variance in the flux is proportional to the sum of the particle numbers; and (4) there are backward flows, where particles flow up a concentration gradient, rather than down it. In addition, in recent years, two key theorems about nonequilibrium systems have been introduced:\u2009 Evans' fluctuation theorem for the distribution of entropies and Jarzynski's work theorem. Here, we introduce a new fluctuation theorem, for the fluxes, and we find that it is confirmed quantitatively by our experiments.", "doi": "10.1021/jp067036j", "pmcid": "PMC3261839", "issn": "1520-6106", "publisher": "American Chemical Society", "publication": "Journal of Physical Chemistry B", "publication_date": "2007-03-08", "series_number": "9", "volume": "111", "issue": "9", "pages": "2288-2292" }, { "id": "authors:r3awa-hvw12", "collection": "authors", "collection_id": "r3awa-hvw12", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180619-104551728", "type": "article", "title": "Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity", "author": [ { "family_name": "Garcia", "given_name": "Hernan G.", "clpid": "Garcia-H-G" }, { "family_name": "Grayson", "given_name": "Paul", "clpid": "Grayson-P" }, { "family_name": "Han", "given_name": "Lin", "clpid": "Han-Lin" }, { "family_name": "Inamdar", "given_name": "Mandar", "clpid": "Inamdar-M-M" }, { "family_name": "Kondev", "given_name": "Jan\u00e9", "clpid": "Kondev-J" }, { "family_name": "Nelson", "given_name": "Philip C.", "clpid": "Nelson-P-C" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Widom", "given_name": "Jonathan", "clpid": "Widom-J" }, { "family_name": "Wiggins", "given_name": "Paul A.", "clpid": "Wiggins-P-A" } ], "abstract": "The mechanical properties of DNA play a critical role in many biological functions. For example, DNA packing in viruses involves confining the viral genome in a volume (the viral capsid) with dimensions that are comparable to the DNA persistence length. Similarly, eukaryotic DNA is packed in DNA\u2013protein complexes (nucleosomes), in which DNA is tightly bent around protein spools. DNA is also tightly bent by many proteins that regulate transcription, resulting in a variation in gene expression that is amenable to quantitative analysis. In these cases, DNA loops are formed with lengths that are comparable to or smaller than the DNA persistence length. The aim of this review is to describe the physical forces associated with tightly bent DNA in all of these settings and to explore the biological consequences of such bending, as increasingly accessible by single\u2010molecule techniques.", "doi": "10.1002/bip.20627", "pmcid": "PMC3496788", "issn": "0006-3525", "publisher": "Wiley", "publication": "Biopolymers", "publication_date": "2007-02-05", "series_number": "2", "volume": "85", "issue": "2", "pages": "115-130" }, { "id": "authors:9s8t1-hxz71", "collection": "authors", "collection_id": "9s8t1-hxz71", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150409-090719819", "type": "article", "title": "High flexibility of DNA on short length scales probed by atomic force microscopy", "author": [ { "family_name": "Wiggins", "given_name": "Paul A.", "clpid": "Wiggins-P-A" }, { "family_name": "van der Heijden", "given_name": "Thijn", "clpid": "van-der-Heijden-T" }, { "family_name": "Moreno-Herrero", "given_name": "Fernando", "clpid": "Moreno-Herrero-F" }, { "family_name": "Spakowitz", "given_name": "Andrew", "clpid": "Spakowitz-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Widom", "given_name": "Jonathan", "clpid": "Widom-J" }, { "family_name": "Dekker", "given_name": "Cees", "orcid": "0000-0001-6273-071X", "clpid": "Dekker-C" }, { "family_name": "Nelson", "given_name": "Philip C.", "clpid": "Nelson-P-C" } ], "abstract": "The mechanics of DNA bending on intermediate length scales (5\u2013100 nm) plays a key role in many cellular processes, and is also important in the fabrication of artificial DNA structures, but previous experimental studies of DNA mechanics have focused on longer length scales than these. We use high-resolution atomic force microscopy on individual DNA molecules to obtain a direct measurement of the bending energy function appropriate for scales down to 5 nm. Our measurements imply that the elastic energy of highly bent DNA conformations is lower than predicted by classical elasticity models such as the worm-like chain (WLC) model. For example, we found that on short length scales, spontaneous large-angle bends are many times more prevalent than predicted by the WLC model. We test our data and model with an interlocking set of consistency checks. Our analysis also shows how our model is compatible with previous experiments, which have sometimes been viewed as confirming the WLC.", "doi": "10.1038/nnano.2006.63", "issn": "1748-3387", "publisher": "Nature Publishing Group", "publication": "Nature Nanotechnology", "publication_date": "2006-11", "series_number": "2", "volume": "1", "issue": "2", "pages": "137-141" }, { "id": "authors:mt673-rte75", "collection": "authors", "collection_id": "mt673-rte75", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180619-083759884", "type": "article", "title": "The effect of genome length on ejection forces in bacteriophage lambda", "author": [ { "family_name": "Grayson", "given_name": "Paul", "clpid": "Grayson-P" }, { "family_name": "Evilevitch", "given_name": "Alex", "clpid": "Evilevitch-A" }, { "family_name": "Inamdar", "given_name": "Mandar M.", "clpid": "Inamdar-M-M" }, { "family_name": "Purohitc", "given_name": "Prashant K.", "clpid": "Purohitc-P-K" }, { "family_name": "Gelbart", "given_name": "William M.", "clpid": "Gelbart-W-M" }, { "family_name": "Knobler", "given_name": "Charles M.", "clpid": "Knobler-C-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "A variety of viruses tightly pack their genetic material into protein capsids that are barely large enough to enclose the genome. In particular, in bacteriophages, forces as high as 60 pN are encountered during packaging and ejection, produced by DNA bending elasticity and self-interactions. The high forces are believed to be important for the ejection process, though the extent of their involvement is not yet clear. As a result, there is a need for quantitative models and experiments that reveal the nature of the forces relevant to DNA ejection. Here, we report measurements of the ejection forces for two different mutants of bacteriophage \u03bb, \u03bbb221cI26 and \u03bbcI60, which differ in genome length by \u223c30%. As expected for a force-driven ejection mechanism, the osmotic pressure at which DNA release is completely inhibited varies with the genome length: we find inhibition pressures of 15 atm and 25 atm, for the short and long genomes, respectively, values that are in agreement with our theoretical calculations.", "doi": "10.1016/j.virol.2006.01.003", "pmcid": "PMC3178461", "issn": "0042-6822", "publisher": "Elsevier", "publication": "Virology", "publication_date": "2006-05-10", "series_number": "2", "volume": "348", "issue": "2", "pages": "430-436" }, { "id": "authors:26rj3-dvj38", "collection": "authors", "collection_id": "26rj3-dvj38", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:PHIpt06", "type": "article", "title": "The Biological Frontier of Physics", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Quake", "given_name": "Stephen R.", "clpid": "Quake-S-R" } ], "abstract": "Problems at the interface between biology and physics offer unique opportunities for physicists to make quantitative contributions to biology. Equally important, they enrich the discipline of physics by challenging its practitioners to think in new ways.", "issn": "0031-9228", "publisher": "Physics Today", "publication": "Physics Today", "publication_date": "2006-05-01", "series_number": "5", "volume": "59", "issue": "5", "pages": "38-43" }, { "id": "authors:s38q9-dv316", "collection": "authors", "collection_id": "s38q9-dv316", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:SEGprl06", "type": "article", "title": "Volume-exclusion effects in tethered-particle experiments: bead size matters", "author": [ { "family_name": "Segall", "given_name": "Darren E.", "clpid": "Segall-D-E" }, { "family_name": "Nelson", "given_name": "Philip C.", "clpid": "Nelson-P-C" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "We give a theoretical analysis of bead motion in tethered-particle experiments, a single-molecule technique that has been used to explore the dynamics of a variety of macromolecules of biological interest. Our analysis reveals that the proximity of the tethered bead to a nearby surface gives rise to a volume-exclusion effect, resulting in an entropic stretching-force on the molecule that changes its statistical properties. In addition, volume exclusion brings about intriguing scaling relations between key observables (statistical moments of the bead) and parameters such as bead size and contour length of the molecule. We present analytic and numerical results for these effects in both flexible and semiflexible tethers. Finally, our results give a precise, experimentally testable prediction for the probability distribution of the bead center measured from the polymer attachment point.", "doi": "10.1103/PhysRevLett.96.088306", "pmcid": "PMC3261840", "issn": "0031-9007", "publisher": "American Physical Society", "publication": "Physical Review Letters", "publication_date": "2006-03-03", "series_number": "8", "volume": "96", "issue": "8", "pages": "Art. No. 088306" }, { "id": "authors:dpsbs-ezm70", "collection": "authors", "collection_id": "dpsbs-ezm70", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:DUPprl05", "type": "article", "title": "Finite-Temperature Quasicontinuum: Molecular Dynamics without All the Atoms", "author": [ { "family_name": "Dupuy", "given_name": "L. M.", "clpid": "Dupuy-L-M" }, { "family_name": "Tadmor", "given_name": "E. B.", "clpid": "Tadmor-E-B" }, { "family_name": "Miller", "given_name": "R. E.", "clpid": "Miller-R-E" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Using a combination of statistical mechanics and finite-element interpolation, we develop a coarse-grained (CG) alternative to molecular dynamics (MD) for crystalline solids at constant temperature. The new approach is significantly more efficient than MD and generalizes earlier work on the quasicontinuum method. The method is validated by recovering equilibrium properties of single crystal Ni as a function of temperature. CG dynamical simulations of nanoindentation reveal a strong dependence on temperature of the critical stress to nucleate dislocations under the indenter.", "doi": "10.1103/PhysRevLett.95.060202", "issn": "0031-9007", "publisher": "American Physical Society", "publication": "Physical Review Letters", "publication_date": "2005-08-05", "series_number": "6", "volume": "95", "issue": "6", "pages": "Art. No. 060202" }, { "id": "authors:ftpag-7k048", "collection": "authors", "collection_id": "ftpag-7k048", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:WIGpre05", "type": "article", "title": "Exact theory of kinkable elastic polymers", "author": [ { "family_name": "Wiggins", "given_name": "Paul A.", "clpid": "Wiggins-P-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Nelson", "given_name": "Philip C.", "clpid": "Nelson-P-C" } ], "abstract": "The importance of nonlinearities in material constitutive relations has long been appreciated in the continuum mechanics of macroscopic rods. Although the moment (torque) response to bending is almost universally linear for small deflection angles, many rod systems exhibit a high-curvature softening. The signature behavior of these rod systems is a kinking transition in which the bending is localized. Recent DNA cyclization experiments by Cloutier and Widom have offered evidence that the linear-elastic bending theory fails to describe the high-curvature mechanics of DNA. Motivated by this recent experimental work, we develop a simple and exact theory of the statistical mechanics of linear-elastic polymer chains that can undergo a kinking transition. We characterize the kinking behavior with a single parameter and show that the resulting theory reproduces both the low-curvature linear-elastic behavior which is already well described by the wormlike chain model, as well as the high-curvature softening observed in recent cyclization experiments.", "doi": "10.1103/PhysRevE.71.021909", "pmcid": "PMC3496790", "issn": "1063-651X", "publisher": "American Physical Society", "publication": "Physical Review E", "publication_date": "2005-02", "series_number": "2", "volume": "71", "issue": "2", "pages": "Art. No. 021909" }, { "id": "authors:03j66-2a878", "collection": "authors", "collection_id": "03j66-2a878", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:OLMmsmse04", "type": "article", "title": "Modelling diffusion in crystals under high internal stress gradients", "author": [ { "family_name": "Olmsted", "given_name": "David L.", "clpid": "Olmsted-D-L" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Curtin", "given_name": "W. A.", "clpid": "Curtin-W-A" } ], "abstract": "Diffusion of vacancies and impurities in metals is important in many processes occurring in structural materials. This diffusion often takes place in the presence of spatially rapidly varying stresses. Diffusion under stress is frequently modelled by local approximations to the vacancy formation and diffusion activation enthalpies which are linear in the stress, in order to account for its dependence on the local stress state and its gradient. Here, more accurate local approximations to the vacancy formation and diffusion activation enthalpies, and the simulation methods needed to implement them, are introduced. The accuracy of both these approximations and the linear approximations are assessed via comparison to full atomistic studies for the problem of vacancies around a Lomer dislocation in Aluminium. Results show that the local and linear approximations for the vacancy formation enthalpy and diffusion activation enthalpy are accurate to within 0.05 eV outside a radius of about 13 \u00c5 (local) and 17 \u00c5 (linear) from the centre of the dislocation core or, more generally, for a strain gradient of roughly up to 6 \u00d7 10^6 m^-1 and 3 \u00d7 10^6 m^-1, respectively. These results provide a basis for the development of multiscale models of diffusion under highly non-uniform stress.", "doi": "10.1088/0965-0393/12/5/003", "issn": "0965-0393", "publisher": "Modelling and Simulation in Materials Science and Engineering", "publication": "Modelling and Simulation in Materials Science and Engineering", "publication_date": "2004-09-01", "series_number": "5", "volume": "12", "issue": "5", "pages": "781-797" }, { "id": "authors:h3axf-hk012", "collection": "authors", "collection_id": "h3axf-hk012", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:WIGpnas04", "type": "article", "title": "Analytic models for mechanotransduction: gating a mechanosensitive channel", "author": [ { "family_name": "Wiggins", "given_name": "Paul", "clpid": "Wiggins-P-A" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Analytic estimates for the forces and free energy generated by bilayer deformation reveal a compelling and intuitive model for MscL channel gating analogous to the nucleation of a second phase. We argue that the competition between hydrophobic mismatch and tension results in a surprisingly rich story that can provide both a quantitative comparison with measurements of opening tension for MscL when reconstituted in bilayers of different thickness, and qualitative insights into the function of the MscL channel and other transmembrane proteins.", "doi": "10.1073/pnas.0307804101", "pmcid": "PMC384697", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2004-03-23", "series_number": "12", "volume": "101", "issue": "12", "pages": "4071-4076" }, { "id": "authors:0v7z6-d0z78", "collection": "authors", "collection_id": "0v7z6-d0z78", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170408-162850097", "type": "article", "title": "Force steps during viral DNA packaging?", "author": [ { "family_name": "Purohit", "given_name": "Prashant K.", "clpid": "Purohit-P-K" }, { "family_name": "Kondev", "given_name": "Jan\u00e9", "clpid": "Kondev-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Biophysicists and structural biologists increasingly acknowledge the role played by the mechanical properties of macromolecules as a critical element in many biological processes. This change has been brought about, in part, by the advent of single molecule biophysics techniques that have made it possible to exert piconewton forces on key macromolecules and observe their deformations at nanometer length scales, as well as to observe the mechanical action of macromolecules such as molecular motors. This has opened up immense possibilities for a new generation of mechanical investigations that will respond to such measurements in an attempt to develop a coherent theory for the mechanical behavior of macromolecules under conditions where thermal and chemical effects are on an equal footing with deterministic forces. This paper presents an application of the principles of mechanics to the problem of DNA packaging, one of the key events in the life cycle of bacterial viruses with special reference to the nature of the internal forces that are built up during the DNA packaging process.", "doi": "10.1016/j.jmps.2003.09.016", "issn": "0022-5096", "publisher": "Elsevier", "publication": "Journal of the Mechanics and Physics of Solids", "publication_date": "2003-11", "series_number": "11-12", "volume": "51", "issue": "11-12", "pages": "2239-2257" }, { "id": "authors:fg5rt-wxg15", "collection": "authors", "collection_id": "fg5rt-wxg15", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:PURpnas03", "type": "article", "title": "Mechanics of DNA packaging in viruses", "author": [ { "family_name": "Purohit", "given_name": "Prashant K.", "clpid": "Purohit-P-K" }, { "family_name": "Kondev", "given_name": "Jan\u00e9", "clpid": "Kondev-J" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "A new generation of single-molecule experiments has opened up the possibility of reexamining many of the fundamental processes of biochemistry and molecular biology from a unique and quantitative perspective. One technique producing a host of intriguing results is the use of optical tweezers to measure the mechanical forces exerted by molecular motors during key processes such as the transcription of DNA or the packing of a viral genome into its capsid. The objective of the current article is to respond to such measurements on viruses and to use the theory of elasticity and a simple model of charge and hydration forces to derive the force required to pack DNA into a viral capsid as a function of the fraction of the viral genome that has been packed. The results are found to be in excellent accord with recent measurements and complement previous theoretical work. Because the packing of DNA in viral capsids occurs under circumstances of high internal pressure, we also compute how much pressure a capsid can sustain without rupture.", "doi": "10.1073/pnas.0737893100", "pmcid": "PMC404299", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2003-03-18", "series_number": "6", "volume": "100", "issue": "6", "pages": "3173-3178" }, { "id": "authors:fmfyq-mvp06", "collection": "authors", "collection_id": "fmfyq-mvp06", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:PHIarmr02", "type": "article", "title": "Quasicontinuum representations of atomic-scale mechanics: From proteins to dislocations", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Dittrich", "given_name": "\u00adMarkus", "clpid": "Dittrich-M" }, { "family_name": "Schulten", "given_name": "\u00adKlaus", "clpid": "Schulten-K" } ], "abstract": "Computation is one of the centerpieces of both the physical and biological sciences. A key thrust in computational science is the explicit mechanistic simulation of the spatiotemporal evolution of materials ranging from macromolecules to intermetallic alloys. However, our ability to simulate such systems is in the end always limited in both the spatial extent of the systems that are considered, as well as the duration of the time that can be simulated. As a result, a variety of efforts have been put forth that aim to finesse these challenges in both space and time through new techniques in which constraint is exploited to reduce the overall computational burden. The aim of this review is to describe in general terms some of the key ideas that have been set forth in both the materials and biological setting and to speculate on future developments along these lines. We begin by developing general ideas on the exploitation of constraint as a systematic tool for degree of freedom thinning. These ideas are then applied to case studies ranging from the plastic deformation of solids to the interactions of proteins and DNA.", "doi": "10.1146/annurev.matsci.32.122001.102202", "issn": "1531-7331", "publisher": "Annual Reviews", "publication": "Annual Review of Materials Research", "publication_date": "2002-08", "volume": "32", "pages": "219-233" }, { "id": "authors:0mkhs-wz494", "collection": "authors", "collection_id": "0mkhs-wz494", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20111102-074723462", "type": "book_section", "title": "Atomistic Simulations of the Motion of an Edge Dislocation in Aluminum Using the Embedded Atom Method", "book_title": "Shock Compression of Condensed Matter 2001", "author": [ { "family_name": "Bhate", "given_name": "N.", "clpid": "Bhate-N" }, { "family_name": "Clifton", "given_name": "R. J.", "clpid": "Clifton-R-J" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "contributor": [ { "family_name": "Furnish", "given_name": "M .D.", "clpid": "Furnish-M-D" }, { "family_name": "Thadhani", "given_name": "N. N.", "clpid": "Thadhani-N-N" }, { "family_name": "Horie", "given_name": "Y.", "clpid": "Horie-Y" } ], "abstract": "The motion of an edge dislocation is analyzed for temperatures ranging from 10 K to 200 K and for stresses up to 5 GPa. The dislocation velocity versus the applied shear stress curve can be divided into four regimes corresponding to successively higher shear stresses. In the first regime,\nthe applied shear stress is below the Peierls stress and the dislocation velocity is nominally zero. In the second regime, the dislocation velocity decreases with increasing temperature indicating the presence of a drag due to thermal phonons. In the third regime, the dislocation reaches a sub-sonic limiting velocity that can be predicted by a two-dimensional lattice-dynamics analysis. Such an\nanalysis predicts a limiting velocity for a moving defect when the phase velocity and the group velocity are equal. If even higher shear stresses are applied, the dislocation travels with a transonic velocity of \u221a2 times the shear wave speed.", "doi": "10.1063/1.1483548", "isbn": "0-7354-0068-7", "publisher": "American Institute of Physics", "place_of_publication": "Melville, N.Y.", "publication_date": "2002-07-08", "pages": "339-342" }, { "id": "authors:c16ng-4mp29", "collection": "authors", "collection_id": "c16ng-4mp29", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20111101-090938695", "type": "article", "title": "Dislocation plasticity in thin metal films", "author": [ { "family_name": "Kraft", "given_name": "O.", "clpid": "Kraft-O" }, { "family_name": "Freund", "given_name": "L. B.", "clpid": "Freund-L-B" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Arzt", "given_name": "E.", "clpid": "Arzt-E" } ], "abstract": "This article describes the current level of understanding of dislocation plasticity in thin\nfilms and small structures in which the film or structure dimension plays an important\nrole. Experimental observations of the deformation behavior of thin films, including\nmechanical testing as well as electron microscopy studies, will be discussed in light of\ntheoretical models and dislocation simulations. In particular, the potential of applying\nstrain-gradient plasticity theory to thin-film deformation is discussed. Although the\nresults of all studies presented follow a \"smaller is stronger\" trend, a clear functional\ndependence has not yet been established.", "doi": "10.1557/mrs2002.17", "issn": "0883-7694", "publisher": "Materials Research Society", "publication": "MRS Bulletin", "publication_date": "2002-01", "series_number": "1", "volume": "27", "issue": "1", "pages": "30-37" }, { "id": "authors:dhhrz-5bh17", "collection": "authors", "collection_id": "dhhrz-5bh17", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:OLMmsmse01", "type": "article", "title": "Lattice resistance and Peierls stress in finite size atomistic dislocation simulations", "author": [ { "family_name": "Olmsted", "given_name": "David L.", "clpid": "Olmsted-D-L" }, { "family_name": "Hardikar", "given_name": "Kedar Y.", "clpid": "Hardikar-K-Y" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "Atomistic computations of the Peierls stress in fcc metals are relatively scarce. By way of contrast, there are many more atomistic computations for bcc metals, as well as mixed discrete-continuum computations of the Peierls-Nabarro type for fcc metals. One of the reasons for this is the low Peierls stresses in fcc metals. Because atomistic computations of the Peierls stress take place in finite simulation cells, image forces caused by boundaries must either be relaxed or corrected for if system size-independent results are to be obtained. One of the approaches that has been developed for treating such boundary forces is by computing them directly and subsequently subtracting their effects, as developed in (Shenoy V B and Phillips R 1997 Phil. Mag. A 76 367). That work was primarily analytic, and limited to screw dislocations and special symmetric geometries. We extend that work to edge and mixed dislocations, and to arbitrary two-dimensional geometries, through a numerical finite element computation. We also describe a method for estimating the boundary forces directly on the basis of atomistic calculations. We apply these methods to the numerical measurement of the Peierls stress and lattice resistance curves for a model aluminium (fcc) system using an embedded-atom potential.", "doi": "10.1088/0965-0393/9/3/308", "issn": "0965-0393", "publisher": "Modelling and Simulation in Materials Science and Engineering", "publication": "Modelling and Simulation in Materials Science and Engineering", "publication_date": "2001-05-01", "series_number": "3", "volume": "9", "issue": "3", "pages": "215-247" }, { "id": "authors:4b7gy-xfe58", "collection": "authors", "collection_id": "4b7gy-xfe58", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171212-153930662", "type": "article", "title": "Hierarchical modeling in the mechanics of materials", "author": [ { "family_name": "Tadmor", "given_name": "E. B.", "clpid": "Tadmor-E-B" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Ortiz", "given_name": "M.", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" } ], "abstract": "Many problems in the mechanics of materials involve the operation of either multiple spatial or temporal scales simultaneously. As a result, an important thrust of recent work in this area has been the development of methods allowing for the consideration of multiple scales simultaneously. In this paper, we examine hierarchical approaches to modeling problems of this kind with special reference to the way in which information can be fed from one scale to the next in models of plasticity.", "doi": "10.1016/S0020-7683(99)00095-5", "issn": "0020-7683", "publisher": "Elsevier", "publication": "International Journal of Solids and Structures", "publication_date": "2000-01", "series_number": "1-2", "volume": "37", "issue": "1-2", "pages": "379-389" }, { "id": "authors:b9z9d-qw090", "collection": "authors", "collection_id": "b9z9d-qw090", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:PHImsmse99", "type": "article", "title": "Hierarchical models of plasticity: dislocation nucleation and interaction", "author": [ { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Rodney", "given_name": "David", "clpid": "Rodney-D" }, { "family_name": "Shenoy", "given_name": "Vivek", "clpid": "Shenoy-V-B" }, { "family_name": "Tadmor", "given_name": "Ellad", "clpid": "Tadmor-E-B" }, { "family_name": "Ortiz", "given_name": "Michael", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" } ], "abstract": "One avenue being pursued in the development of dislocation-based models of plasticity is the explicit simulation of the dynamics of dislocations, based on the recognition that such dislocations are the carriers of plasticity. The construction of models of dislocation dynamics requires insights into both the nucleation and interaction of dislocations, many of the details of which fall outside the domain of validity of the linear theory of elasticity. The aim of the present paper is to show how preliminary steps have been made to elucidate the mechanisms of dislocation nucleation and interaction, and to illustrate how such information can be imported into explicit models of dislocation dynamics. This effort reflects, in part, the research program of Gilles Canova, to whom the present volume is dedicated and to whom the authors dedicate this paper.", "doi": "10.1088/0965-0393/7/5/309", "issn": "0965-0393", "publisher": "Modelling and Simulation in Materials Science and Engineering", "publication": "Modelling and Simulation in Materials Science and Engineering", "publication_date": "1999-09-01", "series_number": "5", "volume": "7", "issue": "5", "pages": "769-780" }, { "id": "authors:vvwwb-ncj77", "collection": "authors", "collection_id": "vvwwb-ncj77", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:SHEmsmse99", "type": "article", "title": "The atomistic structure and energy of nascent dislocation loops", "author": [ { "family_name": "Shenoy", "given_name": "V. B.", "clpid": "Shenoy-V-B" }, { "family_name": "Ortiz", "given_name": "M.", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "An harmonic lattice theory is used, in conjunction with Mura's theory of eigendistorsions, to study the structure and energetics of nascent dislocation loops in face-centred-cubic (FCC) crystals. An analytical expression for the activation energies of such loops is derived. The results obtained herein indicate that thermal activation of small dislocation loops is possible at high stress levels such as those found in the vicinity of a crack tip. The implications of these results in understanding phenomena such as the brittle-ductile transition are discussed.", "doi": "10.1088/0965-0393/7/4/309", "issn": "0965-0393", "publisher": "Modelling and Simulation in Materials Science and Engineering", "publication": "Modelling and Simulation in Materials Science and Engineering", "publication_date": "1999-07-01", "series_number": "4", "volume": "7", "issue": "4", "pages": "603-619" }, { "id": "authors:agdhm-50x60", "collection": "authors", "collection_id": "agdhm-50x60", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:TADjmr99", "type": "article", "title": "Nanoindentation and incipient plasticity", "author": [ { "family_name": "Tadmor", "given_name": "E.B.", "clpid": "Tadmor-E-B" }, { "family_name": "Miller", "given_name": "R.", "clpid": "Miller-R" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Ortiz", "given_name": "M.", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" } ], "abstract": "This paper presents a large-scale atomic resolution simulation of nanoindentation into a thin aluminum film using the recently introduced quasicontinuum method. The purpose of the simulation was to study the initial stages of plastic deformation under the action of an indenter. Two different crystallographic orientations of the film and two different indenter geometries (a rectangular prism and a cylinder) were studied. We obtained both macroscopic load versus indentation depth curves, as well as microscopic quantities, such as the Peierls stress and density of geometrically necessary dislocations beneath the indenter. In addition, we obtain detailed information regarding the atomistic mechanisms responsible for the macroscopic curves. A strong dependence on geometry and orientation is observed. Two different microscopic mechanisms are observed to accommodate the applied loading: (i) nucleation and subsequent propagation into the bulk of edge dislocation dipoles and (ii) deformation twinning.", "issn": "0884-2914", "publisher": "Journal of Materials Research", "publication": "Journal of Materials Research", "publication_date": "1999-06-01", "series_number": "6", "volume": "14", "issue": "6", "pages": "2233-2250" }, { "id": "authors:0gm5a-3j168", "collection": "authors", "collection_id": "0gm5a-3j168", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171213-100338338", "type": "article", "title": "A non-local formulation of the Peierls dislocation model", "author": [ { "family_name": "Miller", "given_name": "Ron", "clpid": "Miller-R" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Beltz", "given_name": "Glen", "clpid": "Beltz-G" }, { "family_name": "Ortiz", "given_name": "Michael", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" } ], "abstract": "Cohesive zone models provide an illuminating and tractable way to include constitutive non-linearity into continuum models of defects. Powerful insights have been gained by studying both dislocations and cracks using such analyses. Recent work has shown that as a result of the locality assumption present in such cohesive zone models, significant errors can be made in the treatment of defect energies. This paper aims to construct a non-local version of the Peierls\u2013Nabarro model in which the atomic level stresses induced at the slip plane depend in a non-local way on the slip degrees of freedom. Our results should be seen as a demonstration in principle of how microscopic calculations can be used to construct insights into constitutive nonlocality. The non-local interplanar kernel used here is computed directly from atomistics and is used to evaluate both the structure and energetics of planar dislocations. The non-local formulation does not significantly change the dislocation core structure from that obtained with the local model, but the new formulation leads to significant improvements in the description of dislocation energetics for dislocations with planar cores.", "doi": "10.1016/S0022-5096(98)00057-X", "issn": "0022-5096", "publisher": "Elsevier", "publication": "Journal of the Mechanics and Physics of Solids", "publication_date": "1998-10-01", "series_number": "10", "volume": "46", "issue": "10", "pages": "1845-1867" }, { "id": "authors:24038-f3c11", "collection": "authors", "collection_id": "24038-f3c11", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:MILmsmse98", "type": "article", "title": "Quasicontinuum simulation of fracture at the atomic scale", "author": [ { "family_name": "Miller", "given_name": "R.", "clpid": "Miller-R" }, { "family_name": "Tadmor", "given_name": "E. B.", "clpid": "Tadmor-E-B" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Ortiz", "given_name": "M.", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" } ], "abstract": "We study the problem of atomic scale fracture using the recently developed quasicontinuum method in which there is a systematic thinning of the atomic-level degrees of freedom in regions where they are not needed. Fracture is considered in two distinct settings. First, a study is made of cracks in single crystals, and second, we consider a crack advancing towards a grain boundary (GB) in its path. In the investigation of single crystal fracture, we evaluate the competition between simple cleavage and crack-tip dislocation emission. In addition, we examine the ability of analytic models to correctly predict fracture behaviour, and find that the existing analytical treatments are too restrictive in their treatment of nonlinearity near the crack tip. In the study of GB-crack interactions, we have found a number of interesting deformation mechanisms which attend the advance of the crack. These include the migration of the GB, the emission of dislocations from the GB, and deflection of the crack front along the GB itself. In each case, these mechanisms are rationalized on the basis of continuum mechanics arguments.", "doi": "10.1088/0965-0393/6/5/008", "issn": "0965-0393", "publisher": "Modelling and Simulation in Materials Science and Engineering", "publication": "Modelling and Simulation in Materials Science and Engineering", "publication_date": "1998-09-01", "series_number": "5", "volume": "6", "issue": "5", "pages": "607-638" }, { "id": "authors:rv4td-vh423", "collection": "authors", "collection_id": "rv4td-vh423", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171213-100643529", "type": "article", "title": "Quasicontinuum models of fracture and plasticity", "author": [ { "family_name": "Miller", "given_name": "R.", "clpid": "Miller-R" }, { "family_name": "Ortiz", "given_name": "M.", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Shenoy", "given_name": "V.", "clpid": "Shenoy-V-B" }, { "family_name": "Tadmor", "given_name": "E. B.", "clpid": "Tadmor-E-B" } ], "abstract": "The development of modeling tools which allow for the simultaneous treatment of scales ranging from \u00c5ngstroms to microns has stood out as one of the main challenges in materials modeling. In this paper we discuss a formulation of the quasicontinuum (QC) method that allows for a treatment of internal interfaces, opening the possibility of simulating the interactions of cracks, dislocations and grain boundaries. The model is applied to crack tip deformation and is shown to account for both brittle fracture and crack tip dislocation emission. A key example of the method is the treatment of a crack propagating into a grain boundary which reveals both migration of the boundary and that the boundary is a source for the emission of dislocations.", "doi": "10.1016/S0013-7944(98)00047-2", "issn": "0013-7944", "publisher": "Elsevier", "publication": "Engineering Fracture Mechanics", "publication_date": "1998-09", "series_number": "3-4", "volume": "61", "issue": "3-4", "pages": "427-444" }, { "id": "authors:2ymz7-jfc17", "collection": "authors", "collection_id": "2ymz7-jfc17", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:SHEprl98", "type": "article", "title": "Quasicontinuum Models of Interfacial Structure and Deformation", "author": [ { "family_name": "Shenoy", "given_name": "V. B.", "clpid": "Shenoy-V-B" }, { "family_name": "Miller", "given_name": "R.", "clpid": "Miller-R" }, { "family_name": "Tadmor", "given_name": "E. B.", "clpid": "Tadmor-E-B" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Ortiz", "given_name": "M.", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" } ], "abstract": "Microscopic models of the interaction between grain boundaries (GBs) and both dislocations and cracks are of importance in understanding the role of microstructure in altering the mechanical properties of a material. A recently developed mixed atomistic and continuum method is reformulated to allow for the examination of the interactions between GBs, dislocations, and cracks. These calculations elucidate plausible microscopic mechanisms for these defect interactions and allow for the quantitative evaluation of critical parameters such as the force needed to induce GB migration.", "doi": "10.1103/PhysRevLett.80.742", "issn": "0031-9007", "publisher": "American Physical Society", "publication": "Physical Review Letters", "publication_date": "1998-01-26", "series_number": "4", "volume": "08", "issue": "4", "pages": "742-745" }, { "id": "authors:5ck70-j6f85", "collection": "authors", "collection_id": "5ck70-j6f85", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171213-092247784", "type": "book_section", "title": "Nanomechanics of Defects in Solids", "book_title": "Advances in Applied Mechanics", "author": [ { "family_name": "Ortiz", "given_name": "Michael", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "contributor": [ { "family_name": "van der Giessen", "given_name": "Erik", "clpid": "van-der-Giessen-E" }, { "family_name": "Wu", "given_name": "Theodore Y.", "clpid": "Wu-T-Y-T" } ], "abstract": "This chapter examines different aspects of nanomechanics of defects in solids. The methods by which the classical boundary-value problems of continuum mechanics can be imbued with atomistic content are reviewed. Microscopic modeling is founded on the fundamental assertion that beneath the details of observed macroscopic phenomenology, there is a set of microscopic processes which, when understood, rationalize the observed macroscopic behavior to the extent of enabling quantitative predictions. The microscopic simulation of materials is based on the evolution of degrees of freedom that are governed by the Schrodinger equation. It is found that either phenomenologically, or through explicit calculational strategies, the electronic degrees of freedom is implicitly subsumed in the effective pair potential. Once the pair potential has been identified, it is a straightforward matter to evaluate radial derivatives and the corresponding force fields. The energy associated with each distortion may be computed explicitly by recourse to direct atomistics. The contribution due to slip may be extracted by subtracting off the bulk elastic energy. As a result, the exact misfit energy is determined from atomistics. The cohesive-zone theories applied to fracture are also elaborated.", "doi": "10.1016/S0065-2156(08)70184-4", "isbn": "978-0-12-002036-2", "publisher": "Academic Press", "place_of_publication": "San Diego, CA", "publication_date": "1998", "pages": "1-79" }, { "id": "authors:20avj-ts985", "collection": "authors", "collection_id": "20avj-ts985", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160607-114810939", "type": "article", "title": "Mixed Atomistic and Continuum Models of Deformation in Solids", "author": [ { "family_name": "Tadmor", "given_name": "E. B.", "clpid": "Tadmor-E-B" }, { "family_name": "Phillips", "given_name": "Rob", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Ortiz", "given_name": "M.", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" } ], "abstract": "The modeling of processes involving multiple length scales is an area of pressing concern, especially in problems such as nanoidentation and crack tip dislocation activity. In these cases, there is more than one characteristic dimension with the nanometer scale arising due to the presence of extended defects such as dislocations and a second length scale at least 2 orders of magnitude larger set by the scale of the indenter or the crack tip itself. To properly model such processes, both scales must be treated explicitly, which is normally beyond the scope of conventional atomistic and continuum analyses alike. This paper describes a quasicontinuum method which seizes upon the strengths of both atomistic and continuum techniques and allows for the simultaneous treatment of multiple scales. The method is based upon a continuum formulation of the problem of interest as a boundary value problem treated within the confines of the finite element method. We part company with traditional approaches by utilizing direct atomistic calculations as the source of the constitutive input used in the finite element analysis. The method is illustrated through application to the case of the structure and energetics of single dislocations. This case is a stringent test as it represents an extreme limit for the model since dislocation core structures are primarily dictated by lattice effects. It is then shown how the method may be applied to problems of tribological concern such as nanoindentation, where it is found that dislocations are initiated beneath the indenter.", "doi": "10.1021/la9508912", "issn": "0743-7463", "publisher": "American Chemical Society", "publication": "Langmuir", "publication_date": "1996-09-18", "series_number": "19", "volume": "12", "issue": "19", "pages": "4529-4534" }, { "id": "authors:bjp9j-xq184", "collection": "authors", "collection_id": "bjp9j-xq184", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171213-150724557", "type": "article", "title": "Quasicontinuum analysis of defects in solids", "author": [ { "family_name": "Tadmor", "given_name": "E. B.", "clpid": "Tadmor-E-B" }, { "family_name": "Ortiz", "given_name": "M.", "orcid": "0000-0001-5877-4824", "clpid": "Ortiz-M" }, { "family_name": "Phillips", "given_name": "R.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "abstract": "We develop a method which permits the analysis of problems requiring the simultaneous resolution of continuum and atomistic length scales-and associated deformation processes-in a unified manner. A finite element methodology furnishes a continuum statement of the problem of interest and provides the requisite multiple-scale analysis capability by adaptively refining the mesh near lattice defects and other highly energetic regions. The method differs from conventional finite element analyses in that interatomic interactions are incorporated into the model through a crystal calculation based on the local state of deformation. This procedure endows the model with crucial properties, such as slip invariance, which enable the emergence of dislocations and other lattice defects. We assess the accuracy of the theory in the atomistic limit by way of three examples: a stacking fault on the (111) plane, and edge dislocations residing on (111) and (100) planes of an aluminium single crystal. The method correctly predicts the splitting of the (111) edge dislocation into Shockley partials. The computed separation of these partials is consistent with results obtained by direct atomistic simulations. The method predicts no splitting of the Al Lomer dislocation, in keeping with observation and the results of direct atomistic simulation. In both cases, the core structures are found to be in good agreement with direct lattice statics calculations, which attests to the accuracy of the method at the atomistic scale.", "doi": "10.1080/01418619608243000", "issn": "0141-8610", "publisher": "Taylor & Francis", "publication": "Philosophical Magazine A", "publication_date": "1996-06", "series_number": "6", "volume": "73", "issue": "6", "pages": "1529-1563" } ]