Natural cell death pathways such as apoptosis and pyroptosis play dual roles: they eliminate harmful cells and modulate the immune system by dampening or stimulating inflammation. Synthetic protein circuits capable of triggering specific death programs in target cells could similarly remove harmful cells while appropriately modulating immune responses. However, cells actively influence their death modes in response to natural signals, making it challenging to control death modes. Here, we introduce naturally inspired “synpoptosis” circuits that proteolytically regulate engineered executioner proteins and mammalian cell death. These circuits direct cell death modes, respond to combinations of protease inputs, and selectively eliminate target cells. Furthermore, synpoptosis circuits can be transmitted intercellularly, offering a foundation for engineering synthetic killer cells that induce desired death programs in target cells without self-destruction. Together, these results lay the groundwork for programmable control of mammalian cell death.
\nA system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently and selectively package and secrete target RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (Controlled Output and Uptake of RNA for Interrogation, Expression, and Regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.
", "doi": "10.1016/j.cell.2023.06.013", "pmcid": "PMC10528933", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "2023-08-17", "series_number": "17", "volume": "186", "issue": "17", "pages": "3642-3658.e32" }, { "id": "authors:a7vwv-mhw66", "collection": "authors", "collection_id": "a7vwv-mhw66", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220712-283791000", "type": "monograph", "title": "A synthetic protein-level neural network in mammalian cells", "author": [ { "family_name": "Chen", "given_name": "Zibo", "orcid": "0000-0003-2990-2895", "clpid": "Chen-Zibo" }, { "family_name": "Linton", "given_name": "James M.", "clpid": "Linton-James-M" }, { "family_name": "Zhu", "given_name": "Ronghui", "orcid": "0000-0001-8171-482X", "clpid": "Zhu-Ronghui" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "abstract": "Artificial neural networks provide a powerful paradigm for information processing that has transformed diverse fields. Within living cells, genetically encoded synthetic molecular networks could, in principle, harness principles of neural computation to classify molecular signals. Here, we combine de novo designed protein heterodimers and engineered viral proteases to implement a synthetic protein circuit that performs winner-take-all neural network computation. This \"perceptein\" circuit includes modules that compute weighted sums of input protein concentrations through reversible binding interactions, and allow for self-activation and mutual inhibition of protein components using irreversible proteolytic cleavage reactions. Altogether, these interactions comprise a network of 310 chemical reactions stemming from 8 expressed protein species. The complete system achieves signal classification with tunable decision boundaries in mammalian cells. These results demonstrate how engineered protein-based networks can enable programmable signal classification in living cells.", "doi": "10.1101/2022.07.10.499405", "publication_date": "2022-07-12" }, { "id": "authors:4jqdv-nrv37", "collection": "authors", "collection_id": "4jqdv-nrv37", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201210-094756085", "type": "article", "title": "The context-dependent, combinatorial logic of BMP signaling", "author": [ { "family_name": "Klumpe", "given_name": "Heidi E.", "orcid": "0000-0001-8938-2006", "clpid": "Klumpe-Heidi-E" }, { "family_name": "Langley", "given_name": "Matthew A.", "orcid": "0000-0003-2890-5584", "clpid": "Langley-Matthew-A" }, { "family_name": "Linton", "given_name": "James M.", "clpid": "Linton-James-M" }, { "family_name": "Su", "given_name": "Christina J.", "orcid": "0000-0002-9223-9777", "clpid": "Su-Christina-J" }, { "family_name": "Antebi", "given_name": "Yaron E.", "orcid": "0000-0002-5771-6814", "clpid": "Antebi-Yaron-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "abstract": "Cell-cell communication systems typically comprise families of ligand and receptor variants that function together in combinations. Pathway activation depends on the complex way in which ligands are presented extracellularly and receptors are expressed by the signal-receiving cell. To understand the combinatorial logic of such a system, we systematically measured pairwise bone morphogenetic protein (BMP) ligand interactions in cells with varying receptor expression. Ligands could be classified into equivalence groups based on their profile of positive and negative synergies with other ligands. These groups varied with receptor expression, explaining how ligands can functionally replace each other in one context but not another. Context-dependent combinatorial interactions could be explained by a biochemical model based on the competitive formation of alternative signaling complexes with distinct activities. Together, these results provide insights into the roles of BMP combinations in developmental and therapeutic contexts and establish a framework for analyzing other combinatorial, context-dependent signaling systems.", "doi": "10.1016/j.cels.2022.03.002", "pmcid": "PMC9127470", "issn": "2405-4712", "publisher": "Cell Press", "publication": "Cell Systems", "publication_date": "2022-05-18", "series_number": "5", "volume": "13", "issue": "5", "pages": "388-407" }, { "id": "authors:4a7y9-nzb92", "collection": "authors", "collection_id": "4a7y9-nzb92", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201210-134335099", "type": "article", "title": "Ligand-receptor promiscuity enables cellular addressing", "author": [ { "family_name": "Su", "given_name": "Christina J.", "orcid": "0000-0002-9223-9777", "clpid": "Su-Christina-J" }, { "family_name": "Murugan", "given_name": "Arvind", "orcid": "0000-0001-5464-917X", "clpid": "Murugan-Arvind" }, { "family_name": "Linton", "given_name": "James M.", "clpid": "Linton-James-M" }, { "family_name": "Yeluri", "given_name": "Akshay", "orcid": "0000-0001-8654-1673", "clpid": "Yeluri-Akshay" }, { "family_name": "Bois", "given_name": "Justin S.", "orcid": "0000-0001-7137-8746", "clpid": "Bois-J-S" }, { "family_name": "Klumpe", "given_name": "Heidi", "orcid": "0000-0001-8938-2006", "clpid": "Klumpe-Heidi-E" }, { "family_name": "Langley", "given_name": "Matthew A.", "orcid": "0000-0003-2890-5584", "clpid": "Langley-Matthew-A" }, { "family_name": "Antebi", "given_name": "Yaron E.", "orcid": "0000-0002-5771-6814", "clpid": "Antebi-Yaron-E" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "abstract": "In multicellular organisms, secreted ligands selectively activate, or \"address,\" specific target cell populations to control cell fate decision-making and other processes. Key cell-cell communication pathways use multiple promiscuously interacting ligands and receptors, provoking the question of how addressing specificity can emerge from molecular promiscuity. To investigate this issue, we developed a general mathematical modeling framework based on the bone morphogenetic protein (BMP) pathway architecture. We find that promiscuously interacting ligand-receptor systems allow a small number of ligands, acting in combinations, to address a larger number of individual cell types, defined by their receptor expression profiles. Promiscuous systems outperform seemingly more specific one-to-one signaling architectures in addressing capability. Combinatorial addressing extends to groups of cell types, is robust to receptor expression noise, grows more powerful with increases in the number of receptor variants, and is maximized by specific biochemical parameter relationships. Together, these results identify design principles governing cellular addressing by ligand combinations.", "doi": "10.1016/j.cels.2022.03.001", "pmcid": "PMC10897978", "issn": "2405-4712", "publisher": "Cell Press", "publication": "Cell Systems", "publication_date": "2022-05-18", "series_number": "5", "volume": "13", "issue": "5", "pages": "408-425" }, { "id": "authors:x8ffe-pb390", "collection": "authors", "collection_id": "x8ffe-pb390", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190920-163128957", "type": "article", "title": "In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription", "author": [ { "family_name": "Askary", "given_name": "Amjad", "orcid": "0000-0002-2913-8498", "clpid": "Askary-Amjad" }, { "family_name": "S\u00e1nchez-Guardado", "given_name": "Luis", "orcid": "0000-0001-5598-8608", "clpid": "S\u00e1nchez-Guardado-Luis" }, { "family_name": "Linton", "given_name": "James M.", "clpid": "Linton-James-M" }, { "family_name": "Chadly", "given_name": "Duncan M.", "orcid": "0000-0002-8417-1522", "clpid": "Chadly-Duncan-M" }, { "family_name": "Budde", "given_name": "Mark W.", "orcid": "0000-0002-4359-1424", "clpid": "Budde-Mark-W" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Lois", "given_name": "Carlos", "orcid": "0000-0002-7305-2317", "clpid": "Lois-C" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "abstract": "Molecular barcoding technologies that uniquely identify single cells are hampered by limitations in barcode measurement. Readout by sequencing does not preserve the spatial organization of cells in tissues, whereas imaging methods preserve spatial structure but are less sensitive to barcode sequence. Here we introduce a system for image-based readout of short (20-base-pair) DNA barcodes. In this system, called Zombie, phage RNA polymerases transcribe engineered barcodes in fixed cells. The resulting RNA is subsequently detected by fluorescent in situ hybridization. Using competing match and mismatch probes, Zombie can accurately discriminate single-nucleotide differences in the barcodes. This method allows in situ readout of dense combinatorial barcode libraries and single-base mutations produced by CRISPR base editors without requiring barcode expression in live cells. Zombie functions across diverse contexts, including cell culture, chick embryos and adult mouse brain tissue. The ability to sensitively read out compact and diverse DNA barcodes by imaging will facilitate a broad range of barcoding and genomic recording strategies.", "doi": "10.1038/s41587-019-0299-4", "pmcid": "PMC6954335", "issn": "1087-0156", "publisher": "Nature Publishing Group", "publication": "Nature Biotechnology", "publication_date": "2020-01", "series_number": "1", "volume": "38", "issue": "1", "pages": "66-75" }, { "id": "authors:m8axx-x2s82", "collection": "authors", "collection_id": "m8axx-x2s82", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170908-115917940", "type": "article", "title": "Combinatorial Signal Perception in the BMP Pathway", "author": [ { "family_name": "Antebi", "given_name": "Yaron E.", "orcid": "0000-0002-5771-6814", "clpid": "Antebi-Yaron-E" }, { "family_name": "Linton", "given_name": "James M.", "clpid": "Linton-James-M" }, { "family_name": "Klumpe", "given_name": "Heidi", "orcid": "0000-0001-8938-2006", "clpid": "Klumpe-Heidi-E" }, { "family_name": "Bintu", "given_name": "Bogdan", "orcid": "0000-0001-9096-5858", "clpid": "Bintu-Bogdan" }, { "family_name": "Gong", "given_name": "Mengsha", "clpid": "Gong-Mengsha" }, { "family_name": "Su", "given_name": "Christina", "orcid": "0000-0002-9223-9777", "clpid": "Su-Christina-J" }, { "family_name": "McCardell", "given_name": "Reed", "clpid": "McCardell-Reed" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "abstract": "The bone morphogenetic protein (BMP) signaling pathway comprises multiple ligands and receptors that interact promiscuously with one another and typically appear in combinations. This feature is often explained in terms of redundancy and regulatory flexibility, but it has remained unclear what signal-processing capabilities it provides. Here, we show that the BMP pathway processes multi-ligand inputs using a specific repertoire of computations, including ratiometric sensing, balance detection, and imbalance detection. These computations operate on the relative levels of different ligands and can arise directly from competitive receptor-ligand interactions. Furthermore, cells can select different computations to perform on the same ligand combination through expression of alternative sets of receptor variants. These results provide a direct signal-processing role for promiscuous receptor-ligand interactions and establish operational principles for quantitatively controlling cells with BMP ligands. Similar principles could apply to other promiscuous signaling pathways.", "doi": "10.1016/j.cell.2017.08.015", "pmcid": "PMC5612783", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "2017-09-07", "series_number": "6", "volume": "170", "issue": "6", "pages": "1184-1196" }, { "id": "authors:h1tvx-s6y87", "collection": "authors", "collection_id": "h1tvx-s6y87", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20161102-141950462", "type": "article", "title": "Synthetic recording and in situ readout of lineage information in single cells", "author": [ { "family_name": "Frieda", "given_name": "Kirsten L.", "clpid": "Frieda-Kirsten-L" }, { "family_name": "Linton", "given_name": "James M.", "clpid": "Linton-James-M" }, { "family_name": "Hormoz", "given_name": "Sahand", "clpid": "Hormoz-Sahand" }, { "family_name": "Choi", "given_name": "Joonhyuk", "clpid": "Choi-Joonhyuk" }, { "family_name": "Chow", "given_name": "Ke-Huan K.", "orcid": "0000-0002-7317-2669", "clpid": "Chow-Ke-Huan-K" }, { "family_name": "Singer", "given_name": "Zachary S.", "clpid": "Singer-Zachary-S" }, { "family_name": "Budde", "given_name": "Mark W.", "orcid": "0000-0002-4359-1424", "clpid": "Budde-Mark-W" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here, we describe a new synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out in single cells in situ. This system, termed Memory by Engineered Mutagenesis with Optical In situ Readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by Cas9-based targeted mutagenesis, and read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). To demonstrate a proof of principle of MEMOIR, we engineered mouse embryonic stem (ES) cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of the lineage trees of cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which ES cells switch between two gene expression states. Finally, using simulations, we showed how parallel MEMOIR systems operating in the same cell can enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single cell readout across diverse biological systems.", "doi": "10.1038/nature20777", "pmcid": "PMC6487260", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2017-01-05", "series_number": "7635", "volume": "541", "issue": "7635", "pages": "107-111" }, { "id": "authors:9kv8f-88h40", "collection": "authors", "collection_id": "9kv8f-88h40", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20161128-114511280", "type": "article", "title": "Inferring Cell-State Transition Dynamics from Lineage Trees and Endpoint Single-Cell Measurements", "author": [ { "family_name": "Hormoz", "given_name": "Sahand", "clpid": "Hormoz-Sahand" }, { "family_name": "Singer", "given_name": "Zakary S.", "clpid": "Singer-Zakary-S" }, { "family_name": "Linton", "given_name": "James M.", "clpid": "Linton-James-M" }, { "family_name": "Antebi", "given_name": "Yaron E.", "orcid": "0000-0002-5771-6814", "clpid": "Antebi-Yaron-E" }, { "family_name": "Shraiman", "given_name": "Boris I.", "clpid": "Shraiman-Boris-I" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "abstract": "As they proliferate, living cells undergo transitions between specific molecularly and developmentally distinct states. Despite the functional centrality of these transitions in multicellular organisms, it has remained challenging to determine which transitions occur and at what rates without perturbations and cell engineering. Here, we introduce kin correlation analysis (KCA) and show that quantitative cell-state transition dynamics can be inferred, without direct observation, from the clustering of cell states on pedigrees (lineage trees). Combining KCA with pedigrees obtained from time-lapse imaging and endpoint single-molecule RNA-fluorescence in situ hybridization (RNA-FISH) measurements of gene expression, we determined the cell-state transition network of mouse embryonic stem (ES) cells. This analysis revealed that mouse ES cells exhibit stochastic and reversible transitions along a linear chain of states ranging from 2C-like to epiblast-like. Our approach is broadly applicable and may be applied to systems with irreversible transitions and non-stationary dynamics, such as in cancer and development.", "doi": "10.1016/j.cels.2016.10.015", "pmcid": "PMC5142829", "issn": "2405-4712", "publisher": "Cell Press", "publication": "Cell Systems", "publication_date": "2016-11-23", "series_number": "5", "volume": "3", "issue": "5", "pages": "419-433" } ]