The mammalian nucleus is compartmentalized by diverse subnuclear structures. These subnuclear structures, marked by nuclear bodies and histone modifications, are often cell-type specific and affect gene regulation and 3D genome organization¹, ², ³. Understanding their relationships rests on identifying the molecular constituents of subnuclear structures and mapping their associations with specific genomic loci and transcriptional levels in individual cells, all in complex tissues. Here, we introduce two-layer DNA seqFISH+, which enables simultaneous mapping of 100,049 genomic loci, together with the nascent transcriptome for 17,856 genes and subnuclear structures in single cells. These data enable imaging-based chromatin profiling of diverse subnuclear markers and can capture their changes at genomic scales ranging from 100–200 kilobases to approximately 1 megabase, depending on the marker and DNA locus. By using multi-omics datasets in the adult mouse cerebellum, we showed that repressive chromatin regions are more variable by cell type than are active regions across the genome. We also discovered that RNA polymerase II-enriched foci were locally associated with long, cell-type-specific genes (bigger than 200 kilobases) in a manner distinct from that of nuclear speckles. Furthermore, our analysis revealed that cell-type-specific regions of heterochromatin marked by histone H3 trimethylated at lysine 27 (H3K27me3) and histone H4 trimethylated at lysine 20 (H4K20me3) are enriched at specific genes and gene clusters, respectively, and shape radial chromosomal positioning and inter-chromosomal interactions in neurons and glial cells. Together, our results provide a single-cell high-resolution multi-omics view of subnuclear structures, associated genomic loci and their effects on gene regulation, directly within complex tissues.
", "doi": "10.1038/s41586-025-08838-x", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2025-05-22", "volume": "641", "pages": "1037\u20131047" }, { "id": "authors:0j638-jx111", "collection": "authors", "collection_id": "0j638-jx111", "cite_using_url": "https://authors.library.caltech.edu/records/0j638-jx111", "type": "article", "title": "Spatial transcriptomics defines injury specific microenvironments and cellular interactions in kidney regeneration and disease", "author": [ { "family_name": "Polonsky", "given_name": "Michal", "orcid": "0000-0003-3871-460X", "clpid": "Polonsky-Michal" }, { "family_name": "Gerhardt", "given_name": "Louisa M. S." }, { "family_name": "Yun", "given_name": "Jina", "clpid": "Yun-Chi-H-Jina" }, { "family_name": "Koppitch", "given_name": "Kari" }, { "family_name": "Col\u00f3n", "given_name": "Katsuya Lex", "orcid": "0000-0002-7347-6128", "clpid": "Col\u00f3n-Katsuya-Lex" }, { "family_name": "Amrhein", "given_name": "Henry", "orcid": "0000-0002-4264-140X", "clpid": "Amrhein-H" }, { "family_name": "Wold", "given_name": "Barbara", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Zheng", "given_name": "Shiwei" }, { "family_name": "Yuan", "given_name": "Guo-Cheng", "orcid": "0000-0002-2283-4714" }, { "family_name": "Thomson", "given_name": "Matt", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "McMahon", "given_name": "Andrew P.", "orcid": "0000-0002-3779-1729", "clpid": "McMahon-Andrew-P" } ], "abstract": "Kidney injury disrupts the intricate renal architecture and triggers limited regeneration, together with injury-invoked inflammation and fibrosis. Deciphering the molecular pathways and cellular interactions driving these processes is challenging due to the complex tissue structure. Here, we apply single cell spatial transcriptomics to examine ischemia-reperfusion injury in the mouse kidney. Spatial transcriptomics reveals injury-specific and spatially-dependent gene expression patterns in distinct cellular microenvironments within the kidney and predicts Clcf1-Crfl1 in a molecular interplay between persistently injured proximal tubule cells and their neighboring fibroblasts. Immune cell types play a critical role in organ repair. Spatial analysis identifies cellular microenvironments resembling early tertiary lymphoid structures and associated molecular pathways. Collectively, this study supports a focus on molecular interactions in cellular microenvironments to enhance understanding of injury, repair and disease.
", "doi": "10.1038/s41467-024-51186-z", "pmcid": "PMC11377535", "issn": "2041-1723", "publisher": "Nature Publishing Group", "publication": "Nature Communications", "publication_date": "2024-09-05", "series_number": "1", "volume": "15", "issue": "1", "pages": "7010" }, { "id": "authors:rpkrg-s2933", "collection": "authors", "collection_id": "rpkrg-s2933", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201123-133350121", "type": "article", "title": "Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesis", "author": [ { "family_name": "Lohoff", "given_name": "T.", "orcid": "0000-0001-9333-842X", "clpid": "Lohoff-Tim" }, { "family_name": "Ghazanfar", "given_name": "S.", "orcid": "0000-0001-7861-6997", "clpid": "Ghazanfar-Shila" }, { "family_name": "Missarova", "given_name": "A.", "orcid": "0000-0001-9472-2095", "clpid": "Missarova-Alsu" }, { "family_name": "Koulena", "given_name": "N.", "orcid": "0000-0002-9419-5712", "clpid": "Koulena-Noushin" }, { "family_name": "Pierson", "given_name": "N.", "orcid": "0000-0002-2451-0633", "clpid": "Pierson-Nico-G" }, { "family_name": "Griffiths", "given_name": "J. A.", "orcid": "0000-0002-2010-2296", "clpid": "Griffiths-Jonathan-A" }, { "family_name": "Bardot", "given_name": "E. S.", "orcid": "0000-0002-0872-4957", "clpid": "Bardot-Evan-S" }, { "family_name": "Eng", "given_name": "C.-H. L.", "orcid": "0000-0002-2521-9696", "clpid": "Eng-Chee-Huat-Linus" }, { "family_name": "Tyser", "given_name": "R. C. V.", "orcid": "0000-0001-7884-1756", "clpid": "Tyser-Rivhard-C-V" }, { "family_name": "Argelaguet", "given_name": "R.", "orcid": "0000-0003-3199-3722", "clpid": "Argelaguet-Ricard" }, { "family_name": "Guibentif", "given_name": "C.", "orcid": "0000-0001-8457-456X", "clpid": "Guibentif-Carolina" }, { "family_name": "Srinivas", "given_name": "S.", "orcid": "0000-0001-5726-7791", "clpid": "Srinivas-Shankar" }, { "family_name": "Briscoe", "given_name": "J.", "orcid": "0000-0002-1020-5240", "clpid": "Briscoe-James" }, { "family_name": "Simons", "given_name": "B. D.", "orcid": "0000-0002-3875-7071", "clpid": "Simons-Benjamin-D" }, { "family_name": "Hadjantonakis", "given_name": "A.-K.", "orcid": "0000-0002-7580-5124", "clpid": "Hadjantonakis-Anna-Katerina" }, { "family_name": "G\u00f6ttgens", "given_name": "B.", "orcid": "0000-0001-6302-5705", "clpid": "G\u00f6ttgens-Berthold" }, { "family_name": "Reik", "given_name": "W.", "orcid": "0000-0003-0216-9881", "clpid": "Reik-Wolf" }, { "family_name": "Nichols", "given_name": "J.", "clpid": "Nichols-J" }, { "family_name": "Cai", "given_name": "L.", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Marioni", "given_name": "J. C.", "orcid": "0000-0001-9092-0852", "clpid": "Marioni-John-C" } ], "abstract": "Molecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8\u201312 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain\u2013hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal\u2013ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.", "doi": "10.1038/s41587-021-01006-2", "issn": "1087-0156", "publisher": "Nature Publishing Group", "publication": "Nature Biotechnology", "publication_date": "2022-01", "series_number": "1", "volume": "40", "issue": "1", "pages": "74-85" }, { "id": "authors:wp4n2-mww55", "collection": "authors", "collection_id": "wp4n2-mww55", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211008-183538677", "type": "article", "title": "Single cell biology\u2014a Keystone Symposia report", "author": [ { "family_name": "Cable", "given_name": "Jennifer", "clpid": "Cable-Jennifer" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Domingos", "given_name": "Ana I." }, { "family_name": "Habib", "given_name": "Naomi", "orcid": "0000-0002-6049-2487" }, { "family_name": "Itzkovitz", "given_name": "Shalev", "orcid": "0000-0003-0685-2522" }, { "family_name": "Hamidzada", "given_name": "Homaira" }, { "family_name": "Balzer", "given_name": "Michael S.", "orcid": "0000-0003-0508-1260" }, { "family_name": "Yanai", "given_name": "Itai", "orcid": "0000-0002-8438-2741" }, { "family_name": "Liberali", "given_name": "Prisca", "orcid": "0000-0003-0695-6081" }, { "family_name": "Whited", "given_name": "Jessica", "orcid": "0000-0002-3709-6515" }, { "family_name": "Streets", "given_name": "Aaron", "orcid": "0000-0002-3909-8389" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Stergachis", "given_name": "Andrew B.", "orcid": "0000-0002-1299-3674" }, { "family_name": "Hong", "given_name": "Clarice Kit Yee", "orcid": "0000-0002-9485-1425" }, { "family_name": "Keren", "given_name": "Leeat", "orcid": "0000-0002-6799-6303" }, { "family_name": "Guilliams", "given_name": "Martin", "orcid": "0000-0003-3525-7570" }, { "family_name": "Alon", "given_name": "Uri" }, { "family_name": "Shalek", "given_name": "Alex K.", "orcid": "0000-0001-5670-8778" }, { "family_name": "Hamel", "given_name": "Regan", "orcid": "0000-0003-0013-6226" }, { "family_name": "Pfau", "given_name": "Sarah J.", "orcid": "0000-0001-7528-0666" }, { "family_name": "Raj", "given_name": "Arjun" }, { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809" }, { "family_name": "Zhang", "given_name": "Nancy R." }, { "family_name": "Fan", "given_name": "Jean", "orcid": "0000-0002-0212-5451" }, { "family_name": "Trapnell", "given_name": "Cole", "orcid": "0000-0002-8105-4347" }, { "family_name": "Wang", "given_name": "Bo" }, { "family_name": "Greenwald", "given_name": "Noah F.", "orcid": "0000-0002-7836-4379" }, { "family_name": "Vento-Tormo", "given_name": "Roser" }, { "family_name": "Santos", "given_name": "Silvia D. M.", "orcid": "0000-0002-2906-7888" }, { "family_name": "Spencer", "given_name": "Sabrina L.", "orcid": "0000-0002-5798-3007" }, { "family_name": "Garcia", "given_name": "Hernan G." }, { "family_name": "Arekatla", "given_name": "Geethika" }, { "family_name": "Gaiti", "given_name": "Federico", "orcid": "0000-0001-5111-8816" }, { "family_name": "Arbel-Goren", "given_name": "Rinat", "orcid": "0000-0002-7253-2036" }, { "family_name": "Rulands", "given_name": "Steffen", "orcid": "0000-0001-6398-1553" }, { "family_name": "Junker", "given_name": "Jan Philipp", "orcid": "0000-0002-2826-8290" }, { "family_name": "Klein", "given_name": "Allon M.", "orcid": "0000-0001-8913-7879" }, { "family_name": "Morris", "given_name": "Samantha A." }, { "family_name": "Murray", "given_name": "John I." }, { "family_name": "Galloway", "given_name": "Kate E.", "orcid": "0000-0001-7416-3193" }, { "family_name": "Ratz", "given_name": "Michael", "orcid": "0000-0002-9795-8033" }, { "family_name": "Romeike", "given_name": "Merrit", "orcid": "0000-0002-5890-2213" } ], "abstract": "Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state\u2014transcriptional, epigenetic, and other characteristics\u2014can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17\u201319, 2021, experts in single cell biology met virtually for the Keystone eSymposium \"Single Cell Biology\" to discuss advances both in single cell applications and technologies.", "doi": "10.1111/nyas.14692", "issn": "0077-8923", "publisher": "New York Academy of Sciences", "publication": "Annals of the New York Academy of Sciences", "publication_date": "2021-12", "volume": "1506", "pages": "74-97" }, { "id": "authors:qdxms-1z506", "collection": "authors", "collection_id": "qdxms-1z506", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211004-203742730", "type": "article", "title": "Single-cell nuclear architecture across cell types in the mouse brain", "author": [ { "family_name": "Takei", "given_name": "Yodai", "orcid": "0000-0002-7226-5185", "clpid": "Takei-Yodai" }, { "family_name": "Zheng", "given_name": "Shiwei", "orcid": "0000-0001-6682-6743", "clpid": "Zheng-Shiwei" }, { "family_name": "Yun", "given_name": "Jina", "orcid": "0000-0002-7792-3749", "clpid": "Yun-Jina" }, { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Pierson", "given_name": "Nico", "orcid": "0000-0002-2451-0633", "clpid": "Pierson-Nico-G" }, { "family_name": "White", "given_name": "Jonathan", "orcid": "0000-0002-7009-3332", "clpid": "White-Joanthan-A" }, { "family_name": "Schindler", "given_name": "Simone", "orcid": "0000-0003-1028-3115", "clpid": "Schindler-Simone" }, { "family_name": "Tischbirek", "given_name": "Carsten H.", "orcid": "0000-0002-6876-3448", "clpid": "Tischbirek-Carsten-H" }, { "family_name": "Yuan", "given_name": "Guo-Cheng", "orcid": "0000-0002-2283-4714", "clpid": "Yuan-Guo-Cheng" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Diverse cell types in tissues have distinct gene expression programs, chromatin states, and nuclear architectures. To correlate such multimodal information across thousands of single cells in mouse brain tissue sections, we use integrated spatial genomics, imaging thousands of genomic loci along with RNAs and epigenetic markers simultaneously in individual cells. We reveal that cell type\u2013specific association and scaffolding of DNA loci around nuclear bodies organize the nuclear architecture and correlate with differential expression levels in different cell types. At the submegabase level, active and inactive X chromosomes access similar domain structures in single cells despite distinct epigenetic and expression states. This work represents a major step forward in linking single-cell three-dimensional nuclear architecture, gene expression, and epigenetic modifications in a native tissue context.", "doi": "10.1126/science.abj1966", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2021-09-30", "series_number": "6567", "volume": "374", "issue": "6567", "pages": "586-594" }, { "id": "authors:ejqar-fpe07", "collection": "authors", "collection_id": "ejqar-fpe07", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210226-074209469", "type": "article", "title": "Spatial transcriptomics of planktonic and sessile bacterial populations at single-cell resolution", "author": [ { "family_name": "Dar", "given_name": "Daniel", "orcid": "0000-0002-6650-5488", "clpid": "Dar-Daniel" }, { "family_name": "Dar", "given_name": "Nina", "clpid": "Dar-Nina" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "abstract": "Within any community of organisms, gene expression is heterogeneous, which can manifest in genetically identical individuals having a different phenotype. One has to look at individuals in context and analyze patterns in both space and time to see the full picture. Aiming to fill a gap in current methods, Dar et al. developed a transcriptome-imaging method named parallel sequential fluorescence in situ hybridization (par-seqFISH). They applied this technique to the opportunistic pathogen Pseudomonas aeruginosa, focusing on biofilms where growth conditions can change at microscopic scale. Development of these communities, as revealed by mRNA composition, were followed in space and time. The results revealed a heterogeneous phenotypic landscape, with oxygen availability shaping the metabolism at a spatial scale of microns within a single contiguous biofilm segment. This tool should be applicable to complex microbial communities in the environment and the human microbiome.", "doi": "10.1126/science.abi4882", "pmcid": "PMC8454218", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2021-08-13", "series_number": "6556", "volume": "373", "issue": "6556", "pages": "Art. No. eabi4882" }, { "id": "authors:tf90z-d3h15", "collection": "authors", "collection_id": "tf90z-d3h15", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210527-161220137", "type": "article", "title": "Deep Tropism Profiling of Barcoded AAV Capsid and Cargo Pools in Intact Tissue Using High-Throughput Ultrasensitive Sequential FISH", "author": [ { "family_name": "Jang", "given_name": "Min Jee", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Coughlin", "given_name": "Gerard M.", "clpid": "Coughlin-Gerard-M" }, { "family_name": "Zhang", "given_name": "Yameng", "clpid": "Zhang-Yameng" }, { "family_name": "Koulena", "given_name": "Noushin", "orcid": "0000-0002-9419-5712", "clpid": "Koulena-Noushin" }, { "family_name": "Schindler", "given_name": "Simone", "clpid": "Schindler-Simone" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Genetic access to specific cell types through minimally invasive routes\nis of particular interest in basic research and clinical applications.\nExtensive efforts have been made in engineering gene delivery\nvectors, such as recombinant adeno-associated viruses (rAAVs),\nand gene regulatory elements to achieve this goal. Despite many\ninteresting candidates, revealed for example from directed evolution\nvia M-CREATE (Sripriya Ravindra Kumar et al., Nature Methods,\n2020), histology-based characterization presents a bottleneck due to\nthe limited number of variants and/or cell types that can be investigated\nat once. To address this, we have developed ultrasensitive sequential\nFISH (useqFISH) for multiplexed detection of both endogenous and\nbarcoded transgene transcripts in intact tissue with single-molecule\nresolution. By combining two amplification strategies (rolling circle\namplification, RCA, and hybridization chain reaction, HCR), we\nachieved a 2.7- or 6.7-fold increased signal-to-background ratio of\nuseqFISH in comparison to one with RCA or HCR only amplification,\nrespectively. UseqFISH allowed us to detect endogenous genes with\na single probe pair (20-nucleotide (nt) for each) and, in transfected\ncell cultures, to distinguish capsid variants with genomes differing by\nonly 7-mer peptide modification. We further improved useqFISH by\nestablishing an automated single-molecule imaging and microfluidic\nsolution exchange system and an analytical pipeline for 3D imaging\ndata. To demonstrate the applicability of useqFISH for in vivo AAV\nprofiling, we employed this method to further characterize a pool of\n6 AAV capsid variants that we found to be highly efficient for brainwide\nand/or cell-type biased transduction in the mouse brain following\nsystemic delivery. We designed unique nucleic acid barcodes (160-nt)\nin the 3'UTR of each viral genome and retro-orbitally injected the\npooled AAVs into 2 C57BL6/J mice at a dose of 5e10 viral genomes\n(vg) per variant (total 3e11 vg/mouse). For transcript detection, 11\ncanonical cell-type markers (e.g., Slc17a7, Gad1, Pvalb, SST, VIP, etc)\nwere used together with probes against the viral genome barcodes, to\ncharacterize the cell-type tropisms of each variant. Next, we designed\na pool of 103 barcoded AAV genomes carrying 4 tandem repeats of a\nunique miRNA target site. We packaged these genomes into AAV-PHP.\neB and delivered to 3 C57BL6/J mice at a dose of 1e10 vg/variant (total\n~1e12 vg/mouse). Using useqFISH, we were able to assess the ability of\neach miRNA target site to dampen transgene expression in different cell\ntypes, thereby revealing useful intersectional strategies to refine celltype-\nspecific transgene expression with capsid/cargo combinations.\nThese results demonstrate that useqFISH allows for high-throughput\ncharacterization of pooled genetic variants of viral capsids and gene\nregulatory elements in intact tissue and thus enables comprehensive\nprofiling of genetic toolkits for precise access to targets of interest.", "doi": "10.1016/j.ymthe.2021.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2021-04-27", "series_number": "4, S1", "volume": "29", "issue": "4, S1", "pages": "142" }, { "id": "authors:zjfrp-a9k69", "collection": "authors", "collection_id": "zjfrp-a9k69", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200225-135944187", "type": "article", "title": "Imaging cell lineage with a synthetic digital recording system", "author": [ { "family_name": "Chow", "given_name": "Ke-Huan K.", "orcid": "0000-0002-7317-2669", "clpid": "Chow-Ke-Huan-K" }, { "family_name": "Budde", "given_name": "Mark W.", "orcid": "0000-0002-4359-1424", "clpid": "Budde-Mark-W" }, { "family_name": "Granados", "given_name": "Alejandro A.", "orcid": "0000-0002-6275-9800", "clpid": "Granados-Alejandro-A" }, { "family_name": "Cabrera", "given_name": "Maria", "orcid": "0000-0001-7026-1132", "clpid": "Cabrera-Maria" }, { "family_name": "Yoon", "given_name": "Shinae", "clpid": "Yoon-Shinae" }, { "family_name": "Cho", "given_name": "Soomin", "orcid": "0000-0003-2971-9337", "clpid": "Cho-Soomin" }, { "family_name": "Huang", "given_name": "Ting-hao", "orcid": "0000-0002-2546-3525", "clpid": "Huang-Ting-Hao" }, { "family_name": "Koulena", "given_name": "Noushin", "orcid": "0000-0002-9419-5712", "clpid": "Koulena-Noushin" }, { "family_name": "Frieda", "given_name": "Kirsten L.", "clpid": "Frieda-Kirsten-L" }, { "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": "Cell lineage plays a pivotal role in cell fate determination. Chow et al. demonstrate the use of an integrase-based synthetic barcode system called intMEMOIR, which uses the serine integrase Bxb1 to perform irreversible nucleotide edits. Inducible editing either deletes or inverts its target region, thus encoding information in three-state memory elements, or trits, and avoiding undesired recombination events. Using intMEMOIR combined with single-molecule fluorescence in situ hybridization, the authors were able to identify clonal structures as well as gene expression patterns in the fly brain, enabling both clonal analysis and expression profiling with intact spatial information. The ability to visualize cell lineage relationships directly within their native tissue context provides insights into development and disease.", "doi": "10.1126/science.abb3099", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2021-04-09", "series_number": "6538", "volume": "372", "issue": "6538", "pages": "Art. No. eabb3099" }, { "id": "authors:jf35w-na941", "collection": "authors", "collection_id": "jf35w-na941", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190716-114136984", "type": "article", "title": "Giotto: a toolbox for integrative analysis and visualization of spatial expression data", "author": [ { "family_name": "Dries", "given_name": "Ruben", "clpid": "Dries-Ruben" }, { "family_name": "Zhu", "given_name": "Qian", "clpid": "Zhu-Qian" }, { "family_name": "Dong", "given_name": "Rui", "clpid": "Dong-Rui" }, { "family_name": "Eng", "given_name": "Chee-Huat Linus", "clpid": "Eng-Chee-Huat-Linus" }, { "family_name": "Li", "given_name": "Huipeng", "clpid": "Li-Huipeng" }, { "family_name": "Liu", "given_name": "Kan", "clpid": "Liu-Kan" }, { "family_name": "Fu", "given_name": "Yuntian", "clpid": "Fu-Yuntian" }, { "family_name": "Zhao", "given_name": "Tianxiao", "clpid": "Zhao-Tianxiao" }, { "family_name": "Sarkar", "given_name": "Arpan", "clpid": "Sarkar-Arpan" }, { "family_name": "Bao", "given_name": "Feng", "clpid": "Bao-Feng" }, { "family_name": "George", "given_name": "Rani E.", "clpid": "George-Rani-E" }, { "family_name": "Pierson", "given_name": "Nico", "orcid": "0000-0002-2451-0633", "clpid": "Pierson-Nico-G" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Yuan", "given_name": "Guo-Cheng", "orcid": "0000-0002-2283-4714", "clpid": "Yuan-Guo-Cheng" } ], "abstract": "Spatial transcriptomic and proteomic technologies have provided new opportunities to investigate cells in their native microenvironment. Here we present Giotto, a comprehensive and open-source toolbox for spatial data analysis and visualization. The analysis module provides end-to-end analysis by implementing a wide range of algorithms for characterizing tissue composition, spatial expression patterns, and cellular interactions. Furthermore, single-cell RNAseq data can be integrated for spatial cell-type enrichment analysis. The visualization module allows users to interactively visualize analysis outputs and imaging features. To demonstrate its general applicability, we apply Giotto to a wide range of datasets encompassing diverse technologies and platforms.", "doi": "10.1186/s13059-021-02286-2", "issn": "1465-6906", "publisher": "BioMed Central", "publication": "Genome Biology", "publication_date": "2021-03-08", "volume": "22", "pages": "Art. No. 78" }, { "id": "authors:hfqhd-6qx30", "collection": "authors", "collection_id": "hfqhd-6qx30", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201207-124413570", "type": "article", "title": "Integrated spatial genomics reveals global architecture of single nuclei", "author": [ { "family_name": "Takei", "given_name": "Yodai", "orcid": "0000-0002-7226-5185", "clpid": "Takei-Yodai" }, { "family_name": "Yun", "given_name": "Jina", "orcid": "0000-0002-7792-3749", "clpid": "Yun-Jina" }, { "family_name": "Zheng", "given_name": "Shiwei", "orcid": "0000-0001-6682-6743", "clpid": "Zheng-Shiwei" }, { "family_name": "Ollikainen", "given_name": "Noah", "orcid": "0000-0002-1174-2400", "clpid": "Ollikainen-Noah" }, { "family_name": "Pierson", "given_name": "Nico", "orcid": "0000-0002-2451-0633", "clpid": "Pierson-Nico-G" }, { "family_name": "White", "given_name": "Jonathan", "orcid": "0000-0002-7009-3332", "clpid": "White-Joanthan-A" }, { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Thomassie", "given_name": "Julian", "clpid": "Thomassie-Julian" }, { "family_name": "Suo", "given_name": "Shengbao", "clpid": "Suo-Shengbao" }, { "family_name": "Eng", "given_name": "Chee-Huat Linus", "orcid": "0000-0002-2521-9696", "clpid": "Eng-Chee-Huat-Linus" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Yuan", "given_name": "Guo-Cheng", "orcid": "0000-0002-2283-4714", "clpid": "Yuan-Guo-Cheng" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Identifying the relationships between chromosome structures, nuclear bodies, chromatin states and gene expression is an overarching goal of nuclear-organization studies. Because individual cells appear to be highly variable at all these levels, it is essential to map different modalities in the same cells. Here we report the imaging of 3,660 chromosomal loci in single mouse embryonic stem (ES) cells using DNA seqFISH+, along with 17 chromatin marks and subnuclear structures by sequential immunofluorescence and the expression profile of 70 RNAs. Many loci were invariably associated with immunofluorescence marks in single mouse ES cells. These loci form 'fixed points' in the nuclear organizations of single cells and often appear on the surfaces of nuclear bodies and zones defined by combinatorial chromatin marks. Furthermore, highly expressed genes appear to be pre-positioned to active nuclear zones, independent of bursting dynamics in single cells. Our analysis also uncovered several distinct mouse ES cell subpopulations with characteristic combinatorial chromatin states. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3 trimethylation at lysine 27 (H3K27me3) and macroH2A1 (mH2A1), are heritable over at least 3\u20134 generations, whereas other marks fluctuate on a faster time scale. This seqFISH+-based spatial multimodal approach can be used to explore nuclear organization and cell states in diverse biological systems.", "doi": "10.1038/s41586-020-03126-2", "pmcid": "PMC7878433", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2021-02-11", "series_number": "7845", "volume": "590", "issue": "7845", "pages": "344-350" }, { "id": "authors:9bp34-5kq98", "collection": "authors", "collection_id": "9bp34-5kq98", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210120-154339392", "type": "article", "title": "Transcriptome-Scale Super-Resolved Imaging in Tissues by RNA SeqFISH", "author": [ { "family_name": "Cai", "given_name": "L.", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Imaging the transcriptome in situ with high accuracy has been a major challenge in single cell biology, particularly hindered by the limits of optical resolution and the density of transcripts in single cells. We developed seqFISH+, that can image the mRNAs for 10,000 genes in single cells with high accuracy and sub-diffraction-limit resolution, in the mouse brain cortex, subventricular zone, and the olfactory bulb, using a standard confocal microscope. The transcriptome level profiling of seqFISH+ allows unbiased identification of cell classes and their spatial organization in tissues. In addition, seqFISH+ reveals subcellular mRNA localization patterns in cells and ligand-receptor pairs across neighboring cells. This technology demonstrates the ability to generate spatial cell atlases and to perform discovery-driven studies of biological processes in situ.", "doi": "10.1038/s41431-020-00740-6", "issn": "1018-4813", "publisher": "Nature Publishing Group", "publication": "European Journal of Human Genetics", "publication_date": "2020-12", "series_number": "S1", "volume": "28", "issue": "S1", "pages": "10" }, { "id": "authors:7rv46-gr940", "collection": "authors", "collection_id": "7rv46-gr940", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200803-104852533", "type": "article", "title": "Detecting protein and post-translational modifications in single cells with iDentification and qUantification sEparaTion (DUET)", "author": [ { "family_name": "Zhang", "given_name": "Yandong", "clpid": "Zhang-Yandong" }, { "family_name": "Sohn", "given_name": "Changho", "orcid": "0000-0002-7585-1841", "clpid": "Sohn-Changho" }, { "family_name": "Lee", "given_name": "Seoyeon", "clpid": "Lee-Seoyeon" }, { "family_name": "Ahn", "given_name": "Heejeong", "clpid": "Ahn-Heejeong" }, { "family_name": "Seo", "given_name": "Jinyoung", "orcid": "0000-0002-3260-4963", "clpid": "Seo-Jinyoung" }, { "family_name": "Cao", "given_name": "Junyue", "orcid": "0000-0003-4097-489X", "clpid": "Cao-Junyue" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "While technologies for measuring transcriptomes in single cells have matured, methods for measuring proteins and their post-translational modification (PTM) states in single cells are still being actively developed. Unlike nucleic acids, proteins cannot be amplified, making detection of minute quantities from single cells difficult. Here, we develop a strategy to detect targeted protein and its PTM isoforms in single cells. We barcode the proteins from single cells by tagging them with oligonucleotides, pool barcoded cells together, run bulk gel electrophoresis to separate protein and its PTM isoform and quantify their abundances by sequencing the oligonucleotides associated with each protein species. We used this strategy, iDentification and qUantification sEparaTion (DUET), to measure histone protein H2B and its monoubiquitination isoform, H2Bub, in single yeast cells. Our results revealed the heterogeneities of H2B ubiquitination levels in single cells from different cell-cycle stages, which is obscured in ensemble measurements.", "doi": "10.1038/s42003-020-01132-8", "issn": "2399-3642", "publisher": "Springer Nature", "publication": "Communications Biology", "publication_date": "2020-08-03", "volume": "3", "pages": "Art. No. 420" }, { "id": "authors:81b9d-wyt68", "collection": "authors", "collection_id": "81b9d-wyt68", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201103-105218022", "type": "article", "title": "Analysis of luminal breast cancer tumor microenvironment using seqFISH", "author": [ { "family_name": "Polonsky", "given_name": "Michal", "clpid": "Polonsky-M" }, { "family_name": "Round", "given_name": "Krista", "clpid": "Round-Krista" }, { "family_name": "Seewaldt", "given_name": "Victoria", "clpid": "Seewaldt-V-L" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Women with luminal breast cancers generally have a good prognosis (92% five-year survival). However, Latina/Hispanic women with luminal breast cancer are 30% more likely to present with advanced luminal breast cancer and twice as likely to die. Disparities persist even when accounting for socioeconomic factors. Whether this is due to a more aggressive biologic subtype remains unclear. Here, we aim to identify the signaling networks and intercellular interactions that drive poor prognosis in luminal breast cancer patients and use this data to tailor therapeutic interventions to improve survival. The tumor microenvironment, which is highly heterogeneous, plays an essential role in cancer progression. An additional layer of information lies in the spatial organization of the tissue, with cells coming in close proximity in order to exchange signals over short distances. Thus, to understand the network of interactions and signaling between cells, there is a need for single-cell measurements of multiple cellular features in the intact tissue. Sequential Fluorescence in Situ Hybridization (seqFISH), developed in the lab of Dr. Long Cai, enables precise quantification of several hundreds to several thousands of mRNA transcripts in single cells, preserving the structure of the tissue. In this methodology, fluorescently labeled probes are hybridized onto the fixed tissue, giving rise to a fluorescent signal whenever the corresponding mRNA is present. The tissue is sequentially imaged, washed and re-hybridized, resulting in a unique barcode for each mRNA. seqFISH has been demonstrated both on cell cultures and on whole organs and has been shown to be highly accurate and repetitive. We will use this methodology and combine it with multiplex antibody staining to line mRNA expression with protein localization and modifications. Combining these experimental methodologies with advanced image analysis tools developed by the Long lab will allow an in-depth investigating of the cellular stated and signaling pathways within malignant tissues. Correlating findings between patients, as well as between samples from the same patient taken over time, could potentially provide a deep understanding of the factors leading to poor prognosis, which can in turn improve diagnostics of luminal breast cancer.", "doi": "10.1158/1538-7755.disp18-b117", "issn": "1055-9965", "publisher": "American Association for Cancer Research", "publication": "Cancer Epidemiology, Biomarkers and Prevention", "publication_date": "2020-06", "series_number": "6", "volume": "29", "issue": "6", "pages": "Art. No. B117" }, { "id": "authors:n1hwc-rqm20", "collection": "authors", "collection_id": "n1hwc-rqm20", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200604-074008042", "type": "article", "title": "Method for High-Throughput, In Situ Characterization of AAV Variant Pools in Intact Tissue Using Ultrasensitive Sequential FISH", "author": [ { "family_name": "Jang", "given_name": "Min Jee", "orcid": "0000-0002-1536-7177", "clpid": "Jang-Min-Jee" }, { "family_name": "Coughlin", "given_name": "Gerard M.", "clpid": "Coughlin-G-M" }, { "family_name": "Zhang", "given_name": "Yameng", "clpid": "Zhang-Yameng" }, { "family_name": "Koulena", "given_name": "Noushin", "orcid": "0000-0002-9419-5712", "clpid": "Koulena-N" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Extensive efforts have been made to engineer adeno-associated viruses (AAVs) with desirable characteristics, such as enhanced transduction efficiency and tissue- or cell-type specific tropisms. In-vivo selection, followed by next-generation sequencing (NGS)-based screening, has enabled us to uncover novel viral capsid variants, such as the AAV-PHP series (Deverman et al., Nat Biotech, 2016; Chan et al., Nat Neurosci, 2017; Kumar et al., Nat Methods, 2020). Despite successful library-based selections, the characterization of viral tropisms is slow and labor-intensive and is thus limited to only a handful of variants. To overcome this bottleneck and allow for high-throughput screening, we introduce an imaging-based approach that detects viral transcripts in intact tissue by using ultrasensitive, sequential fluorescence in situ hybridization (FISH). We first developed a new FISH method to enable detection of relatively low abundance viral transcripts compared to endogenous genes in tissue. Compared to two signal amplification methods, rolling-circle amplification (RCA) and hybridization chain reaction (HCR), our method resulted in a 2.7- or 6.7-fold higher signal-to-background ratio, respectively, with the same number of probes. The high sensitivity of our method also allowed us to detect RNA transcripts with 1 probe and distinguish capsid variants packaging an identical viral genome with a short mutated region (7 amino acids, equivalent to 21 base pairs) transduced in HEK293T cells. We also developed an efficient two-step probe stripping method to enable multiple rounds of labeling (up to 8), which increases the number of targets that can be characterized in the same tissue beyond the spectral limit (e.g., 4 colors x 8 rounds = 32 variants). The high sensitivity and ability for sequential labeling allowed us to examine the cell-type tropism of capsid variants and/or gene regulatory elements in intact tissue. For this purpose, we generated AAV pools, comprising a combination of novel AAV-PHP.B-like capsids and cell-type specific promoters, that package the same coding sequence with a unique barcode in the 3'UTR. The pool was injected into one animal at a low dose (~1e10 for each), and after 3-4 weeks of injection, the transcripts of each variant were detected with a custom probe set targeting the unique barcodes. As a proof-of-concept, we were able to characterize the cell-type tropism of 6 variants in one tissue within 4 hours. Further refinement of barcode designs (e.g., temporal barcoding or in situ sequencing) and single-molecule imaging will allow us to either reduce the screening time or increase the number of variants that can be characterized to hundreds. These approaches enable high-throughput characterization of virally delivered transgenes in intact tissue, thus complementing the active field of viral vector engineering with scalable tropism identification or validation. Moreover, visualizing the distribution of many variants while preserving spatial context will offer insights into AAV biology, which can include entry mechanisms as well as cell- and tissue-type associated expression.", "doi": "10.1016/j.ymthe.2020.04.019", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2020-04-28", "series_number": "4", "volume": "28", "issue": "4", "pages": "78-79" }, { "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:2qmmp-7q537", "collection": "authors", "collection_id": "2qmmp-7q537", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191016-124948167", "type": "article", "title": "Single-Cell Analysis Reveals Regulatory Gene Expression Dynamics Leading to Lineage Commitment in Early T Cell Development", "author": [ { "family_name": "Zhou", "given_name": "Wen", "orcid": "0000-0003-0357-2744", "clpid": "Zhou-Wen" }, { "family_name": "Yui", "given_name": "Mary A.", "orcid": "0000-0002-3136-2181", "clpid": "Yui-Mary-A" }, { "family_name": "Williams", "given_name": "Brian A.", "clpid": "Williams-B-A" }, { "family_name": "Yun", "given_name": "Jina", "clpid": "Yun-Jina" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "abstract": "Intrathymic T cell development converts multipotent precursors to committed pro-T cells, silencing progenitor genes while inducing T cell genes, but the underlying steps have remained obscure. Single-cell profiling was used to define the order of regulatory changes, employing single-cell RNA sequencing (scRNA-seq) for full-transcriptome analysis, plus sequential multiplexed single-molecule fluorescent in situ hybridization (seqFISH) to quantitate functionally important transcripts in intrathymic precursors. Single-cell cloning verified high T cell precursor frequency among the immunophenotypically defined \"early T cell precursor\" (ETP) population; a discrete committed granulocyte precursor subset was also distinguished. We established regulatory phenotypes of sequential ETP subsets, confirmed initial co-expression of progenitor with T cell specification genes, defined stage-specific relationships between cell cycle and differentiation, and generated a pseudotime model from ETP to T lineage commitment, supported by RNA velocity and transcription factor perturbations. This model was validated by developmental kinetics of ETP subsets at population and clonal levels. The results imply that multilineage priming is integral to T cell specification.", "doi": "10.1016/j.cels.2019.09.008", "pmcid": "PMC6932747", "issn": "2405-4712", "publisher": "Cell Press", "publication": "Cell Systems", "publication_date": "2019-10-23", "series_number": "4", "volume": "9", "issue": "4", "pages": "321-337" }, { "id": "authors:e3g77-5qr43", "collection": "authors", "collection_id": "e3g77-5qr43", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191017-094121433", "type": "article", "title": "Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior", "author": [ { "family_name": "Kim", "given_name": "Dong-Wook", "orcid": "0000-0002-5497-5853", "clpid": "Kim-Dong-Wook" }, { "family_name": "Yao", "given_name": "Zizhen", "orcid": "0000-0002-9361-5607", "clpid": "Yao-Zizhen" }, { "family_name": "Graybuck", "given_name": "Lucas T.", "orcid": "0000-0002-8814-6818", "clpid": "Graybuck-Lucas-T" }, { "family_name": "Kim", "given_name": "Tae Kyung", "clpid": "Kim-Tae-Kyung" }, { "family_name": "Nguyen", "given_name": "Thuc Nghi", "clpid": "Nguyen-Thuc-Nghi" }, { "family_name": "Smith", "given_name": "Kimberly A.", "clpid": "Smith-Kimberly-A" }, { "family_name": "Fong", "given_name": "Olivia", "clpid": "Fong-Olivia" }, { "family_name": "Yi", "given_name": "Lynn", "orcid": "0000-0003-4575-0158", "clpid": "Yi-Lynn" }, { "family_name": "Koulena", "given_name": "Noushin", "orcid": "0000-0002-9419-5712", "clpid": "Koulena-Noushin" }, { "family_name": "Pierson", "given_name": "Nico", "orcid": "0000-0002-2451-0633", "clpid": "Pierson-Nico-G" }, { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Lo", "given_name": "Liching", "clpid": "Lo-Liching" }, { "family_name": "Pool", "given_name": "Allan-Hermann", "orcid": "0000-0002-0811-9861", "clpid": "Pool-Allan-Hermann" }, { "family_name": "Oka", "given_name": "Yuki", "orcid": "0000-0003-2686-0677", "clpid": "Oka-Yuki" }, { "family_name": "Pachter", "given_name": "Lior", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Tasic", "given_name": "Bosiljka", "orcid": "0000-0002-6861-4506", "clpid": "Tasic-Bosiljka" }, { "family_name": "Zeng", "given_name": "Hongkui", "orcid": "0000-0002-0326-5878", "clpid": "Zeng-Hongkui" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" } ], "abstract": "The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains \u223c4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms\u2014SMART-seq (\u223c4,500 neurons) and 10x (\u223c78,000 neurons)\u2014and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity.", "doi": "10.1016/j.cell.2019.09.020", "pmcid": "PMC7534821", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "2019-10-17", "series_number": "3", "volume": "179", "issue": "3", "pages": "713-728" }, { "id": "authors:nyhw5-8rc67", "collection": "authors", "collection_id": "nyhw5-8rc67", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191009-112519199", "type": "article", "title": "The human body at cellular resolution: the NIH Human Biomolecular Atlas Program", "author": [ { "family_name": "Snyder", "given_name": "Michael P.", "clpid": "Snyder-M-P" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Shendure", "given_name": "Jay", "clpid": "Shendure-J" }, { "family_name": "Trapnell", "given_name": "Cole", "clpid": "Trapnell-C" }, { "family_name": "Lin", "given_name": "Shin", "clpid": "Lin-Shin" }, { "family_name": "Jackson", "given_name": "Dana", "clpid": "Jackson-D" }, { "family_name": "Yuan", "given_name": "Guo-Cheng", "orcid": "0000-0002-2283-4714", "clpid": "Yuan-Guo-Cheng" }, { "family_name": "Zhu", "given_name": "Qian", "clpid": "Zhu-Qian" }, { "family_name": "Dries", "given_name": "Ruben", "clpid": "Dries-R" }, { "literal": "HuBMAP Consortium" } ], "abstract": "Transformative technologies are enabling the construction of three-dimensional maps of tissues with unprecedented spatial and molecular resolution. Over the next seven years, the NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) intends to develop a widely accessible framework for comprehensively mapping the human body at single-cell resolution by supporting technology development, data acquisition, and detailed spatial mapping. HuBMAP will integrate its efforts with other funding agencies, programs, consortia, and the biomedical research community at large towards the shared vision of a comprehensive, accessible three-dimensional molecular and cellular atlas of the human body, in health and under various disease conditions.", "doi": "10.1038/s41586-019-1629-x", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2019-10-10", "series_number": "7777", "volume": "574", "issue": "7777", "pages": "187-192" }, { "id": "authors:1ejwh-1ka64", "collection": "authors", "collection_id": "1ejwh-1ka64", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190129-144119260", "type": "article", "title": "Transcriptome-scale super-resolved imaging in tissues by RNA seqFISH+", "author": [ { "family_name": "Eng", "given_name": "Chee-Huat Linus", "clpid": "Eng-Chee-Huat-Linus" }, { "family_name": "Lawson", "given_name": "Michael", "orcid": "0000-0002-2868-733X", "clpid": "Lawson-Michael-J" }, { "family_name": "Zhu", "given_name": "Qian", "clpid": "Zhu-Qian" }, { "family_name": "Dries", "given_name": "Ruben", "clpid": "Dries-Ruben" }, { "family_name": "Koulena", "given_name": "Noushin", "orcid": "0000-0002-9419-5712", "clpid": "Koulena-Noushin" }, { "family_name": "Takei", "given_name": "Yodai", "clpid": "Takei-Yodai" }, { "family_name": "Yun", "given_name": "Jina", "clpid": "Yun-Jina" }, { "family_name": "Cronin", "given_name": "Christopher", "clpid": "Cronin-Christopher-J" }, { "family_name": "Karp", "given_name": "Christoph", "clpid": "Karp-Christoph-L" }, { "family_name": "Yuan", "given_name": "Guo-Cheng", "orcid": "0000-0002-2283-4714", "clpid": "Yuan-Guo-Cheng" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Imaging the transcriptome in situ with high accuracy has been a major challenge in single-cell biology, which is particularly hindered by the limits of optical resolution and the density of transcripts in single cells. Here we demonstrate an evolution of sequential fluorescence in situ hybridization (seqFISH+). We show that seqFISH+ can image mRNAs for 10,000 genes in single cells\u2014with high accuracy and sub-diffraction-limit resolution\u2014in the cortex, subventricular zone and olfactory bulb of mouse brain, using a standard confocal microscope. The transcriptome-level profiling of seqFISH+ allows unbiased identification of cell classes and their spatial organization in tissues. In addition, seqFISH+ reveals subcellular mRNA localization patterns in cells and ligand\u2013receptor pairs across neighbouring cells. This technology demonstrates the ability to generate spatial cell atlases and to perform discovery-driven studies of biological processes in situ.", "doi": "10.1038/s41586-019-1049-y", "pmcid": "PMC6544023", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2019-04-11", "series_number": "7751", "volume": "568", "issue": "7751", "pages": "235-239" }, { "id": "authors:164wm-qwp59", "collection": "authors", "collection_id": "164wm-qwp59", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20181023-084128561", "type": "article", "title": "The adult human testis transcriptional cell atlas", "author": [ { "family_name": "Guo", "given_name": "Jingtao", "clpid": "Guo-Jingtao" }, { "family_name": "Grow", "given_name": "Edward J.", "clpid": "Grow-Edward-J" }, { "family_name": "Mlcochova", "given_name": "Hana", "clpid": "Mlcochova-Hana" }, { "family_name": "Maher", "given_name": "Geoffrey J.", "orcid": "0000-0001-7135-4824", "clpid": "Maher-Geoffrey-J" }, { "family_name": "Lindskog", "given_name": "Cecilia", "clpid": "Lindskog-Cecilia" }, { "family_name": "Nie", "given_name": "Xichen", "clpid": "Nie-Xichen" }, { "family_name": "Guo", "given_name": "Yixuan", "clpid": "Guo-Yixuan" }, { "family_name": "Takei", "given_name": "Yodai", "clpid": "Takei-Yodai" }, { "family_name": "Yun", "given_name": "Jina", "clpid": "Yun-Jina" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Kim", "given_name": "Robin", "clpid": "Kim-Robin" }, { "family_name": "Carrell", "given_name": "Douglas T.", "clpid": "Carrell-Douglas-T" }, { "family_name": "Goriely", "given_name": "Anne", "orcid": "0000-0001-9229-7216", "clpid": "Goriely-Anne" }, { "family_name": "Hotaling", "given_name": "James M.", "clpid": "Hotaling-James-M" }, { "family_name": "Cairns", "given_name": "Bradley R.", "orcid": "0000-0002-9864-8811", "clpid": "Cairns-Bradley-R" } ], "abstract": "Human adult spermatogenesis balances spermatogonial stem cell (SSC) self-renewal and differentiation, alongside complex germ cell-niche interactions, to ensure long-term fertility and faithful genome propagation. Here, we performed single-cell RNA sequencing of ~6500 testicular cells from young adults. We found five niche/somatic cell types (Leydig, myoid, Sertoli, endothelial, macrophage), and observed germline-niche interactions and key human-mouse differences. Spermatogenesis, including meiosis, was reconstructed computationally, revealing sequential coding, non-coding, and repeat-element transcriptional signatures. Interestingly, we identified five discrete transcriptional/developmental spermatogonial states, including a novel early SSC state, termed State 0. Epigenetic features and nascent transcription analyses suggested developmental plasticity within spermatogonial States. To understand the origin of State 0, we profiled testicular cells from infants, and identified distinct similarities between adult State 0 and infant SSCs. Overall, our datasets describe key transcriptional and epigenetic signatures of the normal adult human testis, and provide new insights into germ cell developmental transitions and plasticity.", "doi": "10.1038/s41422-018-0099-2", "pmcid": "PMC6274646", "issn": "1001-0602", "publisher": "Nature Publishing Group", "publication": "Cell Research", "publication_date": "2018-12", "series_number": "12", "volume": "28", "issue": "12", "pages": "1141-1157" }, { "id": "authors:p4q31-4g613", "collection": "authors", "collection_id": "p4q31-4g613", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180821-132916530", "type": "article", "title": "Identification of spatially associated subpopulations by combining scRNA-seq and sequential fluorescence in situ hybridization", "author": [ { "family_name": "Zhu", "given_name": "Qian", "clpid": "Zhu-Qian" }, { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Dries", "given_name": "Ruben", "clpid": "Dries-Ruben" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Yuan", "given_name": "Guo-Cheng", "orcid": "0000-0002-2283-4714", "clpid": "Yuan-Guo-Cheng" } ], "abstract": "How intrinsic gene-regulatory networks interact with a cell's spatial environment to define its identity remains poorly understood. We developed an approach to distinguish between intrinsic and extrinsic effects on global gene expression by integrating analysis of sequencing-based and imaging-based single-cell transcriptomic profiles, using cross-platform cell type mapping combined with a hidden Markov random field model. We applied this approach to dissect the cell-type- and spatial-domain-associated heterogeneity in the mouse visual cortex region. Our analysis identified distinct spatially associated, cell-type-independent signatures in the glutamatergic and astrocyte cell compartments. Using these signatures to analyze single-cell RNA sequencing data, we identified previously unknown spatially associated subpopulations, which were validated by comparison with anatomical structures and Allen Brain Atlas images.", "doi": "10.1038/nbt.4260", "pmcid": "PMC6488461", "issn": "1087-0156", "publisher": "Nature Publishing Group", "publication": "Nature Biotechnology", "publication_date": "2018-12", "series_number": "12", "volume": "36", "issue": "12", "pages": "1183-1190" }, { "id": "authors:xrfk6-20h25", "collection": "authors", "collection_id": "xrfk6-20h25", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180607-101054915", "type": "article", "title": "Higher-Order Inter-chromosomal Hubs Shape 3D Genome Organization in the Nucleus", "author": [ { "family_name": "Quinodoz", "given_name": "Sofia A.", "orcid": "0000-0003-1862-5204", "clpid": "Quinodoz-Sofia-A" }, { "family_name": "Ollikainen", "given_name": "Noah", "orcid": "0000-0002-1174-2400", "clpid": "Ollikainen-Noah" }, { "family_name": "Tabak", "given_name": "Barbara", "clpid": "Tabak-Barbara" }, { "family_name": "Palla", "given_name": "Ali", "clpid": "Palla-Ali" }, { "family_name": "Schmidt", "given_name": "Jan Marten", "clpid": "Schmidt-Jan-Marten" }, { "family_name": "Detmar", "given_name": "Elizabeth", "clpid": "Detmar-Elizabeth" }, { "family_name": "Lai", "given_name": "Mason M.", "clpid": "Lai-Mason-M" }, { "family_name": "Shishkin", "given_name": "Alexander A.", "clpid": "Shishkin-Alexander-A" }, { "family_name": "Bhat", "given_name": "Prashant", "orcid": "0000-0003-3832-4871", "clpid": "Bhat-Prashant" }, { "family_name": "Takei", "given_name": "Yodai", "orcid": "0000-0002-7226-5185", "clpid": "Takei-Yodai" }, { "family_name": "Trinh", "given_name": "Vickie", "clpid": "Trinh-Vickie" }, { "family_name": "Aznauryan", "given_name": "Erik", "clpid": "Aznauryan-Erik" }, { "family_name": "Russell", "given_name": "Pamela", "clpid": "Russell-Pamela" }, { "family_name": "Cheng", "given_name": "Christine", "clpid": "Cheng-Christine" }, { "family_name": "Jovanovic", "given_name": "Marko", "clpid": "Jovanovic-Marko" }, { "family_name": "Chow", "given_name": "Amy", "clpid": "Chow-Amy-Y" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "McDonel", "given_name": "Patrick", "clpid": "McDonel-Patrick-E" }, { "family_name": "Garber", "given_name": "Manuel", "orcid": "0000-0001-8732-1293", "clpid": "Garber-Manuel" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "abstract": "Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.", "doi": "10.1016/j.cell.2018.05.024", "pmcid": "PMC6548320", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "2018-07-26", "series_number": "3", "volume": "174", "issue": "3", "pages": "744-757" }, { "id": "authors:9x905-bc055", "collection": "authors", "collection_id": "9x905-bc055", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180607-131657607", "type": "article", "title": "Dynamics and Spatial Genomics of the Nascent Transcriptome by Intron seqFISH", "author": [ { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Takei", "given_name": "Yodai", "orcid": "0000-0002-7226-5185", "clpid": "Takei-Yodai" }, { "family_name": "Zhou", "given_name": "Wen", "orcid": "0000-0003-0357-2744", "clpid": "Zhou-Wen" }, { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-Eric" }, { "family_name": "Yun", "given_name": "Jina", "orcid": "0000-0002-7792-3749", "clpid": "Yun-Jina" }, { "family_name": "Eng", "given_name": "Chee-Huat Linus", "clpid": "Eng-Chee-Huat-Linus" }, { "family_name": "Koulena", "given_name": "Noushin", "orcid": "0000-0002-9419-5712", "clpid": "Koulena-Noushin" }, { "family_name": "Cronin", "given_name": "Christopher", "clpid": "Cronin-Christopher-J" }, { "family_name": "Karp", "given_name": "Christoph", "clpid": "Karp-Christoph-L" }, { "family_name": "Liaw", "given_name": "Eric J.", "clpid": "Liaw-Eric-J" }, { "family_name": "Amin", "given_name": "Mina", "clpid": "Amin-Mina" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Visualization of the transcriptome and the nuclear organization in situ has been challenging for single-cell analysis. Here, we demonstrate a multiplexed single-molecule in situ method, intron seqFISH, that allows imaging of 10,421 genes at their nascent transcription active sites in single cells, followed by mRNA and lncRNA seqFISH and immunofluorescence. This nascent transcriptome-profiling method can identify different cell types and states with mouse embryonic stem cells and fibroblasts. The nascent sites of RNA synthesis tend to be localized on the surfaces of chromosome territories, and their organization in individual cells is highly variable. Surprisingly, the global nascent transcription oscillated asynchronously in individual cells with a period of 2 hr in mouse embryonic stem cells, as well as in fibroblasts. Together, spatial genomics of the nascent transcriptome by intron seqFISH reveals nuclear organizational principles and fast dynamics in single cells that are otherwise obscured.", "doi": "10.1016/j.cell.2018.05.035", "pmcid": "PMC6046268", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "2018-07-12", "series_number": "2", "volume": "174", "issue": "2", "pages": "363-376" }, { "id": "authors:nbx8x-z0253", "collection": "authors", "collection_id": "nbx8x-z0253", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171002-110633228", "type": "article", "title": "Profiling the transcriptome with RNA SPOTs", "author": [ { "family_name": "Eng", "given_name": "Chee-Huat Linus", "clpid": "Eng-Chee-Huat-Linus" }, { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Thomassie", "given_name": "Julian", "clpid": "Thomassie-Julian" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Single-molecule FISH (smFISH) has been the gold standard for quantifying individual transcript abundances. Here, we scale up multiplexed smFISH to the transcriptome level and profile 10,212 different mRNAs from mouse fibroblast and embryonic stem cells. This method, called RNA sequential probing of targets (SPOTs), provides an accurate, flexible, and low-cost alternative to sequencing for profiling transcriptomes.", "doi": "10.1038/nmeth.4500", "pmcid": "PMC5819366", "issn": "1548-7091", "publisher": "Nature Publishing Group", "publication": "Nature Methods", "publication_date": "2017-12", "series_number": "12", "volume": "14", "issue": "12", "pages": "1153-1155" }, { "id": "authors:030t5-ngz04", "collection": "authors", "collection_id": "030t5-ngz04", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171128-101613048", "type": "article", "title": "Identification of a neural crest stem cell niche by Spatial Genomic Analysis", "author": [ { "family_name": "Lignell", "given_name": "Antti", "orcid": "0000-0001-7664-5583", "clpid": "Lignell-Antti" }, { "family_name": "Kerosuo", "given_name": "Laura", "orcid": "0000-0001-6710-3512", "clpid": "Kerosuo-Laura" }, { "family_name": "Streichan", "given_name": "Sebastian J.", "orcid": "0000-0002-6105-9087", "clpid": "Streichan-Sebastian-J" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" } ], "abstract": "The neural crest is an embryonic population of multipotent stem cells that form numerous defining features of vertebrates. Due to lack of reliable techniques to perform transcriptional profiling in intact tissues, it remains controversial whether the neural crest is a heterogeneous or homogeneous population. By coupling multiplex single molecule fluorescence in situ hybridization with machine learning algorithm based cell segmentation, we examine expression of 35 genes at single cell resolution in vivo. Unbiased hierarchical clustering reveals five spatially distinct subpopulations within the chick dorsal neural tube. Here we identify a neural crest stem cell niche that centers around the dorsal midline with high expression of neural crest genes, pluripotency factors, and lineage markers. Interestingly, neural and neural crest stem cells express distinct pluripotency signatures. This Spatial Genomic Analysis toolkit provides a straightforward approach to study quantitative multiplex gene expression in numerous biological systems, while offering insights into gene regulatory networks via synexpression analysis.", "doi": "10.1038/s41467-017-01561-w", "pmcid": "PMC5705662", "issn": "2041-1723", "publisher": "Nature Publishing Group", "publication": "Nature Communications", "publication_date": "2017-11-28", "volume": "8", "pages": "Art. No. 1830" }, { "id": "authors:mc5hx-x5144", "collection": "authors", "collection_id": "mc5hx-x5144", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170519-092429585", "type": "article", "title": "seqFISH Accurately Detects Transcripts in Single Cells and Reveals Robust Spatial Organization in the Hippocampus", "author": [ { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-E" }, { "family_name": "Zhou", "given_name": "Wen", "orcid": "0000-0003-0357-2744", "clpid": "Zhou-Wen" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "We recently applied multiplexed seqFISH to profile expressions of hundreds of genes at the single-cell level in situ (Shah et al., 2016) and provided a map of spatial heterogeneity within each subregion, reconciling previously contradictory descriptions of CA1 at lower spatial resolutions. The accompanying Matters Arising paper from Cembrowski and Spruston questions the spatial organization described for CA1 and raise concerns that the results were determined only by high expression, non-barcoded genes. In response, we show that the same robust spatial structure is observed when only lower average abundance genes measured by barcoded seqFISH are used. In fact, many genes with low average abundance are informative of cell states because they can be expressed strongly in specific subpopulation of cells. Our discussion highlights the importance of single-cell in situ analysis in resolving cellular and spatial heterogeneities otherwise lost in population-averaged measurements. This Matters Arising Response paper addresses the Cembrowski and Spruston (2017) Matters Arising paper, published concurrently in this issue of Neuron.", "doi": "10.1016/j.neuron.2017.05.008", "issn": "0896-6273", "publisher": "Elsevier", "publication": "Neuron", "publication_date": "2017-05-17", "series_number": "4", "volume": "94", "issue": "4", "pages": "752-758" }, { "id": "authors:fqa6m-y7024", "collection": "authors", "collection_id": "fqa6m-y7024", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170424-085453982", "type": "article", "title": "Multiplexed Dynamic Imaging of Genomic Loci by Combined CRISPR Imaging and DNA Sequential FISH", "author": [ { "family_name": "Takei", "given_name": "Yodai", "clpid": "Takei-Yodai" }, { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Harvey", "given_name": "Sho", "clpid": "Harvey-Sho" }, { "family_name": "Qi", "given_name": "Lei S.", "clpid": "Qi-Lei-S" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Visualization of chromosome dynamics allows the investigation of spatiotemporal chromatin organization and its role in gene regulation and other cellular processes. However, current approaches to label multiple genomic loci in live cells have a fundamental limitation in the number of loci that can be labeled and uniquely identified. Here we describe an approach we call \"track first and identify later\" for multiplexed visualization of chromosome dynamics by combining two techniques: CRISPR imaging and DNA sequential fluorescence in situ hybridization. Our approach first labels and tracks chromosomal loci in live cells with the CRISPR-Cas9 system, then barcodes those loci by DNA sequential fluorescence in situ hybridization in fixed cells and resolves their identities. We demonstrate our approach by tracking telomere dynamics, identifying 12 unique subtelomeric regions with variable detection efficiencies, and tracking back the telomere dynamics of respective chromosomes in mouse embryonic stem cells.", "doi": "10.1016/j.bpj.2017.03.024", "pmcid": "PMC5425380", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2017-05-09", "series_number": "9", "volume": "112", "issue": "9", "pages": "1773-1776" }, { "id": "authors:he21r-5z560", "collection": "authors", "collection_id": "he21r-5z560", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170515-103755842", "type": "article", "title": "Challenges and emerging directions in single-cell analysis", "author": [ { "family_name": "Yuan", "given_name": "Guo-Cheng", "orcid": "0000-0002-2283-4714", "clpid": "Yuan-Guo-Cheng" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Elowitz", "given_name": "Michael", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Enver", "given_name": "Tariq", "clpid": "Enver-Tariq" }, { "family_name": "Fan", "given_name": "Guoping", "clpid": "Fan-Guoping" }, { "family_name": "Guo", "given_name": "Guoji", "clpid": "Guo-Guoji" }, { "family_name": "Irizarry", "given_name": "Rafael", "clpid": "Irizarry-Rafael" }, { "family_name": "Kharchenko", "given_name": "Peter", "clpid": "Kharchenko-Peter" }, { "family_name": "Kim", "given_name": "Junhyong", "clpid": "Kim-Junhyong" }, { "family_name": "Orkin", "given_name": "Stuart", "clpid": "Orkin-Stuart" }, { "family_name": "Quackenbush", "given_name": "Assieh", "clpid": "Quackenbush-Assieh" }, { "family_name": "Saadatpour", "given_name": "Assieh", "clpid": "Saadatpour-Assieh" }, { "family_name": "Schroeder", "given_name": "Timm", "clpid": "Schroeder-Timm" }, { "family_name": "Shivdasani", "given_name": "Ramesh", "clpid": "Shivdasani-Ramesh" }, { "family_name": "Tirosh", "given_name": "Itay", "clpid": "Tirosh-Itay" } ], "abstract": "Single-cell analysis is a rapidly evolving approach to characterize genome-scale molecular information at the individual cell level. Development of single-cell technologies and computational methods has enabled systematic investigation of cellular heterogeneity in a wide range of tissues and cell populations, yielding fresh insights into the composition, dynamics, and regulatory mechanisms of cell states in development and disease. Despite substantial advances, significant challenges remain in the analysis, integration, and interpretation of single-cell omics data. Here, we discuss the state of the field and recent advances and look to future opportunities.", "doi": "10.1186/s13059-017-1218-y", "pmcid": "PMC5421338", "issn": "1474-760X", "publisher": "BioMed Central", "publication": "Genome Biology", "publication_date": "2017-05-08", "series_number": "1", "volume": "18", "issue": "1", "pages": "Art. No. 84" }, { "id": "authors:eyt49-dzw75", "collection": "authors", "collection_id": "eyt49-dzw75", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170307-082231020", "type": "article", "title": "Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore", "author": [ { "family_name": "Herwig", "given_name": "Lukas", "clpid": "Herwig-Lukas" }, { "family_name": "Rice", "given_name": "Austin J.", "clpid": "Rice-Austin-J" }, { "family_name": "Bedbrook", "given_name": "Claire N.", "orcid": "0000-0003-3973-598X", "clpid": "Bedbrook-Claire-N" }, { "family_name": "Zhang", "given_name": "Ruijie K.", "orcid": "0000-0002-7251-5527", "clpid": "Zhang-Ruijie-K" }, { "family_name": "Lignell", "given_name": "Antti", "orcid": "0000-0001-7664-5583", "clpid": "Lignell-Antti" }, { "family_name": "Cahn", "given_name": "Jackson K. B.", "clpid": "Cahn-Jackson-K-B" }, { "family_name": "Renata", "given_name": "Hans", "orcid": "0000-0003-2468-2328", "clpid": "Renata-Hans" }, { "family_name": "Dodani", "given_name": "Sheel C.", "clpid": "Dodani-Sheel-C" }, { "family_name": "Cho", "given_name": "Inha", "orcid": "0000-0002-7564-5378", "clpid": "Cho-Inha" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Arnold", "given_name": "Frances H.", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "abstract": "By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore, we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems.", "doi": "10.1016/j.chembiol.2017.02.008", "pmcid": "PMC5357175", "issn": "2451-9456", "publisher": "Cell Press", "publication": "Cell Chemical Biology", "publication_date": "2017-03-16", "series_number": "3", "volume": "24", "issue": "3", "pages": "415-425" }, { "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:j1hkz-vms23", "collection": "authors", "collection_id": "j1hkz-vms23", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20161021-132836401", "type": "article", "title": "In Situ Transcription Profiling of Single Cells Reveals Spatial Organization of Cells in the Mouse Hippocampus", "author": [ { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-Eric" }, { "family_name": "Zhou", "given_name": "Wen", "orcid": "0000-0003-0357-2744", "clpid": "Zhou-Wen" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Identifying the spatial organization of tissues at cellular resolution from single-cell gene expression profiles is essential to understanding biological systems. Using an in situ 3D multiplexed imaging method, seqFISH, we identify unique transcriptional states by quantifying and clustering up to 249 genes in 16,958 cells to examine whether the hippocampus is organized into transcriptionally distinct subregions. We identified distinct layers in the dentate gyrus corresponding to the granule cell layer and the subgranular zone and, contrary to previous reports, discovered that distinct subregions within the CA1 and CA3 are composed of unique combinations of cells in different transcriptional states. In addition, we found that the dorsal CA1 is relatively homogeneous at the single cell level, while ventral CA1 is highly heterogeneous. These structures and patterns are observed using different mice and different sets of genes. Together, these results demonstrate the power of seqFISH in transcriptional profiling of complex tissues.", "doi": "10.1016/j.neuron.2016.10.001", "pmcid": "PMC5087994", "issn": "0896-6273", "publisher": "Cell Press", "publication": "Neuron", "publication_date": "2016-10-19", "series_number": "2", "volume": "92", "issue": "2", "pages": "342-357" }, { "id": "authors:npxan-57878", "collection": "authors", "collection_id": "npxan-57878", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160628-085406782", "type": "article", "title": "Single-molecule RNA detection at depth via hybridization chain reaction and tissue hydrogel embedding and clearing", "author": [ { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-Eric" }, { "family_name": "Schwarzkopf", "given_name": "Maayan", "orcid": "0000-0001-8128-1059", "clpid": "Schwarzkopf-Maayan" }, { "family_name": "He", "given_name": "Ting-Fang", "clpid": "He-Ting-Fang" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-Alon" }, { "family_name": "Sohn", "given_name": "Chang Ho", "clpid": "Sohn-Chang-Ho" }, { "family_name": "Lignell", "given_name": "Antti", "orcid": "0000-0001-7664-5583", "clpid": "Lignell-Antti" }, { "family_name": "Choi", "given_name": "Harry M. T.", "orcid": "0000-0002-1530-0773", "clpid": "Choi-Harry-M-T" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas, from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA, imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (PAssive CLARITY Technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving \u223c20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.", "doi": "10.1242/dev.138560", "pmcid": "PMC5004914", "issn": "0950-1991", "publisher": "Company of Biologists", "publication": "Development", "publication_date": "2016-08-01", "series_number": "15", "volume": "143", "issue": "15", "pages": "2862-2867" }, { "id": "authors:7rpkw-jjt62", "collection": "authors", "collection_id": "7rpkw-jjt62", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160907-080424816", "type": "article", "title": "Noncommutative Biology: Sequential Regulation of Complex Networks", "author": [ { "family_name": "Letsou", "given_name": "William", "clpid": "Letsou-William" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Single-cell variability in gene expression is important for generating distinct cell types, but it is unclear how cells use the same set of regulatory molecules to specifically control similarly regulated genes. While combinatorial binding of transcription factors at promoters has been proposed as a solution for cell-type specific gene expression, we found that such models resulted in substantial information bottlenecks. We sought to understand the consequences of adopting sequential logic wherein the time-ordering of factors informs the final outcome. We showed that with noncommutative control, it is possible to independently control targets that would otherwise be activated simultaneously using combinatorial logic. Consequently, sequential logic overcomes the information bottleneck inherent in complex networks. We derived scaling laws for two noncommutative models of regulation, motivated by phosphorylation/neural networks and chromosome folding, respectively, and showed that they scale super-exponentially in the number of regulators. We also showed that specificity in control is robust to the loss of a regulator. Lastly, we connected these theoretical results to real biological networks that demonstrate specificity in the context of promiscuity. These results show that achieving a desired outcome often necessitates roundabout steps.", "doi": "10.1371/journal.pcbi.1005089", "pmcid": "PMC4999240", "issn": "1553-734X", "publisher": "Public Library of Science", "publication": "PLoS Computational Biology", "publication_date": "2016-08", "series_number": "8", "volume": "12", "issue": "8", "pages": "Art. No. e1005089" }, { "id": "authors:4zeyt-4zf66", "collection": "authors", "collection_id": "4zeyt-4zf66", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160509-170833016", "type": "article", "title": "Dense transcript profiling in single cells by image correlation decoding", "author": [ { "family_name": "Coskun", "given_name": "Ahmet F.", "orcid": "0000-0002-5797-1524", "clpid": "Coskun-Ahmet-F" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Sequential barcoded fluorescent in situ hybridization (seqFISH) allows large numbers of molecular species to be accurately detected in single cells, but multiplexing is limited by the density of barcoded objects. We present correlation FISH (corrFISH), a method to resolve dense temporal barcodes in sequential hybridization experiments. Using corrFISH, we quantified highly expressed ribosomal protein genes in single cultured cells and mouse thymus sections, revealing cell-type-specific gene expression.", "doi": "10.1038/nmeth.3895", "pmcid": "PMC4965285", "issn": "1548-7091", "publisher": "Nature Publishing Group", "publication": "Nature Methods", "publication_date": "2016-08", "series_number": "8", "volume": "13", "issue": "8", "pages": "657-660" }, { "id": "authors:72nva-kvd34", "collection": "authors", "collection_id": "72nva-kvd34", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150805-144646254", "type": "article", "title": "Combinatorial gene regulation by modulation of relative pulse timing", "author": [ { "family_name": "Lin", "given_name": "Yihan", "orcid": "0000-0002-2763-5538", "clpid": "Lin-Yihan" }, { "family_name": "Sohn", "given_name": "Chang Ho", "orcid": "0000-0002-7585-1841", "clpid": "Sohn-Chang-Ho" }, { "family_name": "Dalal", "given_name": "Chiraj K.", "orcid": "0000-0002-3624-8409", "clpid": "Dalal-Chiraj-K" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "abstract": "Studies of individual living cells have revealed that many transcription factors activate in dynamic, and often stochastic, pulses within the same cell. However, it has remained unclear whether cells might exploit the dynamic interaction of these pulses to control gene expression. Here, using quantitative single-cell time-lapse imaging of Saccharomyces cerevisiae, we show that the pulsatile transcription factors Msn2 and Mig1 combinatorially regulate their target genes through modulation of their relative pulse timing. The activator Msn2 and repressor Mig1 showed pulsed activation in either a temporally overlapping or non-overlapping manner during their transient response to different inputs, with only the non-overlapping dynamics efficiently activating target gene expression. Similarly, under constant environmental conditions, where Msn2 and Mig1 exhibit sporadic pulsing, glucose concentration modulated the temporal overlap between pulses of the two factors. Together, these results reveal a time-based mode of combinatorial gene regulation. Regulation through relative signal timing is common in engineering and neurobiology, and these results suggest that it could also function broadly within the signalling and regulatory systems of the cell.", "doi": "10.1038/nature15710", "pmcid": "PMC4870307", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2015-11-05", "series_number": "7576", "volume": "527", "issue": "7576", "pages": "54-58" }, { "id": "authors:e4qmn-syf18", "collection": "authors", "collection_id": "e4qmn-syf18", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150914-123933415", "type": "article", "title": "Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping", "author": [ { "family_name": "Treweek", "given_name": "Jennifer B.", "clpid": "Treweek-J-B" }, { "family_name": "Chan", "given_name": "Ken Y.", "orcid": "0000-0002-8853-5186", "clpid": "Chan-Ken-Y" }, { "family_name": "Flytzanis", "given_name": "Nicholas C.", "orcid": "0000-0002-7921-9392", "clpid": "Flytzanis-N-C" }, { "family_name": "Yang", "given_name": "Bin", "clpid": "Yang-Bin" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Greenbaum", "given_name": "Alon", "orcid": "0000-0002-2897-876X", "clpid": "Greenbaum-A" }, { "family_name": "Lignell", "given_name": "Antti", "orcid": "0000-0001-7664-5583", "clpid": "Lignell-Antti" }, { "family_name": "Xiao", "given_name": "Cheng", "orcid": "0000-0001-9649-7450", "clpid": "Xiao-Cheng" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Ladinsky", "given_name": "Mark S.", "orcid": "0000-0002-1036-3513", "clpid": "Ladinsky-M-S" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Fowlkes", "given_name": "Charless C.", "orcid": "0000-0002-2990-1780", "clpid": "Fowlkes-C-C" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1\u20132 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.", "doi": "10.1038/nprot.2015.122", "pmcid": "PMC4917295", "issn": "1754-2189", "publisher": "Nature Publishing Group", "publication": "Nature Protocols", "publication_date": "2015-11", "series_number": "11", "volume": "10", "issue": "11", "pages": "1860-1896" }, { "id": "authors:ey2f8-hnp09", "collection": "authors", "collection_id": "ey2f8-hnp09", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140909-172029209", "type": "article", "title": "Pulsatile Dynamics in the Yeast Proteome", "author": [ { "family_name": "Dalal", "given_name": "Chiraj K.", "orcid": "0000-0002-3624-8409", "clpid": "Dalal-C-K" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Lin", "given_name": "Yihan", "clpid": "Lin-Yihan" }, { "family_name": "Rahbar", "given_name": "Kasra", "clpid": "Rahbar-K" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "abstract": "The activation of transcription factors in response to environmental conditions is fundamental to cellular regulation. Recent work has revealed that some transcription factors are activated in stochastic pulses of nuclear localization, rather than at a constant level, even in a constant environment. In such cases, signals control the mean activity of the transcription factor by modulating the frequency, duration, or amplitude of these pulses. Although specific pulsatile transcription factors have been identified in diverse cell types, it has remained unclear how prevalent pulsing is within the cell, how variable pulsing behaviors are between genes, and whether pulsing is specific to transcriptional regulators or is employed more broadly. To address these issues, we performed a proteome-wide movie-based screen to systematically identify localization-based pulsing behaviors in Saccharomyces cerevisiae. The screen examined all genes in a previously developed fluorescent protein fusion library of 4,159 strains in multiple media conditions. This approach revealed stochastic pulsing in ten proteins, all transcription factors. In each case, pulse dynamics were heterogeneous and unsynchronized among cells in clonal populations. Pulsing is the only dynamic localization behavior that we observed, and it tends to occur in pairs of paralogous and redundant proteins. Taken together, these results suggest that pulsatile dynamics play a pervasive role in yeast and may be similarly prevalent in other eukaryotic species.", "doi": "10.1016/j.cub.2014.07.076", "pmcid": "PMC4203654", "issn": "0960-9822", "publisher": "Cell Press", "publication": "Current Biology", "publication_date": "2014-09-22", "series_number": "18", "volume": "24", "issue": "18", "pages": "2189-2194" }, { "id": "authors:k57mg-mbq97", "collection": "authors", "collection_id": "k57mg-mbq97", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140801-121634734", "type": "article", "title": "Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing", "author": [ { "family_name": "Yang", "given_name": "Bin", "clpid": "Yang-Bin" }, { "family_name": "Treweek", "given_name": "Jennifer B.", "clpid": "Treweek-J-B" }, { "family_name": "Kulkarni", "given_name": "Rajan P.", "clpid": "Kulkarni-R-P" }, { "family_name": "Deverman", "given_name": "Benjamin E.", "orcid": "0000-0002-6223-9303", "clpid": "Deverman-B-E" }, { "family_name": "Chen", "given_name": "Chun-Kan", "clpid": "Chen-Chun-Kan" }, { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-E" }, { "family_name": "Shah", "given_name": "Sheel", "clpid": "Shah-Sheel" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Gradinaru", "given_name": "Viviana", "orcid": "0000-0001-5868-348X", "clpid": "Gradinaru-V" } ], "abstract": "Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.", "doi": "10.1016/j.cell.2014.07.017", "pmcid": "PMC4153367", "issn": "0092-8674", "publisher": "Elsevier", "publication": "Cell", "publication_date": "2014-08-14", "series_number": "4", "volume": "158", "issue": "4", "pages": "945-958" }, { "id": "authors:77t57-k8q45", "collection": "authors", "collection_id": "77t57-k8q45", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140729-074318148", "type": "article", "title": "Dynamic Heterogeneity and DNA Methylation in Embryonic Stem Cells", "author": [ { "family_name": "Singer", "given_name": "Zakary S.", "clpid": "Singer-Z-S" }, { "family_name": "Yong", "given_name": "John", "clpid": "Yong-John" }, { "family_name": "Tischler", "given_name": "Julia", "clpid": "Tischler-J" }, { "family_name": "Hackett", "given_name": "Jamie A.", "clpid": "Hackett-J-A" }, { "family_name": "Altinok", "given_name": "Alphan", "clpid": "Altinok-A" }, { "family_name": "Surani", "given_name": "M. Azim", "clpid": "Surani-M-A" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "abstract": "Cell populations can be strikingly heterogeneous, composed of multiple cellular states, each exhibiting stochastic noise in its gene expression. A major challenge is to disentangle these two types of variability and to understand the dynamic processes and mechanisms that control them. Embryonic stem cells (ESCs) provide an ideal model system to address this issue because they exhibit heterogeneous and dynamic expression of functionally important regulatory factors. We analyzed gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. These data discriminated stochastic switching between two coherent (correlated) gene expression states and burst-like transcriptional noise. We further showed that the \"2i\" signaling pathway inhibitors modulate both types of variation. Finally, we found that DNA methylation plays a key role in maintaining these metastable states. Together, these results show how ESC gene expression states and dynamics arise from a combination of intrinsic noise, coherent cellular states, and epigenetic regulation.", "doi": "10.1016/j.molcel.2014.06.029", "pmcid": "PMC4104113", "issn": "1097-2765", "publisher": "Elsevier", "publication": "Molecular Cell", "publication_date": "2014-07-17", "series_number": "2", "volume": "55", "issue": "2", "pages": "319-331" }, { "id": "authors:10534-ax963", "collection": "authors", "collection_id": "10534-ax963", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140228-201820558", "type": "article", "title": "Single-cell in situ RNA profiling by sequential hybridization", "author": [ { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-E" }, { "family_name": "Coskun", "given_name": "Ahmet F.", "orcid": "0000-0002-5797-1524", "clpid": "Coskun-Ahmet-F" }, { "family_name": "Zhiyentayev", "given_name": "Timur", "clpid": "Zhiyentayev-T" }, { "family_name": "Ahmad", "given_name": "Mubhij", "clpid": "Ahmad-Mubhij" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "In our previous paper, Lubeck and Cai, we used super-resolution microscopy to resolve a large number of mRNAs in single cells. In this Correspondence, we present a sequential barcoding scheme to multiplex different mRNAs.", "doi": "10.1038/nmeth.2892", "pmcid": "PMC4085791", "issn": "1548-7091", "publisher": "Nature Publishing Group", "publication": "Nature Methods", "publication_date": "2014-04", "series_number": "4", "volume": "11", "issue": "4", "pages": "360-361" }, { "id": "authors:m8kea-ywp29", "collection": "authors", "collection_id": "m8kea-ywp29", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140710-095942704", "type": "article", "title": "Imaging Chromosome Structure in Bacteria by Super-Resolution Microscopy", "author": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral separability of fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. The basis for this new approach are the following: given the 10 nanometers resolution of a super-resolution microscope and a typical cell a size of (10um)3, individual cells contains effectively 109 super-resolution pixels or bits of information. Most eukaryotic cells have 104 genes and cellular abundances of 10-100 copies per transcript. Thus, under a super-resolution microscope, an individual cell has 1000 times more pixel volume or information capacities than is needed to encode all transcripts within that cell. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using Fluorescence in situ Hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured the mRNA levels of 32 genes simultaneously in single cells. In addition, we have performed DNA-FISH experiments simultaneously with RNA-FISH to image both the chromosome structure and transcription in single E.coli cells.", "doi": "10.1016/j.bpj.2013.11.2120", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2014-01-28", "series_number": "2", "volume": "106", "issue": "2", "pages": "375A" }, { "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:tcvn9-17174", "collection": "authors", "collection_id": "tcvn9-17174", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130507-073209344", "type": "article", "title": "Turning single cells into microarrays by super-resolution barcoding", "author": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "In this review, we discuss a strategy to bring genomics and proteomics into single cells by super-resolution microscopy. The basis for this new approach are the following: given the 10\u2009nm resolution of a super-resolution microscope and a typical cell with a size of (10\u2009\u00b5m)^3, individual cells contain effectively 10^9 super-resolution pixels or bits of information. Most eukaryotic cells have 10^4 genes and cellular abundances of 10\u2013100 copies per transcript. Thus, under a super-resolution microscope, an individual cell has 1000 times more pixel volume or information capacities than is needed to encode all transcripts within that cell. Individual species of mRNA can be uniquely identified by labeling them each with a distinct combination of fluorophores by fluorescence in situ hybridization. With at least 15 fluorophores available in super-resolution, hundreds of genes in can be barcoded with a three-color barcode (_3C_(15)\u2009=\u2009455). These calculations suggest that by combining super-resolution microscopy and barcode labeling, single cells can be turned into informatics platforms denser than microarrays and that molecular species in individual cells can be profiled in a massively parallel fashion.", "doi": "10.1093/bfgp/els054", "pmcid": "PMC3609437", "issn": "2041-2649", "publisher": "Oxford University Press", "publication": "Briefings in Functional Genomics", "publication_date": "2013-03", "series_number": "2", "volume": "12", "issue": "2", "pages": "75-78" }, { "id": "authors:yfw60-z2p09", "collection": "authors", "collection_id": "yfw60-z2p09", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130429-080544935", "type": "article", "title": "Towards Single-Cell Systems Biology through Super-Resolution Imaging and Molecular Barcoding", "author": [ { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-E" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral overlap between fluorophores. We have demonstrated a simple but general strategy to dramatically increase the capacity for multiplex detection in single cells by labeling with unique combinations of fluorophores using fluorescence in situ hybridization (FISH) and resolving these barcodes using optical super-resolution microscopy (SRM). We have used this technique to measure mRNA levels of 32 genes simultaneously in single Saccharomyces cerevisiae cells. Ongoing work to scale this methodology up for the high-throughput analysis of gene regulatory networks in single cells will be presented.", "doi": "10.1016/j.bpj.2012.11.2062", "issn": "0006-3495", "publisher": "Biophysical Society", "publication": "Biophysical Journal", "publication_date": "2013-01-29", "series_number": "2", "volume": "104", "issue": "2", "pages": "371A" }, { "id": "authors:dm56d-f5r69", "collection": "authors", "collection_id": "dm56d-f5r69", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120801-094824257", "type": "article", "title": "Single-cell systems biology by super-resolution imaging and combinatorial labeling", "author": [ { "family_name": "Lubeck", "given_name": "Eric", "orcid": "0000-0002-5457-0258", "clpid": "Lubeck-E" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" } ], "abstract": "Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral overlap between fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using fluorescence in situ hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured mRNA levels of 32 genes simultaneously in single Saccharomyces cerevisiae cells. These experiments demonstrate that combinatorial labeling and super-resolution imaging of single cells is a natural approach to bring systems biology into single cells.", "doi": "10.1038/nmeth.2069", "pmcid": "PMC3418883", "issn": "1548-7091", "publisher": "Nature Publishing Group", "publication": "Nature Methods", "publication_date": "2012-07", "series_number": "7", "volume": "9", "issue": "7", "pages": "743-748" }, { "id": "authors:v07d1-09860", "collection": "authors", "collection_id": "v07d1-09860", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20100720-094603442", "type": "article", "title": "Stochasticity in Gene Expression as Observed by Single-molecule Experiments in Live Cells", "author": [ { "family_name": "Friedman", "given_name": "Nir", "orcid": "0000-0002-9678-3550", "clpid": "Friedman-N" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Xie", "given_name": "X. Sunney", "clpid": "Xie-X-Sunney" } ], "abstract": "The process of gene expression has two seemingly opposite characteristics: it is highly regulated on one hand, but on the other hand it is inherently random, due to the low copy number of molecules involved. Recent advances in detection techniques allow for direct observations of stochastic molecular events in live cells, with single molecule sensitivity. Here we describe the main methods used for dynamic single molecule detection of mRNA and protein production in live cells. Random bursts of protein production were observed, as well as of mRNA production in some cases. In all experiments to date, bursts occur at random times and the number of molecules per burst is exponentially distributed. We discuss these results using a theoretical model which relates the dynamic process of protein production in bursts to the distribution of protein levels in a population of cells. We propose the gamma distribution as a useful tool for analysis of protein level distributions, both in and out of steady-state. This model can provide quantitative information on the dynamic parameters describing protein production based on measured distributions of protein levels in populations of cells, which are much easier to obtain than dynamic data.", "doi": "10.1560/IJC.49.3-4.333", "issn": "0021-2148", "publisher": "Laser Pages Publishing Ltd", "publication": "Israel Journal of Chemistry", "publication_date": "2009-04", "series_number": "3-4", "volume": "49", "issue": "3-4", "pages": "333-342" }, { "id": "authors:dd3vc-5fa50", "collection": "authors", "collection_id": "dd3vc-5fa50", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:CHOsci08", "type": "article", "title": "A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell", "author": [ { "family_name": "Choi", "given_name": "Paul J.", "clpid": "Choi-Paul-J" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Frieda", "given_name": "Kirsten", "clpid": "Frieda-K-L" }, { "family_name": "Xie", "given_name": "Sunney", "clpid": "Xie-X-Sunney" } ], "abstract": "By monitoring fluorescently labeled lactose permease with single-molecule sensitivity, we investigated the molecular mechanism of how an Escherichia coli cell with the lac operon switches from one phenotype to another. At intermediate inducer concentrations, a population of genetically identical cells exhibits two phenotypes: induced cells with highly fluorescent membranes and uninduced cells with a small number of membrane-bound permeases. We found that this basal-level expression results from partial dissociation of the tetrameric lactose repressor from one of its operators on looped DNA. In contrast, infrequent events of complete dissociation of the repressor from DNA result in large bursts of permease expression that trigger induction of the lac operon. Hence, a stochastic single-molecule event determines a cell's phenotype.", "doi": "10.1126/science.1161427", "pmcid": "PMC2819113", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2008-10-17", "series_number": "5900", "volume": "322", "issue": "5900", "pages": "442-446" }, { "id": "authors:5b636-xfq63", "collection": "authors", "collection_id": "5b636-xfq63", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:CAInat08", "type": "article", "title": "Frequency-modulated nuclear localization bursts coordinate gene regulation", "author": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Dalal", "given_name": "Chiraj K.", "orcid": "0000-0002-3624-8409", "clpid": "Dalal-C-K" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" } ], "abstract": "In yeast, the transcription factor Crz1 is dephosphorylated and translocates into the nucleus in response to extracellular calcium. Here we show, using time-lapse microscopy, that Crz1 exhibits short bursts of nuclear localization (typically lasting 2 min) that occur stochastically in individual cells and propagate to the expression of downstream genes. Strikingly, calcium concentration controls the frequency, but not the duration, of localization bursts. Using an analytic model, we also show that this frequency modulation of bursts ensures proportional expression of multiple target genes across a wide dynamic range of expression levels, independent of promoter characteristics. We experimentally confirm this theory with natural and synthetic Crz1 target promoters. Another stress-response transcription factor, Msn2, exhibits similar, but largely uncorrelated, localization bursts under calcium stress suggesting that frequency-modulation regulation of localization bursts may be a general control strategy used by the cell to coordinate multi-gene responses to external signals.", "doi": "10.1038/nature07292", "pmcid": "PMC2695983", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2008-09-25", "series_number": "7212", "volume": "455", "issue": "7212", "pages": "485-491" }, { "id": "authors:pa8ty-yj072", "collection": "authors", "collection_id": "pa8ty-yj072", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120726-073337126", "type": "article", "title": "Linking Stochastic Dynamics to Population Distribution: An Analytical Framework of Gene Expression", "author": [ { "family_name": "Friedman", "given_name": "Nir", "orcid": "0000-0002-9678-3550", "clpid": "Friedman-N" }, { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Xie", "given_name": "X. Sunney", "clpid": "Xie-X-Sunney" } ], "abstract": "We present an analytical framework describing the steady-state distribution of protein concentration in live cells, considering that protein production occurs in random bursts with an exponentially distributed number of molecules. We extend this framework for cases of transcription autoregulation and noise propagation in a simple genetic network. This model allows for the extraction of kinetic parameters of gene expression from steady-state distributions of protein concentration in a cell population, which are available from single cell data obtained by flow cytometry or fluorescence microscopy.", "doi": "10.1103/PhysRevLett.97.168302", "issn": "0031-9007", "publisher": "American Physical Society", "publication": "Physical Review Letters", "publication_date": "2006-10-20", "series_number": "16", "volume": "97", "issue": "16", "pages": "Art. No. 168302" }, { "id": "authors:n9k4d-v9z66", "collection": "authors", "collection_id": "n9k4d-v9z66", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120726-072703757", "type": "article", "title": "Stochastic protein expression in individual cells at the single molecule level", "author": [ { "family_name": "Cai", "given_name": "Long", "orcid": "0000-0002-7154-5361", "clpid": "Cai-Long" }, { "family_name": "Friedman", "given_name": "Nir", "orcid": "0000-0002-9678-3550", "clpid": "Friedman-N" }, { "family_name": "Xie", "given_name": "X. Sunney", "clpid": "Xie-X-Sunney" } ], "abstract": "In a living cell, gene expression\u2014the transcription of DNA to messenger RNA followed by translation to protein\u2014occurs stochastically, as a consequence of the low copy number of DNA and mRNA molecules involved. These stochastic events of protein production are difficult to observe directly with measurements on large ensembles of cells owing to lack of synchronization among cells. Measurements so far on single cells lack the sensitivity to resolve individual events of protein production. Here we demonstrate a microfluidic-based assay that allows real-time observation of the expression of \u03b2-galactosidase in living Escherichia coli cells with single molecule sensitivity. We observe that protein production occurs in bursts, with the number of molecules per burst following an exponential distribution. We show that the two key parameters of protein expression\u2014the burst size and frequency\u2014can be either determined directly from real-time monitoring of protein production or extracted from a measurement of the steady-state copy number distribution in a population of cells. Application of this assay to probe gene expression in individual budding yeast and mouse embryonic stem cells demonstrates its generality. Many important proteins are expressed at low levels, and are thus inaccessible by current genomic and proteomic techniques. This microfluidic single cell assay opens up possibilities for system-wide characterization of the expression of these low copy number proteins.", "doi": "10.1038/nature04599", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2006-03-16", "series_number": "7082", "volume": "440", "issue": "7082", "pages": "358-362" } ]