Tsung-Li Liu, Srigokul Upadhyayula, Daniel E. Milkie, Ved Singh, Kai Wang, Ian A. Swinburne, Kishore R. Mosaliganti, Zach M. Collins, Tom W. Hiscock, Jamien Shea, Abraham Q. Kohrman, Taylor N Medwig, Daphne Dambournet, Ryan Forster, Brian Cunniff, Yuan Ruan, Hanako Yashiro, Steffen Scholpp, Elliot M. Meyerowitz, Dirk Hockemeyer, David G. Drubin, Benjamin L. Martin, David Q. Matus, Minoru Koyama, Sean G. Megason, Tom Kirchhausen, Eric Betzig

True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution without inducing undue stress on either. We combined lattice light sheet microscopy with two-channel adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages, and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.

Nicholas Schaum, Jim Karkanias, Norma F. Neff, Andrew P May, Stephen R. Quake, Tony Wyss-Coray, Spyros Darmanis, Joshua Batson, Olga Botvinnik, Michelle B Chen, Steven Chen, Foad Green, Robert Jones, Ashley Maynard, Lolita Penland, Rene V Sit, Geoffrey M. Stanley, James T Webber, Fabio Zanini, Ankit S. Baghel, Isaac Bakerman, Ishita Bansal, Daniela Berdnik, Biter Bilen, Douglas Brownfield, Corey Cain, Min Cho, Giana Cirolia, Stephanie D Conley, Aaron Demers, Kubilay Demir, Antoine de Morree, Tessa Divita, Haley du Bois, Laughing Bear Torrez Dulgeroff, Hamid Ebadi, F. Hernan Espinoza, Matt Fish, Qiang Gan, Benson M. George, Astrid Gillich, Geraldine Genetiano, Xueying Gu, Gunsagar S. Gulati, Yan Hang, Shayan Hosseinzadeh, Albin Huang, Tal Iram, Taichi Isobe, Feather Ives, Kevin S Kao, Guruswamy Karnam, Aaron M Kershner, Bernhard Kiss, William Kong, Maya E. Kumar, Jonathan Lam, Davis P Lee, Song E Lee, Guang Li, Qingyun Li, Ling Liu, Annie Lo, Wan-Jin Lu, Anoop Manjunath, Kaia L. May, Oliver L. May, Marina McKay, Ross J Metzger, Marco Mignardi, Dullei Min, Ahmad N. Nabhan, Katharine M. Ng, Joseph Noh, Rasika Patkar, Weng Chuan Peng, Robert Puccinelli, Eric J. Rulifson, Shaheen S. Sikandar, Rahul Sinha, Krzysztof Szade, Weilun Tan, Cristina Tato, Krissie Tellez, Kyle J. Travaglini, Carolina Tropini, Lucas Waldburger, Linda J. van Weele, Michael N. Wosczyna, Jinyi Xiang, Soso Xue, Justin Youngyunpipatkul, Macy E Zardeneta, Fan Zhang, Lu Zhou, Paola Castro, Derek Croote, Joseph L. DeRisi, Angela Pisco, Bernhard M. Kiss, Christin S Kuo, Benoit Lehallier, Patricia K. Nguyen, Serena Y. Tan, Bruce M. Wang, Hanadie Yousef, Philip A. Beachy, Charles K. F. Chan, Kerwyn Casey Huang, Kenneth Weinberg, Sean Wu, Ben A. Barres, Michael F Clarke, Seung K Kim, Mark A Krasnow, Norma Neff, Roel Nusse, Thomas A. Rando, Justin Sonnenburg, Irving L. Weissman, Sean M. Wu

We have created a compendium of single cell transcriptome data from the model organism Mus musculus comprising more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, revealing gene expression in poorly characterized cell populations and allowing for direct and controlled comparison of gene expression in cell types shared between tissues, such as T-lymphocytes and endothelial cells from distinct anatomical locations. Two distinct technical approaches were used for most tissues: one approach, microfluidic droplet-based 3-end counting, enabled the survey of thousands of cells at relatively low coverage, while the other, FACS-based full length transcript analysis, enabled characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.

Aviv Regev, Sarah Teichmann, Eric S Lander, Ido Amit, Christophe Benoist, Ewan Birney, Bernd Bodenmiller, Peter Campbell, Piero Carninci, Menna Clatworthy, Hans Clevers, Bart Deplancke, Ian Dunham, James Eberwine, Roland Eils, Wolfgang Enard, Andrew Farmer, Lars Fugger, Berthold Göttgens, Nir Hacohen, Muzlifah Haniffa, Martin Hemberg, Seung Kim, Paul Klenerman, Arnold Kriegstein, Ed S. Lein, Sten Linnarsson, Joakim Lundeberg, Partha Majumder, John C. Marioni, Miriam Merad, Musa Mhlanga, Martijn Nawijn, Mihai Netea, Garry Nolan, Dana Pe’er, Anthony Phillipakis, CP Ponting, Steve Quake, Wolf Reik, Orit Rozenblatt-Rosen, Joshua Sanes, Rahul Satija, Ton N. Schumacher, Alex Shalek, Ehud Shapiro, Padmanee Sharma, Jay W Shin, Oliver Stegle, Michael Stratton, Michael J. T. Stubbington, Alexander van Oudenaarden, Allon Wagner, Fiona Watt, Jonathan Weissman, Barbara Wold, Ramnik Xavier, Nir Yosef, the Human Cell Atlas Meeting Participants

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

Joan C. Smith, Erin L. Sausville, Vishruth Girish, Monet Lou Yuan, Kristen M. John, Jason M. Sheltzer

The factors mediating fatal SARS-CoV-2 infections are poorly understood. Here, we show that cigarette smoke causes a dose-dependent upregulation of Angiotensin Converting Enzyme 2 (ACE2), the SARS-CoV-2 receptor, in rodent and human lungs. Using single-cell sequencing data, we demonstrate that ACE2 is expressed in a subset of secretory cells in the respiratory tract. Chronic smoke exposure triggers the expansion of this cell population and a concomitant increase in ACE2 expression. In contrast, quitting smoking decreases the abundance of these secretory cells and reduces ACE2 levels. Finally, we demonstrate that ACE2 expression is responsive to inflammatory signaling and can be upregulated by viral infections or interferon treatment. Taken together, these results may partially explain why smokers are particularly susceptible to severe SARS-CoV-2 infections. Furthermore, our work identifies ACE2 as an interferon-stimulated gene in lung cells, suggesting that SARS-CoV-2 infections could create positive-feedback loops that increase ACE2 levels and facilitate viral dissemination. ### Competing Interest Statement J.C.S. is a co-founder of Meliora Therapeutics and is an employee of Google, Inc. This work was performed outside of her affiliation with Google and used no proprietary knowledge or materials from Google. J.M.S. has received consulting fees from Ono Pharmaceuticals, is a member of the Advisory Board of Tyra Biosciences, and is a co-founder of Meliora Therapeutics.

Dieter Egli, Michael V. Zuccaro, Michal Kosicki, George M Church, Allan Bradley, Maria Jasin

Many human diseases have an underlying genetic component. The development and application of methods to prevent the inheritance of damaging mutations through the human germline could have significant health benefits, and currently include preimplantation genetic diagnosis and carrier screening. Ma et al. take this a step further by attempting to remove a disease mutation from the human germline through gene editing (1). They assert the following advances: (i) the correction of a pathogenic gene mutation responsible for hypertrophic cardiomyopathy in human embryos using CRISPR-Cas9 and (ii) the avoidance of mosaicism in edited embryos. In the case of correction, the authors conclude that repair using the homologous chromosome was as or more frequent than mutagenic nonhomologous end-joining (NHEJ). Their conclusion is significant, if validated, because such a self-repair mechanism would allow gene correction without the introduction of a repair template. While the authors analyses relied on the failure to detect mutant alleles, here we suggest approaches to provide direct evidence for inter-homologue recombination and discuss other events consistent with the data. We also review the biological constraints on inter-homologue recombination in the early embryo. (1) Ma, H. et al. Correction of a pathogenic gene mutation in human embryos. Nature, doi:10.1038/nature23305 (2017).

Hitoshi Niwa

In January 2014, it was reported that strong external stimuli, such as a transient low-pH stressor, was capable of inducing the reprogramming of mammalian somatic cells, resulting in the generation of pluripotent cells (Obokata et al. 2014a, b). This cellular reprograming event was designated 'stimulus-triggered acquisition of pluripotency' (STAP) by the authors of these reports. However, after multiple instances of scientific misconduct in the handling and presentation of the data were brought to light, both reports were retracted. To investigate the actual scientific significance of the purported STAP phenomenon, we sought to repeat the original experiments based on the methods presented in the retracted manuscripts and other relevant information. As a result, we have concluded that the STAP phenomenon as described in the original studies is not reproducible.

Furqan M. Fazal, Shuo Han, Pornchai Kaewsapsak, Kevin R Parker, Jin Xu, Alistair N Boettiger, Howard Y. Chang, Alice Y Ting

We introduce APEX-seq, a method for RNA sequencing based on spatial proximity to the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome, revealing extensive and exquisite patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.

The Tabula Muris Consortium, Angela Oliveira Pisco, Aaron McGeever, Nicholas Schaum, Jim Karkanias, Norma F. Neff, Spyros Darmanis, Tony Wyss-Coray, Stephen R. Quake

Aging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death[1][1]. Despite rapid advances over recent years, many of the molecular and cellular processes which underlie progressive loss of healthy physiology are poorly understood[2][2]. To gain a better insight into these processes we have created a single cell transcriptomic atlas across the life span of Mus musculus which includes data from 23 tissues and organs. We discovered cell-specific changes occurring across multiple cell types and organs, as well as age related changes in the cellular composition of different organs. Using single-cell transcriptomic data we were able to assess cell type specific manifestations of different hallmarks of aging, such as senescence[3][3], genomic instability[4][4] and changes in the organism’s immune system[2][2]. This Tabula Muris Senis provides a wealth of new molecular information about how the most significant hallmarks of aging are reflected in a broad range of tissues and cell types. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4

Dominique Chrétien, Paule Bénit, Hyung-Ho Ha, Susanne Keipert, Riyad El-Khoury, Young-Tae Chang, Martin Jastroch, Howard T. Jacobs, Pierre Rustin, Malgorzata Rak

In endothermic species, heat released as a product of metabolism ensures stable internal temperature throughout the organism, despite varying environmental conditions. Mitochondria are major actors in this thermogenic process. Part of the energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport, while a noticeable proportion is released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured mitochondrial temperature in situ under different physiological conditions. At a constant external temperature of 38 C, mitochondria were more than 10 C warmer when the respiratory chain was fully functional, both in HEK293cells and primary skin fibroblasts. This differential was abolished in cells lacking mitochondrial DNA or by respiratory inhibitors, but preserved or enhanced by expressing thermogenic enzymes such as the alternative oxidase or the uncoupling protein 1. The activity of various RC enzymes was maximal at, or slightly above, 50 C. Our study prompts a re-examination of the literature on mitochondria, taking account of the inferred high temperature.

Feria Hikmet, Loren Méar, Åsa Edvinsson, Patrick Micke, Mathias Uhlén, Cecilia Lindskog

The novel SARS-coronavirus 2 (SARS-CoV-2) poses a global challenge on healthcare and society. For understanding the susceptibility for SARS-CoV-2 infection, the cell type-specific expression of the host cell surface receptor is necessary. The key protein suggested to be involved in host cell entry is Angiotensin I converting enzyme 2 (ACE2). Here, we report the expression pattern of ACE2 across >150 different cell types corresponding to all major human tissues and organs based on stringent immunohistochemical analysis. The results were compared with several datasets both on the mRNA and protein level. ACE2 expression was mainly observed in enterocytes, renal tubules, gallbladder, cardiomyocytes, male reproductive cells, placental trophoblasts, ductal cells, eye and vasculature. In the respiratory system, the expression was limited, with no or only low expression in a subset of cells in a few individuals, observed by one antibody only. Our data constitutes an important resource for further studies on SARS-CoV-2 host cell entry, in order to understand the biology of the disease and to aid in the development of effective treatments to the viral infection. ### Competing Interest Statement The authors have declared no competing interest.

Shan Zhao, Mihail Ivilinov Todorov, Ruiyao Cai, Hanno Steinke, Elisabeth Kemter, Eckhard Wolf, Jan Lipfert, Ingo Bechmann, Ali Ertürk

Optical tissue transparency permits cellular and molecular investigation of complex tissues in 3D, a fundamental need in biomedical sciences. Adult human organs are particularly challenging for this approach, owing to the accumulation of dense and sturdy molecules in decades-aged human tissues. Here, we introduce SHANEL method utilizing a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to generate the first intact transparent adult human brain and kidney, and perform 3D histology using antibodies and dyes in centimeters depth. Thereby, we revealed structural details of the sclera, iris and suspensory ligament in the human eye, and the vessels and glomeruli in the human kidney. We also applied SHANEL on transgenic pig organs to map complex structures of EGFP expressing beta cells in >10 cm size pancreas. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of intact large mammalian organs.

James L Daly, Boris Simonetti, Carlos Antón-Plágaro, Maia Kavanagh Williamson, Deborah K. Shoemark, Lorena Simón-Gracia, Katja Klein, Michael Bauer, Reka Hollandi, Urs F. Greber, Peter Horvath, Richard B. Sessions, Ari Helenius, Julian A Hiscox, Tambet Teesalu, David A. Matthews, Andrew D. Davidson, Peter J. Cullen, Yohei Yamauchi

SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than 6.6 million people and caused over 390,000 deaths worldwide. The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2. For SARS-CoV-2 the cleavage is catalysed by furin, a host cell protease, which cleaves the S protein precursor at a specific sequence motif that generates a polybasic Arg-Arg-Ala-Arg (RRAR) C-terminal sequence on S1. This sequence motif conforms to the C-end rule (CendR), which means that the C-terminal sequence may allow the protein to associate with cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors. Here we demonstrate using immunoprecipitation, site-specific mutagenesis, structural modelling, and antibody blockade that, in addition to engaging the known receptor ACE2, S1 can bind to NRP1 through the canonical CendR mechanism. This interaction enhances infection by SARS-CoV-2 in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection, and provides a therapeutic target for COVID-19. ### Competing Interest Statement The authors have declared no competing interest.

Ruilin Tian, Mariam A Gachechiladze, Connor H Ludwig, Matthew T. Laurie, Jason Y Hong, Diane Nathaniel, Anika V Prabhu, Michael S Fernandopulle, Rajan Patel, Mehrnoosh Abshari, Michael E Ward, Martin Kampmann

CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines, rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons, and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons.

Chris D Richardson, Katelynn R. Kazane, Sharon J. Feng, Nicholas L Bray, Axel J Schäfer, Stephen Floor, Jacob E Corn

CRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double stranded DNA donor (dsDonor). The high efficacy of SSTR makes it a promising avenue for the treatment of genetic diseases, but the genetic basis of SSTR editing is still unclear, leaving its use a mostly empiric process. To determine the pathways underlying SSTR in human cells, we developed a coupled knockdown-editing screening system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. Unexpectedly, we found that SSTR requires multiple components of the Fanconi Anemia (FA) repair pathway, but does not require Rad51-mediated homologous recombination, distinguishing SSTR from repair using dsDonors. Knockdown of FA genes impacts SSTR without altering break repair by non-homologous end joining (NHEJ) in multiple human cell lines and in neonatal dermal fibroblasts. Our results establish an unanticipated and central role for the FA pathway in templated repair from single stranded DNA by human cells. Therapeutic genome editing has been proposed to treat genetic disorders caused by deficiencies in DNA repair, including Fanconi Anemia. Our data imply that patient genotype and/or transcriptome profoundly impact the effectiveness of gene editing treatments and that adjuvant treatments to bias cells towards FA repair pathways could have considerable therapeutic value.

Sinem K. Saka, Yu Wang, Jocelyn Y. Kishi, Allen Zhu, Yitian Zeng, Wenxin Xie, Koray Kirli, Clarence Yapp, Marcelo Cicconet, Brian J. Beliveau, Sylvain W Lapan, Siyuan Yin, Millicent Lin, Ed Boyden, Pascal S Kaeser, German Pihan, George M Church, Peng Yin

Probing the molecular organization of tissues requires in situ analysis by microscopy. However current limitations in multiplexing, sensitivity, and throughput collectively constitute a major barrier for comprehensive single-cell profiling of proteins. Here, we report Immunostaining with Signal Amplification By Exchange Reaction (Immuno-SABER), a rapid, highly multiplexed signal amplification method that simultaneously tackles these key challenges. Immuno-SABER utilizes DNA-barcoded antibodies and provides a method for highly multiplexed signal amplification via modular orthogonal DNA concatemers generated by Primer Exchange Reaction. This approach offers the capability to preprogram and control the amplification level independently for multiple targets without in situ enzymatic reactions, and the intrinsic scalability to rapidly amplify and image a large number of protein targets. We validated our approach in diverse sample types including cultured cells, cryosections, FFPE sections, and whole mount tissues. We demonstrated independently tunable 5-180-fold amplification for multiple targets, covering the full signal range conventionally achieved by secondary antibodies to tyramide signal amplification, as well as simultaneous signal amplification for 10 different proteins using standard equipment and workflow. We further combined Immuno-SABER with Expansion Microscopy to enable rapid and highly multiplexed super-resolution tissue imaging. Overall, Immuno-SABER presents an effective and accessible platform for rapid, multiplexed imaging of proteins across scales with high sensitivity.

Mirjana Efremova, Miquel Vento-Tormo, Sarah Teichmann, Roser Vento-Tormo

Cell-cell communication mediated by receptor-ligand complexes is crucial for coordinating diverse biological processes, such as development, differentiation and responses to infection. In order to understand how the context-dependent crosstalk of different cell types enables physiological processes to proceed, we developed CellPhoneDB, a novel repository of ligands, receptors and their interactions. Our repository takes into account the subunit architecture of both ligands and receptors, representing heteromeric complexes accurately. We integrated our resource with a statistical framework that predicts enriched cellular interactions between two cell types from single-cell transcriptomics data. Here, we outline the structure and content of our repository, the procedures for inferring cell-cell communication networks from scRNA-seq data and present a practical step-by-step guide to help implement the protocol. CellPhoneDB v2.0 is a novel version of our resource that incorporates additional functionalities to allow users to introduce new interacting molecules and reduce the time and resources needed to interrogate large datasets. CellPhoneDB v2.0 is publicly available at https://github.com/Teichlab/cellphonedb and as a user-friendly web interface at http://www.cellphonedb.org/. In our protocol, we demonstrate how to reveal meaningful biological discoveries from CellPhoneDB v2.0 using published data sets.

Norman Sachs, Domenique D. Zomer-van Ommen, Angelos Papaspyropoulos, Inha Heo, Lena Böttinger, Dymph Klay, Fleur Weeber, Guizela Huelsz-Prince, Nino Iakobachvili, Marco C. Viveen, Anna Lyubimova, Luc Teeven, Sepideh Derakhshan, Jeroen Korving, Harry Begthel, Kuldeep Kumawat, Emilio Ramos, Matthijs F.M. van Oosterhout, Eduardo P. Olimpio, Joep de Ligt, Krijn K. Dijkstra, Egbert F. Smit, Maarten van der Linden, Emile E. Voest, Coline H.M. van Moorsel, Cornelis K. van der Ent, Edwin Cuppen, Alexander van Oudenaarden, Frank E. Coenjaerts, Linde Meyaard, Louis J. Bont, Peter J. Peters, Sander Tans, Jeroen S. van Zon, Sylvia F. Boj, Robert G. Vries, Jeffrey M. Beekman, Hans Clevers

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar biopsies or lavage material. The pseudostratified airway organoid epithelium consists of basal cells, functional multi-ciliated cells, mucus-producing goblet cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoid culture conditions also allow gene editing as well as the derivation of various types of lung cancer organoids. Respiratory syncytial virus (RSV) infection recapitulated central disease features and dramatically increases organoid cell motility, found to be driven by the non-structural viral NS2 protein. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

Jonathan B. Grimm, Brian P. English, Anand K Muthusamy, Brian P Mehl, Peng Dong, Timothy A. Brown, Zhe Liu, Timothée Lionnet, Luke D. Lavis

Small molecule fluorophores are important tools for advanced imaging experiments. The development of self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in live-cell microscopy. We recently described a general method for improving the brightness and photostability of small, cell-permeable fluorophores, resulting in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of the JF dyes by synthesizing photoactivatable derivatives that are compatible with established live cell labeling strategies. These compounds retain the superior brightness of the JF dyes but their facile photoactivation enables improved single-particle tracking and localization microscopy experiments.

Paul M. Müller, Juliane Rademacher, Richard D. Bagshaw, Keziban M. Alp, Girolamo Giudice, Louise E Heinrich, Carolin Barth, Rebecca L Eccles, Marta Sanchez-Castro, Lennart Brandenburg, Geraldine Mbamalu, Monika Tucholska, Lisa Spatt, Celina Wortmann, Maciej T Czajkowski, Robert-William Welke, Sunqu Zhang, Vivian Nguyen, Trendelina Rrustemi, Philipp Trnka, Kiara Freitag, Brett Larsen, Oliver Popp, Philipp Mertins, Chris Bakal, Anne-Claude Gingras, Olivier Pertz, Frederick P Roth, Karen Colwill, Tony Pawson, Evangelia Petsalaki, Oliver Rocks

Rho GTPases control cell shape formation and thus fundamental physiological processes in all eukaryotes. Their functions are regulated by 145 RhoGEF and RhoGAP multi-domain proteins in humans. To provide the framework for a systems-level understanding of how these regulators orchestrate cellular morphogenesis, we comprehensively characterized their substrate specificities, localization and interactome. The resulting resource places the RhoGEFs/RhoGAPs in functional context, serving as a foundation for targeted and integrated studies. Our data reveals their critical role in the spatial organization of Rho signaling. They localize to multiple compartments to provide positional information, are extensively interconnected to jointly coordinate their signaling networks and are widely autoinhibited to remain sensitive to local activation. RhoGAPs exhibit lower substrate specificity than RhoGEFs and may contribute to preserving Rho activity gradients. We demonstrate the utility of our integrated data by detailing a multi-RhoGEF complex downstream of G-protein-coupled receptors in which the enzymes mutually regulate their activities.

Chawin Ounkomol, Sharmishtaa Seshamani, Mary M Maleckar, Forrest Collman, Gregory R. Johnson

Understanding cells as integrated systems is a challenge central to modern biology. While different microscopy approaches may be used to probe diverse aspects of biological organization, each method presents limitations which ultimately restrict a view into unified cellular organization. For example, while fluorescence microscopy can resolve subcellular structure in living cells, it is expensive, slow, and can damage cells. Here, we present a label-free method for predicting 3D fluorescence directly from transmitted light images and demonstrate that it can be used to generate multi-structure, integrated images. We then demonstrate that this same method can be used to predict immunofluorescence from electron micrograph inputs, extending the method to a wider range of bioimaging applications.