Most downloaded biology preprints, all time
in category cell biology
5,819 results found. For more information, click each entry to expand.
2,873 downloads bioRxiv cell biology
Morgan Delarue, Greg Brittingham, S. Pfeffer, Ivan Surovstev, S. Ping-lay, K.J. Kennedy, M. Schaffer, J. Ignacio Gutiérrez, D. Sang, G. Poterewicz, J.K. Chung, J. Plitzko, Jay T Groves, Christine Jacobs-Wagner, B.D. Engel, L.J. Holt
Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed Genetically Encoded Multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein. GEMs self-assemble into bright, stable fluorescent particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can tune the effective diffusion coefficient of macromolecules ≥15 nm in diameter more than 2-fold without any discernable effect on the motion of molecules ≤5 nm. These mTORC1-dependent changes in crowding and rheology affect phase-separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the biophysical properties of the cytoplasm and the phase-separation of biopolymers.
2,829 downloads bioRxiv cell biology
Yes-associated Protein (YAP) is a transcriptional co-activator that regulates cell proliferation and survival by binding to a selective set of enhancers for potent target gene activation, but how YAP coordinates these transcriptional responses is unknown. Here, we demonstrate that YAP forms liquid-like condensates in the nucleus in response to macromolecular crowding. Formed within seconds of hyperosmotic stress, YAP condensates compartmentalized YAP’s DNA binding cofactor TEAD1 along with other YAP-related transcription co-activators, including TAZ, and subsequently induced transcription of YAP-specific proliferation genes. Super-resolution imaging using Assay for Transposase Accessible Chromatin with photoactivated localization microscopy (ATAC-PALM) revealed that YAP nuclear condensates were areas enriched in accessible chromatin domains organized as super-enhancers. Initially devoid of RNA Polymerase II (Pol II), the accessible chromatin domains later acquired Pol II, producing newly transcribed RNA. Removal of YAP’s intrinsically-disordered transcription activation domain (TAD) prevented YAP condensate formation and diminished downstream YAP signaling. Thus, dynamic changes in genome organization and gene activation during YAP reprogramming is mediated by liquid-liquid phase separation.
2,825 downloads bioRxiv cell biology
Andres M Cardozo Gizzi, Diego I. Cattoni, Jean-Bernard Fiche, Sergio Espinola, Julian Gurgo, Olivier Messina, Christophe Houbron, Yuki Ogiyama, Giorgio-Lucio Papadopoulos, Giacomo Cavalli, Mounia Lagha, Marcelo Nöllmann
Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here, we present a multiplexed, sequential imaging approach (Hi-M) that permits the simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome un-pairing during the awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understand the mechanisms and consequences of the 4D organization of the genome.
2,808 downloads bioRxiv cell biology
Adult tissue repair and regeneration require the activation of resident stem/progenitor cells that can self-renew and generate differentiated progeny. The regenerative capacity of skeletal muscle relies on muscle satellite cells (MuSCs) and their interplay with different cell types within the niche. Yet, our understanding of the cells that compose the skeletal muscle tissue is limited and molecular definitions of the principal cell types are lacking. Using a combined approach of single-cell RNA-sequencing and mass cytometry, we precisely mapped the different cell types in adult skeletal muscle tissue and highlighted previously overlooked populations. We identified known functional populations, characterized their gene signatures, and determined key markers. Among the ten main cell populations present in skeletal muscle, we found an unexpected complexity in the interstitial compartment and identified two new cell populations. One express the transcription factor Scleraxis and generate tenocyte-like cells. The second express smooth muscle and mesenchymal cell markers (SMMCs). While distinct from MuSCs, SMMCs are endowed with myogenic potential and promote MuSC engraftment following transplantation. Our high-dimensional single-cell atlas uncovers principles of an adult tissue composition and can be exploited to reveal unknown cellular sub-fractions that contribute to tissue regeneration.
2,802 downloads bioRxiv cell biology
Sandra Ruiz Garcia, Marie Deprez, Kevin Lebrigand, Agnès Paquet, Amélie Cavard, Marie-Jeanne Arguel, Virginie Magnone, Ignacio Caballero, Sylvie Leroy, Charles-Hugo Marquette, Brice Marcet, Pascal Barbry, Laure-Emmanuelle Zaragosi
Background: It is usually considered that the upper airway epithelium is composed of multiciliated, goblet, secretory and basal cells, which collectively constitute an efficient first line of defense against inhalation of noxious substances. Upon injury, regeneration of this epithelium through proliferation and differentiation can restore a proper mucociliary function. However, in chronic airway diseases, the injured epithelium frequently displays defective repair leading to tissue remodeling, characterized by a loss of multiciliated cells and mucus hyper-secretion. Delineating drivers of differentiation dynamics and cell fate in the human airway epithelium is important to preserve homeostasis. Results: We have used single cell transcriptomics to characterize the sequence of cellular and molecular processes taking place during human airway epithelium regeneration. We have characterized airway subpopulations with high resolution and lineage inference algorithms have unraveled cell trajectories from basal to luminal cells, providing markers for specific cell populations, such as deuterosomal cells, i.e. precursors of multiciliated cells. We report that goblet cells, like secretory cells, can act as precursors of multiciliated cells. Our study provides a repertoire of molecules involved in key steps of the regeneration process, either keratins or components of the Notch, Wnt or BMP/TGFbeta signaling pathways. Our findings were confirmed in independent experiments performed on fresh human and pig airway samples, and on mouse tracheal epithelial cells. Conclusions: Our single-cell RNA-seq study provides novel insights about airway epithelium differentiation dynamics, clarifies cell trajectories between secretory, goblet and multiciliated cells, identifies novel cell subpopulations, and maps the activation and repression of key signaling pathways.
2,800 downloads bioRxiv cell biology
The accurate timing and execution of organelle biogenesis is crucial for cell physiology. Centriole biogenesis is regulated by Polo-like kinase 4 (Plk4) and initiates in S-phase when a daughter centriole grows from the side of a pre-existing mother. Here we show that Plk4 forms an adaptive oscillator at the base of the growing centriole to initiate and time centriole biogenesis, ensuring that centrioles grow at the right time and to the right size. The Plk4 oscillator is normally entrained to the cell-cycle oscillator, but can run autonomously of it - explaining how centrioles can duplicate independently of cell cycle progression under certain conditions. Mathematical modelling indicates that this autonomously oscillating system is generated by a time-delayed negative-feedback loop in which Plk4 inactivates its centriolar receptor through multiple rounds of phosphorylation. We postulate that such organelle-specific autonomous oscillators could regulate the timing and execution of organelle biogenesis more generally.
2,638 downloads bioRxiv cell biology
Diana Mahdessian, Anthony J. Cesnik, Christian Gnann, Frida Danielsson, Lovisa Stenström, Muhammad Arif, Cheng Zhang, Rutger Shutten, Anna Bäckström, Peter Thul, Nathan H Cho, Oana Carja, Mathias Uhlén, Adil Mardinoglu, Charlotte Stadler, Cecilia Lindskog, Burcu Ayoglu, Manuel D. Leonetti, Fredrik Pontén, Devin Sullivan, Emma Lundberg
Cellular division is a fundamental source of cell-to-cell variability, and studies of transcript and protein abundances have revealed several hundred genes that are regulated by the cell cycle–. However, none of these studies provide single-cell resolution of protein expression, leaving an incomplete understanding of cell-to-cell heterogeneity and the roles of cycling transcripts and proteins. Here, we present the first comprehensive map of spatiotemporal heterogeneity of the human proteome by integrating proteomics at subcellular resolution, single-cell transcriptomics, and pseudotime measurements of individual cells within the cell cycle. We identify that 17% of the human proteome displays cell-to-cell variability, of which 26% is correlated to cell cycle progression, and we present the first evidence of cell cycle association for 235 proteins. Only 15% of proteomic cell cycle regulation is due to transcriptomic cycling, which points to other means of regulation such as post-translational modifications. For proteins regulated at the transcript level, we observe a 7.7 hour delay between peak expression of transcript and protein on average. This spatially resolved proteomic map of the cell cycle has been integrated into the Human Protein Atlas and serves as a valuable resource for accelerating molecular studies of the human cell cycle and cell proliferation. : #ref-1 : #ref-8
2,621 downloads bioRxiv cell biology
Immunofluorescence (IF) imaging using antibodies to visualize specific biomolecules is a widely used technique in both biological and clinical laboratories. Standard IF imaging methods using primary antibodies followed by secondary antibodies have low multiplexing capability due to limited availability of primary antibodies raised in different animal species. Here, we used a DNA-based signal amplification method, Hybridization Chain Reaction (HCR), to replace secondary antibodies to achieve multiplexed imaging using primary antibodies of the same species with superior signal intensity. To enable imaging with DNA-conjugated antibodies, we developed a new antibody staining protocol to minimize nonspecific binding of antibodies caused by conjugated DNA oligonucleotides. We also expanded the HCR hairpin pool from previously published 5 to 13 for highly multiplexed in situ imaging. We finally demonstrated multiplexed in situ protein imaging using the technique in both cultured cells and mouse retina sections. ### Competing Interest Statement G.M.C. is a cofounder of Readcoor, Inc. and P.Y. is a cofounder of Utivue, Inc.
2,616 downloads bioRxiv cell biology
Tapash Jay Sarkar, Marco Quarta, Shravani Mukherjee, Alex Colville, Patrick Paine, Linda Doan, Christopher M. Tran, Constance R. Chu, Steve Horvath, Nidhi Bhutani, Thomas A. Rando, Vittorio Sebastiano
Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging is associated with the progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. The technology of nuclear reprogramming to pluripotency, through over-expression of a small number of transcription factors, can revert both the age and the identity of any cell to that of an embryonic cell by driving epigenetic reprogramming. Recent evidence has shown that transient transgenic reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of epigenetic rejuvenation would apply to physiologically aged cells and, importantly, how it might translate to human cells. Here we show that transient reprogramming, mediated by transient expression of mRNAs, promotes a rapid reversal of both cellular aging and of epigenetic clock in human fibroblasts and endothelial cells, reduces the inflammatory profile in human chondrocytes, and restores youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity. Our method, that we named Epigenetic Reprogramming of Aging (ERA), paves the way to a novel, potentially translatable strategy for ex vivo cell rejuvenation treatment. In addition, ERA holds promise for in vivo tissue rejuvenation therapies to reverse the physiological manifestations of aging and the risk for the development of age-related diseases.
2,613 downloads bioRxiv cell biology
The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy. We developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of previously undetected sub-organellar actin dynamics with high spatiotemporal resolution. These probes reveal that ER-associated actin drives fission of multiple organelles including mitochondria, endosomes, lysosomes, peroxisomes, and the Golgi. ![Figure]</img> : pending:yes
2,574 downloads bioRxiv cell biology
A key limitations to understand kidney function and disease development has been that specific cell types responsible for specific homeostatic kidney function or disease phenotypes have not been defined at the molecular level. To fill this gap, we characterized 57,979 cells from healthy mouse kidneys using unbiased single-cell RNA sequencing. We show that genetic mutations that present with similar phenotypes mostly affect genes that are expressed in a single unique differentiated cell type. On the other hand, we found unexpected cell plasticity of epithelial cells in the final segment of the kidney (collecting duct) that is responsible for final composition of the urine. Using computational cell trajectory analysis and in vivo linage tracing, we found that, intercalated cells (that secrete protons) and principal cells (that maintain salt, water and potassium balance) undergo a Notch mediated interconversion via a newly identified transitional cell type. In disease states this transition is shifted towards the principal cell fate. Loss of intercalated cells likely contributes to metabolic acidosis observed in kidney disease. In summary, single cell analysis advanced a mechanistic description of kidney diseases by identifying a defective homeostatic cell lineage.
2,548 downloads bioRxiv cell biology
Frederik J Verweij, Celine Revenu, Guillaume Arras, Florent Dingli, Damarys Loew, Gautier Follain, Guillaume Allio, Jacky G. Goetz, Jeroen den Hertog, Filippo Del Bene, Graca Raposo, Guillaume Van Niel
Extracellular vesicles (EVs) are released by most cell types but the definitive demonstration of their functional relevance remains challenging due to the lack of appropriate model organisms. Here we developed an in vivo model to study EV physiology by expressing CD63-pHluorin in zebrafish embryos. A combination of microscopy techniques and proteomic analysis allowed us to study the biogenesis, composition, transfer, uptake and fate of individual endogenous EVs in vivo. We identified an exosome population released in a syntenin-dependent manner from the Yolk Syncytial Layer into the blood circulation. These exosomes were specifically captured, endocytosed and degraded by patrolling macrophages and endothelial cells in the Caudal Vein Plexus (CVP) in a scavenger receptor and dynamin-dependent manner. Interference with exosome secretion affected CVP growth, supporting their trophic role. Altogether, our work provides a unique model to track in vivo inter-organ communication by endogenous exosomes at individual vesicle level and high spatio-temporal accuracy.
2,542 downloads bioRxiv cell biology
We present here an improved protocol for expansion microscopy, which increases the expansion factor from ~4x to ~10x. This protocol, which we termed X10 microscopy, achieves multi-colour imaging at a resolution of at least 30 nm, using off-the-shelf reagents and conventional epifluorescence microscopes. This enables a level of imaging detail rivalling that of much more challenging methods, such as STED, STORM and iExM, in both cell cultures and tissues.
2,517 downloads bioRxiv cell biology
Eszter Posfai, John Paul Schell, Adrian Janiszewski, Isidora Rovic, Alexander Murray, Brian Bradshaw, Tine Pardon, Mouna El Bakkali, Irene Talon, Natalie De Geest, Pankaj Kumar, San Kit To, Sophie Petropoulos, Andrea Jurisicova, Vincent Pasque, Fredrik Lanner, Janet Rossant
Totipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.
2,501 downloads bioRxiv cell biology
Neurons are highly polarized cells with complex neurite morphology. Spatial organization and local translation of RNAs in dendrites and axons play an important role in many neuronal functions. Here we performed super-resolution spatial profiling of RNAs inside individual neurons at the genome scale using multiplexed error-robust fluorescence in situ hybridization (MERFISH), and mapped the spatial organization of up to ~4,200 RNA species (genes) across multiple length scales, ranging from sub-micrometer to millimeters. Our data generated a quantitative intra-neuronal atlas of RNAs with distinct transcriptome compositions in somata, dendrites, and axons, and revealed diverse sub-dendritic distribution patterns of RNAs. Moreover, our spatial analysis identified distinct groups of genes exhibiting specific spatial clustering of transcripts at the sub-micrometer scale that were dependent on protein synthesis and differentially dependent on synaptic activity. Overall, these data provide a rich resource for characterizing the subcellular organization of the transcriptome in neurons with high spatial resolution.
2,494 downloads bioRxiv cell biology
Non-random, dynamic three-dimensional organization of the nucleus is important for regulation of gene expression. Numerous studies using chromosome conformation capture strategies have uncovered ensemble organizational principles of individual chromosomes, including organization into active (A) and inactive (B) compartments. In addition, large inactive regions of the genome appear to be associated with the nuclear lamina, the so-called Lamina Associated Domains (LADs). However, the interrelationship between overall chromosome conformation and association of domains with the nuclear lamina remains unclear. In particular, the 3D organization of LADs within the context of the entire chromosome has not been investigated. In this study, we describe “chromosome conformation paints” to determine the relationship in situ between LAD and non-LAD regions of the genome in single cells. We find that LADs organize into constrained and compact regions at the nuclear lamina, and these findings are supported by an integrated analysis of both DamID and Hi-C data. Using a refined algorithm to identify active (A) and inactive (B) compartments from Hi-C data, we demonstrate that the LADs correspond to the B compartment. We demonstrate that in situ single cell chromosome organization is strikingly predicted by integrating both Hi-C and DamID data into a chromosome conformation model. In addition, using the chromosome conformation paints, we demonstrate that LAD (and B-compartment) organization is dependent upon both chromatin state and Lamin A/C. Finally, we demonstrate that small regions within LADs escape the repressive regime at the peripheral zone to interact with the A-compartment and are enriched for both transcription start sites (TSSs) and active enhancers.
2,476 downloads bioRxiv cell biology
Genetic knock-in using homology directed repair is an inefficient process, requiring selection of few modified cells and hindering its application to primary cells. Here we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% transfected cells with one single electroporation. The technique has proven effective in various cell types, can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor levels of a protein of interest by a luciferase assay in primary cells.
2,463 downloads bioRxiv cell biology
Fluorescence microscopy has enabled imaging of key subcellular structures in living cells; however, the use of fluorescent dyes and proteins is often expensive, time-consuming, and damaging to cells. Here, we present a tool for the prediction of fluorescently labeled structures in live cells solely from 3D brightfield microscopy images. We show the utility of this approach in predicting several structures of interest from the same static 3D brightfield image, and show that the same tool can prospectively be used to predict the spatiotemporal position of these structures from a bright-field time series. This approach could also be useful in a variety of application areas, such as cross-modal image registration, quantification of live cell imaging, and determination of cell state changes.
2,463 downloads bioRxiv cell biology
Lanlan Zhou, Kelsey Huntington, Shengliang Zhang, Lindsey Carlsen, Eui-Young So, Cassandra Parker, Ilyas Sahin, Howard Safran, Suchitra Kamle, Chang-Min Lee, Chun Geun Lee, Jack A. Elias, Kerry S. Campbell, Mandar T. Naik, Walter J. Atwood, Emile Youssef, Jonathan A. Pachter, Arunasalam Navaraj, Attila A. Seyhan, Olin Liang, Wafik S. El-Deiry
COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. In serum-deprived and stimulated cells treated with remdesivir and MEKi we observed correlations between pRB, pERK, and ACE2 expression further supporting role of proliferative state and MAPK pathway in ACE2 regulation. We show elevated cytokines in COVID-19-(+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1α, IL-6 and MCP-1 are suppressed by MEKi alone or with remdesivir. We observed MEKi stimulation of NK-cell killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. Pseudotyped SARS-CoV-2 virus with a lentiviral core and SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope infected human bronchial epithelial cells, small airway epithelial cells, or lung cancer cells and MEKi suppressed infectivity of the pseudovirus. We show a drug class-effect with MEKi to stimulate NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells. MEKi may attenuate SARS-CoV-2 infection to allow immune responses and antiviral agents to control disease progression. ### Competing Interest Statement E.Y. and J.A.P. are employees and stockholders of Verastem Oncology. None of the other co-authors have relevant disclosures for this work.
2,459 downloads bioRxiv cell biology
Selective degradation of organelles via autophagy is critical for cellular differentiation, homeostasis, and organismal health. Autophagy of the ER (ER-phagy) is implicated in human neuropathy but is poorly understood beyond a few specialized autophagosomal receptors and remodelers. Using an ER-phagy reporter and genome-wide CRISPRi screening, we identified 200 high-confidence factors involved in human ER-phagy. We mechanistically investigated two pathways unexpectedly required for ER-phagy. First, reduced mitochondrial metabolism represses ER-phagy, which reverses the logic of general autophagy. Mitochondrial crosstalk with ER-phagy bypasses the energy sensor AMPK, instead directly impacting ULK1. Second, ER-localized UFMylation is required for ER-phagy that represses the unfolded protein response. The UFL1 ligase is brought to the ER surface by DDRGK1, analogous to PINK1-Parkin regulation during mitophagy. Our data provide insight into the unique cellular logic of ER-phagy, reveal parallels between organelle autophagies, and provide an entry point to the relatively unexplored process of degrading the ER network.
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