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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2021

A genome-wide genetic screen uncovers novel determinants of human pigmentation

The skin color is one of the most diverse human traits and is determined by the quantity, type and distribution of melanin. Here, we leverage light scattering properties of melanin to conduct a genome-wide CRISPR-Cas9 screen for novel regulators of melanogenesis. We identify functionally diverse genes converging on melanosome biogenesis, endosomal transport and transcriptional/posttranscriptional gene regulation, most of which represent novel associations with pigmentation. A survey of transcriptomes from diversely pigmented individuals reveals that the majority of genes discovered in our screen are upregulated in dark skin melanocytes, in agreement with their melanin-promoting function and potential contribution to skin color variation. This association is further buttressed by the significant skin color heritability enrichment in the vicinity of these genes. Taken together, our study presents a novel approach to assay pigmentation and uncovers a plethora of melanogenesis regulators, with broad implications for human variation, cell biology and medicine. One Sentence SummaryGenetic screen uncovers genes involved in human melanogenesis, many of which are differentially expressed in individuals of diverse skin color.

cell biology↗

bioRxiv2018

Identifying the genetic basis of variation in cell behaviour in human iPS cell lines from healthy donors

Large cohorts of human iPSCs from healthy donors are potentially a powerful tool for investigating the relationship between genetic variants and cellular phenotypes. Here we integrate high content imaging, gene expression and DNA sequence datasets for over 100 human iPSC lines to identify the genetic basis of inter-individual variability in cell behaviour. By applying a dimensionality reduction approach, Probabilistic Estimation of Expression Residuals (PEER), we identified genes that correlated in expression with intrinsic (genetic) and extrinsic (ECM) factors. However, variation in mRNA levels could not account for outlier cell behaviour. Instead, we identified rare, deleterious SNVs in the coding sequence of genes involved in ECM adhesion that occurred in cell lines that were outliers for one or more phenotypes such as cell spreading. These also correlated with altered germ layer differentiation on micropatterned surfaces. Our study thus establishes a strategy for integrating genetic and cell biological measurements for high-throughput analysis.

cell biology↗

bioRxiv2022

Integrated optical device for Structured Illumination Microscopy

Structured Illumination Microscopy (SIM) is a key technology for high resolution and super-resolution imaging of biological cells and molecules. The spread of portable and easy-to-align SIM systems requires the development of novel methods to generate a light pattern and to shift it across the field of view of the microscope. Here we show a miniaturized chip that incorporates optical waveguides, splitters, and phase shifters, to generate a 2D structured illumination pattern suitable for SIM microscopy. The chip creates three point-sources, coherent and controlled in phase, without the need for further alignment. Placed in the pupil of a microscopes objective, the three sources generate a hexagonal illumination pattern on the sample, which is spatially translated thanks to thermal phase shifters. We validate and use the chip, upgrading a commercial inverted fluorescence microscope to a SIM setup and we image biological sample slides, extending the resolution of the microscope.

cell biology↗

bioRxiv2022

Growth cone advance requires EB1 as revealed by genomic replacement with a light-sensitive variant

A challenge in analyzing dynamic intracellular cell biological processes is the dearth of methodologies that are sufficiently fast and specific to perturb intracellular protein activities. We previously developed a light-sensitive variant of the microtubule plus end tracking protein EB1 by inserting a blue light-controlled protein dimerization module between functional domains. Here we describe an advanced method to replace endogenous EB1 with this light-sensitive variant in a single genome editing step enabling this approach in human induced pluripotent stem cells and derived neurons. We demonstrate that acute and local optogenetic EB1 inactivation in developing cortical neurons induces microtubule depolymerization in the growth cone periphery and subsequent neurite retraction. In addition, advancing growth cones are repelled from areas of blue light exposure. These phenotypes were independent of the neuronal EB1 homolog EB3, revealing a direct dynamic role of EB1-mediated microtubule plus end interactions in neuron morphogenesis and neurite guidance.

cell biology↗

bioRxiv2019

Multiplexed electron microscopy by fluorescent barcoding allows screening for ultrastructural phenotype

Genetic screens performed using high-throughput fluorescent microscopes have generated large datasets that have contributed many insights into cell biology. However, such approaches typically cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) is not subject to this resolution limit, generating detailed images of cellular ultrastructure, but requires time consuming preparation of individual samples, limiting its throughput. Here we overcome this obstacle and describe a robust method for screening by high-throughput electron microscopy. Our approach uses combinations of fluorophores as barcodes to mark the genotype of each cell in mixed populations, and correlative light and electron microscopy to read the fluorescent barcode of each cell before it is imaged by electron microscopy. Coupled with an easy-to-use software workflow for correlation, segmentation and computer image analysis, our method allows to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate the method on several organelles with samples that each contain up to 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher-throughput, and can be utilized in multiple ways to enable electron microscopy to become a powerful screening methodology.

cell biology↗

bioRxiv2026

Mesenchymal stroma cell-derived stem cell factor mediates cross-species compatibility of the hematopoietic stem cell niche.

The regulation of human hematopoietic stem cell (HSC) function within its native bone marrow microenvironment remains poorly understood due to the limitations of existing humanized models. Here, we present a non-conditioned human HSC xenotransplantation platform that serves as a physiologically relevant in vivo surrogate to study these complex cellular interactions. Using this system, we uncover a dynamic, cross-species communication between human HSCs and the murine niche, revealing a profound cellular and molecular plasticity of the bone marrow microenvironment in response to humanization. Upon engraftment, platelet-derived growth factor receptor alpha positive (Pdgfra+) mesenchymal stromal cells (MSCs) undergo significant numerical expansion and a major transcriptional shift, transitioning from a mixed adipo- and osteo-primed state toward a leptin receptor positive (Lepr+) predominantly adipo-primed, hematopoiesis-supporting signature. Functional validation demonstrates that this niche plasticity is a critical determinant of stem cell engraftment: the targeted depletion of Lepr+ MSCs or the genetic deletion of stem cell factor results in the rapid mobilization and lack of engraftment of human HSCs, respectively. Our findings establish niche plasticity as a primary regulator of human HSC function and demonstrate the utility of this cross-species platform as a modular genetic toolbox for precise in vivo engineering of the bone marrow microenvironment. Ultimately, this adaptable system provides a powerful framework for elucidating human stem cell biology and advancing bone marrow transplantation therapies.

cell biology↗

bioRxiv2026

Systematic real-time profiling of Salmonella type III effector translocation provides quantitative resolution of the T3SS-1/T3SS-2 secretion dichotomy

Type III secretion systems (T3SSs) enable Salmonella to modulate host cell biology by delivering a diverse repertoire of effector proteins. Yet, the temporal dynamics of effector translocation during prolonged infection remain poorly defined. Here, we establish a systematic real-time framework for quantifying effector secretion and translocation by combining endogenous HiBiT-tagging with split NanoLuc-based detection. Using this approach, we monitored the translocation kinetics of all 39 currently annotated type III effectors (T3Es) in Salmonella enterica serovar Typhimurium SL1344 over a 24-hour infection of epithelial cells. This comprehensive analysis revealed temporally distinct translocation patterns, widespread mid- and late-stage effector delivery, and a quantitatively resolved functional overlap between T3SS-1 and T3SS-2-mediated secretion than previously appreciated. These findings provide a more refined view of effector deployment throughout infection and illuminate previously overlooked aspects of effector dynamics. Altogether, this work offers a detailed temporal map of effector dynamics during epithelial infection and establishes a scalable strategy for dissecting secretion system function across host-pathogen systems.

cell biology↗

bioRxiv2022

Proteostatic regulation of caveolins avoids premature oligomerisation and preserves ER homeostasis

Caveolin-1 (CAV1) and CAV3 are membrane sculpting proteins driving formation of plasma membrane caveolae. Caveola formation is unique as it requires oligomerisation of newly synthesised caveolins through the biosynthetic-secretory pathway. Here, we combine structural, biochemical, and microscopy analyses to examine the early proteostasis of caveolin family members and mutants. We describe striking trafficking differences between newly synthesised caveolins, with CAV1 rapidly exported to the Golgi but CAV3 showing ER retention and targeting to lipid droplets. Only monomeric/low oligomeric caveolins are efficiently exported from the ER, with oligomers assembling beyond the cis-Golgi and disease-causing mutations leading to detrimental non-functional complexes. Caveolins in the ER are maintained at low levels by active proteasomal degradation, avoiding premature oligomerisation and ER stress. Increasing lipid availability, cholesterol for CAV1 and fatty acids for CAV3, enhances trafficking and reduces proteasomal degradation. In conclusion, we identify proteostatic mechanisms that modulate stability and trafficking of newly synthesised caveolins, protecting cells against ER stress but perturbed in caveolin-related disease. SummaryUnderstanding the unique proteostasis of caveolins has important implications for cell biology and physiopathology. Combining structural, microscopy, and biochemical analyses, we uncover new insights into the mechanisms that differentiate the early biosynthetic steps of caveolin family members, isoforms, and pathogenic mutants.

cell biology↗

bioRxiv2023

Localization of PPM1H phosphatase tunes Parkinsons disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis

PPM1H phosphatase reverses Parkinsons disease-associated, LRRK2-mediated Rab GTPase phosphorylation. We show here that PPM1H relies on an N-terminal amphipathic helix for Golgi localization. The amphipathic helix enables PPM1H to bind to liposomes in vitro, and small, highly curved liposomes stimulate PPM1H activity. We artificially anchored PPM1H to the Golgi, mitochondria, or mother centriole. Our data show that regulation of Rab10 GTPase phosphorylation requires PPM1H access to Rab10 at or near the mother centriole. Moreover, poor co-localization of Rab12 explains in part why it is a poor substrate for PPM1H in cells but not in vitro. These data support a model in which localization drives PPM1H substrate selection and centriolar PPM1H is critical for regulation of Rab GTPase-regulated ciliogenesis. Moreover, Golgi localized PPM1H maintains active Rab GTPases on the Golgi to carry out their non-ciliogenesis-related functions in membrane trafficking. Significance StatementPathogenic, hyperactive LRRK2 kinase is strongly linked to Parkinsons disease and LRRK2 phosphorylates a subset of Rab GTPases that are master regulators of membrane trafficking. PPM1H phosphatase specifically dephosphorylates Rab8A and Rab10, the major LRRK2 substrates. Here we provide novel cell biological and biochemical insight related to the localization and activation of PPM1H phosphatase. Understanding how PPM1H modulates LRRK2 activity is of fundamental interest and also important, as activators of PPM1H may eventually benefit Parkinsons disease patients.

cell biology↗

bioRxiv2019

Optimising DNA-Coated Gold Nanoparticles to Enrich Skeletal Stem Cells from Human Bone Marrow Samples

Human bone marrow (BM) derived stromal cells contain a population of skeletal stem cells (SSCs), with the capacity to differentiate along the osteogenic, adipogenic and chondrogenic lineages enabling their application to clinical therapies. However, current methods, to isolate and enrich SSCs from human tissues remain, at best, challenging in the absence of a specific SSC marker. Unfortunately, none of the current proposed markers, alone, can isolate a homogenous cell population with the ability to form bone, cartilage, and adipose tissue in humans. Here, we have designed DNA-gold nanoparticles able to identify and sort SSCs displaying specific mRNA signatures. The current approach demonstrates the significant enrichment attained in the isolation of SSCs, with potential therein to enhance our understanding of bone cell biology and translational applications. TABLE OF CONTENTS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/882563v3_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@48c5acorg.highwire.dtl.DTLVardef@1a9d096org.highwire.dtl.DTLVardef@1bd4da2org.highwire.dtl.DTLVardef@133dd1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Naked mole-rat hematopoietic stem and progenitors are highly quiescent with an inherent myeloid bias

Naked mole-rats (NMRs) are the longest-lived rodents yet their stem cell characteristics remain enigmatic. Here we comprehensively mapped the NMR hematopoietic landscape and identified unique features likely contributing to longevity. Adult NMRs form red blood cells in spleen and marrow, which is a neotenic trait. A myeloid bias towards granulopoiesis in concert with decreased B-lymphopoiesis defines the marrow composition, resembling fetal leukopoiesis. Very similar to primates, the primitive stem cell compartment is marked by CD34 and THY1. Remarkably, stem and progenitor respiration rates are as low as in human cells, while NMR cells show a strong expression signature for fatty acid metabolism. The pool of quiescent stem cells is higher than in mice, and the cell cycle of hematopoietic cells is prolonged. Our work provides a platform to study immunology and stem cell biology in an animal model of exceptional longevity. TeaserJuvenile features of hematopoiesis shape the blood system of the longest-lived rodent.

cell biology↗

bioRxiv2026

The Unexpected Visibility of the SARS-CoV-2 Nucleocapsid Protein Reveals a Hidden Route of Surface Trafficking.

The SARS-CoV-2 Nucleocapsid (N) protein, long regarded as an internal structural component of the virion, unexpectedly localizes to the plasma membrane of infected cells. Here, we show that N is actively trafficked to the cell surface via a ceramide-dependent unconventional secretory pathway. Live-stained imaging and kinetic analyses revealed that N surface association begins early in infection, before Spike (S) expression and viral release, and persists after enzymatic removal of heparan sulfate. Pharmacological disruption of phosphoinositide or phosphatidylserine interactions had minimal effect, whereas inhibition of neutral sphingomyelinase with GW4869 markedly reduced surface N, identifying a ceramide-regulated route as essential for its export. This mechanism distinguishes N from canonical transmembrane viral proteins and explains how N-specific antibodies mediate potent Fc-effector responses across SARS-CoV-2 variants. Our findings redefine the spatiotemporal dynamics of coronavirus structural proteins and reveal an unanticipated axis of immune visibility within the infected cell. IMPORTANCEInternal viral proteins are generally thought to remain confined to intracellular compartments, yet several viruses display such proteins at the surface of infected cells through mechanisms that remain incompletely defined. In this study, we characterize a host-regulated trafficking route that contributes to the delivery of the SARS-CoV-2 nucleocapsid (N) protein, a non-membrane viral protein, to the plasma membrane. Our findings indicate that N protein surface expression occurs independently of virion assembly, membrane integration, or extracellular rebinding, and instead involves host vesicular processes outside the classical secretory pathway. By elucidating how a leaderless coronavirus protein can access the cell surface, this work addresses a key gap in coronavirus cell biology and provides a framework for understanding how internal viral proteins may exploit host trafficking pathways. More broadly, these results highlight unconventional host trafficking pathways as determinants of viral protein localization and provide a framework for understanding how viruses exploit cellular export mechanisms beyond the classical secretory system.

cell biology↗

bioRxiv2020

Single-cell RNA sequencing deconvolutes the in vivo heterogeneity of human bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells, which have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat found in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte cells that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271+ BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPRhiCD45low BM-MSCs within the CD271+ BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of transcriptomes suggest that osteoblast precursors may induce angiogenesis coupled with osteogenesis, and chondrocyte precursors may have the potential to differentiate into myocytes. We discovered transcripts for several cluster of differentiation (CD) markers that were highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs and could be novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing insight into the extent of their cellular heterogeneity and bone homeostasis.

cell biology↗

bioRxiv2025

Visualizing sub-organellar lipid distribution using correlative light and electron microscopy

Lipids and proteins compartmentalize biological membranes into nanoscale domains which are crucial for signaling, intracellular trafficking and many other cellular processes. Studying nanodomain function requires the ability to measure protein and lipid localization at the nanoscale. Current methods for visualizing lipid localization do not meet this requirement. Here, we introduce a correlative light and electron microscopy workflow to image lipids (Lipid-CLEM), combining near-native lipid probes and on-section labeling by click chemistry. This approach enables the quantification of relative lipid densities in membrane nanodomains. We find differential partitioning of sphingomyelin into intraluminal vesicles, recycling tubules, and the boundary membrane of the early endosome, representing a degree of nanoscale organization previously observed only for proteins. We anticipate that our Lipid-CLEM workflow will greatly facilitate the mechanistic analysis of lipid functions in cell biology, allowing for the simultaneous investigation of proteins and lipids during membrane nanodomain assembly and function.

cell biology↗

bioRxiv2025

STAR/STARD1: a mitochondrial intermembrane space cholesterol shuttle degraded through mitophagy

The import of cholesterol to the inner mitochondrial membrane by the steroidogenic acute regulatory protein (STAR/STARD1) is essential for de novo steroid hormone biosynthesis and the acidic pathway of bile acid synthesis. This robust system, evolved to start and stop colossal cholesterol movement, ensures pulsatile yet swift mitochondrial steroid metabolism in cells. Nonetheless, the proposed mechanism and components involved in this process has remained a topic of ongoing debate. In this study, we elucidate the mitochondrial import machinery and structural aspects of STAR, revealing its role as an intermembrane space cholesterol shuttle that subsequently undergoes rapid degradation by mitophagy. This newfound mechanism illuminates a fundamental process in cell biology and provides precise interpretations for the full range of human STAR mutation-driven lipoid congenital adrenal hyperplasia in patients. One Sentence SummarySTAR activates mitochondrial steroid metabolism as a cholesterol shuttle in the intermembrane space and is destroyed by mitophagy.

cell biology↗

bioRxiv2019

Long-term in vivo imaging of Drosophila larvae

The Drosophila larva has been used to investigate many processes in cell biology, including morphogenesis, physiology, responses to drugs and new therapeutic compounds. Despite its enormous potential as a model system, it has technical limitations in cases where longer-term live imaging is necessary, because of the lack of efficient methods for immobilising larvae for extended periods. We describe here a simple procedure for anaesthetisation and long-term in vivo imaging of the epidermis and other larval organs including gut, imaginal discs, neurons, fat body, tracheae and haemocytes, and show a procedure for probing cell properties by laser ablation. We include a survey of different anaesthetics, showing that short exposure to diethyl ether is the most effective for long-term immobilisation of larvae. This method does not require specific expertise beyond basic Drosophila genetics and husbandry, and confocal microscopy. It enables high-resolution studies of many systemic and sub-cellular processes in larvae.

cell biology↗

bioRxiv2020

Optimized iLID membrane anchors for local optogenetic protein recruitment

Optogenetic protein dimerization systems are powerful tools to investigate the biochemical networks that cells use to make decisions and coordinate their activities. These tools, including the improved Light-Inducible Dimer (iLID) system, offer the ability to selectively recruit components to subcellular locations, such as micron-scale regions of the plasma membrane. In this way, the role of individual proteins within signaling networks can be examined with high spatiotemporal resolution. Currently, consistent recruitment is limited by heterogeneous optogenetic component expression, and spatial precision is diminished by protein diffusion, especially over long timescales. Here, we address these challenges within the iLID system with alternative membrane anchoring domains and fusion configurations. Using live cell imaging and mathematical modeling, we demonstrate that the anchoring strategy affects both component expression and diffusion, which in turn impact recruitment strength, kinetics, and spatial dynamics. Compared to the commonly used C-terminal iLID fusion, fusion proteins with large N-terminal anchors show stronger local recruitment, slower diffusion of recruited components, and efficient recruitment over wider gene expression ranges. We also define guidelines for component expression regimes for optimal recruitment for both cell-wide and subcellular recruitment strategies. Our findings highlight key sources of imprecision within light-inducible dimer systems and provide tools that allow greater control of subcellular protein localization across diverse cell biological applications. SignificanceOptogenetic light-inducible dimer systems, such as iLID, offer the ability to examine cellular signaling networks on second timescales and micrometer spatial scales. Confined light stimulation can recruit proteins to subcellular regions of the plasma membrane, and local signaling effects can be observed. Here, we report alternative iLID fusion proteins that display stronger and more spatially confined membrane recruitment. We also define optogenetic component expression regimes for optimal recruitment and show that slow-diffusing iLID proteins allow more robust recruitment in cell populations with heterogenous expression. These tools should improve the spatiotemporal control and reproducibility of optogenetic protein recruitment to the plasma membrane.

cell biology↗

bioRxiv2025

Proximity interactomics identifies RAI14, EPHA2 and PHACTR4 as essential components of Wnt/planar cell polarity pathway in vertebrates

Wnt/planar cell polarity (Wnt/PCP) pathway is an evolutionarily conserved signaling cascade playing an inevitable role in cell biology. Deregulation of Wnt/PCP leads to severe developmental defects or cancer progression. Here, we applied proximity-dependent biotinylation (BioID) to capture the intracellular interactome of key Wnt/PCP components: transmembrane ROR1, ROR2 and VANGL2, and cytoplasmic DVL3 and PRICKLE1. Mapping of individual preys across the baits and subcellular compartments identified a group of 30 proteins that we tested by loss-of-function in zebrafish. Among those rai14, epha2 and phactr4 were essential for several Wnt/PCP-dependent processes such as zebrafish convergent extension, orientation of lateral organ cells, or migration of melanoma cells. Mechanistically, RAI14, EPHA2 and PHACTR4 connect the receptor complex to effector actomyosin. In summary, this study identified novel essential components of vertebrate WNT/PCP pathway and provides a detailed characterization of PCP complex composition that can serve as comprehensive resource for further analysis of Wnt/PCP function.

cell biology↗