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Results for “cell biology”

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

Hemodynamic forces can be accurately measured in vivo with optical tweezers

Force sensing and generation at the tissular and cellular scale is central to many biological events. There is a growing interest in modern cell biology for methods enabling force measurements in vivo. Optical trapping allows non-invasive probing of pico-Newton forces and thus emerged as a promising mean for assessing biomechanics in vivo. Nevertheless, the main obstacles rely in the accurate determination of the trap stiffness in heterogeneous living organisms, at any position where the trap is used. A proper calibration of the trap stiffness is thus required for performing accurate and reliable force measurements in vivo. Here, we introduce a method that overcomes these difficulties by accurately measuring hemodynamic profiles in order to calibrate the trap stiffness. Doing so, and using numerical methods to assess the accuracy of the experimental data, we measured flow profiles and drag forces imposed to trapped red blood cells of living zebrafish embryos. Using treatments enabling blood flow tuning, we demonstrated that such method is powerful in measuring hemodynamic forces in vivo with high accuracy and confidence. Altogether, this study demonstrates the power of optical tweezing in measuring low range hemodynamic forces in vivo and offers an unprecedented tool in both cell and developmental biology.

biophysics↗

bioRxiv2020

High-temperature live-cell imaging of cytokinesis, cell motility and cell-cell adhesion in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

Significant technical challenges have limited the study of extremophile cell biology. For example, the absence of methods for performing high-resolution, live-cell imaging at high temperatures (>50{degrees}C) has impeded the study of cell motility and cell division in thermophilic archaea such as model organisms from the genus Sulfolobus. Here we describe a system for imaging samples at 75{degrees}C using high numerical aperture, oil-immersion lenses. With this system we observed and quantified the dynamics of cell division in the model thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. In addition, we observed previously undescribed dynamic cell shape changes, cell motility, and cell-cell interactions, shedding significant new light on the high-temperature lifestyle of this organism.

cell biology↗

bioRxiv2025

The Kelch 3 motif on gigaxonin mediates the interaction with NUDCD3 and regulates vimentin filament morphology

Gigaxonin is an intermediate filament (IF)-interacting partner belonging to the Kelch-like (KLHL) protein family. Gigaxonin is encoded by the KLHL16 gene, which is mutated in Giant Axonal Neuropathy (GAN). The lack of functional gigaxonin in GAN patient cells impairs IF proteostasis, leading to focal abnormal accumulations of IFs and compromised neuronal function. We hypothesized that gigaxonin forms molecular interactions via specific sequence motifs to regulate IF proteostasis. The goal of this study was to examine how distinct Kelch motifs on gigaxonin regulate IF protein degradation and filament morphology. We analyzed vimentin IFs in HEK293 cells overexpressing wild type (WT) gigaxonin, or gigaxonin lacking each of the six individual Kelch motifs: K1 (aa274-326), K2 (aa327-374), K3 (aa376-421), K4 (aa422-468), K5 (aa470-522), and K6 (aa528-574). All six gigaxonin deletion mutants ({Delta}K1-{Delta}K6) promoted the degradation of soluble vimentin. The {Delta}K3 gigaxonin mutant exhibited soluble vimentin degradation and promoted the bundling of vimentin IFs relative to WT gigaxonin. Using mass spectrometry proteomic analysis we found that, relative to WT gigaxonin, {Delta}K3 gigaxonin had increased associations with ubiquitination-associated and mitochondrial proteins and lost the association with the NudC domain-containing protein 3 (NUDCD3), a molecular chaperone enriched in the nervous system. Collectively, our cell biological data show the induction of an abnormal GAN-like IF phenotype in cells expressing {Delta}K3-gigaxonin, while our mass spectrometry profiling links the loss of gigaxonin-NUDCD3 interactions with defective IF proteostasis, revealing NUDCD3 as a potential new target in GAN.

cell biology↗

bioRxiv2025

Offsetting ROS-mediated arrest of endothelial fenestration dynamics permits long-term optical super-resolution- and AFM-imaging

Advances in cell biology demand methods that resolve the structure and dynamics of subcellular organelles in living cells. Live-cell super-resolution fluorescence microscopy meets this need but is constrained by phototoxicity, which disturbs cellular function and biases interpretation. Liver sinusoidal endothelial cells (LSECs), with their physiologically critical and highly dynamic fenestrations, represent a particularly challenging model system. We show that photoactivation-generated reactive oxygen species (ROS) are the primary cause of fenestration arrest during fluorescence imaging. Using three-dimensional structured illumination microscopy (3D SR-SIM), we systematically evaluated fluorophores and ROS scavengers to optimize imaging conditions. A combination of BioTracker staining and CO2-independent medium supplemented with N-acetylcysteine (NAC) preserved fenestration dynamics without altering fenestration number or size. Complementary atomic force microscopy (AFM) confirmed ROS-dependent impairment of fenestration dynamics and revealed nanomechanical changes upon illumination. These findings establish the mechanism underlying imaging-induced artefacts in LSECs and provide a broadly applicable strategy to extend live-cell super-resolution microscopy.

cell biology↗

bioRxiv2026

Pancreatic Gαs ablation disrupts tissue architecture and YAP signaling and unveils a compensatory regenerative response

Diabetes mellitus is characterized by chronic hyperglycemia and loss of pancreatic {beta}-cell function and mass. Current therapies focus on {beta}-cell protection and regeneration, led by GLP-1 receptor agonists. The G protein -subunit (Gs) acts as a key signaling node downstream of numerous GPCRs, integrating diverse signals that impact {beta}-cell mass and function. Elucidating the integrative role of pancreatic Gs signaling is thus crucial for understanding {beta}-cell biology. Our map of the pancreatic Gs-coupled GPCR landscape reveals sophisticated, cell-type-specific networks, positioning Gs as a central hub for intra-pancreatic communication. Previous studies in mice with {beta}-cell-specific or whole-pancreatic Gs deletion demonstrated reduced {beta}-cell mass, impaired insulin secretion, and glucose intolerance. The stronger phenotype in the whole-pancreas model--marked by -cell expansion and abnormal distribution--points to a crucial role for Gs in differential control of postnatal - and {beta}-cell proliferation. Here, we analyze the organ-wide consequences of Gs deletion using pancreas-specific Gs knockout mice (PGsKO). Consistent with prior findings, PGsKO mice exhibit reduced weight gain from four weeks and severe diabetes due to decreased {beta}-cell mass and concomitant -cell expansion. Furthermore, Gs loss induces profound architectural and functional defects in the exocrine pancreas, linked to YAP reactivation in acinar cells. Importantly, we observed attempted {beta}-cell regeneration in PGsKO mice. Although insufficient to reverse diabetes, our results delineate the full pancreatic phenotype that may facilitate these regenerative efforts and suggest that strategically biasing GPCR signaling network away from Gs could be a viable strategy to promote {beta}-cell regeneration from other pancreatic cell types. ARTICLE HIGHLIGHTSO_LIGs is a central signaling hub that integrates diverse GPCR inputs across pancreatic cell types, yet its organ-wide role remained poorly defined. C_LIO_LIWe addressed how pancreas-wide Gs deletion disrupts both endocrine and exocrine compartments, and whether regenerative programs are engaged. C_LIO_LIGs loss caused severe diabetes through {beta}-cell loss and -cell expansion, induced profound exocrine defects with YAP reactivation, and triggered attempted {beta}-cell regeneration from ducts and potentially other cell types. C_LIO_LIOur findings suggest that strategically biasing GPCR signaling away from Gs could promote regeneration from non-{beta}-cell sources, offering new therapeutic avenues for diabetes. C_LI

cell biology↗

bioRxiv2026

Recipient-driven metabolic and epigenetic remodeling of donor hematopoietic cells after transplantation in Fanconi Anemia.

Background Haematopoietic cell transplantation (HCT) restores donor-derived haematopoiesis in Fanconi anaemia (FA) but leaves recipient stromal and other non-haematopoietic cells genetically uncorrected. We investigated to what extent the persistent FA recipient environment influences the metabolic, redox, inflammatory and epigenetic phenotype of donor-derived haematopoietic cells after HCT. Methods Peripheral blood and, when available, bone marrow samples were analysed from healthy donors, patients with FA before and after HCT, and severe aplastic anaemia (SAA) patients after HCT as transplant controls. Mitochondrial function, cellular energy status, oxidative damage, antioxidant responses and circulating cytokines were assessed. Healthy donor mononuclear cells were exposed to conditioned media from FANCA-deficient mesenchymal stromal cells or FA fibroblasts, with FANCA-corrected fibroblasts as controls. DNA methylation profiling, acute inflammatory stimulation and pharmacological targeting of inflammatory pathways or mitochondrial oxidative stress were also performed. Results Despite stable full donor chimerism, FA recipients retained a pro-inflammatory and pro-oxidative circulating environment after HCT that was not observed in SAA transplant controls. Donor-derived mononuclear cells exposed to this environment acquired a distinct dysfunctional phenotype characterized by impaired mitochondrial oxidative phosphorylation and energy homeostasis, compensatory antioxidant and stress-response activation, and persistent lipid and protein oxidation. These alterations remained detectable after 96 h of ex vivo culture, suggesting relatively stable cellular remodelling, and were accompanied by a DNA methylation profile that, although globally shifted toward the donor state, retained an intermediate FA-associated episignature. Mechanistically, conditioned media from FANCA-deficient stromal cells and FA fibroblasts reproduced mitochondrial and oxidative abnormalities in healthy donor cells, whereas FANCA-corrected fibroblasts did not. Pharmacological targeting of inflammatory pathways or mitochondrial oxidative stress improved energy homeostasis and reduced oxidative damage, supporting a contributory role for chronic inflammation and oxidative stress in the microenvironment-driven phenotype acquired by donor-derived cells. Conclusions HCT corrects FA haematopoiesis without fully normalising the recipient environment. Persistent exposure to the FA milieu promotes metabolic, redox, signalling and epigenetic remodelling of donor-derived haematopoietic cells after transplantation. These findings identify the recipient environment as a determinant of post-HCT cell biology and highlight chronic inflammation and oxidative stress as mechanisms contributing to donor-cell remodelling in FA.

cell biology↗

bioRxiv2022

An EpCAM/Trop2 mechanostat differentially regulates individual and collective migration of human carcinoma cells

EpCAM and its close relative Trop2 are well-known markers of carcinoma, but the potential role of these cell surface proteins in cancer metastasis remains unclear. They are known, however, to downregulate myosin-dependent contractility, a key parameter involved in cell adhesion and migration. We investigate here the morphogenetic impact of the high EpCAM and Trop2 levels typically found in epithelial breast cancer cells, using spheroids of MCF7 cells as an in vitro model. Intriguingly, EpCAM depletion stimulated spheroid cohesive spreading, while Trop2 depletion had the opposite effect. Combining cell biological and biophysical approaches, we demonstrate that while EpCAM and Trop2 both contribute to moderate cell contractility, their depletions differentially impact on the process of "wetting" a substrate, here both matrix and neighboring cells, by affecting the balance of cortical tension at cell and tissue interfaces. These distinct phenotypes can be explained by partial enrichment at specific interfaces. Differential distribution and antagonistic loss-of-function phenotypes are also observed in two other breast cancer cell lines with very different characteristics, suggesting that these are general properties of these two regulators. Our data are consistent with a simple model in which the EpCAM-Trop2 pair acts as a mechanostat that tunes local cortical tension, thus influencing a spectrum of adhesive and migratory behaviours.

cell biology↗

bioRxiv2022

Inferring cell diversity in single cell data using consortium-scale epigenetic data as a biological anchor for cell identity

Methods for cell clustering and gene expression from single-cell RNA sequencing (scRNA-seq) data are essential for biological interpretation of cell processes. Here we present TRIAGE-Cluster which uses genome-wide epigenetic data from diverse bio-samples to identify genes demarcating cell diversity in scRNA-seq data. TRIAGE-Cluster integrates patterns of repressive chromatin deposited across diverse cell types with weighted density estimation to determine cell type clusters in a 2D UMAP space. We then present TRIAGE-ParseR, a machine learning method that evaluates gene expression rank lists to define gene groups governing the identity and function of cell types. We demonstrate the utility of this two-step approach using atlases of in vivo and in vitro cell diversification and organogenesis. We also provide a web accessible dashboard for analysis and download of data and software. Collectively, genome-wide epigenetic repression provides a versatile strategy to define cell diversity and study gene regulation of scRNA-seq data.

bioinformatics↗

bioRxiv2023

Molecular Regulation of Invasive Protrusion Formation at the Mammalian Fusogenic Synapse

Invasive membrane protrusions play a central role in a variety of cellular processes. Unlike filopodia, invasive protrusions are mechanically stiff and propelled by branched actin polymerization. However, how branched actin filaments are organized to create finger-like invasive protrusions remains a longstanding question in cell biology. Here, by examining the mammalian fusogenic synapse, where invasive protrusions are generated to promote cell membrane juxtaposition and fusion, we have uncovered the mechanism underlying invasive protrusion formation. We show that two Arp2/3 nucleation promoting factors (NPFs), WAVE and N-WASP, exhibit distinct and complementary localization patterns in the protrusions. While WAVE is at the leading edge, N-WASP is recruited by its interacting protein, WIP, to the shaft of the protrusion. During protrusion growth, new branched actin filaments are polymerized at the periphery of the shaft and crosslinked to preexisting actin bundles by the "pioneer" actin-bundling protein dynamin. The thickened actin bundles are further stabilized by WIP, which functions as a WH2 domain-mediated actin-bundling protein. Disrupting any of these components results in defective protrusions and myoblast fusion in cultured cells and/or in mouse embryos. Thus, our study has revealed the intricate spatiotemporal coordination between two NPFs and two actin-bundling proteins in creating invasive protrusions and has general implications in understanding protrusion formation in many cellular processes beyond cell-cell fusion.

cell biology↗

bioRxiv2024

3D-Aligner: An advanced computational tool designed to correct image distortion in expansion microscopy for precise 3D reconstitution and quantitative analysis

Expansion Microscopy (ExM) is an innovative and cost-effective super-resolution microscopy technique that has become popular in cell biology research. It achieves super-resolution by physically expanding specimens. Since its introduction, ExM has undergone continuous methodological developments to enhance its resolution and labeling capabilities. However, ExM imaging often encounters sample drift during image acquisition due to the physical movement of the expanded hydrogel, posing a significant challenge for accurate image reconstruction. Despite many proposed experimental solutions to mitigate sample drift, a universal solution has yet to be established. In response to this challenge, we developed 3D-Aligner, an advanced and user-friendly image analysis tool designed to computationally correct drift in ExM images for precise three-dimensional image reconstruction and downstream quantification. We demonstrate that 3D-Aligner effectively determines and corrects drift in ExM images with different expansion rates and various fluorescently labeled biological targets, showcasing its capabilities and robustness in drift correction. Additionally, we validate the precision of 3D-Aligner by comparing drift values across different labeled targets and highlight the importance of drift correction in quantification of biological structures.

cell biology↗

bioRxiv2024

Model supports asymmetric regulation across the intercellular junction for collective cell polarization

Symmetry breaking, which is ubiquitous in biological cells, functionally enables directed cell movement and organized embryogenesis. Prior to movement, cells break symmetry to form a well-defined cell front and rear in a process called polarization. In developing and regenerating tissues, collective cell movement requires the coordination of the polarity of the migration machineries of neighboring cells. Though several works shed light on the molecular basis of polarity, fewer studies have focused on the regulation across the cell-cell junction required for collective polarization, thus limiting our ability to connect subcellular interactions to tissue-level dynamics. Here, we investigate how polarity signals are communicated from one cell to its neighbor to ensure coordinated front-to-rear symmetry breaking with the same orientation across the group. In a theoretical setting, we exhaustively search a variety of intercellular interactions and identify the conditions required for the Rho GTPase signaling module and/or cytoskeletal dynamics to achieve either co-alignment arrangement or supracellular arrangement of the polarity axes in a group of 2 and 4 cells. Our work shows that only asymmetric regulations are favorable - such interactions involve either up-regulation of the kinetic rate of complementary polarity components or opposite regulation of the kinetic rates of the same polarity components across the cell-cell junction. Surprisingly, our results hold if we further assume the presence of an external stimulus, intrinsic cellular variability, or larger group size. The results underline the potential of using quantitative models to probe the molecular interactions required for macroscopic biological phenomena. Lastly, we posit that asymmetric regulation is achieved through junction proteins and predict that in the absence of cytoplasmic tails of such linker proteins, the likeliness of doublet co-polarity is greatly diminished. Author summaryCells of the developing embryo undergo a highly complex chain of events that define their correct shape and positioning. Among these events, a crucial role belongs to coordinated cell movement of cells of different lineages over short and long distances to give rise to mature organs and organ systems. During collective movement, individual cells typically engage their autonomous polarity machinery, while being connected to their neighbors through adhesive cell-cell interactions. Despite advances in revealing the cell-cell interactions required for collective cell migration, a comprehensive picture of the molecular basis of intercellular communication for collective guidance is missing. To address this question, we devise a generalized mechanochemical model for cell polarity in a doublet and investigate how polarity signals are transmitted from one cell to another across seemingly symmetrical junctions. We have chosen to screen through all possible intercellular conditions of the Rho GTPase signaling circuit and/or cytoskeletal dynamics. Our systematic approach provides information on over 300 distinct conditions and reveals the intercellular regulation provided by junction proteins. In addition to predicting that only asymmetric interactions favor co-polarization, ensuring movement of the group in the same direction, our analysis also highlights the need for additional regulatory mechanisms for larger cell groups.

biophysics↗

bioRxiv2022

Efficient and rapid fluorescent protein knock-in with universal donors in mammalian stem cells

Fluorescent protein (FP) tagging is a foundational approach in cell biology because it allows observation of protein distribution, dynamics, and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been standard in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous fluorescent protein tagging has not been widely used in mammalian cells due to inefficient homology-directed repair (HDR). Here, we describe a streamlined method for efficient and fast integration of FP tags into native loci via non-homologous end joining (NHEJ) in mouse embryonic stem cells. Our protocol minimizes cloning with universal donors, allows for N or C-terminal tagging of endogenous proteins, and requires less than 2 weeks from transfection to imaging, thereby improving the applicability of FP knock-in in mammalian cells.

cell biology↗

bioRxiv2023

An integrated transcriptomic cell atlas of human endoderm-derived organoids

Human stem cells can generate complex, multicellular epithelial tissues of endodermal origin in vitro that recapitulate aspects of developing and adult human physiology. These tissues, also called organoids, can be derived from pluripotent stem cells or tissue-resident fetal and adult stem cells. However, it has remained difficult to understand the precision and accuracy of organoid cell states through comparison with primary counterparts, and to comprehensively assess the similarity and differences between organoid protocols. Advances in computational single-cell biology now allow the integration of datasets with high technical variability. Here, we integrate single-cell transcriptomes from 218 samples covering organoids of diverse endoderm-derived tissues including lung, pancreas, intestine, liver, biliary system, stomach, and prostate to establish an initial version of a human endoderm organoid cell atlas (HEOCA). The integration includes nearly one million cells across diverse conditions, data sources and protocols. We align and compare cell types and states between organoid models, and harmonize cell type annotations by mapping the atlas to primary tissue counterparts. To demonstrate utility of the atlas, we focus on intestine and lung, and clarify ontogenic cell states that can be modeled in vitro. We further provide examples of mapping novel data from new organoid protocols to expand the atlas, and showcase how integrating organoid models of disease into the HEOCA identifies altered cell proportions and states between healthy and disease conditions. The atlas makes diverse datasets centrally available, and will be valuable to assess organoid fidelity, characterize perturbed and diseased states, and streamline protocol development.

cell biology↗

bioRxiv2026

Magnetic nanobiopsy: A minimal technology for live cell biomolecular sampling

Cellular heterogeneity is a fundamental determinant of precision medicine, governing transcriptional plasticity, lineage commitment, and adaptive programs that drive disease progression and therapeutic resistance. However, most molecular interrogation technologies remain inherently destructive, relying on bulk measurements that average cellular signals and systematically obscure rare but functionally decisive subpopulations. As a result, current approaches provide only static snapshots of complex cellular systems, preventing longitudinal analyses of dynamic molecular states and masking the contributions of relapse-initiating, phenotypically plastic, or therapy-resistant cells that ultimately dictate population fate. Here, we present magnetic nanobiopsy, a simple and scalable platform for minimally invasive, repeatable intracellular molecular sampling from living cells. Magnetically actuated nanocomposites (200 nm-1.4 {micro}m) enable efficient intracellular access and stable biomolecular capture while preserving cellular structural and functional integrity. High-resolution imaging and biochemical analyses confirm robust internalization and anchoring of intracellular biomolecules, while flow cytometry demonstrates that the retrieved cargo remains cell-specific and quantitatively representative of the parental heterogeneous population. By enabling longitudinal, single-cell-resolved molecular profiling within intact living populations, magnetic nanobiopsy bridges the gap between static bulk analyses and technically complex single-cell methods. This platform establishes a new framework for real-time investigation of cellular heterogeneity, adaptive responses, lineage diversification, and transient cell-state transitions, with broad applicability in cell biology, oncology, and biobanking. HighlightsO_LIMagnetic nanobiopsy enables non-destructive, longitudinal molecular sampling of living cell populations with high throughput and minimal operational complexity. C_LIO_LIThe nanoprobes bypass endo-lysosomal sequestration, directly interfacing with the cytosol to capture representative protein and RNA cargo via a membrane-preserving budding exit. C_LIO_LIQuantitative validation confirms that the retrieved molecular profiles faithfully mirror the heterogeneity and relative abundance of the parental cell population. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/705970v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1e0bee0org.highwire.dtl.DTLVardef@13a5b6org.highwire.dtl.DTLVardef@1e48f5dorg.highwire.dtl.DTLVardef@805b08_HPS_FORMAT_FIGEXP M_FIG C_FIG A magnetic nanobiopsy platform is presented for the longitudinal, non-destructive molecular sampling of living cell populations. Magnetically actuated nanocomposites directly access the cytosol--bypassing endo-lysosomal sequestration--to recruit a representative cargo of proteins and RNA through surface interactions and mechanical dragging. Guided by a changing external magnetic field, the molecularly loaded nanoparticles reversibly traverse the plasma membrane and exit via a controlled, budding-like mechanism that ensures cellular integrity. By preserving cell viability and population-level heterogeneity, this approach enables the retrieval of representative biomolecular cargo and the continuous monitoring of dynamic cellular states.

cell biology↗

bioRxiv2026

The microprotein Dafcin resembles influenza HA fusion peptide and regulates the size of storage lysosomes in the germline

Microproteins translated from short open reading frames are increasingly understood to play important roles in cell biology and development. Here, we describe a microprotein in Drosophila that is expressed in ovarian follicle cells which surround the developing oocyte. The Dafcin microprotein is predicted to form an amphipathic alpha-helix, a structure known to interact with lipid bilayers. Dafcins structure most resembles the influenza HA fusion peptide, which induces negative curvature of endosomal membranes. Dafcin tagged with GFP localizes to the Golgi and is ultimately secreted from the follicle cells. Remarkably, this occurs without the microprotein having a secretory signal sequence. The protein is taken up into the oocyte by endocytosis, localizing to the inner face of storage lysosomes called yolk granules. Mutant analysis shows that Dafcin is required to limit the size of yolk granules. This may occur by inducing negative membrane curvature like HA peptide. In support, liposomes formed in vitro with both Dafcin and HA peptides are smaller in size.

cell biology↗

bioRxiv2019

High-performance analysis of biomolecular containers to measure small-molecule transport, transbilayer lipid diffusion, and protein cavities

Compartmentalization is a central theme in biology. Cells are composed of numerous membrane-enclosed structures, evolved to facilitate specific biochemical processes; viruses act as containers of genetic material, optimized to drive infection. Molecular dynamics simulations provide a mechanism to study biomolecular containers and the influence they exert on their environments; however, trajectory analysis software generally lacks knowledge of container interior versus exterior. Further, many relevant container analyses involve large-scale particle tracking endeavors, which may become computationally prohibitive with increasing system size. Here, a novel method based on 3-D ray casting is presented, which rapidly classifies the space surrounding biomolecular containers of arbitrary shape, enabling fast determination of the identities and counts of particles (e.g., solvent molecules) found inside and outside. The method is broadly applicable to the study of containers and enables high-performance characterization of properties such as solvent density, small-molecule transport, transbilayer lipid diffusion, and topology of protein cavities. The method is implemented in VMD, a widely used simulation analysis tool that supports personal computers, clouds, and parallel supercomputers, including ORNLs Summit and Titan and NCSAs Blue Waters, where the method can be employed to efficiently analyze trajectories encompassing millions of particles. The ability to rapidly characterize the spatial relationships of particles relative to a biomolecular container over many trajectory frames, irrespective of large particle counts, enables analysis of containers on a scale that was previously unfeasible, at a level of accuracy that was previously unattainable.\n\nAuthor summaryThe cell is the basic unit of life. Within the container of the cell, the many chemical reactions and biological processes essential to life are carried out simultaneously. Human and other eukaryotic cells include a variety of sub-containers, namely organelles, that provide separation between reactions and processes, and engender the chemical environments conducive to them. In order to understand how the cell works, researchers must study the functions of these containers. Molecular dynamics simulations can reveal important information about how biomolecular containers behave and control their enclosed environments, but the latter can be particularly challenging and expensive to measure. The challenge arises because simulation analysis software lacks awareness of the concepts of container \"inside\" and \"outside.\" The expense arises because tracking the many solvent molecules that make up a containers environment requires significant computing power. We have developed a method that allows the simulation analysis software VMD to automatically detect the interior versus exterior of a container and quickly identify the solvent molecules found in each location. This versatile new feature enables researchers to characterize essential container properties using a relatively inexpensive calculation. Further, the method performs efficiently on supercomputers, allowing researchers to study massive container systems that include millions of particles.

biophysics↗

bioRxiv2017

A cell based, high throughput assay for quantitative analysis of Hedgehog pathway activation using a Smoothened phosphorylation sensor

The Hedgehog (Hh) signalling cascade is conserved across evolution and plays an important role in development and disease. In the absence of Hh, activity of the key signal transducer Smoothened (Smo) is downregulated by the Hh receptor Patched (Ptc). However, the mechanisms underlying this inhibition, and especially its release upon ligand stimulation, are still poorly understood, in part because tools for directly following Smo activation at the subcellular level were long lacking. Here we present a high throughput, cell culture assay based on a fluorescent sensor for Drosophila Smo phosphorlyation. Using this approach we could first demonstrate that the graded response to increasing Hh levels observed at the population level can be traced back to threshold responses of individual cells exhibiting differential Hh sensitivity. Second, we screened a small molecule inhibitor library for regulators of Smo phosphorylation. We observed increased Smo sensor fluorescence with compounds aimed at two major target groups, the MAPK signalling cascade and polo and aurora kinases. Biochemical validation confirmed the screen results for selected inhibitors (dobrafenib, tak-733, volasertib) and revealed differences in the mode of Smo activation, demonstrating that the assay is in principle suitable for dissecting the cell biological basis of Hh pathway activation.

cell biology↗

bioRxiv2017

Pattern analysis of pluripotency reveals the molecular basis of naïve, primed and formative states

The molecular regulatory network underlying stem cell pluripotency has been intensively studied, and we now have a reliable ensemble model for the average pluripotent cell. However, evidence of significant cell-to-cell variability suggests that the activity of this network varies within individual stem cells, leading to differential processing of environmental signals and variability in cell fates. Here, we adapt a method originally designed for face recognition to infer regulatory network patterns within individual cells from single-cell expression data. Using this method we identify three distinct network configurations in cultured mouse embryonic stem cells - corresponding to naive and formative pluripotent states and an early primitive endoderm state - and associate these configurations with particular combinations of regulatory network activity archetypes that govern different aspects of the cells response to environmental stimuli, cell cycle status and core information processing circuitry. These results show how variability in cell identities arise naturally from alterations in underlying regulatory network dynamics and demonstrate how methods from machine learning may be used to better understand single cell biology, and the collective dynamics of cell communities.

systems biology↗