SEARCH · Search bioRxiv

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 721 records · Page 40Linked to original sources

bioRxiv2019

BactMAP: an R package for integrating, analyzing and visualizing bacterial microscopy data

High-throughput analyses of single-cell microscopy data is a critical tool within the field of bacterial cell biology. Several programs have been developed to specifically segment bacterial cells from phase-contrast images. Together with spot and object detection algorithms, these programs offer powerful approaches to quantify observations from microscopy data, ranging from cell-to-cell genealogy to localization and movement of proteins. Most segmentation programs contain specific post-processing and plotting options, but these options vary between programs and possibilities to optimize or alter the outputs are often limited. Therefore, we developed BactMAP (Bacterial toolbox for Microscopy Analysis & Plotting), a software package that allows researchers to transform cell segmentation and spot detection data generated by different programs automatically into various plots. Furthermore, BactMAP makes it possible to perform custom analyses and change the layout of the output. Because BactMAP works independently of segmentation and detection programs, inputs from different sources can be compared within the same analysis pipeline. BactMAP runs in R, which enables the use of advanced statistical analysis tools as well as easily adjustable plot graphics in every operating system. Using BactMAP we visualize key cell cycle parameters in Bacillus subtilis and Staphylococcus aureus, and demonstrate that the DNA replication forks in Streptococcus pneumoniae dissociate and associate before splitting of the cell, after the Z-ring is formed at the new quarter positions. BactMAP is available from https://veeninglab.com/bactmap.

microbiology↗

bioRxiv2019

The lysine methyltransferase DOT1L controls CD4+ T cell differentiation and plasticity

CD4+ T helper (Th) cell differentiation is controlled by lineage-specific expression of transcription factors and effector proteins, as well as silencing of lineage-promiscuous genes. Lysine methyltransferases (KMTs) comprise a major class of epigenetic enzymes that are emerging as important regulators of Th cell biology. Here, we show that the KMT DOT1L regulates Th cell function and lineage integrity. DOT1L-dependent dimethylation of lysine 79 of histone H3 (H3K79me2) is associated with lineage-specific gene expression. However, DOT1L-deficient Th cells overproduce IFN-{gamma} under lineage-specific and lineage-promiscuous conditions. Consistent with the increased IFN-{gamma} response, mice with a T cell-specific deletion of DOT1L are susceptible to infection with the helminth parasite Trichuris muris and resistant to the development of allergic lung inflammation. These results identify a central role for DOT1L in Th cell lineage commitment and stability, and suggest that inhibition of DOT1L may provide a novel therapeutic strategy to limit type 2 immune responses.

immunology↗

bioRxiv2026

Evaluating Single-Cell Perturbation Response Models Is Far from Straightforward

Predicting cellular responses to genetic and chemical perturbations remains a central challenge in single-cell biology and a key step toward building in silico virtual cells. The rapid growth of perturbation datasets and advances in deep-learning models have raised expectations for accurate and generalizable prediction. We show that these expectations are overly optimistic, largely due to the failure modes of existing evaluation metrics. In this study, using cross-splitting, controlled noise experiments, and synthetic data, we systematically evaluate both prediction models and evaluation metrics. We demonstrate that widely used metrics, including correlation-based measures and common distributional distances, are strongly influenced by scale, sparsity, and dimensionality, often misrepresenting model performance. In particular, the Wasserstein distance fails in high-dimensional gene expression spaces under variance scaling, while the Energy distance can overlook disruptions in gene-gene dependencies. Our analyses further reveal that complex deep learning models often underperform simple baselines and remain far from empirical performance bounds across multiple chemical perturbation datasets. Together, our framework exposes critical pitfalls, establishes robust evaluation guidelines, and provides a foundation for trustworthy benchmarking toward reliable virtual-cell models.

bioinformatics↗

bioRxiv2021

Characterizing stochastic cell cycle dynamics in exponential growth

Two powerful and complementary experimental approaches are commonly used to study the cell cycle and cell biology: One class of experiments characterizes the statistics (or demographics) of an unsynchronized exponentially-growing population, while the other captures cell cycle dynamics, either by time-lapse imaging of full cell cycles or in bulk experiments on synchronized populations. In this paper, we study the subtle relationship between observations in these two distinct experimental approaches. We begin with an existing model: a single-cell deterministic description of cell cycle dynamics where cell states (i.e. periods or phases) have precise lifetimes. We then generalize this description to a stochastic model in which the states have stochastic lifetimes, as described by arbitrary probability distribution functions. Our analyses of the demographics of an exponential culture reveal a simple and exact correspondence between the deterministic and stochastic models: The corresponding state lifetimes in the deterministic model are equal to the exponential mean of the lifetimes in the stochastic model. An important implication is therefore that the demographics of an exponential culture will be well-fit by a deterministic model even if the state timing is stochastic. Although we explore the implications of the models in the context of the Escherichia coli cell cycle, we expect both the models as well as the significance of the exponential-mean lifetimes to find many applications in the quantitative analysis of cell cycle dynamics in other biological systems.

biophysics↗

bioRxiv2019

A Novel Taxon of RNA Viruses Endemic to Planarian Flatworms

The phylum Platyhelminthes is composed of both parasitic and non-parasitic flatworms. While the parasitic species have drawn attention for their wide effects on human and livestock heath, free-living flatworms, such as freshwater planarians, have become molecular models of regeneration and stem cell biology in the laboratory. However, one aspect of planarian biology that remains understudied is the relationship between host and any endemic viruses. Here we used searches of multiple transcriptomes from Schmidtea mediterranea asexual strain CIW4 and detected a novel, double-stranded RNA (dsRNA) virus, named S. mediterranea tricladivirus (SmedTV), which represents a distinct taxon (proposed new genus) within a larger taxon of monosegmented dsRNA viruses of diverse hosts. Experimental evidence for SmedTV in S. mediterranea CIW4 was obtained through whole-mount in situ hybridization (WISH). SmedTV "expression" (detected by both sense and anti-sense probes) was discrete yet variable from worm to worm and cell type to cell type, suggesting a persistent infection. Single-cell RNA sequencing (scRNAseq) further supported that SmedTV expression was low in stem cells, but substantially higher in multiple, though not all, differentiated tissues, with notable neural enrichment. Interestingly, knockdown of SmedTV by RNA-interference resulted in a "cure" of SmedTV after 10 RNAi doses, and expression remained undetectable by WISH even after 90 days. Due to being able to evade host defenses and the endogenous RNAi pathway, we believe SmedTV represents a novel animal model to study host-virus evolution. Statement of significancePlanarians are freshwater flatworms and emerging models to study the molecular mechanisms of adult stem cell and regenerative biology. However, they also live in aquatic environments with high amounts of viruses, bacteria, fungi, and protist pathogens. How the planarian immune system copes with all of these is largely unknown and only 2 types of virus have been described. Here we find a novel dsRNA virus, endemic to multiple types of flatworms. We show that it is a persistent infection, and likely transmits from stem cell to differentiated cell in the planarian, while avoiding endogenous RNA-interference machinery and mechanisms used to suppress viruses. We present this as a new model to study host-virus defense and evolution.

microbiology↗

bioRxiv2021

Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights

Bacillus subtilis is a model Gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, we demonstrate broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.

synthetic biology↗

bioRxiv2018

Stochastic models of cell invasion with fluorescent cell cycle indicators

Fluorescent cell cycle labelling in cell biology experiments provides real time information about the location of individual cells, as well as the phase of the cell cycle of individual cells. We develop a stochastic, lattice-based random walk model of a two-dimensional scratch assay where the total population is composed of three distinct subpopulations which we visualise as red, yellow and green subpopulations. Our model mimics FUCCI technology in which cells in the G1 phase of the cell cycle fluoresce red, cells in the early S phase fluoresce yellow, and cells in the S/G2/M phase fluoresce green. The model is an exclusion process so that any potential motility or proliferation event that would place an agent on an occupied lattice site is aborted. Using experimental images and previous experimental measurements, we explain how to apply the stochastic model to simulate a scratch assay initialised with a low to moderate density monolayer of human melanoma cell line. We obtain additional mathematical insight by deriving an approximate partial differential equation (PDE) description of the stochastic model, leading to a novel system of three coupled nonlinear reaction diffusion equations. Comparing averaged simulation data with the solution of the continuum limit model confirms that the PDE description is accurate for biologically-relevant parameter combinations.

biophysics↗

bioRxiv2026

Subcellular spatial proteomics of the close animal relative, Capsaspora owczarzaki

Capsaspora owczarzaki is one of the closest relatives of animals and an increasingly tractable model for investigating the evolution of multicellularity. However, very little cell biological work has been done on the sister linages to animals. Subcellular spatial proteomics (SSP) is a technique that can localize thousands of proteins to subcellular compartments in a single experiment by combining cell fractionation with sensitive mass spectrometry. To provide broad insight into the localization of the Capsaspora proteome, we performed SSP based on a label-free version of LOPIT-DC (Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation). We detected 6,466 proteins, corresponding to 68.4% of the predicted Capsaspora proteome, and assigned high-confidence localizations to 3,106 of them across 13 subcellular compartments. This study serves as a proteomic dataset to resolve cellular compartmentalization in a close relative of animals, enabling downstream evolutionary, biological, and functional analyses. Investigators interested in using these data prior to publication are encouraged to contact us to avoid overlap and to facilitate coordinated and collaborative use of this resource.

cell biology↗

bioRxiv2024

The mechanoreceptor PIEZO1 is a novel oncogene in glioma by promoting astrocyte reactivity

Glioblastoma is the most common and aggressive brain tumour in adults. Despite advances in its molecular characterization, there is a gap-of-knowledge in the identification of bona fide drivers and potential therapeutic targets that could change the clinical picture. Mechanoreception, the sensing of mechanical cues by cells, has proven to be an important factor in cell biology, particularly in cancer. Piezo1 is a mechanoreceptor found in all cells and seems to play a role in different cancer types, such as gastric, breast or lung cancer. However, there is still lack of understanding about its role in the onset and progression of glioblastoma. Here, we show that Piezo1 acts as an oncogene in glioma by potentially promoting chronic astrocyte reactivity. We developed a novel transgenic murine model of Piezo1 overexpression in astrocytes. These animals had a significant reduction in overall survival and developed glioma with a penetrance of 30%. We also developed a PIEZO1-overexpressing U251 cell line and found that it had a more aggressive and reactive-like phenotype. Finally, we correlated the levels of PIEZO1 with the clinical outcome of a cohort of glioblastoma patients and observed that PIEZO1 is a biomarker of worse prognosis. However, PIEZO1 only correlated with worse prognosis in male patients, suggesting a sexual dimorphism. In conclusion, we identified Piezo1 as a bona fide driver of glioma, revealing its implication in astrocyte reactivity and identifying it as a biomarker for glioma in the clinic.

cancer biology↗

bioRxiv2020

Nanoscale Organization of FasL on DNA Origami as a Versatile Platform to Tune Apoptosis Signaling in Cells

Nanoscale probes with fine-tunable properties are of key interest in cell biology and nanomedicine to elucidate and eventually control signaling processes in cells. A critical, still challenging issue is to conjugate these probes with molecules in a number- and spatially-controlled manner. Here, DNA origami-based nanoagents as nanometer precise scaffolds presenting Fas ligand (FasL) in well-defined arrangements to cells are reported. These nanoagents activate receptor molecules in the plasma membrane initiating apoptosis signaling in cells. Signaling for apoptosis depends sensitively on FasL geometry: fastest time-to-death kinetics are obtained for FasL nanoagents representing predicted structure models of hexagonal receptor ordering with 10 nm inter-molecular spacing. Slower kinetics are observed for one to two FasL on DNA origami or FasL coupled with higher flexibility. Nanoagents with FasL arranged in hexagons with small (5 nm) and large (30 nm) spacing impede signal transduction. Moreover, for predicted hexagonal FasL nanoagents, signaling efficiency is faster and 100× higher compared to naturally occurring soluble FasL. Incubation of the FasL-origami nanoagent in solution exhibited an EC50 value of only 90 pM. These studies present DNA origami as versatile signaling platforms to probe the significance of molecular number and nanoscale ordering for signal initiation in cells.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2022

Cell-Sized Lipid Vesicles as Artificial Antigen-Presenting Cells for Antigen-Specific T Cell Activation

In this study, efficient T cell activation is demonstrated using cell-sized artificial antigen-presenting cells (aAPCs) with protein-conjugated bilayer lipid membranes that mimic biological cell membranes. The highly uniform aAPCs are generated by a facile method based on standard droplet microfluidic devices. These aAPCs are able to activate the T cells in peripheral blood mononuclear cells (PBMCs), showing a 28-fold increase in IFN{gamma} secretion, a 233-fold increase in antigen-specific CD8 T cells expansion, and a 16-fold increase of CD4 T cell expansion. The aAPCs do not require repetitive boosting or additional stimulants and can function at a relatively low aAPC-to-T cell ratio (1-to-17). The research presents strong evidence that the surface fluidity and size of the aAPCs are critical to the effective formation of immune synapses essential for T cell activation. The findings demonstrate that the microfluidic-generated aAPCs can be instrumental in investigating the physiological conditions and mechanisms for T cell activation. Finally, this method demonstrates the feasibility of customizable aAPCs for a cost-effective off-the-shelf approach to immunotherapy.

bioengineering↗

bioRxiv2023

Single-cell profiling uncovers regulatory programs of pathogenic Th2 cells in allergic asthma

Lung pathogenic T helper type 2 (pTh2) cells are important drivers of allergic asthma, but fundamental questions remain regarding their regulation and heterogeneity. The differentiation and effector functions of immune cells are tightly regulated by epigenetic processes. Histone deacetylase 1 (HDAC1) is an important epigenetic regulator of T cells, however, its role in pTh2 cells is yet to be determined. Here we investigate immune regulation in allergic asthma by single-cell RNA sequencing (scRNA-seq) in mice challenged with house dust mite, in the presence and absence of HDAC1 function. Our analyses reveal two distinct subsets of lung pTh2 cells: pathogenic effector Th2 (peTh2) and pathogenic Th2 tissue-resident memory (Th2 Trm) cells. Both pTh2 cell subsets are highly proinflammatory and exhibit distinct transcriptional and phenotypic signatures as compared with other lung Th subsets. Based on our scRNA-seq analysis, we identify conditions to generate pTh2 cells in vitro and confirm that these in vitro generated pTh2 cells have a similar transcriptional profile as lung peTh2 cells. Using our new in vitro model, we demonstrate that the p38 mitogen-activated protein kinase pathway is critical for interleukin-5 (IL-5) and IL-13 expression in pTh2 cells. Our data further underline the importance of HDAC1 in limiting the pathogenicity of lung and in vitro pTh2 cells and in the formation of lung Th2 Trm cells. In summary, we have generated novel insights into pTh2 cell biology and established a new in vitro model for investigating pTh2 cells that will be useful for discovering molecular mechanisms involved in pTh2-mediated allergic asthma.

immunology↗

bioRxiv2024

Mapping single-cell developmental potential in health and disease with interpretable deep learning

Single-cell RNA sequencing (scRNA-seq) has transformed our understanding of cell fate in developmental systems. However, identifying the molecular hallmarks of potency - the capacity of a cell to differentiate into other cell types - has remained challenging. Here, we introduce CytoTRACE 2, an interpretable deep learning framework for characterizing potency and differentiation states on an absolute scale from scRNA-seq data. Across 31 human and mouse scRNA-seq datasets encompassing 28 tissue types, CytoTRACE 2 outperformed existing methods for recovering experimentally determined potency levels and differentiation states covering the entire range of cellular ontogeny. Moreover, it reconstructed the temporal hierarchy of mouse embryogenesis across 62 timepoints; identified pan-tissue expression programs that discriminate major potency levels; and facilitated discovery of cellular phenotypes in cancer linked to survival and immunotherapy resistance. Our results illuminate a fundamental feature of cell biology and provide a broadly applicable platform for delineating single-cell differentiation landscapes in health and disease.

genomics↗

bioRxiv2024

Structure of the Flotillin Complex in a Native Membrane Environment

In this study we used cryo-electron microscopy to determine the structures of the Flotillin protein complex, part of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, from cell-derived vesicles without detergents. It forms a right-handed helical barrel consisting of 22 pairs of Flotillin1 and Flotillin2 subunits, with a diameter of 32 nm its wider end and 19 nm at its narrower end. Oligomerization is stabilized by the C-terminus, which forms two helical layers linked by a {beta}-strand, and coiled-coil domains that enable strong charge-charge inter-subunit interactions. Flotillin interacts with membranes at both ends; through its SPFH1 domains at the wide end and the C-terminus at the narrow end, facilitated by hydrophobic interactions and lipidation. The inward tilting of the SPFH domain, likely triggered by phosphorylation, suggests its role in membrane curvature induction, which could be connected to its proposed role in clathrin-independent endocytosis. The structure suggests a shared architecture across the family of SPFH proteins and will promote further research into Flotillins roles in cell biology. Significance statementIt is well known that many biochemical processes in cells must occur in localized regions. There are many different ideas about how cells keep processes localized. In this study we demonstrate that Flotillin1 and Flotillin2 co-assemble to form a large, truncated cone shaped cage whose wide end is always attached to a membrane surface and whose narrow end is sometimes attached to a separate membrane. The entire wall of the cage is without holes and is likely impervious even to small molecules, forming a diffusion barrier that can connect membrane systems. The Flotillin cage is thus well suited to isolate biochemical processes. Through membrane attachment, it also alters local membrane curvature, which could influence endocytic and mechanosensory processes.

biochemistry↗

bioRxiv2024

Segregation between an ornamental and a disease driver gene provides insights into pigment cell regulation

Genetic interactions are adaptive within a species. Hybridization can disrupt such species-specific genetic interactions and creates novel interactions that alter the hybrid progeny overall fitness. Hybrid incompatibility, which refers to degenerative genetic interactions that decrease the overall hybrid survival, is one of the results from combining two diverged genomes in hybrids. The discovery of spontaneous lethal tumorigenesis and underlying genetic interactions in select hybrids between diverged Xiphophorus species showed that lethal pathological process can result from degenerative genetic interactions. Such genetic interactions leading to lethal phenotype are thought to shield gene flow between diverged species. However, hybrids between certain Xiphophorus species do not develop such tumors. Here we report the identification of a locus residing in the genome of one Xiphophorus species that represses an oncogene from a different species. Our finding provides insights into normal and pathological pigment cell development, regulation and molecular mechanism in hybrid incompatibility. SignificanceThe Dobzhansky-Muller model states epistatic interactions occurred between genes in diverged species underlies hybrid incompatibility. There are a few vertebrate interspecies hybrid cases that support the Dobzhansky-Muller model. This study reports a fish hybrid system where incompatible genetic interactions are involved in neuronal regulation of pigment cell biology, and also identified a novel point of regulation for pigment cells.

genetics↗

bioRxiv2024

LC3-associated endocytosis facilitates extracellular Tau aggregate internalization and degradation in microglia

BackgroundMicroglia, which are resident phagocytic cells in the brain, are involved in the active clearance of microbes, misfolded proteins, and cell debris, among others. In Alzheimers disease, microglia play a pivotal role in clearing extracellular amyloid-{beta} (A{beta}) plaques and intracellular Tau aggregates from the brain. Microglial cells have several mechanisms for Tau internalization, including macropinocytosis, heparan sulfate proteoglycans (HSPGs), dynamin- dependent endocytosis, and receptor-mediated endocytosis. Internalized Tau seeds either undergo proteasomal or lysosomal degradation or are exocytosed into the extracellular space via exosomes. Microtubule-associated protein 1 light chain 3 (LC3)-associated endocytosis (LANDO) is a recently discovered microglial mechanism for an effective clearance of A{beta} aggregates and alleviates neurodegeneration in murine Alzheimers disease. Several microglial receptors are reported to be involved in misfolded A{beta} and Tau aggregate internalization such as triggering receptor expressed on myeloid cell 2 (TREM-2), P2Y purinoceptor 12 (P2Y12R), C- X3-C motif chemokine receptor 1 (CX3CR1), etc. MethodsIn this study, we report the LANDO of Tau monomers and aggregates by murine microglial cells, which are further degraded by lysosomal fusion. LANDO of extracellular Tau is demonstrated by several biochemical and cell-biology studies including western blotting, fluorescence and confocal imaging of microglial cells. ResultsWe analyzed microglial activation by measuring the upregulation of Ionized Calcium- binding Adapter Molecule 1 (Iba-1) expression. Later, we demonstrated the LANDO of human full-length Tau species, where LC3 colocalizes with internalized Tau, followed by lysosomal degradation by microglia. The accumulation of internalized Tau in the perinuclear region of the cell in the presence of chloroquine supports phagolysosomal fusion and degradation. ConclusionHence, we concluded that microglia internalize extracellular Tau species via LANDO, which further undergoes degradation via the lysosomal pathway.

neuroscience↗

bioRxiv2025

Early resident NK cell response to local HIV infection in lymphoid tissue

Natural killer (NK) cells are critical mediators of antiviral immunity, yet their role in lymphoid tissues--key reservoirs of HIV persistence--remains poorly defined. Here, we uncover a distinct cytotoxic signature of tonsil-resident NK cells essential for targeting HIV-infected CD4 T cells. Using a human tonsillar model of HIV infection and extracellular matrix-based functional assays, we identify a subset of NK cells co-expressing CD69, CD49a, CD103, and the adaptive marker NKG2C as potent effectors against autologous HIV-infected tissue CD4 T cells. Both CD16 and CD16- NK subsets exhibited cytotoxic antiviral activity. However, HIV infection induced profound functional alterations in these NK cells, including dysregulated expression of CD9, TRAF2, ITGA1, suggesting disrupted activation, signaling, and tissue residency. Functional assays corroborated a significant impairment in the cytotoxic capacity, indicating HIV-driven NK cell dysfunction. Intriguingly, a subset of immature CD16-CD69 NK cells underwent functional reprogramming, transitioning into a migration-competent, and metabolically primed state, driven by the upregulation of NUMAI, LCP1, SLC38A1, MT-ND2, NUMA1, MYH9, and CD44. Functional assays confirmed this transition, revealing changes in the expression of immune checkpoint receptors and a gain of cytotoxic function in these reprogrammed cells. These findings advance our understanding of NK cell biology in HIV infection and highlight novel avenues for NK-cell-based therapeutic strategies.

immunology↗

bioRxiv2025

Fabrication of microcompartments with controlled size and shape for encapsulating active matter

In all living systems, the cytoplasm is separated from the external environment by membranes. This confinement imposes spatial constraints on the self-organization of internal components, filaments and organelles. While reconstituted systems are instrumental for understanding fundamental biological principles, traditional experiments often utilize volumes vastly larger than actual cells. In recent studies, water-in-oil droplets or giant unilamellar vesicles have been widely used to impose confinement. However, these compartments present imaging challenges and make precise protein content control difficult. To address these limitations, we have developed versatile microwells that are straightforward to implement, compatible with different types of imaging, suitable for long-term experiments, and capable of generating large amounts of data. These microwells are compatible with several surface treatments and a wide range of experimental techniques (such as micropatterning) making them a powerful tool for answering key questions in cell biology. We present here a detailed protocol of the fabrication of the microwells as well as characterization of the method to ensure quality throughout the manufacturing process. These microwells support various cytoskeleton-based processes including actin polymerization, dynamic steady-state actin networks, and composite actin-microtubule networks. More broadly, they can be used to encapsulate and study over time any kind of active matter.

biochemistry↗