SEARCH · bioRxiv Science

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 937 records · Page 52Linked to original sources

bioRxiv2025

Asymmetry and redundancy of STAT5 paralogs across CD8+ T cell differentiation states

Fostering STAT5 signaling is key to immunotherapies that leverage CD8+ T cell biology. Using mouse models, we demonstrate that the two mammalian STAT5 paralogs, STAT5A and STAT5B, are at once redundant and functionally distinct in CD8+ T cells. Specifically, we establish that they are asymmetric paralogs, exhibiting both widespread homology at molecular level and functional asymmetry at cellular level, with STAT5B emerging as dominant. In fact, compared to STAT5A, STAT5B deficiency had greater impact on nearly all parameters tested. As a mechanism, we determined STAT5B is twice as abundant, accounting for two-thirds of the total STAT5 pool. We also defined both cytokine- and cell state-restricted STAT5B functions, and a core gene signature that highlights universal effects. Together, these studies affirm the centrality of STAT5 in CD8+ T cells, reveal common and circumscribed activities, and present a unifying model for paralog redundancy that foregrounds and explains the dominance of STAT5B. Summary: STAT5 paralog dominance and redundancy in CD8+ T cells

immunology↗

bioRxiv2020

Pancreatic cancer stem cells (CSCs) are regulated via a feedback-loop of telomerase activity and stemness factors

To date, it is still unclear how cancer stem cells (CSCs) regulate their stemness properties, and to what extent they share common features with normal stem cells. Telomerase regulation is a key factor in stem cell maintenance. In this study, we investigate how telomerase regulation affects cancer stem cell biology in pancreatic ductal adenocarcinoma (PDAC), and delineate the mechanisms by which telomerase activity and CSC properties are linked. Using primary patient-derived pancreatic cancer cells, we show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic TERT-knockdown or pharmacological inhibitor (BIBR1532) resulted in CSC marker depletion in vitro, and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (KLF4, SOX2, OCT3/4, NANOG) and telomerase, which is essential for the self-renewal of pancreatic CSCs. Disruption the balance between telomerase activity and stemness factors, eliminates CSCs via induction of DNA damage and apoptosis, opening future perspectives to avoid CSC driven therapy resistance and tumor relapse in PDAC patients.

cancer biology↗

bioRxiv2019

Titrating gene expression with series of systematically compromised CRISPR guide RNAs

Biological phenotypes arise from the degrees to which genes are expressed, but the lack of tools to precisely control gene expression limits our ability to evaluate the underlying expression-phenotype relationships. Here, we describe a readily implementable approach to titrate expression of human genes using series of systematically compromised sgRNAs and CRISPR interference. We empirically characterize the activities of compromised sgRNAs using large-scale measurements across multiple cell models and derive the rules governing sgRNA activity using deep learning, enabling construction of a compact sgRNA library to titrate expression of [~]2,400 genes involved in central cell biology and a genome-wide in silico library. Staging cells along a continuum of gene expression levels combined with rich single-cell RNA-seq readout reveals gene-specific expression-phenotype relationships with expression level-specific responses. Our work provides a general tool to control gene expression, with applications ranging from tuning biochemical pathways to identifying suppressors for diseases of dysregulated gene expression.

genomics↗

bioRxiv2026

Building virtual cell dynamics that evolve with biological measurements

Virtual cell models built from single-cell measurements need to predict how cells change over time, but new biological measurements often reveal dynamical relationships that existing models cannot express. Translating such measurements into dynamics therefore requires deciding what biological relation should be modeled, where it should enter the formulation and how its recovery should be validated. Here we present CellCompass, an agentic AI system that couples a scientific agent with an extensible dynamical engine for modeling state transitions and population change, along with fate and molecular readout. CellCompass reuses existing formulations when sufficient and designs, implements and refines new ones when experimental measurements introduce additional constraints. To assess this capability, we developed DynBench100, a benchmark of 100 simulated single-cell time-course scenarios with hidden dynamical ground truth. CellCompass achieved the highest aggregate performance among the tested general-purpose and biomedical agents across six dynamical tasks. Across four real single-cell studies, it retained an existing velocity-growth model to reconstruct context-dependent epithelial-mesenchymal transition and developed three new dynamical formulations for distinct experimental measurements. In drug-response data, incorporating metabolic RNA labeling reduced pulse-control identifiability error by 31.1%, revealing staged response programs and generating testable predictions for perturbing a candidate adaptive program. In hematopoietic lineage tracing, incorporating clonal barcodes improved clone-fate agreement by 54% over the strongest dynamical baseline, identifying early lineage priming among transcriptionally similar progenitors. Finally, in time-resolved CITE-seq data, CellCompass reduced future surface-protein distribution error by 13.2% for RNA-initialized trajectories, uncovering lineage-specific temporal offsets between transcriptional and protein programs. Together, CellCompass provides a framework in which virtual-cell dynamics can expand with the biological measurements used to constrain them.

bioinformatics↗

bioRxiv2024

Differences in RAD51 transcriptional response and cell cycle dynamics reveal varying sensitivity to DNA damage among Arabidopsis thaliana root cell types

O_LIThroughout their lifecycle, plants are subjected to DNA damage from various sources, both environmental and endogenous. Investigating the mechanisms of the DNA damage response (DDR) is essential to unravel how plants adjust to the changing environment that can elicit varying amounts of DNA damage. C_LIO_LIUsing a combination of state-of-the-art cell biology methods including whole-mount single-molecule RNA fluorescence in situ hybridization (WM-smFISH), allowing detection of individual mRNA molecules in intact plant tissue and plant cell cycle reporter lines we investigated how the transcriptional activation of a key homologous recombination (HR) gene, RAD51, occurs in response to increasing amounts of DNA damage in Arabidopsis thaliana roots. C_LIO_LIThe results uncover consistent variations in RAD51 transcriptional response and cell cycle arrest among distinct cell types and developmental zones. Furthermore, we demonstrate that DNA damage induced by genotoxic stress results in RAD51 transcription throughout the whole cell cycle, dissociating its traditional link with S/G2 phases. C_LIO_LIThis work advances the current comprehension of DNA damage response in plants showing quantitative differences in DDR activation. In addition, it reveals new associations with the cell cycle and cell types, providing crucial insights for further studies of the broader response mechanisms in plants. C_LI

plant biology↗

bioRxiv2025

The phosphatases TCPTP, PTPN22, and SHP1 play unique roles in T cell phosphotyrosine maintenance and feedback regulation of the TCR

The protein tyrosine phosphatases (PTPs) TCPTP, PTPN22, and SHP1 are critical regulators of the activating phosphotyrosine (pY) site on the initiating T cell kinase, LckY394. Still, the broader implications of these phosphatases in T cell receptor (TCR) signalling and T cell biology remain unclear. By combining CRISPR/Cas9 gene editing and mass spectrometry, we evaluate the protein- and pY-level effects of TCPTP, PTPN22, and SHP1 in the Jurkat T cell model system. We find that deletion of each phosphatase corresponds to unique changes in the proteome of T cells, with few large-scale changes to TCR signalling proteins. Notably, PTPN22 and SHP1 deletions have opposing effects on pY abundance globally, while TCPTP deletion modestly elevates pY levels. Finally, we show that TCPTP is indirectly involved in Erk1/2 positive feedback to the TCR. Overall, our work provides evidence for alternative functions of three T cell phosphatases long thought to be redundant.

systems biology↗

bioRxiv2019

Photorhabdus Luminescens Toxin complex (TCC) a recombinant injection nano-machine - delivery of recombinant Yersinia enterocolitica YopT

Engineering delivery systems for proteins and peptides into mammalian cells is an ongoing challenge for cell biological studies as well as for therapeutic approaches. Photorhabdus luminescens toxin complex (PTC) is a heterotrimeric protein complex able to deliver diverse protein toxins into mammalian cells. We engineered the syringe like nano-machine for delivery of protein toxins from different species. Additionally, we loaded the highly active copepod luciferase Metridia longa M-Luc7 for accurate quantification of injected molecules. We suggest that besides the size also the charge of the cargo defines the efficiency of packing and transport into mammalian cells. Our data show that the Photorhabdus luminescens toxin complex constitutes a powerful system to inject recombinant proteins, peptides and potentially other molecules like aptamers into mammalian cells. In contrast to other protein transporters based on pore formation, the cargo is protected from degradation. The system opens new perspectives for cell research and pharmacology.

bioengineering↗

bioRxiv2026

ExPert infers causal perturbations from cell states across biological and clinical contexts

Pooled single-cell CRISPR screens have driven substantial effort toward models predicting the response to a perturbation. We posed the inverse question: which perturbation produced an observed cell state? Here we developed ExPert (Extracting Perturbations), which learns one prototype per perturbation in a latent space shared by 12 cellular contexts, trained on 8,964 perturbations and 19.5 million cells, and ranks 19,895 candidate genes per query, over half absent from training. ExPert more than doubled held-out-context recall over the best existing method with 1,000-fold less compute, and transferred without retraining to drugs, protein degradation and combinations. In alcohol-associated liver disease it implicated mRNA export, unchanged in expression, and we experimentally confirmed nuclear poly(A) retention and isoform switching. In primary mitochondrial disease it recovered the affected complex from blood, localized the signal to monocytes, and flagged a misdiagnosed control. Perturbation atlases can thus be queried in reverse, from state to likely driver.

systems biology↗

bioRxiv2019

How prolonged expression of Hunchback, a temporal transcription factor, re-wires locomotor circuits

AbstractIn many CNS regions, neuronal birth timing is associated with circuit membership. In Drosophila larvae, we show U motor neurons are a temporal cohort--a set of non-identical, contiguously-born neurons from a single neuronal stem cell that contribute to the same circuit. We prolong expression of a temporal transcription factor, Hunchback, to increase the number of U motor neurons with early-born molecular identities. On the circuit level, this expands and re-wires the U motor neuron temporal cohort. On the cell biological level, we find novel roles for Hunchback in motor neuron target selection, neuromuscular synapse formation, dendrite morphogenesis, and behavior. These data provide insight into the relationship between stem cell and circuit, show that Hunchback is a potent regulator of circuit assembly, and suggest that temporal transcription factors are molecules that could be altered during evolution or biomedical intervention for the generation of novel circuits.

developmental biology↗

bioRxiv2020

Open source software tool for the automated detection and characterization of epithelial cells from trace biological samples

This paper presents a strategy for an unsupervised workflow for identifying epithelial cells in microscopic images and characterizing their morphological and/or optical properties. The proposed method can be used on cells that have been stained with fluorescent dyes and imaged using conventional optical microscopes. The workflow was tested on cell populations that were imaged directly on touch/contact surfaces and stained with nucleic acid dyes to visualize genetic content. Our results show that this approach could be a useful strategy for characterizing differences in staining efficiency and/or morphological properties of individual cells or aggregate populations within a biological sample. Further, they can potentially reduce the laborious nature of microscopic analysis and increase throughput and reproducibility of similar studies.

cell biology↗

bioRxiv2022

Curvature-induced cell rearrangements in biological tissues

On a curved surface, epithelial cells can adapt to geometric constraints by tilting and by exchanging their neighbors from apical to basal sides, known as an apicobasal T1 (AB-T1) transition. The relationship between cell tilt, AB-T1 transitions, and tissue curvature still lacks a unified understanding. Here, we propose a general framework for cell packing in curved environments and explain the formation of AB-T1 transitions under different conditions. We find that steep curvature gradients can lead to cell tilting and induce AB-T1 transitions. Conversely, large curvature anisotropy can drive AB-T1 transitions by hydrostatic pressure. The two mechanisms compete to determine the impact of tissue geometry and mechanics on optimized cell rearrangements in 3D.

biophysics↗

bioRxiv2018

Characterization of fluorescent proteins, promoters, and selectable markers for applications in the Lyme disease spirochete Borrelia burgdorferi

Lyme disease is the most widely reported vector-borne disease in the United States. Its incidence is rapidly increasing and disease symptoms can be debilitating. The need to understand the biology of the disease agent, the spirochete Borrelia burgdorferi, is thus evermore pressing. Despite important advances in B. burgdorferi genetics, the array of molecular tools available for use in this organism remains limited, especially for cell biological studies. Here, we adapt a palette of bright and mostly monomeric fluorescent proteins for versatile use and multi-color imaging in B. burgdorferi. We also characterize two novel antibiotic selection markers and establish the feasibility of their use in conjunction with extant markers. Lastly, we describe a set of constitutively active promoters of low and intermediate strengths that allow fine-tuning of gene expression levels. These molecular tools complement and expand current experimental capabilities in B. burgdorferi, which will facilitate future investigation of this important human pathogen. To showcase the usefulness of these reagents, we used them to investigate the subcellular localization of BB0323, a B. burgdorferi lipoprotein essential for survival in the host and vector environments. We show that BB0323 accumulates at the cell poles and future division sites of B. burgdorferi cells, highlighting the complex subcellular organization of this spirochete.\n\nIMPORTANCEGenetic manipulation of the Lyme disease spirochete B. burgdorferi remains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest, B. burgdorferi displays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacteriums transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use in B. burgdorferi by generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and constitutively active promoters of different strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

microbiology↗

bioRxiv2021

Obesity and adiposity have opposing genetic impacts on human blood traits

Obesity, hyperlipidemia, and truncal adiposity concordantly elevate cardiovascular disease risks, but have unknown genetic effects on blood trait variation. Using Mendelian randomization, we define unexpectedly opposing roles for generalized obesity and truncal adiposity on blood traits. Elevated genetically determined body mass index (BMI) and lipid levels decreased hemoglobin and hematocrit levels, explaining consistent with clinical observations associating obesity and anemia. However, lipid-related effects were confined to erythroid traits, whereas BMI affected multiple blood lineages, indicating broad effects on hematopoiesis. BMI-related effects were unexpectedly opposed by truncal adipose distribution, which increased hemoglobin and blood cell counts across lineages. Conditional analyses indicated genes, pathways, and cell types responsible for these effects, including Leptin Receptor and other blood cell-extrinsic factors in adipocytes and endothelium, which regulate hematopoietic stem and progenitor cell biology. Our findings identify novel roles for obesity and adipose distribution on hematopoiesis and show that genetically determined adiposity plays a previously underappreciated role in determining blood cell formation and function.

genetics↗

bioRxiv2022

Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we rigorously compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides the best balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

genomics↗

bioRxiv2024

Splicing-aware resolution of scRNA-Seq data

Single-cell RNA sequencing (scRNA-Seq) provides invaluable insights in cell biology. Current scRNA-Seq analytic approaches do not distinguish between spliced and unspliced mRNA. RNA velocity paradigm suggests that the presence of unspliced mRNA reflects transitional cell states, informative for studies of dynamic processes such as embryogenesis or tissue regeneration. Alternatively, stable cell subsets may also maintain unspliced mRNA reservoirs for prompt initiation of transcription-independent expression. Based on the latter paradigm, we have developed a method called SANSARA (Splicing-Aware scrNa-Seq AppRoAch) for the splicing-aware analysis of scRNA-Seq data. We employed SANSARA to characterize peripheral blood regulatory T cell (Treg) subsets, revealing the complex interplay between FoxP3 and Helios master transcription factors and other unexpected splicing-informed features. For Th1 and cytotoxic CD4+ T cell subsets, SANSARA also revealed substantial splicing heterogeneity across crucial subset-specific genes. SANSARA is straightforward to implement in current data analysis pipelines and opens new dimensions in scRNA-Seq-based discoveries.

immunology↗

bioRxiv2024

Image-based Strain Analysis Reveals Intracellular Strain Controlled by Nucleo-Cytoskeletal Coupling

Cells can sense and transduce mechanical forces, such as stretching, and convert these signals into diverse cell biological events. While much effort has been devoted to identifying the downstream biochemical and cellular responses, it is equally crucial to pinpoint the mechanical stimuli within a cell driving these responses. Specifically, much remains unknown about how intracellular strains are distributed and controlled during mechanical deformation. In this study, we developed a microscopy-based intracellular strain measurement technique. Utilizing the intrapopulation mechanical heterogeneity of epithelial monolayers, we observed an inverse relationship between cytoplasmic and nuclear strains. We found that this anti-correlation is abolished by the inhibition of Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, suggesting that nucleo-cytoskeletal coupling controls intracellular strain distribution. We discovered a direct connection between cytoplasmic strain and stretch-induced nucleus size changes, implying that molecular events arising from cytoplasmic deformation may drive nuclear remodeling during stretching. By conducting multivariable analyses, we found that the intracellular strain can be inferred from cell morphology. Overall, our experimental platform and findings provide a foundation for unraveling the relationship between mechanotransduction pathways and upstream intracellular strain. SIGNIFICANCEMechanical stimuli exert influence on epithelial cells, not only orchestrating embryogenesis and regeneration, but also regulating cancer progression and inflammatory conditions. Despite efforts to identify mechanically activated molecular events, understanding how deformation is distributed within cells to induce subcellular responses remains limited. Specifically, the control of subcellular strain distribution during mechanical stretch is unclear. In this study, we developed a microscopy-based method to measure subcellular strain and observed an inverse relationship between cytoplasmic and nuclear strains. Disrupting nucleo-cytoplasmic coupling abolished this relationship, suggesting its role in controlling strain distribution. Additionally, we found that cytoplasmic strain correlates with nucleus size changes during stretching, indicating cytoplasmic events influence nucleus remodeling.

biophysics↗

bioRxiv2025

Encoded cell-material interactions to reroute cytokine signaling for regenerative medicine

Regenerative engineering harnesses materials science and stem cell biology to develop strategies to repair damaged and diseased tissue. Despite advances in designer materials, few techniques effectively provide auto-regulated feedback mechanisms that govern how cells sense and respond to discrete microenvironmental changes. Here, we demonstrate that the artificial, juxtacrine-like receptor synthetic Notch (synNotch) can be activated by endogenous multimeric cytokines in solution, without immobilizing materials, revealing a previously unreported activation modality and yielding up to 24-fold dynamic range. To broaden synNotch sensing to monomeric cytokines, we developed nMATRIX, a co-engineered material-cell platform that detects endogenous, soluble ligands and routes them to programmed gene circuits with spatially confined effects. nMATRIX can be tuned to recognize the interleukins IL-1{beta} and IL-6 using synNotch receptors plus cognate biomaterials, yielding up to 68-fold dynamic range and converting these inflammatory inputs into orthogonal outputs that reprogram nearby cell phenotypes. nMATRIX functions across multiple cell types and can incorporate the synNotch-related SNIPR synthetic receptor platform. nMATRIX repurposed inflammatory signals and converted them into anti-inflammatory cues to polarize macrophages (increased CD163, CD206; decreased CD86). Thus, nMATRIX couples native soluble cues to customized cellular responses with tunable sensitivity, offering a flexible materials-based approach for self-regulating regenerative therapies.

bioengineering↗

bioRxiv2026

Regucalcin-containing extracellular vesicles suppress M2 macrophage polarization and attenuate tumor progression in vivo

Regucalcin (RGN) plays diverse roles in cell biology, highlighting its importance in both physiological and pathological conditions. Prostate cancer patients with higher RGN expression exhibited significantly longer disease-free survival. Although RGN is a cell signaling suppressor, the molecular mechanisms underlying tumor suppression by RGN in the tumor microenvironment through cell-cell communication remain unclear. PC3 prostate cancer cell lines stably expressing RGN or a control vector were generated for this study. Extracellular vesicles (EVs) were isolated from these cell lines using differential ultracentrifugation. The murine macrophage cell line J7441 was treated with isolated EVs, and effects on M2 polarization were evaluated using qRT-PCR and western blot analysis. To assess the potential anti-tumor effects of EVs, PC3 parental cells were subcutaneously implanted at two sites per mouse, followed by intratumoral injection of the respective EVs. Tumor volume was monitored. Harvested fresh frozen tumor tissues underwent immunofluorescence staining for CD206, an M2 macrophage marker. RGN was detected in EVs from RGN-expressing cells, and treatment with these RGN-containing EVs was associated with reduced tumor growth and reduced M2 macrophage polarization in vitro and in vivo. Furthermore, recombinant RGN protein reduced the levels of p-AKT1 and p-ERK1/2. Moreover, the suppression of M2 macrophage polarization by RGN-containing EVs was accompanied by decreased p-AKT1 and p-ERK1/2 in vitro. This study describes an EV-associated mechanism that may contribute to the regulation of macrophage polarization and indicates that RGN-containing EVs merit further evaluation as a candidate approach for cancer treatment. Causal validation, such as macrophage depletion or CD206 knockdown, and evaluation in additional models remain to be addressed in future studies.

cancer biology↗