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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 433 records · Page 24Linked to original sources

bioRxiv2019

Wash and the WASH Regulatory Complex function in Nuclear Envelope budding

Nuclear envelope budding is a recently described phenomenon wherein large macromolecular complexes can be packaged inside the nucleus and be extruded through the nuclear membranes, completely bypassing nuclear pores. While factors have been identified both as cargos or actively involved in this process, much remains unknown about the molecules that generate the forces and membrane deformations which appear inherent. Using fluorescence and electron microscopy, biochemical and cell biological assays, and genetic perturbations in the Drosophila model, we identify Wash, its regulatory complex, and Arp2/3 as novel players in NE-budding. Surprisingly, Washs role in this process is bipotent and, independent of SHRC/Arp2/3, its perturbation disrupts the normal homotypic Lamin A/B meshworks that are necessary for NE-budding to occur. In addition to NE-budding emerging as important in additional cellular processes and organisms, its incredible similarity to herpesvirus egress suggests new avenues for exploration in both normal and disease biology.

cell biology↗

bioRxiv2024

Quantitative phase imaging with temporal kinetics predicts hematopoietic stem cell diversity

Innovative identification technologies for hematopoietic stem cells (HSCs) have advanced the frontiers of stem cell biology. However, most analytical techniques capture only a single snapshot, disregarding the temporal context. A comprehensive understanding of the temporal heterogeneity of HSCs necessitates live-cell, real-time and non-invasive analysis. Here, we developed a prediction system for HSC diversity by integrating single-HSC ex vivo expansion technology with quantitative phase imaging (QPI)-driven machine learning. By analyzing single-cell kinetics with QPI, we discovered previously undetectable diversity among HSCs that snapshot analysis fails to capture. Our QPI-driven algorithm quantitatively evaluates the stemness of individual HSCs and incorporates temporal information to significantly improve prediction accuracy. This platform marks a paradigm shift from "identification" to "prediction", enabling us to forecast HSC status by analyzing their past temporal kinetics.

cell biology↗

bioRxiv2018

Akt kinases are required for efficient feeding by macropinocytosis in Dictyostelium

Macropinocytosis is an actin-driven process of large-scale, non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum. In Dictyostelium, macropinocytic cups are organised by macropinocytic patches in the plasma membrane. These contain activated Ras, Rac and PI(3,4,5)P3 and direct actin polymerisation to their periphery. Here, we show that a classical (PkbA) and a variant (PkbR1) Akt protein kinase acting downstream of PI(3,4,5)P3 are together are near-essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Akt targets identified by phosphoproteomics were highly enriched in small G-protein regulators, including the RhoGAP GacG. GacG knockout mutants make few macropinosomes but instead redeploy their cytoskeleton from macropinocytosis to motility, moving rapidly but taking up little fluid. The function of Akt in cell feeding through control of macropinosome size has implications for cancer cell biology.\n\nSummary statementDictyostelium amoebae feed by macropinocytosis in a PIP3 dependent manner. In the absence of PI3-kinases or the downstream Akt protein kinases, cells have smaller macropinosomes and nearly abolished fluid uptake.

cell biology↗

bioRxiv2019

Pac1/LIS1 promotes an uninhibited conformation of dynein that coordinates its localization and activity

Cytoplasmic dynein is a minus end-directed microtubule motor that transports myriad cargos in various cell types and contexts. How dynein is regulated to perform all these activities with a high degree of spatial and temporal precision is unclear. Recent studies have revealed that human dynein-1 and dynein-2 can be regulated by a mechanism of autoinhibition, whereby intermolecular contacts limit motor activity. Whether this autoinhibitory mechanism is conserved throughout evolution, whether it can be affected by extrinsic factors, and its precise role in regulating cellular dynein activity remain unknown. Here, we use a combination of negative stain EM, single molecule motility assays, genetic, and cell biological techniques to show that the autoinhibitory conformation is conserved in budding yeast, and it plays an important role in coordinating dynein localization and function in cells. Moreover, we find that the Lissencephaly-related protein, LIS1 (Pac1 in yeast) plays an important role in regulating this autoinhibitory conformation of dynein. Specifically, our studies demonstrate that rather than inhibiting dynein motility, Pac1/LIS1 promotes dynein activity by stabilizing the uninhibited conformation, which ensures appropriate localization and activity of dynein in cells.

cell biology↗

bioRxiv2021

Enabling high throughput target-based drug discovery in 3D cell cultures through novel bioprinting workflow

Advanced three-dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter Rastrum with the high throughput screening assay AlphaLISA, we demonstrate the utility of the workflow in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3{beta} phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.

cell biology↗

bioRxiv2022

Improved methods for bulk cultivation and fixation of Loxodes ciliates for fluorescence microscopy

Loxodes is one of the best ecologically characterized ciliate genera with numerous intriguing physiological abilities, including gravity-sensing organelles and nitrate respiration. However, these cells have been considered challenging to cultivate in bulk, and are poorly preserved by conventional fixatives used for fluorescence microscopy. Here we describe methods to grow and harvest Loxodes cells in bulk with liquid soil extract medium, as well as a new fixative called ZFAE (zinc sulfate, formaldehyde, acetic acid, ethanol) that can fix Loxodes cells more effectively than buffered formaldehyde or methanol. We show that ZFAE is compatible with immunofluorescence and the nuclear stain DAPI. Loxodes is thus now amenable to long-term maintenance, large-scale growth, and modern cell biology investigations of monoclonal strains in laboratory conditions.

cell biology↗

bioRxiv2019

Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors

KIF1A is a kinesin-family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules. In humans, more than ten point mutations in KIF1A are associated with the motor neuron disease, hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus, a clear molecular explanation for how KIF1A mutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found that KIF1A mutations associated with the pure form of spastic paraplegia hyperactivate motility of the KIF1A motor. Introduction of the corresponding mutations into Caenorhabditis elegans KIF1A homologue unc-104 revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyper-activation of kinesin motor activity, rather than its loss-of-function, is a novel cause of motor neuron disease in humans.\n\nSignificance StatementAnterograde axonal transport supplies organelles and protein complexes throughout axonal processes to support neuronal morphology and function. It has been observed that reduced anterograde axonal transport is associated with neuronal diseases. In contrast, here we show that particular disease-associated mutations in KIF1A, an anterograde axonal motor for synaptic vesicle precursors, induce hyperactivation of KIF1A motor activity and increased axonal transport of synaptic vesicle precursors. Our results advance the knowledge of the regulation of motor proteins and axonal transport and cell biology of motor neuron diseases.

cell biology↗

bioRxiv2019

ER transmembrane protein TMTC3 contributes to O-mannosylation of E-cadherin, Cellular Adherence and Embryonic Gastrulation

Protein glycosylation plays essential roles in protein structure, stability and activity such as cell adhesion. The cadherin superfamily of adhesion molecules carry O-linked mannose glycans at conserved sites and it was recently demonstrated that the TMTC1-4 genes contribute to the addition of these O-linked mannoses. Here, biochemical, cell biological and organismal analysis was used to determine that TMTC3 supports the O-mannosylation of E-cadherin, cellular adhesion and embryonic gastrulation. Using genetically engineered cells lacking all four TMTC genes, overexpression of TMTC3 rescued O-linked glycosylation of E-cadherin and cell adherence. The knockdown of the Tmtcs in Xenopus laevis embryos caused a delay in gastrulation that was rescued by the addition of human TMTC3. Mutations in TMTC3 have been linked to neuronal cell migration diseases including Cobblestone lissencephaly. Analysis of TMTC3 mutations associated with Cobblestone lissencephaly found that three of the variants exhibit reduced stability and missence mutations were unable to complement TMTC3 rescue of gastrulation in Xenopus embryo development. Our study demonstrates that TMTC3 regulates O-linked glycosylation and cadherin-mediated adherence, providing insight into its effect on cellular adherence and migration, as well the basis of TMTC3-associated Cobblestone lissencephaly.

cell biology↗

bioRxiv2025

MAGNET: an all-in-one foundation model for cross-modal and cross-dimensional microscopic image restoration

The application of emerging Foundation models in current microscopy has remained limited due to task-specific model designs, strong modality dependence and insufficient robustness under complex degradations. Here, we introduce MAGNET (Microscopic All-in-one General fouNdation model for imagE resToration), the first end-to-end All-in-One foundation model designed for universal microscopic image restoration. MAGNET integrates multi-task, cross-modal and cross-dimensional (2D + 3D) image restoration capabilities within a single unified architecture, and it is specifically designed to handle composite degradations in practical imaging systems. The framework comprises three components: (i) a task-aware, prompt-guided feature enhancement module for adaptive learning across diverse degradations; (ii) an LIIF-based reconstruction module enabling continuous, resolution-adaptive restoration; and (iii) a dimension-compatible triple-plane projection module supporting both 2D and 3D data. Trained on large-scale datasets spanning 5 major microscopy modalities (structured illumination, confocal, light-sheet, wide-field and two-photon) and 8 representative restoration tasks, MAGNET achieves state-of-the-art performance across super-resolution, denoising, deblurring, background suppression, isotropic reconstruction, aberration correction, de-scattering and virtual staining. Beyond supervised training, MAGNET supports direct inference on unseen systems, efficient few-shot fine-tuning and self-supervised test-time optimization, enabling robust performance across diverse data and imaging conditions. MAGNET serves as a unified, generalizable, and scalable computational framework for microscopy, enabling more efficient and reliable downstream analyses in cell biology, neuroscience, and pathology.

cell biology↗

bioRxiv2026

Fast single molecule localization microscopy in 3D

Super-resolution methods, such as single molecule localisation microscopy (SMLM), can determine the distribution of specific proteins at the nanometre scale. However, three-dimensional (3D) SMLM is typically limited to thin sections and requires prohibitively long acquisition times to image complete cells. Here we introduce a 3D SMLM imaging modality, Intrinsic3D, that combines the speed and optical sectioning of oblique plane microscopy (OPM) with the nanometre resolution of SMLM to determine the position of single molecule emitters throughout complete volumes more than an order-of-magnitude faster over regular DNA-PAINT. By scanning the oblique light sheet across the volume and combining localization information from two or more adjacent planes, an emitter's x, y and z position is determined, eliminating the need for post-processing steps to stitch multiple image stacks together. We demonstrate resolution and speed by imaging nuclear pore complexes in U2OS cells and Drosophila egg chambers. As a proof-of-concept, we map the 3D distribution of the cell-adhesion protein DE-cadherin in the Drosophila follicular epithelium and show that spot adherens junctions are enriched near tricellular junctions and contain more E-cadherin than the apical adherens junctions, suggesting that spot junctions play a wider role in cell-cell adhesion than previously appreciated. Finally, we examine the organization of Gliotactin, a tricellular septate junction protein relative to E-cadherin using exchange-PAINT. These investigations demonstrate that Intrinsic3D delivers rapid 3D protein mapping in whole cells. Combining Intrinsic3D with existing multiplex and resolution enhancing methods (e.g. exchange-PAINT and RESI) will enable multicolour, 3D in situ spatial proteomics in tissue samples and bridge the gap between SMLM and systems-level cell biology.

cell biology↗

bioRxiv2020

Wnt-and Glutamate-receptors orchestrate stem cell dynamics and asymmetric cell division

The Wnt-pathway is part of a signalling network that regulates many aspects of cell biology. Recently we discovered crosstalk between AMPA/Kainate-type ionotropic glutamate receptors (iGluRs) and the Wnt-pathway during the initial Wnt3a-interaction at the cytonemes of embryonic stem cells (ESCs). Here, we demonstrate that this crosstalk persists throughout the Wnt3a-response in ESCs. Both AMPA- and Kainate-receptors regulate early Wnt3a-recruitment, dynamics on the cell membrane, and orientation of the spindle towards a Wnt3a-source at mitosis. AMPA-receptors specifically are required for segregating cell fate components during Wnt3a-mediated asymmetric cell division (ACD). Using Wnt-pathway component knockout lines, we determine that Wnt co-receptor Lrp6 has particular functionality over Lrp5 in cytoneme formation, and in facilitating ACD. Both Lrp5 and 6, alongside pathway effector {beta}-catenin act in concert to mediate the positioning of the dynamic interaction with, and spindle orientation to, a localized Wnt3a-source. Wnt-iGluR crosstalk may prove pervasive throughout embryonic and adult stem cell signalling.

cell biology↗

bioRxiv2022

The C. elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator

How serine/threonine phosphatases are spatially and temporally tuned by regulatory subunits is a fundamental question in cell biology. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins (ASPPs) are protein phosphatase 1 (PP1) binding partners associated with cardiocutaneous diseases. ASPPs localize PP1 to cell-cell junctions, but how ASPPs localize and whether they regulate PP1 activity in vivo is unclear. Through a C. elegans genetic screen, we find that loss of the ASPP homolog, APE-1, suppresses a pathology called jowls, providing us with an in vivo assay for APE-1 activity. Using structure-function analysis, we discover that APE-1s N-terminal half directs the APE-1-PP1 complex to intercellular junctions. Additionally, we isolated mutations in highly conserved residues of APE-1s ankyrin repeats that suppress jowls yet do not preclude PP1 binding, implying ASPPs do more than simply localize PP1. Indeed, in vivo reconstitution of APE-1 suggests the ankyrin repeats modulate phosphatase output, a function we find to be conserved among vertebrate homologs.

cell biology↗

bioRxiv2022

Establishment of a novel fetal ovine heart cell line by spontaneous cell fusion

We established a unique immortal cell line designated, FOH-SA, by serial passage of fetal ovine heart cells. In a novel phenomenon in cell biology, we demonstrated that the immortalization of the line supervened as a result of spontaneous cellular and nuclear fusion of two morphologically distinct cardiocytes in passage 29. Fused cells gave progeny cells which grew into multicellular filaments. Trypsinization of the filamentous multicellular growth gave the immortal heart cell line. Comparative single nucleotide polymorphism (SNP) genotyping of the cell line before and after cell fusion revealed a large scale genetic conversion resulting in 96% homozygosity in SNPs genotypes in the progeny cells. Partial sequencing of the mitochondrial (mt) genome of the cell line revealed the occurrence of large mutational events in the control region, the tRNA-Phe and 12S rRNA genes of the mt genome of progeny cells. The cell line was found permissive to sheep pox, peste des petits ruminants (PPR), lumpy skin disease (LSD), rift valley fever (RVF), and camel pox viruses. This study would resolve the over half a century mystery of how the African Green Monkey cell line (VERO) had evolved. We authenticated the cell line at the European Collection of Authenticated Cell Cultures (ECACC) and deposited it at the American Type Culture Collection (ATCC) for the purpose of patenting under the Budapest Treaty.

cell biology↗

bioRxiv2018

RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction point

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 bound to its target loci, where it opened chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closed these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal was constitutively activated, RUNX3 inhibited cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.

cell biology↗

bioRxiv2019

Comparative analysis of diverse cell states establishes an epigenetic basis for inferring regulatory genes governing cell identity

Determining genes orchestrating cell differentiation in development and disease remains a fundamental goal of cell biology. This study establishes a genome-wide metric based on the gene-repressive tri-methylation of histone 3 lysine 27 (H3K27me3) across hundreds of diverse cell types to identify genetic regulators of cell differentiation. We introduce a computational method, TRIAGE, that uses discordance between gene-repressive tendency and expression to identify genetic drivers of cell identity. We apply TRIAGE to millions of genome-wide single-cell transcriptomes, diverse omics platforms, and eukaryotic cells and tissue types. Using a wide range of data, we validate TRIAGEs performance for identifying cell-type specific regulatory factors across diverse species including human, mouse, boar, bird, fish, and tunicate. Using CRISPR gene editing, we use TRIAGE to experimentally validate RNF220 as a regulator of Ciona cardiopharyngeal development and SIX3 as required for differentiation of endoderm in human pluripotent stem cells. A record of this papers Transparent Peer Review process is included in the Supplemental Information.

cell biology↗

bioRxiv2020

High-Efficiency Electroporation of Chytrid Fungi

Two species of parasitic fungi from the phylum Chytridiomycota (chytrids) are annihilating global amphibian populations. These chytrid species--Batrachochytrium dendrobatidis and B. salamandrivorans--have high rates of mortality and transmission. Upon establishing infection in amphibians, chytrids rapidly multiply within the skin and disrupt their hosts vital homeostasis mechanisms. Current disease models suggest that chytrid fungi locate and infect their hosts during a motile, unicellular zoospore life stage. Moreover, other chytrid species parasitize organisms from across the tree of life, making future epidemics in new hosts a likely possibility. Efforts to mitigate the damage and spread of chytrid disease have been stymied by the lack of knowledge about basic chytrid biology and tools with which to test molecular hypotheses about disease mechanisms. To overcome this bottleneck, we have developed high-efficiency delivery of molecular payloads into chytrid zoospores using electroporation. Our electroporation protocols result in payload delivery to between 75-97% of living cells of three species: B. dendrobatidis, B. salamandrivorans, and a non-pathogenic relative, S. punctatus. This method lays the foundation for molecular genetic tools needed to establish ecological mitigation strategies and answer broader questions in evolutionary and cell biology.

cell biology↗

bioRxiv2020

Overlapping roles of JIP3 and JIP4 in promoting axonal transport of lysosomes in human iPSC-derived neurons

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimers disease-related amyloid precursor protein (APP)-derived A{beta}42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC-derived neurons for the investigation of neuronal cell biology and pathology.

cell biology↗

bioRxiv2020

Automated tracking of S. pombe spindle elongation dynamics

The mitotic spindle is a microtubule-based machine that pulls the two identical sets of chromosomes to opposite ends of the cell during cell division. The fission yeast Schizosaccharomyces pombe is an important model organism for studying mitosis due to its simple, stereotyped spindle structure and well-established genetic toolset. S. pombe spindle length is a useful metric for mitotic progression, but manually tracking spindle ends in each frame to measure spindle length over time is laborious and can limit experimental throughput. We have developed an ImageJ plugin that can automatically track S. pombe spindle length over time and replace manual or semi-automated tracking of spindle elongation dynamics. Using an algorithm that detects the principal axis of the spindle and then finds its ends, we reliably track the length and angle of the spindle as the cell divides. The plugin integrates with existing ImageJ features, exports its data for further analysis outside of ImageJ, and does not require any programming by the user. Thus, the plugin provides an accessible tool for quantification of S. pombe spindle length that will allow automatic analysis of large microscopy data sets and facilitate screening for effects of cell biological perturbations on mitotic progression.

cell biology↗