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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2024

AKT-mediated phosphorylation of TSC2 controls stimulus and tissue-specific mTORC1 signaling and organ growth

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates diverse intracellular and extracellular growth signals to regulate cell and tissue growth. How the molecular mechanisms regulating mTORC1 signaling established through biochemical and cell biological studies function under physiological states in specific mammalian tissues are unknown. Here, we characterize a genetic mouse model lacking the 5 phosphorylation sites on the tuberous sclerosis complex 2 (TSC2) protein through which the growth factor-stimulated protein kinase AKT can active mTORC1 signaling in cell culture models. These phospho-mutant mice (TSC2-5A) are developmentally normal but exhibit reduced body weight and the weight of specific organs, such as brain and skeletal muscle, associated with cell intrinsic decreases in growth factor-stimulated mTORC1 signaling. The TSC2-5A mouse model demonstrates that TSC2 phosphorylation is a primary mechanism of mTORC1 activation in some, but not all, tissues and provides a genetic tool to facilitate studies on the physiological regulation of mTORC1.

cell biology↗

bioRxiv2026

A Glucan Synthase-Remodeler Module Organizes Branched Glucan Assembly in the Fungal Cell Wall

The fungal cell wall is an essential extracellular matrix that underpins growth, morphogenesis, and pathogenesis. Cell wall construction requires numerous enzymes that synthesize and remodel extracellular polymers, yet the principles governing their spatial and functional organization remain unclear. In the fission yeast Schizosaccharomyces pombe, we identify Ghs2, a predicted glycoside hydrolase 16 (GH16) domain-containing transmembrane protein, as an obligate binding partner of the {beta}-1,3-glucan synthase Bgs3. Ghs2 and Bgs3 co-localize at sites of polarized growth and physically associate in vivo. Structure-guided modeling positions the Ghs2 GH16 domain proximal to the predicted Bgs3 glucan extrusion pore, suggesting coordinated polymer synthesis and remodeling. Solid-state NMR analyses demonstrate that both Ghs2 and Bgs3 are required for the proper accumulation of branched {beta}-1,3-glucan. Together with genetic and cell biological evidence, these findings support a model in which Bgs3 synthesizes linear {beta}-1,3-glucan and Ghs2 subsequently introduces {beta}-1,6-linked branch points onto the nascent polymer. More broadly, we propose that synthase-modifier pairs may act together to shape polymer architecture during cell wall assembly.

cell biology↗

bioRxiv2024

Metadichol induces CD14 Glycoprotein Expression in Human Embryonic Stem Cells and Fibroblasts

CD14, or cluster of differentiation 14, is a glycoprotein essential to the immune system and is found primarily on monocytes, macrophages, and other immune cells. Despite its importance, there are no examples in the literature of small compounds that can induce multifold expression of CD14 in human embryonic stem cells (hESCs) or fibroblasts. This study addresses this gap by exploring the potential of metadichol, a nanoemulsion of long-chain fatty alcohols, to induce CD14 expression in hESCs. Using quantitative real-time PCR (qRT{square}PCR) and Western blotting techniques, we showed that metadichol significantly upregulated CD14 expression by seventeen -fold in hESCs but downregulated it in fibroblasts. This novel finding indicates that metadichol can modulate CD14 expression in a cell type-specific manner, highlighting its potential for enhancing stem cell-based therapeutics and advancing our understanding of stem cell biology. The implications of these findings are substantial, suggesting new directions for research into the immune modulatory functions of hESCs and their potential applications in regenerative medicine. Our work highlights the potential of metadichol as a powerful tool for modulating CD14 expression in stem as well as somatic cells marking a significant step forward in the field of stem cell research and therapeutic development.

cell biology↗

bioRxiv2019

Changes in endolysosomal organization define a pre-degenerative state in the crumbs mutant Drosophila retina

Mutations in the epithelial polarity gene crumbs (crb) lead to retinal degeneration in Drosophila and in humans. The overall morphology of the retina and its deterioration in Drosophila crb mutants has been well-characterized, but the cell biological origin of the degeneration is not well understood. Degenerative conditions in the retina and elsewhere in the nervous system often involve defects in degradative intracellular trafficking pathways. So far, however, effects of crb on the endolysosomal system, or on the spatial organization of these compartments in photoreceptor cells have not been described. We therefore asked whether photoreceptors in crb mutants exhibit alterations in endolysosomal compartments under pre-degenerative conditions, where the retina is still morphologically intact. Data presented here show that, already well before the onset of degeneration, Arl8, Rab7, and Atg8-carrying endolysosomal and autophagosomal compartments undergo changes in morphology and positioning with respect to each other in crb mutant retinas. We propose that these changes may be early signs of the degeneration-prone condition in crb retinas.

cell biology↗

bioRxiv2024

Parameters for sensing lipid loosening at the inner nuclear membrane by amphipathic helices

Amphipathic helices (AHs) are ubiquitous protein motifs that modulate targeting to organellar membranes by sensing differences in bulk membrane properties. However, the adaptation between membrane-targeting AHs and the nuclear membrane environment that surrounds the genome is poorly understood. Here, we computationally screened for candidate AHs in a curated list of characterized and putative human inner nuclear membrane (INM) proteins. Cell biological and in vitro experimental assays combined with computational calculations demonstrated that AHs detect lipid packing defects over electrostatics to bind to the INM, indicating that the INM is loosely packed under basal conditions. Membrane tension resulting from hypotonic shock further promoted AH binding to the INM, whereas cell-substrate stretch did not enhance recruitment of membrane tension-sensitive AHs. Together, our work demonstrates the rules driving lipid-protein interactions at the INM, and its implications in the response of the nucleus to different stimuli.

cell biology↗

bioRxiv2023

A Narrow Range of Transcript-error Rates Across the Tree of Life

The expression of genomically-encoded information is not error-free. Transcript-error rates are dramatically higher than DNA-level mutation rates, and despite their transient nature, the steady-state load of such errors must impose some burden on cellular performance. However, a broad perspective on the degree to which transcript-error rates are constrained by natural selection and diverge among lineages remains to be developed. Here, we present a genome-wide analysis of transcript-error rates across the Tree of Life using a modified rolling-circle sequencing method, revealing that the range in error rates is remarkably narrow across diverse species. Transcript errors tend to be randomly distributed, with little evidence supporting local control of error rates associated with gene-expression levels. A majority of transcript errors result in missense errors if translated, and as with a fraction of nonsense transcript errors, these are underrepresented relative to random expectations, suggesting the existence of mechanisms for purging some such errors. To quantitatively understand how natural selection and random genetic drift might shape transcript-error rates across species, we present a model based on cell biology and population genetics, incorporating information on cell volume, proteome size, average degree of exposure of individual errors, and effective population size. However, while this model provides a framework for understanding the evolution of this highly conserved trait, as currently structured it explains only 20% of the variation in the data, suggesting a need for further theoretical work in this area.

evolutionary biology↗

bioRxiv2024

da_Tracker: Automated workflow for high throughput single cell and single phagosome tracking in infected cells.

Time-lapse microscopy has emerged as a crucial tool in cell biology, facilitating a deeper understanding of dynamic cellular processes. While existing tracking tools have proven effective in detecting and monitoring objects over time, the quantification of signals within these tracked objects often faces implementation constraints. In the context of infectious diseases, the quantification of signals at localized compartments within the cell and around intracellular pathogens can provide even deeper insight into the interactions between the pathogen and host cell organelles. Existing quantitative analysis at a single-phagosome level remains limited and dependent on manual tracking methods. We developed a near-fully automated workflow that performs with limited bias, high-throughput cell segmentation and quantitative tracking of both single cell and single bacterium/phagosome within multi-channel, z-stack, time-lapse confocal microscopy videos. We took advantage of the PyImageJ library to bring Fiji functionality into a Python environment and combined deep-learning-based segmentation from Cellpose with tracking algorithms from Trackmate. Our workflow provides a versatile toolkit of functions for measuring relevant signal parameters at the single-cell level (such as velocity or bacterial burden) and at the single-phagosome level (i.e. assessment of phagosome maturation over time). Its capabilities in both single-cell and single-phagosome quantification, its flexibility and open-source nature should assist studies that aim to decipher for example the pathogenicity of bacteria and the mechanism of virulence factors that could pave the way for the development of innovative therapeutic approaches.

cell biology↗

bioRxiv2026

A multimodal perturbation atlas defines the phenotypic resolution of cellular morphology.

Modeling cellular behavior requires measurements that capture how cells evolve across time, environments, and interventions. Microscopy is uniquely suited to this goal: it is non-destructive and can be applied to living cells in their native context. Yet its phenotypic resolving power remains incompletely characterized relative to molecular assays. Here, we present a multimodal perturbation atlas of 1,000 pooled CRISPR knockouts in A549 cells, profiled by fluorescence microscopy (42 live, 13 fixed markers), label-free quantitative phase imaging of the same live cells (at single timepoints), and single-cell RNA sequencing (scRNA-seq). We develop deep learning frameworks to interpret the rich cell-biological signatures in these ~65M single-cell profiles. At matched reagent cost, phase imaging exceeds the phenotypic resolution of both fluorescence imaging and scRNA-seq, and more reliably recovers higher-order pathway organization. These results establish intrinsic morphology as a high-precision readout of cellular state, and lay a foundation for live-cell profiling of phenotypic trajectories.

systems biology↗

bioRxiv2023

The Effect of E-Cigarettes on the Human Heart Studied using Cardiomyocytes Generated from Induced Pluripotent Stem Cells

Although many dangerous effects of smoking e-cigarettes on lungs have come to light, limited and only qualitative efforts have been made to analyze the impact of e-cigarettes on the human heart directly. In this study, we experimentally determined e-liquid cardiotoxicity in both healthy cells and cells with long QT syndrome by treating healthy and diseased human cardiomyocytes with e-liquids with varying nicotine concentrations. These cardiomyocytes were generated from human induced pluripotent stem cells. The cardiomyocytes were divided into 5 groups, a control group and 4 test groups, each treated with e-liquid containing varying amounts of nicotine between 0% and 70%. The cells biological indicators such as heart rate, pulse pressure, essential protein concentration, and metabolic activity, were measured and characterized using three different functional assays: contractility, Western blot, and viability, and tracked over 4 days. The results demonstrated that acute exposure to e-liquid led to tachycardia, hypertension, decreased protein levels, and cell death. The rate of cardiotoxicity increases with higher nicotine concentrations. The basal fluid also showed non-negligible toxicity. Under identical conditions, the functionality of the diseased heart cells declined at a faster rate compared to healthy cells. Overall, this work establishes the harmful physiological effects of e-cigarettes on the human heart quantitatively.

cell biology↗

bioRxiv2019

Defined extracellular ionic solutions to study and manipulate the cellular resting membrane potential

All cells possess an electric potential across their plasma membranes. While familiar in the context of excitable cells such as neurons, healthy non-excitable cells are also able to generate and receive bioelectric signals. The cellular resting membrane potential (RMP) regulates many factors in cell homeostasis, such as cell proliferation, differentiation and apoptosis. It is therefore critical to develop simple strategies to measure, manipulate and characterize this feature. Current studies to evaluate RMP rely on the patch clamp approach, which is technically challenging, low-throughput and not widely available to the scientific community. Here, we present a relatively simple methodology to functionally study the role of RMP in non-excitable cells by modulating it pharmacologically, and using a voltage-sensitive dye to characterize the contribution of individual ions to the RMP. Specifically, we define protocols for using extracellular solutions in which permeable ions (Na+, Cl- and K+) are substituted with non-permeable ions (N-Methyl-D-glucamine (NMDG), gluconate, choline, SO42-) to study and manipulate RMP in vitro. The resulting RMP modifications were assessed with both patch clamp and a voltage sensitive dye. Using an epithelial and cancer cell line, we demonstrate that the proposed ionic solutions can determine the relative contribution of ionic species in setting the RMP and be used to actively and selectively modify the RMP. The proposed method is simple and reproducible and will make the study of bioelectricity more readily available to the cell biology community by enabling functional modulation of RMP in most cellular assays.\n\nAuthor Disclosure StatementsNo competing financial interests exist.

cell biology↗

bioRxiv2023

An atypical endomembrane localized CNL-type immune receptor with a conserved deletion in the N-terminal signaling domain functions in cell death and immunity

Plants have evolved intracellular nucleotide-binding leucine rich repeat receptors (NLRs) to induce a superior immune response. Upon activation, coiled-coil (CC) domain containing NLRs (CNLs) oligomerize to form apparent cation channels that promote calcium influx and cell death induction, with the alpha-1 helix of the individual CC domains penetrating membranes. Some members of a monophyletic subclass of CNLs, the ancient and autonomous NLRs (ANLs), are characterized by putative N- myristoylation and S-acylation sites at the N-terminus of their CCG10/GA domain, potentially mediating permanent membrane association. Whether these Potentially Membrane Localized NLRs (PMLs) mediate cell death upon activation in a similar way as reported for other CNLs has been unknown. We integrated phylogenetic, cell- biological, and functional studies to uncover the cell death function of an atypical but conserved Arabidopsis PML, PML5, which has a 113 amino acid deletion in its CCG10/GA domain. Active PML5 oligomers localize in Golgi membranes and the tonoplast, changes vacuolar morphology, and induce cell death, with the short N- terminus being sufficient for cell death. Mutant analysis supports a potential key role of PMLs in plant immunity. Similar deletions as in Arabidopsis PML5 are found in several Brassicales paralogs, pointing to the evolutionary importance of this innovation. PML5 is thus a naturally occurring CNL variant with a minimal signaling domain and its further study should help in understanding the functional importance of this minimal domain for NLR signaling.

plant biology↗

bioRxiv2022

The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division

ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems sequential changes in the composition of ESCRT-III polymers induced by the AAA ATPase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organised and remodelled in space and time in cells. Here, taking advantage of the relative simplicity of the ESCRT-III-dependent division system in Sulfolobus acidocaldarius, one of the closest experimentally tractable prokaryotic relative of eukaryotes, we use super-resolution microscopy and computational modelling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III-dependent membrane remodelling.

cell biology↗

bioRxiv2017

Reconstructing regulatory pathways by systematically mapping protein localization interdependency networks

A key goal of functional genomics is to elucidate how genes and proteins act together in space and time, wired as pathways, to control specific aspects of cell biological function. Here, we develop a method to quantitatively determine proteins localization interdependencies at high throughput. We show that this method can be used to systematically obtain weighted, signed and directional pathway relationships, and hence to reconstruct a detailed pathway wiring. As proof-of-principle, we focus on 42 factors that control cell polarity in fission yeast (Schizosaccharomyces pombe) and use high-throughput confocal microscopy and quantitative image analysis to reconstruct their Localization Interdependency Network (LIN). Through this approach we identify 554 pairwise interactions across the factors, including 98% putative new directed links. Validation of an unexpected interaction between two polarity factor subgroups - the polarity landmark proteins and the cell integrity pathway components - by orthogonal phenotyping demonstrates the power of the LIN approach in detecting subtle, systems-level causal connections.

cell biology↗

bioRxiv2025

PB-scope: Contrastive learning of dynamic processing body formation reveals undefined mechanisms of approved compounds

Membraneless organelles (MLOs) formed by liquid-liquid phase separation contribute to intracellular compartmentalization of specific biological functions. The dynamic regulation of MLOs is critical for RNA metabolism, stress response, and signal transduction. While the number and size of MLOs serve as valuable indicators of cellular states, leveraging their spatial distribution and diverse quantitative characteristics to infer cellular conditions has posed significant challenges. Here, we present processing body (PB)-scope: an unsupervised deep learning-based framework designed for imaging-based phenotypic screening of PB, one representative MLO. The model was trained on a substantial dataset comprising over 400,000 single-cell confocal microscopy images from a colon cancer cell line treated with 280 compounds. PB-scope enabled precise drug classification based on their effects on PB characteristics, including number and size. This artificial intelligence framework offers insights previously obscured by the dynamic and subtle nature of PBs. Notably, PB-scope identified drugs with a shared function in the JAK signaling pathway, a previously unrecognized regulator of PB dynamics. PB-scope holds the potential for adapting to the study of other MLOs, offering broad applicability in this emerging field of cell biology.

cell biology↗

bioRxiv2017

LINE-1 and the cell cycle: protein localization and functional dynamics

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology to aid its proliferation. However, a clear understanding of L1s lifecycle and the processes involved in restricting its insertion and its intragenomic spreading remains elusive. Here we identify modes of L1 proteins entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

cell biology↗

bioRxiv2021

Mod3D: A Low-Cost, Flexible Modular System of Live-Cell Microscopy Chambers and Holders

Live-cell microscopy imaging typically involves the use of high-quality glass-bottom chambers that allow cell culture, gaseous buffer exchange and optical properties suitable for microscopy applications. However, commercial sources of these chambers can add significant annual costs to cell biology laboratories. Consumer products in three-dimensional printing technology, for both Filament Deposition Modeling (FDM) and Masked Stereo Lithography (MSLA), have resulted in more biomedical research labs adopting the use of these devices for prototyping and manufacturing of lab plastic-based items, but rarely consumables. Here we describe a modular, live-cell chamber with multiple design options that can be mixed per experiment. Single reusable carriers and the use of biodegradable plastics, in a hybrid of FDM and MSLA manufacturing methods, reduce plastic waste. The system is easy to adapt to bespoke designs, with concept-to-prototype in a single day, offers significant cost savings to the users over commercial sources, and no loss in dimensional quality or reliability.

cell biology↗

bioRxiv2018

Urothelial organoids originate from Cd49f-High stem cells and display Notch-dependent differentiation capacity

The urothelium is a specialized stratified epithelium with unique structural and functional features. Understanding the mechanisms involved in urothelial stem cell biology and differentiation has been limited by the lack of methods for unlimited propagation. Here, we establish normal mouse urothelial organoid (NMU-o) cultures that can be maintained uninterruptedly for >1 year. Organoid growth is dependent on EGF and Wnt activators. High CD49f/ITGA6 expression features a subpopulation of organoid-forming urothelial stem cells expressing basal markers. On induction of differentiation, multilayered organoids show reduced layer number, acquire barrier function, and activate the urothelial program, including expression of uroplakins and tight junction components. Combined pharmacological modulation of PPAR{gamma} and EGFR was most potent driving cell differentiation. Transcriptome analysis of organoids widely validates the system, highlights the transcriptional networks involved, and reveals NOTCH signaling as a novel pathway required for normal urothelial organoid differentiation.

cell biology↗

bioRxiv2024

Calcitonin gene-related peptide and intermedin induce phosphorylation of p44/42 MAPK in primary human lymphatic endothelial cells in vitro

BackgroundCalcitonin gene-related peptide (CGRP) is a 37-amino acid peptide that belongs to the calcitonin family of peptides together with adrenomedullin (AM), adrenomedullin 2 (AM2), also known as intermedin (IMD), amylin (AMY) and calcitonin. Recent reports demonstrated the efficacy of drugs targeting CGRP signalling axis for the treatment of migraine, but also revealed significant side effects such as inflammation and microvascular complications, including aberrant neovascularisation in the skin. These findings highlight the importance of studying cellular targets of CGRP in human tissues. Recent studies using animal models implicate the role of CGRP in lymphangiogenesis and lymphatic vessel function. However, whether CGRP has direct effects on lymphatic endothelial cells (LEC) remains unknown. AimIn this study, we analysed phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) in proliferating primary human dermal LEC (HDLEC) stimulated with CGRP and compared with responses to AM2/IMD and AM. Materials and MethodsPrimary breast HDLEC from two female donors (39 and 46 years old) were cultured in vitro. Cell viability and proliferation of sub-confluent HDLEC were confirmed by MTS assay and Ki67 immunostaining. Phosphorylation of p44/42 MAPK in HDLEC treated with CGRP, AM2/IMD or AM at different time points (1-30 min) and concentrations (10-12-10-6M) was assessed by immunoblotting. All experiments have been performed at least 4 times. Statistical analysis was done using Shapiro-Wilk and Kruskal Wallis tests followed by uncorrected Dunns test, with p<0.05 interpreted as significant. ResultsCGRP induced p44/42 MAPK phosphorylation in a time- and dose-dependent manner in proliferating primary HDLEC from both donors. p44/42 MAPK phosphorylation was induced by all three peptides (10-6M) at 5-15 min (p<0.05), but only CGRP-stimulated effect was sustained at 30 min. CGRP consistently induced p44/42 MAPK phosphorylation at 10-7-10-6M (p<0.05) in HDLEC from both donors, whilst responses to AM2/IMD and AM varied. ConclusionsOur study demonstrates that CGRP and AM2/IMD have direct effects on proliferating primary HDLEC. These new findings reveal CGRP and AM2/IMD as novel regulators of lymphatic endothelial cell biology and warrant further investigation of their roles in pathologies associated with lymphatic function in the skin and other organs.

cell biology↗