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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2020

Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii

The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultra-pure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single cell transcriptomic analysis at 1-3 hours post-infection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the hosts earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent pro-inflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs, (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggests that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes. IMPORTANCEThis work performs the first transcriptomic analysis of U-I cells, captures the earliest stage of a host cells interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.

microbiology↗

bioRxiv2016

Length control of filamentous structures in cells by the limiting pool mechanism

How the size of organelles in cells is controlled despite a constant turnover of their constituent parts is a central problem in cell biology. A general mechanism has been proposed based on the idea that an organelle grows by self-assembly of molecular subunits that freely diffuse in the cytoplasm. Assembly continues until the available pool of subunits is depleted to the point when the stochastic addition and removal of subunits is balanced, leading to a structure of well-defined size. Here we focus on length control of multiple filamentous structures in cells, such as actin cables and flagella. Using queueing theory and computation we show that the limiting pool mechanism leads to three different phases of assembly, starting with a rapid growth phase when all filaments quickly accumulate a large number of available subunits. Then, the slower growing filamentous structures enter a disassembly phase as they gradually lose all of their subunits to the faster growing structures. Finally, when multiple, equivalent fast-growing filaments are present, their lengths undergo protracted diffusive dynamics due to the stochastic swapping of subunits between them. This eventually leads to a broad, power-law distribution of filament lengths in steady state. Our findings demonstrate that the limiting-pool mechanism is incapable of controlling lengths of multiple filamentous structures that are assembled from a common pool of subunits, and at best, can produce only one filament of a well-defined size. Overall, our theoretical results reveal physical limitations of the limiting-pool mechanism of organelle size control.\n\nSignificance StatementWhat determines the size of organelles in cells is a classic problem in cell biology. Recent experiments on mitotic spindles, and nucleolus have singled out the limiting-pool mechanism of size control. As these structures assemble, they deplete a finite pool of subunits present in the cell, thereby reducing the rate of subunit addition. Eventually the stochastic addition and removal of subunits are balanced and a well-defined size is achieved. We find that, while the limiting-pool mechanism does control the size of an individual structure, it fails when multiple structures are competing for the same pool of subunits. In that case we predict large size fluctuations and that the fastest growing structure takes up practically all the subunits from the pool.

Biophysics↗

bioRxiv2024

Evaluation of a Deep Learning Based Approach to Computational Label Free Cell Viability Quantification

One of the most common techniques found in a cell biology or tissue engineering lab is the cytotoxicity assay. This can be performed using a variety of different dyes and stains and various protocols to result in a clear indication of dead and live cells within a culture to quantify the viability of a culture and monitor for sudden drops or increases in viability by a drug, material, viral vector, etc introduced into the culture. This assay helps cell biologists determine the health of their culture and what toxicity added substances may add to the culture and whether they are appropriate and safe to use with human cells. However, many of the dyes and stains used for this process are eventually toxic to cells, rendering the cells useless after testing and preventing real time monitoring of the same culture over a period of hours or days. Computation biology is moving cell biology towards novel and innovative techniques such as in silico labeling and dye free labeling using deep learning algorithms. In this work, we investigate whether it is feasible to train a Resnet CNN model to detect morphological changes in human cells that indicate cell death in order to classify cells as live or dead without utilizing a stain or dye. This work also aims to train one CNN model to count all cells regardless of viability status to get a total cell count, and then one CNN model that specifically identifies and counts all of the dead cells for an accurate dead and live cell total by utilizing both pieces of data to determine a general viability percentage for the culture. Additionally, this work explores the use of various image enhancements to understand if this process helps or impedes the deep learning models in their detection of total cells and dead cells.

cell biology↗

bioRxiv2024

The N-terminus of the Chlamydia trachomatis effector Tarp engages the host Hippo pathway

Chlamydia trachomatis is an obligate, intracellular Gram-negative bacteria and the leading bacterial STI in the United States. Chlamydias developmental cycle involves host cell entry, replication within a parasitophorous vacuole called an inclusion, and induction of host cell lysis to release new infectious particles. During development, Chlamydia manipulates the host cell biology using various secreted bacterial effectors. The early effector Tarp is important for Chlamydia entry via its well-characterized C-terminal region which can polymerize and bundle F-actin. In contrast, not much is known about the function of Tarps N-terminus (N-Tarp), though this N-terminal region is present in many Chlamydia species. To address this, we use Drosophila melanogaster as an in vivo cell biology platform to study N-Tarp-host interactions. Drosophila development is well-characterized such that developmental phenotypes can be traced back to the perturbed molecular pathway. Transgenic expression of N-Tarp in Drosophila tissues results in phenotypes consistent with altered host Hippo signaling. The Salvador-Warts-Hippo pathway is a conserved signaling cascade that regulates host cell proliferation and survival during normal animal development. We studied N-Tarp function in larval imaginal wing discs, which are sensitive to perturbations in Hippo signaling. N-Tarp causes wing disc overgrowth and a concomitant increase in adult wing size, phenocopying overexpression of the Hippo co-activator Yorkie. N-Tarp also causes upregulation of Hippo target genes. Last, N-Tarp-induced phenotypes can be rescued by reducing the levels of Yorkie, or the Hippo target genes CycE and Diap1. Thus, we provide the first evidence that the N-terminal region of the Chlamydia effector Tarp is sufficient to alter host Hippo signaling and acts upstream of the co-activator Yorkie. Chlamydia alters host cell apoptosis during infection, though the exact mechanism remains unknown. Our findings implicate the N-terminal region of Tarp as a way to manipulate the host Hippo signaling pathway, which directly influences cell survival. Author SummaryChlamydia-infected cells are known to be resistant to apoptotic cues, facilitating the successful completion of its infectious cycle. The exact molecular mechanism of apoptosis inhibition is unknown and the search for secreted effectors that mediate this is ongoing. We developed Drosophila melanogaster as a platform to study Chlamydia effector function in vivo without the confounding influence of actual infection. Genetic tools make it easy to generate transgenic flies and express bacterial effectors of interest in any tissue, allowing for discovery of new function based on observed developmental phenotypes. Using this platform, we showed that the N-terminal region of the early effector Tarp intersects with the host Hippo pathway, causing upregulation of Hippo target genes. Interestingly, the Hippo pathway directly controls the expression of potent inhibitors of apoptosis (IAPs). Our findings support a possible link between a secreted effector, Tarp and its N-terminal region, and the Hippo pathway to block apoptosis during infection.

microbiology↗

bioRxiv2023

A Rab6 to Rab11 transition is required for dense-core granule and exosome biogenesis in Drosophila secondary cells

Secretory cells in glands and the nervous system frequently package and store proteins destined for regulated secretion in dense-core granules (DCGs), which disperse when released from the cell surface. Despite the relevance of this dynamic process to diseases such as diabetes and human neurodegenerative disorders, our mechanistic understanding is relatively limited, because of the lack of good cell models to follow the nanoscale events involved. Here, we employ the prostate-like secondary cells (SCs) of the Drosophila male accessory gland to dissect the cell biology and genetics of DCG biogenesis. These cells contain unusually enlarged DCGs, which are assembled in compartments that also form secreted nanovesicles called exosomes. We demonstrate that known conserved regulators of DCG biogenesis, including the small G-protein Arf1 and the coatomer complex AP-1, play key roles in making SC DCGs. Using real-time imaging, we find that the aggregation events driving DCG biogenesis are accompanied by a change in the membrane associated small Rab GTPases which are major regulators of membrane and protein trafficking in the secretory and endosomal systems. Indeed, a transition from trans-Golgi Rab6 to recycling endosomal protein Rab11, which requires conserved DCG regulators like AP-1, is essential for DCG and exosome biogenesis. Our data allow us to develop a model for DCG biogenesis that brings together several previously disparate observations concerning this process and highlights the importance of communication between the secretory and endosomal systems in controlling regulated secretion. Author summaryCells communicate with each other by releasing signalling molecules that bind receptors on target cells and alter their behaviour. Before their release, these signals are typically stored in condensed structures called dense-core granules (DCGs). DCGs are found in many animal species and their dysregulation is linked to several major human diseases, such as diabetes and neurodegenerative disorders. However, the mechanisms controlling DCG formation and secretion are only partly understood. Here we study this process in fruit flies using a secretory cell, which contains unusually large DCGs. We show that known regulators of DCG formation in mammals also control DCG production in these fly cells and identify new DCG assembly steps by following the process in living cells. Furthermore, we show that the cells secretory and recycling endosomal compartments must interact to induce the rapid condensation of proteins into a DCG, and that known regulators of DCG formation are needed for this crucial event to take place. Our work provides a platform from which to work out the molecular mechanisms that enable this critical secretory-endosomal interaction and probe its roles in diseases of secretion.

cell biology↗

bioRxiv2026

A novel role for TRPV1 in macrophage giant cell formation

TRPV1 (transient receptor potential vanilloid 1) is a non-selective cation channel with high permeability to Ca2+ and is best known for its roles in sensory signaling. However, its function in immune cell biology, particularly in macrophage fusion, remains unknown. Cell fusion is a critical process in both physiological and pathological contexts, including development, tissue remodeling, and the foreign body response (FBR) to implanted biomaterials. During FBR, macrophages undergo fusion to form multinucleated foreign body giant cells (FBGCs), which contribute to implant degradation and fibrotic encapsulation. Here, we identify TRPV1 as a key regulator of macrophage multinucleation and FBGC formation. We demonstrate that TRPV1 is endogenously expressed in bone marrow-derived macrophages (BMDMs) and is upregulated in response to fusogenic cytokines and inflammatory stimuli. Functionally, TRPV1 promotes matrix stiffness-dependent macrophage adhesion and spreading, indicating a role in mechanosensitive signaling. We show that TRPV1 is required for efficient macrophage fusion under both cytokine-driven and matrix stiffness-mediated conditions. Mechanistically, TRPV1 links extracellular mechanical cues and cytokine signaling to cytoskeletal remodeling, facilitating the actin reorganization necessary for cell fusion. Importantly, TRPV1 deficiency does not alter TRPV4-mediated Ca2+ signaling, demonstrating that TRPV1 operates independently of TRPV4, a known mechanosensitive channel implicated in FBR and FBGC formation. Collectively, these findings suggest TRPV1 as a previously unrecognized mechanosensitive regulator of macrophage fusion and FBGC formation. This work provides new insight into the molecular mechanisms governing FBR and identifies TRPV1 as a potential therapeutic target for improving biomaterial biocompatibility and mitigating fibrosis.

cell biology↗

bioRxiv2022

Assessment of 3D MINFLUX data for quantitative structural biology in cells revisited

Prakash and Curd provide a re-analysis1 of individual datasets taken from our report2 demonstrating MINFLUX 3D imaging in cells. Their evaluation confirms the unique localization precision provided by MINFLUX2,3 featuring a standard deviation of {sigma} = 1-3 nm. We appreciate their confirmation and also welcome the opportunity to clarify their remaining points. The hitherto almost unconceivable 3D localization precision attained by MINFLUX is likely to hold the key to an all-optical dynamical structural biology.

biophysics↗

bioRxiv2020

A single-cell RNA-seq Training and Analysis Suite using the Galaxy Framework

BackgroundThe vast ecosystem of single-cell RNA-seq tools has until recently been plagued by an excess of diverging analysis strategies, inconsistent file formats, and compatibility issues between different software suites. The uptake of 10x Genomics datasets has begun to calm this diversity, and the bioinformatics community leans once more towards the large computing requirements and the statistically-driven methods needed to process and understand these ever-growing datasets. ResultsHere we outline several Galaxy workflows and learning resources for scRNA-seq, with the aim of providing a comprehensive analysis environment paired with a thorough user learning experience that bridges the knowledge gap between the computational methods and the underlying cell biology. The Galaxy reproducible bioinformatics framework provides tools, workflows and trainings that not only enable users to perform one-click 10x preprocessing, but also empowers them to demultiplex raw sequencing from custom tagged and full-length sequencing protocols. The downstream analysis supports a wide range of high-quality interoperable suites separated into common stages of analysis: inspection, filtering, normalization, confounder removal and clustering. The teaching resources cover an assortment of different concepts from computer science to cell biology. Access to all resources is provided at the singlecell.usegalaxy.eu portal. ConclusionsThe reproducible and training-oriented Galaxy framework provides a sustainable HPC environment for users to run flexible analyses on both 10x and alternative platforms. The tutorials from the Galaxy Training Network along with the frequent training workshops hosted by the Galaxy Community provide a means for users to learn, publish and teach scRNA-seq analysis. Key PointsO_LISingle-cell RNA-seq has stabilised towards 10x Genomics datasets. C_LIO_LIGalaxy provides rich and reproducible scRNA-seq workflows with a wide range of robust tools. C_LIO_LIThe Galaxy Training Network provides tutorials for the processing of both 10x and non-10x datasets. C_LI

bioinformatics↗

bioRxiv2026

Benchmarking single-cell foundation models for aging biology

Single cell foundation models (scFMs) provide representations of cellular states, but their utility across biological questions in aging research remains unclear. We established a benchmark of cellular representations for aging research, evaluating ten general-purpose scFMs, three aging-specific models and conventional methods across five biological questions using more than 2.5 million single cell transcriptomes. Using frozen pretrained representations, Geneformer performed best among scFMs for chronological age prediction and age pseudotime concordance, although 2,000 highly variable genes achieved higher mean performance. Several scFMs captured positive molecular age shifts across three disease contexts, consistent with reported aging-associated changes. SCimilarity performed well for rare cellular state identification across out-of-distribution datasets, exceeding aging specific models and conventional baselines. At the gene level, scGPT showed the highest recovery of reference TF target interactions, including aging-related regulatory hubs. Overall, scFMs supported diverse aging analyses, but performance depended on the biological question, highlighting their utility for rare cellular state identification and regulatory analysis.

bioinformatics↗

bioRxiv2020

Fussing about fission: defining variety among mainstream and exotic apicomplexan cell division modes

Cellular reproduction defines life, yet our textbook-level understanding of cell division is limited to a small number of model organisms centered around humans. The horizon on cell division variants is expanded here by advancing insights on the fascinating cell division modes found in the Apicomplexa, a key group of protozoan parasites. The Apicomplexa display remarkable variation in offspring number, whether karyokinesis follows each S/M-phase or not, and whether daughter cells bud in the cytoplasm or bud from the cortex. We find that the terminology used to describe the various manifestations of asexual apicomplexan cell division emphasizes either the number of offspring or site of budding, which are not directly comparable features and has led to confusion in the literature. Division modes have been primarily studied in two human pathogenic Apicomplexa, malaria-causing Plasmodium spp. and Toxoplasma gondii, a major cause of opportunistic infections. Plasmodium spp. divide asexually by schizogony, producing multiple daughters per division round through a cortical budding process, though at several life-cycle nuclear amplifications are not followed by karyokinesis. T. gondii divides by endodyogeny producing two internally budding daughters per division round. Here we add to this diversity in replication mechanisms by considering the cattle parasite Babesia bigemina and the pig parasite Cystoisospora suis. B. bigemina produces two daughters per division round by a binary fission mechanism whereas C. suis produces daughters through both endodyogeny and multiple internal budding known as endopolygeny. In addition, we provide new data from the causative agent of equine protozoal myeloencephalitis (EPM), Sarcocystis neurona, which also undergoes endopolygeny but differs from C. suis by maintaining a single multiploid nucleus. Overall, we operationally define two principally different division modes: internal budding found in cyst-forming Coccidia (comprising endodyogeny and two forms of endopolygeny) and external budding found in the other parasites studied (comprising the two forms of schizogony, binary fission and multiple fission). Progressive insights into the principles defining the molecular and cellular requirements for internal versus external budding, as well as variations encountered in sexual stages are discussed. The evolutionary pressures and mechanisms underlying apicomplexan cell division diversification carries relevance across Eukaryota. Contribution to the FieldMechanisms of cell division vary dramatically across the Tree of Life, but the mechanistic basis has only been mapped for several model organisms. Here we present cell division strategies across Apicomplexa, a group of obligate intracellular parasites with significant impact on humans and domesticated animals. Asexual apicomplexan cell division is organized around assembly of daughter buds, but division forms differ in the cellular site of budding, number of offspring per division round, whether each S-phase follows karyokinesis and if mitotic rounds progress synchronously. This varies not just between parasites, but also between different life-cycle stages of a given species. We discuss the historical context of terminology describing division modes, which has led to confusion on how different modes relate to each other. Innovations in cell culture and genetics together with light microscopy advances have opened up cell biological studies that can shed light on this puzzle. We present new data for three division modes barely studied before. Together with existing data, we show how division modes are organized along phylogenetic lines and differentiate along external and internal budding mechanisms. We also discuss new insights into how the variations in division mode are regulated at the molecular level, and possess unique cell biological requirements.

microbiology↗

bioRxiv2020

Creating Clear and Informative Image-based Figures for Scientific Publications

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in three fields; plant sciences, cell biology and physiology (n=580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing.

bioinformatics↗

bioRxiv2023

No Transcriptional Compensation for Extreme Gene Dosage Imbalance in Fragmented Bacterial Endosymbionts of Cicadas

Bacteria that form long-term intracellular associations with host cells lose many genes, a process that often results in tiny, gene-dense, and stable genomes. Paradoxically, the same evolutionary processes that drive genome reduction and simplification may also sometimes cause genome expansion and complexification. A bacterial endosymbiont of cicadas, Hodgkinia cicadicola, exemplifies this paradox. In many cicada species, a single Hodgkinia lineage with a tiny, gene-dense genome has split into several interdependent cell and genome lineages. Each new Hodgkinia lineage encodes a unique subset of the ancestral unsplit genome in a complementary way, such that the collective gene contents of all lineages sum to the total found in the ancestral single genome. This splitting process creates genetically distinct Hodgkinia cells that must function together to carry out basic cellular processes, but also creates a gene dosage problem where some genes are encoded by only a small fraction of cells while others are much more abundant. Here, by sequencing DNA and RNA of Hodgkinia from different cicada species with different amounts of splitting - along with those of its structurally stable, unsplit partner endosymbiont Sulcia muelleri - we show that Hodgkinia does not transcriptionally compensate to rescue the wildly unbalanced gene and genome ratios that result from lineage splitting. We also find evidence that Hodgkinia has a reduced capacity for basic transcriptional control independent of the splitting process. Collectively, these findings reveal another layer of degeneration that further pushes the limits of canonical molecular and cell biology in Hodgkinia, and may in part explain its propensity to go extinct through symbiont replacement. SignificanceMany cicadas host two bacterial endosymbionts, Hodgkinia and Sulcia, which produce essential amino acids missing from the insects xylem sap diet. Following 100+ million years of strict host association, both bacteria have lost many genes and posses extremely tiny genomes. In some cicadas, Hodgkinia has split into multiple cell lineages, distributing its genes, with little respect to their function, among separate lineages present at (sometimes wildly) different abundances. We find no transcriptional response to this in Hodgkinia, resulting in similarly imbalanced mRNA abundances. We also find less control of transcription in Hodgkinia compared to Sulcia. Hodgkinias transcriptome embodies an extreme, even relative to other highly-reduced endosymbionts, and raises questions about how cell biology in multi-lineage Hodgkinia can function at all.

evolutionary biology↗

bioRxiv2023

Lipid nanodiscs as a template for high-resolution cryo-EM structures of peripheral membrane proteins

Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient binding to the membrane has a profound impact on protein function, serving to induce conformational changes and alter biochemical and biophysical parameters by increasing the local concentration of factors and restricting diffusion to two dimensions. Despite the centrality of the membrane in serving as a template for cell biology, there are few reported high-resolution structures of peripheral membrane proteins bound to the membrane. We analyzed the utility of lipid nanodiscs to serve as a template for cryo-EM analysis of peripheral membrane proteins. We tested a variety of nanodiscs and we report a 3.3 [A] structure of the AP2 clathrin adaptor complex bound to a 17-nm nanodisc, with sufficient resolution to visualize a bound lipid head group. Our data demonstrate that lipid nanodiscs are amenable to high-resolution structure determination of peripheral membrane proteins and provide a framework for extending this analysis to other systems.

biochemistry↗

bioRxiv2023

Monitoring single root hairs using a micro-chambered hydroponics system MiCHy reveals a new mode of action of the aminosteroid U73122

When using chemical probes and inhibitors for cell biological studies, the uniform and rapid access of the chemical to the sample is key for the accuracy of the analysis. Until now, however, this could only be accomplished by using expensive equipment and labor-intensive and often lengthy procedures. Here, we introduce MiCHy, a micro-chambered hydroponics system, as an accessible and versatile method for chemical treatments and demonstrate its use for imaging roots of Arabidopsis thaliana, the major model system for plant cell biological research. Using lipid biosensors in combination with established chemicals, such as the phosphoinositol 4-kinase (PI4K) inhibitor phenylarsine oxide and the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122, we demonstrate the suitability of MiCHy for studying the relationship between growth and lipid dynamics in root hairs. We further reveal novel effects of U73122 on the plasma membrane localization of phosphatidylinositol-4-phosphate 5-kinase 3 (PIP5K3), which is the first evidence demonstrating a role for U73122 besides its role as a PI-PLC inhibitor in plants.

plant biology↗

bioRxiv2019

Human antiviral protein MxA forms novel metastable membrane-less cytoplasmic condensates exhibiting rapid reversible \"crowding\"-driven phase transitions

Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles in the mammalian cell cytoplasm and nucleus. We report that the interferon (IFN)-inducible human \"myxovirus resistance protein A\" (MxA) forms membrane-less metastable condensates in the cytoplasm. Light and electron microscopy studies revealed that transient expression of HA- or GFP-tagged MxA in Huh7, HEK293T or Cos7 cells, or exposure of Huh7 cells to IFN-2a led to the appearance of MxA in the cytoplasm in membrane-less variably-sized spherical or irregular bodies, in filaments and even a reticulum. 1,6-Hexanediol treatment led to rapid disassembly of these condensates; however, FRAP revealed a relative rigidity with a mobile fraction of only 0.24{+/-}0.02 within condensates. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with GFP-MxA condensates. Remarkably, the cytoplasmic GFP-MxA condensates disassembled within 1-3 min of exposure of cells to hypotonic medium (40-50 milliosmolar) and reassembled within 0.5-2 min of re-exposure of cells to isotonic medium (310-325 milliosmolar) through multiple cycles. Mechanistically, the extent of cytoplasmic \"crowding\" regulated this phase-separation process. GFP-MxA condensates also included the DNA sensor protein cyclic GMP-AMP synthase (cGAS), another protein known to be associated with liquid-like condensates. Functionally, GFP-MxA expression inhibited DNA/cGAS-responsive ISG54-luciferase activity but enhanced relative inducibility of ISG54-luc by IFN-, revealing a physical separation between condensate- and cytosol-based signaling pathways in the cytoplasm. Taken together, the data reveal a new aspect of the cell biology of MxA in the cell cytoplasm.\n\nImportanceThe human interferon-inducible \"myxovirus resistance protein A\" (MxA), which displays antiviral activity against several RNA and DNA viruses, exists in the cytoplasm in phase-separated membrane-less metastable condensates of variably-sized spherical or irregular bodies, in filaments and even in a reticulum. MxA condensate formation appeared necessary but not sufficient for antiviral activity. Remarkably, MxA condensates showed the unique property of rapid (within 1-3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions Mechanistically, these phase transitions were regulated by the extent of cytoplasmic \"crowding.\" Moreover, GFP-MxA condensates included the DNA sensor protein cyclic GMP-AMP synthase (cGAS). Functionally, GFP-MxA expression inhibited DNA/cGAS-responsive ISG54-luciferase activity but enhanced inducibility of ISG54-luc by IFN-, revealing a biological distinction between condensate- and cytosol-based signaling pathways. Since intracellular edema and ionic changes are hallmarks of cytopathic viral effects, the rapid hypotonicity-driven disassembly of MxA condensates may modulate MxA.function during virus infection.

cell biology↗

bioRxiv2024

Spatial and morphological organization of mitochondria across a connectome

Neuronal function depends critically on the cell biological organization of mitochondria, which regulate calcium signals and produce energy, among other roles. However, little is known about how mitochondria are organized within the circuits of neurons that make up each brain. To uncover the systematic rules that govern mitochondria shape and position in a connectome, we analyzed the morphological and spatial organization of more than 100,000 mitochondria in over 1,000 visual projection neurons in the Drosophila connectome. We found that mitochondrial shape and size differ systematically between cell types, and are distinct enough between cell types to serve as an identifying fingerprint. Moreover, we derived three quantitative rules that describe how mitochondria are positioned within neurons relative to synapses and other subcellular features: (1) they are positioned with a precision of 2-3 microns; (2) their relative preference for pre- and postsynaptic sites and other subcellular features differs between axons and dendrites; (3) their positions were specialized to different cell types. These organizing rules correlated with functional and anatomical properties of the cells, including visual responses and input connectivity. We also find that, in the flys olfactory associative learning circuits, mitochondria are enriched at presynapses to particular postsynaptic cells by accumulating in functional sub-compartments of axons. Overall, our findings reveal a robust set of organizing principles for mitochondria within and between cells, uncovering cell biology that maps onto the organization of the connectome and adding new dimensions for understanding circuit function in the connectome.

neuroscience↗

bioRxiv2023

Information- and Communication-Centric Approach in Cell Metabolism for Analyzing Behavior of Microbial Communities

Microorganisms naturally form community ecosystems to improve fitness in diverse environments and conduct otherwise intractable processes. Microbial communities are therefore central to biogeochemical cycling, human health, agricultural productivity, and technologies as nuanced as nanotechnology-enabled devices; however, the combinatorial scaling of exchanges with the environment that predicate community functions are experimentally untenable. Several computational tools have been presented to capture these exchanges, yet, no attempt has been made to understand the total information flow to a community from its environment. We therefore adapted a recently developed model for singular organisms, which blends molecular communication and the Shannon Information theory to quantify information flow, to communities and exemplify this expanded model on idealized communities: one of Escherichia coli (E. coli) and Pseudomonas fluorescens to emulate an ecological community and the other of Bacteroides thetaiotaomicron (B. theta) and Kleb Ciella to emulate a human microbiome interaction. Each of these sample communities exhibit critical syntrophy in certain environmental conditions, which should be evident through our community mutual information model. We further explored alternative frameworks for constructing community genome-scale metabolic models (GEMs) - mixed-bag and compartmentalized. Our study revealed that information flow is greater through communities than isolated models, and that the mixed-bag framework conducts greater information flow than the compartmentalized framework for community GEMs, presumably because the latter is encumbered with transport reactions that are absent in the former. This community Mutual Information model is furthermore wrapped as a KBase Application (Run Flux Mutual Information Analysis, RFMIA) for optimum accessibility to biological investigators. We anticipate that this unique quantitative approach to consider information flow through metabolic systems will accelerate both basic and applied discovery in diverse biological fields. Author SummaryMicroorganisms frequently communicate information via information-bearing molecules, which must be fundamentally understood to engineer biological cells that properly engage with their environments, such as the envisioned Internet of Bio-NanoThings. The study of these molecular communications has employed information and communication theory to analyze the exchanged information via chemical reactions and molecular transport. We introduce an information- and communication-centric computational approach to estimate the information flow in biological cells and its impacts on the behavior of single organisms and communities. This study complements our previous work of cell metabolism by developing an end-to-end perspective of molecular communication based on enzyme-regulated reactions. We explore the mutual information using Shannon information theory, measured in bits, between influential nutrients and cellular growth rate. The developed RFMIA computational tool is deployed in the U.S. Department of Energys Systems Biology Knowledgebase, where it quantitatively estimates information flow in both organism and community metabolic networks and extends recent developments in computer communications to explore and explain a new biology for the open-source community.

bioinformatics↗

bioRxiv2025

Protein trafficking and synaptic demand configure complex and dynamic synaptome architectures of individual neurons

Excitatory synapses are the most abundant synapse type in the brain. Being essential for behaviour and are implicated in hundreds of brain disorders, these synapses exhibit striking structural and functional diversity. Synaptome mapping at single-synapse resolution reveals that synaptic protein diversity is spatially organised along the dendritic tree of individual neurons and varies with age and cell-type. However, the cell biological mechanisms underlying the generation of these complex spatial synaptic patterns remain poorly understood. Potential mechanisms include somatic and dendritic protein synthesis, protein trafficking, and local regulatory mechanisms such as activity-dependent degradation. Here we developed computational models to test how combinations of these processes account for empirical synaptome data. We found that a combination of molecular transport mechanisms and local synaptic demand for proteins was sufficient to explain very complex profiles of synaptic protein distributions observed in young, mature and old mice and in different cell types. Our findings suggest the highly complex and dynamic synaptome architecture of the brain is an emergent property of a minimal set of cell biological processes. Our model sets the stage for simulations of brain tissue incorporating molecularly diverse neuronal and synaptic types in a synaptome and connectome architecture.

neuroscience↗