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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 1,603 records · Page 89Linked to original sources

bioRxiv2019

Identification of genes involved in the differentiation of R7y and R7p photoreceptor cells in Drosophila

The R7 and R8 photoreceptor cells of the Drosophila compound eye mediate color vision. Throughout the majority of the eye, these cells occur in two principal types of ommatidia. Approximately 35% of ommatidia are of the pale type and express Rh3 in R7 cells and Rh5 in R8 cells. The remaining 65% are of the yellow type and express Rh4 in R7 cells and Rh6 in R8 cells. The specification of an R8 cell in a pale or yellow ommatidium depends on the fate of the adjacent R7 cell. However, pale and yellow R7 cells are specified by a stochastic process that requires the genes spineless, tango and klumpfuss. To identify additional genes involved in this process we performed a genetic screen using a collection of 480 P{EP} transposon insertion strains. We identified genes that when inactivated and/or ectopically expressed in R7 cells resulted in a significantly altered percentage of Rh3 expressing R7 cells (Rh3%) from wild-type. 53 strains resulted in altered Rh3% in the heterozygous inactivation arm of the screen. 36 strains resulted in altered Rh3% in the ectopic expression arm of the screen, where the P{EP} insertion strains were crossed to a sevEP-GAL4 driver line. 4 strains showed differential effects between the two screens. Analyses of these results suggest that R7 cell fate specification is sensitive to perturbations in transcription, growth inhibition, glycoprotein ligand binding, WNT signaling, ubiquitin protease activity and Ser/Thr kinase activity, among other diverse signaling and cell biological processes.

genetics↗

bioRxiv2019

Molecular architecture of the DNA-binding sites of the P-loop ATPases MipZ and ParA from Caulobacter crescentus

Two related P-loop ATPases, ParA and MipZ, mediate the spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus. Both of these proteins share the ability to form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization relies on their nucleotide-dependent cycling between a monomeric and a dimeric state, driven by interaction with the chromosome partitioning protein ParB, and on the ability of the dimeric species to associate non-specifically with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to identify the residues mediating the interaction of MipZ with DNA. Our results show that the DNA-binding activity of MipZ relies on a series of positively charged and hydrophobic residues lining both sides of the dimer interface. MipZ thus appears to associate with DNA in a sequence-independent manner through electrostatic interactions with the DNA phosphate backbone. In support of this hypothesis, chromatin immunoprecipitation analyses did not reveal any specific target sites in vivo. When extending our analysis to ParA, we found that the architectures of the MipZ and ParA DNA-binding sites are markedly different, although their relative positions on the dimer surface and their mode of DNA binding are conserved. Importantly, bioinformatic analysis suggests that the same principles apply to other members of the P-loop ATPase family. ParA-like ATPases thus share common mechanistic features, although their modes of action have diverged considerably during the course of evolution.\n\nSIGNIFICANCEParA-like P-loop ATPases are involved in a variety of cellular processes in bacteria, including chromosome and plasmid segregation, chemoreceptor and carboxysome positioning, and division site placement. Many members of this large protein family depend on the ability to bind non-specific DNA for proper function. Although previous studies have yielded insights in the DNA-binding properties of some ParA-like ATPases, a comprehensive view of the underlying mechanisms is still lacking. Here, we combine state-of-the-art cell biological, biochemical and biophysical approaches to localize the DNA-binding regions of the ParA-like ATPases MipZ and ParA from Caulobacter crescentus. We show that the two proteins use the same interface and mode of action to associate with DNA, suggesting that the mechanistic basis of DNA binding may be conserved in the ParA-like ATPase family.

microbiology↗

bioRxiv2019

A dynamic interaction between CD19 and the tetraspanin CD81 controls B cell co-receptor trafficking

CD81 and its binding partner CD19 are core subunits of the B cell co-receptor complex. While CD19 is a single-pass transmembrane protein belonging to the extensively studied Ig superfamily, CD81 belongs to a conserved but poorly understood family of four-pass transmembrane proteins called tetraspanins. These functionally diverse proteins play important roles in a wide variety of different organ systems by controlling protein trafficking and other cellular processes. Here, we show that CD81 relies on its ectodomain to control trafficking of CD19 to the cell surface. Moreover, the anti-CD81 antibody 5A6, which binds selectively to activated B cells, recognizes a conformational epitope on CD81 that is masked when CD81 is in complex with CD19. Mutations of CD81 in this contact interface suppress its CD19 surface-export activity. Taken together, these data indicate that the CD81 - CD19 interaction is dynamically regulated upon B cell activation, suggesting that this dynamism can be exploited to regulate B cell function. These results are not only important for understanding B cell biology, but also have important implications for understanding tetraspanin function more generally.

immunology↗

bioRxiv2019

Unpacking the Allee effect: determining individual-level mechanisms that drive global population dynamics

We present a solid theoretical foundation for interpreting the origin of Allee effects by providing the missing link in understanding how local individual-based mechanisms translate to global population dynamics. Allee effects were originally proposed to describe population dynamics that cannot be explained by exponential and logistic growth models. However, standard methods often calibrate Allee effect models to match observed global population dynamics without providing any mechanistic insight. By introducing a stochastic individual-based model, with proliferation, death, and motility rates that depend on local density, we present a modelling framework that translates particular global Allee effects to specific individual-based mechanisms. Using data from ecology and cell biology, we unpack individual-level mechanisms implicit in an Allee effect model and provide simulation tools for others to repeat this analysis.

biophysics↗

bioRxiv2019

Plexin-B2 is a key regulator of cell mechanics during multicellular organization

During multicellular organization, individual cells need to constantly respond to environmental cues and adjust contractile and adhesive forces in order to maintain tissue integrity. The signaling pathways linking biochemical cues and tissue mechanics are unclear. Here, we show that Plexin-B2 regulates mechanochemical integration during multicellular organization. In human embryonic stem cells (hESCs), Plexin-B2 controls cell shape and tissue geometry in both 2D epithelial colony and 3D spheroid aggregates by regulating actomyosin contractility and junctional/cell-matrix adhesive properties. Atomic force microscopy (AFM) directly demonstrates that Plexin-B2 modulates cell stiffness in hESC colonies, which in turn impacts cell proliferation and cell fate specification through {beta}-catenin signaling and YAP mechanosensing. YAP also functions as a mechanoregulator downstream of Plexin-B2, thus forming a mechanochemical integrative loop. In human neuroprogenitor cells (hNPCs), Plexin-B2 similarly controls cell stiffness and tensile forces, as revealed by AFM and FRET tension sensor studies. Strikingly, Plexin-B2-deficient hNPCs display accelerated neuronal differentiation. From an organogenesis perspective, Plexin-B2 maintains cytoarchitectural integrity of neuroepithelium, as modeled in cerebral organoids. On a signaling level, Plexin-B2 engages extracellular as well as intracellular Ras-GAP and RBD domains for mechanoregulation through Rap and Rac GTPases. Our data unveil a fundamental function of Plexin-B2 for mechanochemical integration during multicellular organization, and shed light on the principle of force-mediated regulation of stem cell biology and tissue morphogenesis.

developmental biology↗

bioRxiv2019

Molecular and Morphological Signatures of Chordate Development: Two Distinct Pathways, One Tunicate

All chordates, including urochordates such as tunicates, develop through embryogenesis. The chordate larvae of colonial tunicates metamorphose to lose all chordate structures such as notochord, neural tube, segmented musculature, and then develop by asexual reproduction [blastogenesis], whereby stem cells form tissues and organs. These two developmental pathways establish the same body axis, morphogenetic patterning and organ formation. It is unknown if this convergent morphology implies convergent cellular and molecular mechanisms, and whether the stem cells that mediate these processes differ. Using the colonial tunicate Botryllus schlosseri, we combined transcriptome sequencing and multiple microscopy techniques to study the molecular and morphological signatures of cells at each developmental stage of embryogenesis and blastogenesis. This revealed that the molecular programs are distinct, but the blastogenic tissue-specific stem cells and embryonic precursor populations share similar molecular profiles. By comparing embryogenesis in other chordates we found shared developmental principles, highlighting transcription factors as key evolutionary conserved elements. This study establishes a platform for advancing the science of stem cell biology and regulation of development and regeneration.

developmental biology↗

bioRxiv2019

NOX4 regulates TGFβ-induced proliferation and self-renewal in glioblastoma stem cells.

Glioblastoma (GBM) is the most aggressive and common glioma subtype with a median survival of 15 months after diagnosis. Current treatments have limited therapeutic efficacy, thus more effective approaches are needed. The glioblastoma tumoral mass is characterized by a small cellular subpopulation, the Glioblastoma stem cells (GSCs), which has been held accountant for initiation, invasion, proliferation, relapse and resistance to chemo- and radiotherapy. Targeted therapies against GSCs are crucial, and so is the understanding of the molecular mechanisms that govern the GSCs. Transforming growth factor {beta} (TGF{beta}), platelet growth factor (PDGF) signalling and Reactive Oxygen Species (ROS) production govern and regulate cancer-stem cell biology. In this work, we focus on the role of the NADPH oxidase 4 (NOX4) downstream of TGF{beta} signalling in the GSCs. NOX4 utilises NADPH to generate ROS; TGF{beta} induces NOX4 expression, thus increasing ROS production. Interestingly, NOX4 itself regulates GSC self-renewal and modulates Since TGF{beta} regulates PDGFB in GSC, we analysed how PDGFB modulates NOX4 expression and increases ROS production. Both TGF{beta} and PDGF signalling regulate GSC proliferation in a NOX4/ROS-dependent manner. The transcription factor NRF2, involved in the transcriptional regulation of antioxidant and metabolic responses, is regulated by both TGF{beta} and NOX4. This results in an antioxidant response, which positively contributes to GSC self-renewal and proliferation. In conclusion, this work functionally establishes NOX4 as a key mediator of GSC biology.

cancer biology↗

bioRxiv2019

Evidence for a role for BK channels in the regulation of ADAM17 activity.

Large-conductance voltage and calcium activated channels, KCa1.1, have a large single conductance (~p250) and are highly selective for potassium ions. As a result they have been termed big potassium channels (BK channels). Because of the channels ability to integrate multiple physical and chemical signals they have received much attention in excitable cells. In comparison they have received relatively little attention in non-excitable cells in those of the immune system. Here we report evidence that the BK channel regulates ADAM17 activity. Upon macrophage activation, BK channels translocate to the cell membrane. Genetic or pharmacological inhibition of the cell membrane BK channels resulted in elevated TNF- release and increased metalloproteinase a disintegrin and metalloproteinase domain 17 (ADAM17) activity. Inhibitors of BK channels also increased IL-6R release, a second ADAM17 substrate. In comparison, a BK channel opener decreases TNF- release. Taken together, our results demonstrate a novel mechanism by which ion channel regulates ADAM17 activity. Given the broad range of ADAM17 substrates, this finding has implications in many fields of cell biology including immunology, neurology and cancer biology.

pharmacology and toxicology↗

bioRxiv2019

Neto-α controls synapse organization and homeostasis at the Drosophila neuromuscular junction

Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and post-synaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology and electrophysiology we demonstrate that Neto- functions on both sides of the NMJ. In muscle, Neto- limits the size of the postsynaptic receptors field. In motor neurons, Neto- controls neurotransmitter release in a KAR-dependent manner. Furthermore, Neto- is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto- is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensured NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.

neuroscience↗

bioRxiv2019

Polarly localized EccE1is required for ESX-1 function and stabilization of ESX-1 membrane proteins in Mycobacterium tuberculosis

Mycobacterium tuberculosis is a slow-growing intracellular bacterium with the ability to induce host cell death and persist indefinitely in the human body. This pathogen uses the specialized ESX-1 secretion system to secrete virulence factors and potent immunogenic effectors required for disease progression. ESX-1 is a multi-subunit apparatus with a membrane complex that is predicted to form a pore in the cytoplasmic membrane. In M. tuberculosis this complex is composed of five membrane proteins: EccB1, EccCa1, EccCb1, EccD1, EccE1. In this study, we have characterized the membrane component EccE1 and found that deletion of eccE1 lowers the levels of EccB1, EccCa1 and EccD1 thereby abolishing ESX-1 secretion and attenuating M. tuberculosis ex vivo. Surprisingly, secretion of EspB was not affected by loss of EccE1. Furthermore, EccE1 was found to be a membrane- and cell-wall associated protein that needs the presence of other ESX-1 components to assemble into a stable complex at the poles of M. tuberculosis. Overall, this investigation provides new insights into the role of EccE1 and its localization in M. tuberculosis.\n\nIMPORTANCETuberculosis (TB), the worlds leading cause of death of humans from an infectious disease, is caused by the intracellular bacterium Mycobacterium tuberculosis. The development of successful strategies to control TB requires better understanding of the complex interactions between the pathogen and human host. We investigated the contribution of EccE1, a membrane protein, to the function of the ESX-1 secretion system, the major virulence determinant of M. tuberculosis. By combining genetic analysis of selected mutants with eukaryotic cell biology and proteomics, we demonstrate that EccE1 is critical for ESX-1 function, secretion of effector proteins and pathogenesis. Our research improves knowledge of the molecular basis of M. tuberculosis virulence and enhances our understanding of pathogenesis.

microbiology↗

bioRxiv2019

ATP Binding Cassette Proteins ABCG37 and ABCG33 are required for potassium-independent cesium uptake in Arabidopsis roots

Radiocesium, accumulated in the soil by nuclear accidents is a major environmental concern. The transport process of cesium (Cs+) is tightly linked to the indispensable plant nutrient potassium (K+) as they both belong to the group I alkali metal with similar chemical properties. Most of the transporters that had been characterized to date as Cs+ transporters are directly or indirectly linked to K+. Using a combinatorial approach of physiology, genetics, cell biology and root uptake assay, here we identified two ATP-Binding Cassette (ABC) proteins, ABCG37 and ABCG33 as facilitators of Cs+ influx. The gain-of-function mutant of ABCG37 (abcg37-1) showed hypersensitive response to Cs+-induced root growth inhibition, while the double knock out mutant of ABCG33 and ABCG37 (abcg33-1abcg37-2) showed resistance. Single loss-of-function mutant of ABCG33 and ABCG37 did not show any alteration in Cs+ response. Short term uptake experiment with radioactive Cs+ revealed reduced Cs+ uptake in abcg33-1abgc37-2 compared with wild type in presence or absence of K+. Potassium response and content were unaffected in the double mutant background confirming that Cs+ uptake by ABCG33 and ABCG37 is independent of K+. Collectively, this work identified two ABC proteins as new Cs+ influx carriers, which act redundantly and independent of K+ uptake pathway.

plant biology↗

bioRxiv2019

Bayesian Modeling Reveals Ultrasensitivity Underlying Metabolic Compensation in the Cyanobacterial Circadian Clock

Mathematical models can enable a predictive understanding of mechanism in cell biology by quantitatively describing complex networks of interactions, but such models are often poorly constrained by available data. Owing to its relative biochemical simplicity, the core circadian oscillator in Synechococcus elongatus has become a prototypical system for studying how collective dynamics emerge from molecular interactions. The oscillator consists of only three proteins, KaiA, KaiB, and KaiC, and near-24-h cycles of KaiC phosphorylation can be reconstituted in vitro. Here, we formulate a molecularly-detailed but mechanistically agnostic model of the KaiA-KaiC subsystem and fit it directly to experimental data within a Bayesian parameter estimation framework. Analysis of the fits consistently reveals an ultrasensitive response for KaiC phosphorylation as a function of KaiA concentration, which we confirm experimentally. This ultrasensitivity primarily results from the differential affinity of KaiA for competing nucleotide-bound states of KaiC. We argue that the ultrasensitive stimulus-response relation is critical to metabolic compensation by suppressing premature phosphorylation at nighttime. SynopsisThis study takes a data-driven kinetic modeling approach to characterizing the interaction between KaiA and KaiC in the cyanobacterial circadian oscillator and understanding how the oscillator responds to changes in cellular metabolic conditions. O_LIAn extensive dataset of KaiC autophosphorylation measurements was gathered and fit to a detailed yet mechanistically agnostic kinetic model within a Bayesian parameter estimation framework. C_LIO_LIKaiA concentration tunes the sensitivity of KaiC autophosphorylation and the period of the full oscillator to %ATP. C_LIO_LIThe model reveals an ultrasensitive dependence of KaiC phosphorylation on KaiA concentration as a result of differential KaiA binding affinity to ADP- vs. ATP-bound KaiC. C_LIO_LIUltrasensitivity in KaiC phosphorylation contributes to metabolic compensation by suppressing premature phosphorylation at nighttime. C_LI

systems biology↗

bioRxiv2019

Effects of surfaces and macromolecular crowding on bimolecular reaction rates

Biological cells are complex environments that are densely packed with macromolecules and subdivided by membranes, both of which affect the rates of chemical reactions. It is well known that crowding reduces the volume available to reactants, which increases reaction rates, and also inhibits reactant diffusion, which decreases reaction rates. This work investigates these effects quantitatively using analytical theory and particle-based simulations. A reaction rate equation based on only these two processes turned out to be inconsistent with simulation results. However, accounting for diffusion inhibition by the surfaces of nearby obstacles, which affects access to reactants, led to perfect agreement for reactions near impermeable planar membranes and improved agreement for reactions in crowded spaces. A separate model that quantified reactant occlusion by crowders, and extensions to a thermodynamic "cavity" model proposed by Berezhkovskii and Szabo (J. Phys. Chem. B 120:5998, 2016), were comparably successful. These results help elucidate reaction dynamics in confined spaces and improve prediction of in vivo reaction rates from in vitro ones.

biophysics↗

bioRxiv2025

DUCs are C2 domain containing plant-specific deubiquitinases stabilizing endocytic cargo at the plasma membrane

Deubiquitinases (DUBs) remove ubiquitin modifications from proteins in a substrate- or linkage-selective manner and regulate numerous cell-biological processes, including endocytosis. By performing homology-agnostic bioinformatical screens for undescribed deubiquitinase classes, we identified the plant-specific DUC (deubiquitinase with C2) family, whose members are highly selective for cleaving K63-linked ubiquitin chains. The crystal structure of the Arabidopsis member AtDUC1 reveals that DUC enzymes display a papain-like fold with a characteristic DUB-like active site, despite the apparent absence of sequence similarity to other deubiquitinase families. The K63 linkage specificity is attributed to the recognition of both distal and proximal ubiquitin units via highly conserved contact residues. Arabidopsis DUC members localize to the plasma membrane by virtue of their lipid-binding C2-like domains. In protoplast experiments, all three Arabidopsis DUC enzymes demonstrate the capacity to stabilize an endocytotic model cargo at the plasma membrane, a process that requires both the C2-mediated membrane association and catalytic DUB activity.

biochemistry↗

bioRxiv2025

Modular to cyclic TCA governs hematopoiesis in Drosophila

TCA cycle is well known for its role in bioenergetics and also a hub for metabolic exchange, here we identify a developmentally programmed, non-uniform TCA architecture that is required for hematopoietic state transitions in the Drosophila lymph gland. Early blood progenitors operate a modular TCA configuration, in which a CS/mAcon1-derived citrate node preserves progenitor identity, while a Gdh/Kdh mediated -ketoglutarate to succinyl-CoA branch, not succinate, supports proliferative potential. As cells exit the progenitor state and initiate lineage commitment, the cells undergo metabolic reorganization and engage a fully cyclic, Pdh- and Idh-dependent oxidative TCA cycle whose cataplerotic activity keeps the levels of TCA metabolites in check as their excess otherwise induces differentiation. Blocking these metabolic transitions disrupts progenitor homeostasis, compromises proliferative competency of transitional progenitors, and distorts balanced differentiation. These findings establish a developmentally regulated alternative to the traditional TCA cycle and reveals that appropriate engagement of modular versus cyclic TCA modes is required for orchestrating hematopoietic fate transitions. This work highlights TCA topology and not flux alone functions as a core determinant of stemness, proliferation, and lineage progression during hematopoiesis. Significance StatementHematopoietic development requires precise metabolic control to maintain progenitors while enabling their transition into differentiated lineages. We show that the Drosophila lymph gland achieves this through a developmentally programmed switch in TCA-cycle configuration where early progenitors rely on a modular TCA architecture that independently sustains identity and proliferation, whereas later stages require a fully cyclic, oxidative TCA mode to maintain homeostasis and balanced differentiation. Disrupting either phase perturbs hematopoietic organization, demonstrating that TCA topology itself regulates cell-state transitions. These findings reveal TCA architecture as the determinant of hematopoietic fate and provides a conceptual framework relevant to hematopoiesis and also stem cell biology.

developmental biology↗

bioRxiv2025

Isolation of Crenothrix bacteria reveals the distinct ecophysiologies of filamentous methanotrophs and adaptations to redox stress

At the dawn of modern microbiology, Cohn observed abundant filamentous bacteria in drinking water wells that he named Crenothrix polyspora. Subsequent research has revealed the methanotrophic metabolism of Crenothrix bacteria and their disproportionately high activity in stratified lakes compared to unicellular strains, yet laboratory cultivation has proven elusive, leaving the ecophysiology of Crenothrix bacteria largely unknown. Here we report the isolation of two methanotroph strains of the "lacustrine Crenothrix" clade from an iron-rich wetland and reveal their highly unique cell biologies and potential ecological roles. Using physiological approaches, we demonstrate that the strains perform a microaerobic methane metabolism while growing distinct filaments having wide and directionally oriented connective structures. The strains further have broad genomic repertoires for addressing redox stress that we show are uniquely associated with lacustrine Crenothrix compared to related methanotrophs based on genome data. Aligning with laboratory observations, we identify lacustrine Crenothrix bacteria along potential redox gradients in the wetland at iron-rich snow sites, and we further detect such bacteria in diverse global ecosystems based on public metagenome searches. Together, our data strongly point to an ecophysiology of lacustrine Crenothrix bacteria that is tightly linked to redox stress, and we propose these bacteria may uniquely store or share metabolic intermediates via their filamentous lifestyle to thrive under such conditions. Our results provide a fresh view of the diversity, evolution, and ecology of aerobic methanotrophs, connecting over 150 years of microbiology research opening vast new opportunities to probe bacterial adaptations that drive global methane cycling under redox stress.

microbiology↗

bioRxiv2025

MONDE·T: A Database and Interactive Webserver for Non-Canonical Amino Acids (ncAAs) in the PDB

SummaryNon-canonical amino acids (ncAAs) are understood as amino acids that are not genetically encoded. They are of wide interest for protein design and applications in pharmaceutical and cell biological research, but their impact on protein structure has not been explored systematically. We therefore collected all ncAAs in the Protein Data Bank (PDB) into the MONDE{middle dot}T database, amounting to 1,875 different chemical types in >10,000 entries. They are made accessible through a webserver that allows for data download, visualization of the structures in which they occur, plots of their backbone torsion angles compared to canonical residues, and exploration of their similarity to these. Analyses can focus on a single ncAA or a specific protein structure, illustrated here by example applications. Availability and implementationMONDE{middle dot}T is hosted at the Max Planck Institute for Biology Tu bingen, accessible online at https://mondet.tuebingen.mpg.de. The database is available for download under the Creative Commons 4.0 License.

bioinformatics↗

bioRxiv2025

Coronavirus membrane protein with a fluorescent protein tag enables tracking of virus particles in live cells

Coronavirus particles assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and exit from host cells via secretory organelles that are not well defined. The interplay between viral components and intracellular transport pathways that facilitate assembly and egress are not fully understood and recent studies suggest that multiple pathways maybe involved. Reverse genetics was used to develop a model system to further understand the assembly and egress processes. Mouse hepatitis coronavirus (MHV-A59) was genetically engineered to express the membrane (M) protein fused to green fluorescent protein (M-GFP), with the chimeric gene cloned in place of the open reading frame (ORF) 4 coding region in the RNA genome. The recovered M-GFP virus also expresses wild-type (WT) M protein (M WT) from its native ORF. The M-GFP virus exhibited morphology and growth properties like WT virus. M-GFP and WT M proteins colocalized early in infection, but less M-GFP trafficked toward the cell surface at later times, suggesting that the fusion protein is incorporated less efficiently into virus particles. M-GFP was stably expressed through at least four virus passages. Early passage virus M-GFP virus particles were visualized by confocal and Total Internal Reflection Fluorescence (TIRF) microscopy in live cells. The fluorescently labeled virus particles represent a new tool for coronavirus intracellular trafficking and egress studies. Such studies can also help provide a more detailed understanding of infection and disease processes to provide new insight for development of new therapeutic strategies. IMPORTANCECoronavirus assembly, intracellular transport, egress, and the virus-host interactions involved in these processes are not fully understood. The M protein is the most abundant virion structural component, which forms the scaffold for assembly of the viral envelope. It facilitates incorporation of the viral nucleocapsid and the other viral membrane proteins, spike (S) and envelope (E). In this study we engineered M protein tagged with GFP, which is expressed along with wild-type (WT) M from the viral genome. M-GFP interacted with WT M protein and co-assembled into virus particles. The resulting fluorescently labeled coronavirus particles open opportunities to use state of the art microscopy to monitor virus assembly, trafficking, and egress in live cells to facilitate deeper understanding of the molecular and cell biology of these processes. The ability to visualize particles also opens opportunities to help further understanding of steps in viral replication and pathogenesis for potential therapeutic targets.

microbiology↗