Biology subjects

Vishwakarma, V.

Publications and source records attributed to Vishwakarma, V..

8 recordsLinked to original sources

bioRxiv2026

Host cell plasma membrane-derived vesicles efficiently inhibit in vitro Influenza A virus infection in a size-dependent manner

The influenza virus poses a significant global health threat due to its continuous evolution, immune evasion, and zoonotic spillover. The rise of drug resistance, reduced susceptibility to existing antiviral medications, and the limited effectiveness of annual vaccines underscore the need for new antiviral strategies. To infect, the influenza virus binds to sialic acid (SA)-containing molecules on host cell membranes through hemagglutinin (HA). Blocking this interaction represents a promising antiviral approach. Herein, we report that SA containing plasma membrane-derived vesicles (PMV) efficiently inhibits in vitro Influenza A virus (IAV) infection. Using orthogonal methods, we demonstrate that PMV derived from A549, MDCK, and HEK cells competitively bind to H1N1 (WSN) and H3N2 (X-31) IAV strains, block entry and infection in human respiratory epithelial cells in a dose-dependent manner, without causing significant toxicity. When the size of the vesicles was reduced through extrusion, the antiviral activity was enhanced, and this was found to be correlated with a size-dependent increase in hemagglutination inhibition and reduced IAV internalisation. Plasma membrane-derived vesicles may serve as a novel antiviral strategy against influenza virus infections due to their simple production method and conserved SA binding site on HA.

biochemistry↗

bioRxiv2025

Shoc2 Deficiency Disrupts Lymphangiogenesis through mTOR-Mediated Mitochondrial Dysfunction

An interplay of growth factors and signaling pathways governs the development and maintenance of the lymphatic vasculature, ensuring proper fluid homeostasis and immune function. Disruption of these regulatory mechanisms can lead to congenital lymphatic disorders and contribute to various pathological conditions. However, the mechanisms underlying the molecular regulation of these processes remain elusive. Here we reveal a critical and previously unappreciated role for the signaling scaffold protein Shoc2 in lymphangiogenesis. We demonstrate that loss of Shoc2 leads to nearly a complete loss of lymphatic vasculature in vivo and senescence of lymphatic endothelial cells in vitro. Mechanistically, Shoc2 is required for balancing signaling through the ERK1/2 pathway, and its loss results in increased mTORC1 signaling. This dysregulation impairs mitochondrial respiration and triggers an IRF/IFN-II response, ultimately leading to cellular senescence. Strikingly, expression of the Noonan Syndrome with Loose anagen Hair (NSLH)-causing Shoc2 variant S2G phenocopies the effects of Shoc2 loss. Together, these studies establish the critical role of Shoc2 in lymphangiogenesis and uncover a novel mechanistic link between Shoc2 signaling, mitochondrial function, innate immune response, and lymphatic development, with significant implications for Ras-pathway-related congenital disorders.

developmental biology↗

bioRxiv2024

Organization of the apical extracellular matrix during tubular organ formation

The apical extracellular matrix (aECM) plays a critical role in epithelial tube morphogenesis during organ formation, but its composition and organization remain poorly understood. Using the Drosophila embryonic salivary gland (SG) as a model, we identify Papss, an enzyme that synthesizes the universal sulfate donor PAPS, as a critical regulator of tube lumen expansion. Papss mutants show a disorganized apical membrane, condensed aECM, and disruptions in Golgi structures and intracellular trafficking. SG-specific expression of wild-type Papss, but not the catalytically inactive form, rescues the defects in Papss mutants, suggesting that defects in sulfation are the underlying cause of the phenotypes. Additionally, we identify two zona pellucida (ZP) domain proteins, Piopio (Pio) and Dumpy (Dpy), as key components of the SG aECM. In the absence of Papss, Pio is gradually lost in the aECM, while the Dpy-positive aECM structure is condensed and dissociates from the apical membrane, leading to a thin lumen. Mutations in dpy or pio, or in Notopleural, which encodes a matriptase that cleaves Pio to form the luminal Pio pool, result in a SG lumen with alternating bulges and constrictions, with the loss of pio leading to the loss of Dpy in the lumen. Our findings underscore the essential role of sulfation in organizing the aECM during tubular organ formation and highlight the mechanical support provided by ZP domain proteins in maintaining luminal diameter.

developmental biology↗

bioRxiv2024

Developing peptide-based fusion inhibitors as an antiviral strategy utilizing Coronin 1 as a template

Enveloped viruses can enter the host cells by endocytosis and subsequently fuse with the endosomal membranes, or fuse with the plasma membrane at the cell surface. The crucial stage of viral infection, regardless of the route taken to enter the host cell, is membrane fusion. The present work aims to develop a peptide-based fusion inhibitor that prevents membrane fusion by modifying the properties of the participating membranes, without targeting a protein. This would allow us to develop a fusion inhibitor that might work against a larger spectrum of enveloped viruses as it does not target any specific viral fusion protein. With this goal, we have designed a novel peptide by modifying a native sequence derived from coronin 1, a phagosomal protein, that helps to avoid lysosomal degradation of mycobacterium-loaded phagosomes. The designed peptide, mTG-23, inhibits [~] 30-40% fusion between small unilamellar vesicles containing varying amounts of cholesterol by modulating the biophysical properties of the participating bilayers. As a proof of principle, we have further demonstrated that the mTG-23 inhibits Influenza A virus infection in A549 and MDCK cells (with[~] EC50 of 20.45 {micro}M and 21.45 {micro}M, respectively), where viral envelope and endosomal membrane fusion is a crucial step. Through a gamut of biophysical and biochemical methods, we surmise that mTG-23 inhibits viral infection by inhibiting viral envelope and endosomal membrane fusion. We envisage that the proposed antiviral strategy can be extended to other viruses that employ a similar modus operandi, providing a novel pan-antiviral approach.

biophysics↗

bioRxiv2024

Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

IntroductionAutophagy is a dynamic, multi-step process that cells use to degrade damaged, abnormal, and potentially harmful cellular substances. While autophagy is maintained at a basal level in all cells, it is activated at a higher level in many cancer cells and promotes tumor growth, anti-tumor immune response, and resistance to cancer therapy. As a result, autophagy is increasingly being recognized to have an important role in cancer progression and emerging as a potential target for cancer therapy. We recently discovered that small GTPase Rab27B, a known regulator of vesicle trafficking and exosome secretion, is also involved in the autophagy process. MethodsRab27B was knocked out using CRISPR/Cas9 in CRC cell line HCT116. Western blotting, Immunofluorescence, MTT assay, spheroid formation assay, soft agar assay and xenograft studies were performed to analyze the effects of Rab27B deletion on CRC cells. ResultsCRISPR/Cas9 deletion or siRNA knockdown of Rab27B in colorectal cancer cells (CRC) showed an abnormal accumulation of autophagy vesicles. Additionally, we observed a significant increase in the autophagy markers LC3-II and p62 by immunocytochemistry and western blot analysis, suggesting a defect in the autophagy flux process. Lysotracker and mCherry-EGFP-LC3 fusion construct indicate an impairment in autophagosome and lysosome fusion when Rab27B is silenced. This defect was rescued by full-length and constitutively active GTP mutant of Rab27B. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that the observed defects in autophagy flux resulting from Rab27B loss would cause reduced stress response and tumor growth. Indeed, Rab27B knockout reduced cell viability in response to starvation and a 94% reduction in soft agar colony formation. Rab27B deletion also prevented spheroid formation in vitro. Finally, to analyze the effect of Rab27B deletion in tumor formation in vivo, we performed a xenograft study with wildtype and Rab27B knockout CRC cells, resulting in a dramatic loss of tumor growth (p<0.0001) in the KO cells. ConclusionsTogether, our results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Future studies will focus on investigating the mechanism of how Rab27B functions in the autophagy pathway and whether Rab27B can be targeted as a potential therapeutic strategy for CRC.

cancer biology↗

bioRxiv2023

Coordination of cell cycle and morphogenesis during organ formation

Organ formation requires precise regulation of cell cycle and morphogenetic events. Using the Drosophila embryonic salivary gland (SG) as a model, we uncover the role of the SP1/KLF transcription factor Huckebein (Hkb) in coordinating cell cycle regulation and morphogenesis. The hkb mutant SG exhibits defects in invagination positioning and organ size due to abnormal death of SG cells. Normal SG development involves distal-to-proximal progression of endoreplication (endocycle), whereas hkb mutant SG cells undergo abnormal cell division, leading to cell death. Hkb represses the expression of key cell cycle and pro-apoptotic genes in the SG. Knockdown of cyclin E or cyclin-dependent kinase 1, or overexpression of fizzy-related rescues most of the morphogenetic defects observed in the hkb mutant SG. These results indicate that Hkb plays a critical role in controlling endoreplication by regulating the transcription of key cell cycle effectors to ensure proper organ formation.

developmental biology↗

bioRxiv2022

Multifunctional role of GPCR signaling in epithelial tube formation

Epithelial tube formation requires Rho1-dependent actomyosin contractility to generate the cellular forces that drive cell shape changes and rearrangement. Rho1 signaling is activated by G protein-coupled receptor (GPCR) signaling at the cell surface. During Drosophila embryonic salivary gland (SG) invagination, the GPCR ligand Folded gastrulation (Fog) activates Rho1 signaling to drive apical constriction. The SG receptor that transduces the Fog signal into Rho1-dependent myosin activation has not been identified. Here, we reveal that the Smog GPCR transduces Fog signal to regulate Rho kinase accumulation and myosin activation in the medioapical region of cells to control apical constriction during SG invagination. We also report on unexpected Fog-independent roles for Smog in maintaining epithelial integrity and organizing cortical actin. Our data supports a model wherein Smog regulates distinct myosin pools and actin cytoskeleton in a ligand-dependent manner during epithelial tube formation.

developmental biology↗

bioRxiv2020

Isoform-specific roles of the Drosophila filamin-type protein Jitterbug (Jbug) during development

Filamins are highly conserved actin-crosslinking proteins that regulate organization of the actin cytoskeleton. As key components of versatile signaling scaffolds, filamins are implicated in developmental anomalies and cancer. Multiple isoforms of filamins exist, raising the possibility of distinct functions for each isoform during development and in disease. Here, we provide an initial characterization of jitterbug (jbug), which encodes one of the two filamin-type proteins in Drosophila. We generate Jbug antiserum that recognizes all of the spliced forms and reveals differential expression of different Jbug isoforms during development, and a significant maternal contribution of Jbug protein. To reveal the function of Jbug isoforms, we create new genetic tools, including a null allele that deletes all isoforms, hypomorphic alleles that affect only a subset, and UAS lines for Gal4-driven expression of the major isoforms. Using these tools, we demonstrate that Jbug is required for viability and that specific isoforms are required in the formation of actin-rich protrusions including thoracic bristles in adults and ventral denticles in the embryo. We also show that specific isoforms of Jbug show differential localization within epithelia and that maternal and zygotic loss of jbug disrupts Crumbs (Crb) localization in several epithelial cell types.

developmental biology↗