Biology subjects

Sivagurunathan, S.

Publications and source records attributed to Sivagurunathan, S..

14 recordsLinked to original sources

bioRxiv2026

Syntaxin-3 regulates Tight Junction Assembly in Human Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) consists of polarized epithelial cells, serving as a support system for photoreceptor maintenance, where the polarity is contributed by the distribution of syntaxins (STX) within the cell. STX3, a known regulator of apical trafficking in epithelial cells, was previously understood to be absent in human RPE cells, with its functions thought to be compensated by STX1A. However, our results on SNARE mRNA expression profile in RPE detected the presence of 2 splice variants of STX3. Further investigation in donor retina, primary hRPE, and ARPE-19 cells revealed detectable levels of STX3 mRNA and protein. STX3 knockdown in ARPE19 resulted in a significant reduction of tight junction (TJ) proteins, compromising TJ assembly, highlighting the critical role of STX3 in maintaining RPE integrity. In addition, immunoprecipitation followed by LC-MS/MS analysis revealed that STX3 and STX1A have a distinct novel protein interactome in RPE. This study identified unique and shared interactants for STX3 and STX1A, suggesting a broader role for RPE beyond its traditional photoreceptor support function. This further emphasises the biological significance of STX1A and STX3 in maintaining retinal homeostasis, which could facilitate the development of novel therapeutic strategies for retinal disorders. SignificanceThis study identified the presence of STX3 in the human RPE cells, which was previously reported to be absent. Further, we demonstrated that STX3 knockdown in ARPE19 cells disrupted TJ assembly, highlighting its potential role in preserving RPE cell polarity and structural integrity, challenging the notion that STX3 functions were thought to be compensated by STX1A. Moreover, immunoprecipitation followed by LC-MS/MS analysis in RPE identified the protein interaction networks of both STX1A and STX3. Interestingly, unique and shared interactants, including proteins associated with neuronal plasticity, indicated unidentified functions of STX3 and STX1A in RPE. This suggests that they might perform both overlapping and distinct functions for maintaining RPE cell integrity and thus retinal homeostasis. Overall, our preliminary findings challenge the established view that STX3 is non-existent in RPE cells and initiate new directions for exploring the multifaceted and potentially non-redundant functions of STX3 in RPE.

cell biology↗

bioRxiv2025

Expression of vimentin intermediate filaments in epithelial cells promotes cell migration and cell matrix interaction in 3D

During a variety of physiological and pathological processes, such as development, wound healing, and tumor progression, epithelial cells collectively invade into their surroundings. Vimentin intermediate filaments (VIFs) are often observed to play a role in the epithelial cells located at the margins of 2D cultures. However, their role in 3D collective cell behavior remains underexplored. Here, we investigate how induced vimentin expression affects 3D multicellular architecture and mechanics in luminal breast cancer cells (MCF-7) that ordinarily express keratin intermediate filaments only. We find that vimentin expression significantly alters 3D cell cluster morphology, inducing protrusions and increasing boundary fluctuations. Furthermore, cells in vimentin-expressing clusters show enhanced, more stochastic migration. In addition, these clusters exert stronger and localized traction forces on the surrounding matrix, indicating increased cell-matrix interactions. Transcriptomic analysis corroborates these biophysical findings, revealing upregulated gene expression for cell migration and matrix adhesion, and downregulated cell-cell adhesion genes. Our results demonstrate that VIFs are critical in modulating 3D multicellular collective morphology and dynamics, promoting invasive-like behavior by enhancing cell migration and cell-matrix interactions. These results provide fundamental insights into understanding tissue morphogenesis and disease progression.

biophysics↗

bioRxiv2025

Nutritional Triggers of PCOS and NAFLD: A Comparative Analysis of Diets in Female C57BL/6J Mice

ObjectiveObesity is a key factor in the development of Polycystic Ovary Syndrome (PCOS) and Non- alcoholic fatty liver disease (NAFLD). Its relationship with reproductive health and metabolic disorders remains an area of interest. Here we aimed to study the effects of two different obesogenic diets in female C57BL/6J mice on their metabolic and reproductive function. MethodsFemale C57BL/6J mice were fed high-fat high-sugar (HFHS) (45% fat and 10% sugar) diet and High fat diet (60% fat) for 21 weeks to investigate the effects on metabolic health and reproductive health. Mice were assessed for body weight, abdominal circumference, glucose tolerance (OGTT) and estrous cyclicity. At the end of the study biochemical analysis was done on isolated serum and, histological assessment of organs including liver and ovarian tissue was done to score for cystic follicle development and steatosis respectively. ResultsMice on the HFHS diet did not gain weight but developed dyslipidaemia, abnormal liver enzyme levels and severe hepatic steatosis. They did not show impaired glucose tolerance and did not develop any PCOS-like ovarian morphology indicating hepatic metabolic disturbances without significant impact on reproductive health. In contrast, mice on the 60% high-fat diet (HFD) showed marginal increase in body weight and developed altered estrous cyclicity and PCOS-like ovarian morphology. They also exhibited impaired glucose tolerance and insulin resistance, confirming a metabolic disturbance associated with PCOS. These mice did not develop significant liver fibrosis, suggesting a more specific impact on the ovaries and glucose metabolism. Our findings suggest that diet composition plays a crucial role in determining the specific metabolic and reproductive outcomes in obesity-related disorders in females.

physiology↗

bioRxiv2025

Alternate dyes for image-based profiling assays

BackgroundCell Painting, the leading image-based profiling assay, involves staining plated cells with six dyes that mark the different compartments in a cell. Such profiles can then be used to discover connections between samples (whether different cell lines, different genetic treatments, or different compound treatments) as well as to assess particular features impacted by each treatment. Researchers may wish to vary the standard dye panel to assess particular phenotypes, or image cells live while maintaining the ability to cluster profiles overall. MethodsIn this study, we evaluate the performance of dyes that can either replace or augment the traditional Cell Painting dyes or enable tracking live cell dynamics. We perturbed U2OS cells with 90 different compounds and subsequently stained them with either standard Cell Painting dyes (Revvity), or with MitoBrilliant (Tocris) replacing MitoTracker or Phenovue phalloidin 400LS (Revvity) replacing phalloidin. We also tested the live-cell compatible ChromaLive dye (Saguaro). ResultsAll dye sets effectively separated biological replicates of the same sample vs. negative controls (phenotypic activity), although separating from replicates of all other compounds (phenotypic distinctiveness) proved challenging for all dye sets. While individual dye substitutions within the standard Cell Painting panel had minimal impact on assay performance, the live cell dye exhibited distinct performance profiles across different compound classes compared to the standard panel, with later time points more distinct than earlier ones. DiscussionSubstituting MitoBrilliant or Phenovue phalloidin 400LS for standard mitochondrial or actin dyes minimally impacted Cell Painting assay performance. Phenovue phalloidin 400LS offers the advantage of isolating actin features from Golgi or plasma membrane while accommodating an additional 568nm dye. Live cell imaging, enabled by ChromaLive dye, provides real-time assessment of compound-induced morphological changes. Combining this with the standard Cell Painting assay significantly expands the feature space for enhanced cellular profiling. Our findings provide data-driven options for researchers selecting dye sets for image-based profiling.

cell biology↗

bioRxiv2025

Rare missense variants of the leukocyte common antigen related receptor (LAR) display reduced activity in transcellular adhesion and synapse formation

The leukocyte common antigen related receptor (LAR) is a member of the LAR receptor protein tyrosine phosphatase (RPTP) family of synaptic adhesion molecules that contribute to the proper alignment and specialization of synaptic connections in the mammalian brain. LAR-RPTP members have been genetically associated with neuropsychiatric disorders, but the molecular consequences of genetic perturbations of LAR remain unstudied. Using exome sequencing data from psychiatric patients and controls, we identify rare missense variants of LAR that render the extracellular domain (ECD) unstable and susceptible to proteolytic cleavage. Using recombinant and cellular systems, we describe three variants that cause disruption of the LAR:NGL-3 interaction, which results in loss of transcellular adhesion and synaptogenic effects. Furthermore, we show that overexpression of two of these variants elicit altered morphological phenotypes in an imaging-based morphological profiling assay compared to wild type LAR, suggesting that destabilization of the LAR ECD has broad effects on LAR function. In conclusion, our study identifies three rare, missense variants in LAR that could provide insights into LAR involvement with psychiatric pathobiology.

neuroscience↗

bioRxiv2024

A Postdoctoral Training Program in Bioimage Analysis

We herein describe a postdoctoral training program designed to train biologists with microscopy experience in bioimage analysis. We detail the rationale behind the program, the various components of the training program, and outcomes in terms of works produced and the career effects on past participants. We analyze the results of an anonymous survey distributed to past and present participants, indicating overall high value of all 12 rated aspects of the program, but significant heterogeneity in which aspects were most important to each participant. Finally, we propose this model as a template for other programs which may want to train experts in professional skill sets, and discuss the important considerations when running such a program. We believe that such programs can have extremely positive impact for both the trainees themselves and the broader scientific community.

cell biology↗

bioRxiv2023

Bridging Imaging Users to Imaging Analysis - A community survey

The "Bridging Imaging Users to Imaging Analysis" survey was conducted in 2022 by the Center for Open Bioimage Analysis (COBA), Bioimaging North America (BINA), and the Royal Microscopical Society Data Analysis in Imaging Section (RMS DAIM) to understand the needs of the imaging community. Through multi-choice and open-ended questions, the survey inquired about demographics, image analysis experiences, future needs, and suggestions on the role of tool developers and users. Participants of the survey were from diverse roles and domains of the life and physical sciences. To our knowledge, this is the first attempt to survey cross-community to bridge knowledge gaps between physical and life sciences imaging. Survey results indicate that respondents overarching needs are documentation, detailed tutorials on the usage of image analysis tools, user-friendly intuitive software, and better solutions for segmentation, ideally in a format tailored to their specific use cases. The tool creators suggested the users familiarize themselves with the fundamentals of image analysis, provide constant feedback, and report the issues faced during image analysis while the users would like more documentation and an emphasis on tool friendliness. Regardless of the computational experience, there is a strong preference for written tutorials to acquire knowledge on image analysis. We also observed that the interest in having office hours to get an expert opinion on their image analysis methods has increased over the years. In addition, the community suggests the need for a common repository for the available image analysis tools and their applications. The opinions and suggestions of the community, released here in full, will help the image analysis tool creation and education communities to design and deliver the resources accordingly.

scientific communication and education↗

bioRxiv2023

Vimentin filaments integrate low complexity domains in a highly complex helical structure

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion beam milling, cryo-electron microscopy and tomography, to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, densely-packed and highly-ordered helical symmetric structure of 40 -helices in cross-section, organized into 5 protofibrils. Surprisingly, the intrinsically disordered head domains form an amyloid-like fiber in the center of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking strength and stretchability.

biochemistry↗

bioRxiv2021

Oral glucose feeding enhances adherence of quiescent lymphocytes to fibronectin via non-canonical insulin signalling.

Impaired glucose metabolism is associated with chronic inflammation, aberrant immunity and anomalous leukocyte trafficking. Conversely, infusion of functional immune cells restores glucose metabolism. Despite being exposed to periodic alterations in blood insulin levels upon fasting and feeding, studies exploring the physiological effects of these hormonal changes on quiescent circulating lymphocytes are missing. Here we find that oral glucose load in healthy men and mice enhance adherence of circulating peripheral blood mononuclear cells (PBMCs) to fibronectin. This led to increased homing of post-load PBMCs to injured blood vessels. Cell culture based experiments on Jurkat-T cells and PBMCs demonstrated that insulin elicits these adhesive effects through a non-canonical signalling involving insulin growth factor-1 receptor (IGF-1R) and phospholipase C gamma-1 (PLC{gamma}-1) mediated activation of integrin {beta}1. Our findings point to the relevance of post-prandial insulin spikes in regulating homing of circulating T-cells to various organs for tissue repair and immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/464163v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@11edce6org.highwire.dtl.DTLVardef@1389835org.highwire.dtl.DTLVardef@90c96eorg.highwire.dtl.DTLVardef@1988a63_HPS_FORMAT_FIGEXP M_FIG Insulin mediates fibronectin adherence of lymphocytes through non-canonical signalling. Insulin mediates auto-phosphorylation of IGF-1 receptor at Tyr1135 leading to activation of PLC-{gamma}1 through Tyr783 phosphorylation, which in turn leads to the activation of integrin 1 through intracellular calcium to ultimately enhance adhesion of quiescent lymphocytes to fibronectin. C_FIG

physiology↗

bioRxiv2021

Vimentin Intermediate Filaments and Filamentous Actin Form Unexpected Interpenetrating Networks That Redefine the Cell Cortex

Vimentin intermediate filaments (VIFs) and F-actin are filamentous cytoskeletal proteins generally thought to form completely independent networks that have vastly different properties and functions. Here, we show that, unexpectedly, there exist both extensive structural and functional interactions between VIFs and F-actin. We show that VIFs and F-actin form an interpenetrating network (IPN) within the cell cortex and interact synergistically at multiple length scales. This IPN structure has important functional consequences in cells: The IPN results in enhanced contractile forces in the cell. In addition, VIFs influence the diffusive behavior of actin monomers, suggesting specific associations between actin and vimentin proteins in the cytoplasm; this facilitates formation of the IPN and has downstream effects on other actin-driven processes. The results suggest that contributions of VIFs and F-actin are strongly correlated. Such interactions counter generally accepted behavior and are broadly significant given the wide range of processes currently attributed to F-actin alone.

cell biology↗

bioRxiv2021

The molecular architecture of vimentin filaments

Intermediate filaments are integral components of the cytoskeleton in metazoan cells. Due to their specific viscoelastic properties they are principal contributors to flexibility and tear strength of cells and tissues. Vimentin, an intermediate filament protein expressed in fibroblasts and endothelial cells, assembles into ~11 nm thick biopolymers, that are involved in a wide variety of cellular functions in health and disease. Here, we reveal the structure of in-situ polymerized vimentin filaments to a subnanometer resolution by applying cryo-electron tomography to mouse embryonic fibroblasts grown on electron microscopy grids. We show that vimentin filaments are tube-like assemblies with a well-defined helical symmetry. Their structure is comprised of five octameric, spring-like protofibrils harboring 40 vimentin polypeptide chains in cross-section. The protofibrils are connected by the intrinsically disordered head and helix 1A domains of vimentin. Individual filaments display two polymerization states characterized by either the presence or absence of a luminal density along the helical axis. The structure of vimentin filaments unveils the generic building plan of the intermediate filament superfamily in molecular details.

cell biology↗

bioRxiv2021

Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy

Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery. Keratin filaments provide cellular resistance to mechanical stresses, ensure cell and tissue integrity in addition to regulatory functions. Mutations in keratin genes are related to a variety of epithelial tissue diseases that mostly affect skin and hair. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Interestingly, two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Reconstitution of filaments of substantial length from keratin segments uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex architecture of keratin filaments, which demonstrate a remarkable degree of heterogeneity, suggesting that they are highly flexible, dynamic cytoskeletal structures.

cell biology↗

bioRxiv2021

Nuclear lamin isoforms differentially contribute to LINC complex-dependent nucleocytoskeletal coupling and whole cell mechanics

The ability of a cell to regulate its mechanical properties is central to its function. Emerging evidence suggests that interactions between the cell nucleus and cytoskeleton influence cell mechanics through poorly understood mechanisms. Here we show that A- and B-type nuclear lamin isoforms distinctively modulate both nuclear and cellular volume and selectively stabilize the linker of nucleoskeleton and cytoskeleton (LINC) complexes that couple the nucleus to cytoskeletal actin and vimentin. We reveal, further, that loss of each of the four-known lamin isoforms in the mouse embryonic fibroblasts differentially affects cortical and cytoplasmic stiffness as well as cellular contractility, and then propose a LINC complex mediated model that explains these impaired mechanical phenotypes. Finally, we demonstrate that loss of each lamin isoform softens the nucleus in a manner that correlates with loss of heterochromatin. Together, these findings uncover distinctive roles for each lamin isoform in maintaining cellular and nuclear mechanics.

biophysics↗

bioRxiv2018

In silico analysis of piRNAs in retina reveals potential targets in intracellular transport and retinal degeneration

Long considered to be active only in germline, PIWI/piRNA pathway is now known to play significant role in somatic cells, especially neurons. Nonetheless, so far there is no evidence for the presence of piRNAs in the neurosensory retina. In this study, we have uncovered 102 piRNAs in human retina and retinal pigment epithelium (RPE) by analysing RNA-seq data. The identified piRNAs were enriched with three motifs predicted to be involved in rRNA processing and sensory perception. Further, expression of piRNAs in donor eyes were assessed by qRT-PCR. Loss of piRNAs in HIWI2 knockdown ARPE19 cells downregulated targets implicated in intracellular transport (SNAREs and Rabs), circadian clock (TIMELESS) and retinal degeneration (LRPAP1 and RPGRIP1). Moreover, piRNAs were dysregulated under oxidative stress indicating their potential role in retinal pathology. Intriguingly, computational analysis revealed complete and partial seed sequence similarity between piR-62011 and sensory organ specific miR-183/96/182 cluster. Furthermore, the expression of retina enriched piR-62011 positively correlated with miR-182 in HIWI2 silenced Y79 cells. Thus, our data provides an evidence for the expression of piRNAs in human retina and RPE. Collectively, our work demonstrates that piRNAs dynamically regulate distinct molecular events in the maintenance of retinal homeostasis.

cell biology↗