Biology subjects

Subramanyam, D.

Publications and source records attributed to Subramanyam, D..

7 recordsLinked to original sources

bioRxiv2025

Pathogenic mutations in Clathrin Heavy Chain associated with intellectual disability impair synaptic architecture and learning in Drosophila

Clathrin-mediated endocytosis is essential for neural development and function. Recent studies have linked de novo mutations in the clathrin heavy chain (Chc) gene to a range of neurodevelopmental disorders. In this study we have modelled two pathogenic mutations: L1047P and W1108R in Drosophila melanogaster and examined their effects on vesicle dynamics, ligand uptake, neuronal development and memory formation. Our data shows that expression of these mutant forms of Chc result in reduced survival and defective learning when expressed ubiquitously or exclusively in neurons. Our analysis also reveals that these mutations have the ability to disrupt vesicle dynamics and reduce ligand uptake in cells. Although we do not see a defect in neuronal morphology and function at the larval neuro-muscular junction, we see an increase in the number of Dlg-negative boutons, and a significant reduction in Spectrin expression, indicative of disruptions in the process of synapse maturation. Overall, this study provides mechanistic insights into the cellular and molecular basis of Chc-related neurodevelopmental disorders.

cell biology↗

bioRxiv2023

Pre-existing tissue mechanical hypertension at adherens junctions disrupts apoptotic extrusion in epithelia

Apical extrusion is a tissue-intrinsic process that allows epithelia to eliminate unfit or surplus cells. This is exemplified by the early extrusion of apoptotic cells, which is critical to maintain the epithelial barrier and prevent inflammation. Apoptotic extrusion is an active mechanical process, which involves mechanotransduction between apoptotic cells and their neighbours, as well as local changes in tissue mechanics. Here we report that the pre-existing mechanical tension at adherens junctions conditions the efficacy of apoptotic extrusion. Specifically, increasing baseline mechanical tension by overexpression of a phosphomimetic Myosin II regulatory light chain (MRLC) compromises apoptotic extrusion. This occurs when tension is increased in either the apoptotic cell or its surrounding epithelium. Further, we find that the pro-inflammatory cytokine, TNF, stimulates Myosin II and increases baseline AJ tension to disrupt apical extrusion, causing apoptotic cells to be retained in monolayers. Importantly, reversal of mechanical tension with an inhibitory MRLC mutant or tropomyosin inhibitors is sufficient to restore apoptotic extrusion in TNF-treated monolayers. Together, these findings demonstrate that baseline levels of tissue tension are important determinants of apoptotic extrusion, which can potentially be co-opted by pathogenetic factors to disrupt the homeostatic response of epithelia to apoptosis.

cell biology↗

bioRxiv2023

Pathogenic aggregates alter actin organization and cellular viscosity resulting in stalled clathrin mediated endocytosis

Protein aggregation is a common underlying feature of neurodegenerative disorders. Cells expressing neurodegeneration-associated mutant proteins show altered uptake of ligands, suggestive of impaired endocytosis, in a manner as yet unknown. Using live cell imaging, we show that clathrin-mediated endocytosis (CME) is affected due to altered actin cytoskeletal organization in the presence of Huntingtin aggregates. Additionally, we find that cells containing Huntingtin aggregates are stiffer and less viscous than their wild-type counterparts due to altered actin conformation, and not merely due to the physical presence of aggregate(s). We further demonstrate that CME and cellular viscosity can be rescued by overexpressing Hip1, Arp2/3 or transient LatrunculinA treatment. Examination of other pathogenic aggregates revealed that only a subset of these display defective CME, along with altered actin organization and increased stiffness. Together, our results point to an intimate connection between functional CME, actin organization and cellular stiffness in the context of neurodegeneration.

cell biology↗

bioRxiv2022

Autophagy-cell death balance is maintained by Polycomb-mediated regulation during stem cell differentiation

Autophagy is a conserved cytoprotective process, aberrations in which, lead to numerous degenerative disorders. While the cytoplasmic components of autophagy have been extensively studied, the epigenetic regulation of autophagy genes, especially in stem cells, is less understood. Deciphering the epigenetic regulation of autophagy genes becomes increasingly relevant given the therapeutic benefits of small-molecule epigenetic inhibitors in novel treatment modalities. We observe that, during retinoic acid-mediated differentiation of mouse embryonic stem cells (mESCs), autophagy is induced, and identify the Polycomb enzyme EZH2 as a regulator of this process. In mESCs, EZH2 represses several autophagy genes including the autophagy regulator Dram1. EZH2 facilitates the formation of a bivalent chromatin domain at the Dram1 promoter, which allows the expression of the gene and induction of autophagy during differentiation, while still retaining the repressive H3K27me3 mark. EZH2 inhibition leads to loss of the bivalent domain, and a consequential "hyper- expression" of Dram1, with extensive cell death. This study shows that Polycomb group proteins help maintain an autophagy-cell death balance during stem cell differentiation, in part, by regulating the expression of the Dram1 gene.

cell biology↗

bioRxiv2021

E-cadherin regulates the stability and transcriptional activity of β-catenin in embryonic stem cells

E-CADHERIN is abundantly expressed in embryonic stem cells (ESCs) and plays an important role in the maintenance of cell-cell adhesions. However, the exact function of this molecule beyond cell adhesion, in the context of cell fate decisions is largely unknown. Using mouse ESCs (mESCs), we demonstrate that E-CADHERIN and {beta}- CATENIN interact at the membrane and continue to do so upon internalization within the cell. Knockout of the gene encoding E-CADHERIN, Cdh1, in mESCs resulted in a failure to form tight colonies, accompanied by altered expression of differentiation markers, and retention of pluripotency factor expression during differentiation. Interestingly, Cdh1-/- mESCs showed a dramatic reduction in {beta}-CATENIN levels. Transcriptional profiling of Cdh1-/- mESCs displayed a significant alteration in the expression of a subset of {beta}-CATENIN targets, in a cell-state dependent manner. While treatment with a pharmacological inhibitor against GSK3{beta} could rescue levels of {beta}-CATENIN in Cdh1-/- mESCs, expression of downstream targets were altered in a context-dependent manner, indicating an additional layer of regulation within this subset. Together, our results reveal the existence of a cell-state-dependent regulation of {beta}-CATENIN and its transcriptional targets in an E-CADHERIN dependent manner. Our findings hint at hitherto unknown roles played by E- CADHERIN in regulating the activity of {beta}-CATENIN in ESCs. Significance StatementAre cell adhesions only responsible for maintaining tissue architecture, or do they also regulate cell fate decisions during early embryonic stages by modulating the output of specific signalling pathways? In this study, we study the role of E- CADHERIN, a crucial component of cell-cell adhesions in the context of mouse embryonic stem cells (mESCs). We find that E-CADHERIN regulates the stability and activity of {beta}-CATENIN in mESCs through physical interactions. However, the loss of E-CADHERIN affected the expression of only a subset of downstream targets of {beta}-CATENIN in a cell-state dependent manner. This study highlights a critical cross-talk between molecules involved in cell-cell adhesion and the underlying signalling network critical for establishing cell fate during early mammalian development.

cell biology↗

bioRxiv2021

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

molecular biology↗

bioRxiv2020

Loss of clathrin heavy chain enhances actin-dependent stiffness of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cell stiffness, and specific responses to features of the underlying substratum. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking the clathrin heavy chain (Cltc), display higher Youngs modulus, indicative of greater cellular stiffness, in comparison to WT mESCs. We have previously shown that mESCs lacking Cltc display a loss of pluripotency, and an initiation of differentiation. The increased stiffness observed in these cells was accompanied by the presence of actin stress fibres and accumulation of the inactive, phosphorylated, actin binding protein, Cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Youngs modulus, to values similar to those obtained with WT mESCs. However, the expression profile of pluripotency factors was not rescued. This indicates that a restoration of mechanical properties, through modulation of the actin cytoskeleton, may not always be accompanied by a change in the expression of critical transcription factors that regulate the state of a stem cell, and that this may be dependent on the presence of active endocytosis in a cell.

cell biology↗