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Mini-Symposium-NCCS-Oct17.Pdf Mini-Symposium on cell biology National Centre for Cell Science, Pune Date: October 26, 2017 Venue: Auditorium, NCCS Schedule Time Name of speaker Title 1 2.30 pm – 2.55 pm Ashwani Kumar Understanding the nature IMTECH, Chandigarh of extracellular polymeric substances of the Mycobacterium tuberculosis biofilms 2 3.00 pm – 3.25 pm Chandrima Das Decoding the Epigenetic Saha Institute of Nuclear Landscape by the Histone Physics, Kolkata Readers: Implications in Human Diseases 3 3.30 pm – 3.55 pm Dipayaman Ganguly A pathogenetic continuum IICB, Kolkata for systemic autoimmunities and metabolic syndrome 4 4.00 pm – 4.25 pm Ranjith Padinhateeri DNA-bending non-histone IIT Bombay proteins can make regular 30-nm chromatin structure unviable 5 4.30 pm – 4.55 pm Shamik Sen MMP-cytoskeletal IIT Bombay crosstalk in cancer invasion Understanding the nature of extracellular polymeric substances of the Mycobacterium tuberculosis biofilms Ashwani Kumar Council of Scientific and Industrial Research-Institute of Microbial Technology, Sector 39 A, Chandigarh, India The capability of Mycobacterium tuberculosis (Mtb) to form the biofilm could explain the phenotypic drug tolerance displayed by Mtb in humans. However, the factors regulating biofilm formation, the physiology of the resident bacteria and composition of EPS largely remain unknown. Here, we show that intracellular thiol reductive stress (TRS) induces Mtb biofilm formation. TRS-induced biofilms harbour drug-tolerant but metabolically active bacteria with comparable levels of ATP/ADP, NAD+/NADH and NADP+/NADPH. Evidence generated through staining with specific dyes followed by confocal microscopy demonstrated that the ECM of Mtb biofilm is primarily composed of extracellular polysaccharides along with proteins, lipids, and extracellular DNA. We further demonstrate that cellulose plays an important structural role in Mtb biofilms. However the genetic pathway involved in the biosynthesis of cellulose in Mtb is not defined. To further build upon these findings, we propose to identify the genes involved in cellulose biosynthesis in Mtb. We will also develop tools for using cellulose as a biomarker for identification of location of Mtb biofilms in infected animals and/or in clinical samples. We further propose to characterize other polysaccharides and proteins that play an important role in the architecture of Mtb biofilms. Decoding the Epigenetic Landscape by the Histone Readers: Implications in Human Diseases Chandrima Das Biophysics and Structural Genomics Division, Saha Institute of Nuclear Physics, Kolkata, West Bengal, India Epigenetic modifications in DNA and protein component of chromatin fine-tune the underlying gene expression programs, genome repair as well as replication. These alterations are causally related to normal homeostasis of the organism and the pathobiological state. The epigenetic modifications are operated through a class of proteins termed as “chromatin reader/effector” which lead to differential recruitment of other regulatory factors. The research focus of Chromatin Dynamics Laboratory is to understand some of these epigenetic readers in the context of cellular functions and their possible connection to the disease, including metabolic as well as infectious diseases. In this context we have recently uncovered the physiological functions of three chromatin readers: (i) ZMYND8 (Zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8), (ii) TCF19 (Transcription Factor 19) and (iii) Sp110 (Speckled 110). (i) We have shown that dual histone reader ZMYND8, an ATRA-responsive transcription factor, maintains normal cell fate and prevents tumorigenicity. (ii) Interestingly, recognition of these epigenetic modifications by TCF19, under nutritional stress in metabolically active liver tissues have also been our area of interest. Such reader proteins are critical in aiding the cells to cope- up with the changing glycemic environment by influencing gene expression, and act like glucose sensors for metabolically active tissues. (iii) Epigenetic landscape of chromatin is modulated during microbial infection. We have recently shown that Sp110, a PML body protein which can also act as a chromatin reader during pathogenic infection, gets hijacked by the oncoprotein HBx of Hepatitis B virus to promote the viral persistence in host cells. Taken together, we propose that chromatin readers are intricately involved in maintenance of normal cellular homeostasis as well as pathological states by reprogramming the epigenetic landscape of chromatin. A pathogenetic continuum for systemic autoimmunities and metabolic syndrome Dipyaman Ganguly CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India A critical role of plasmacytoid dendritic cells (pDCs) and type I interferons is well established in several systemic autoimmune disorders, e.g. systemic lupus erythematosus, psoriasis, type I diabetes, sjogren syndrome and systemic sclerosis. A dominant type I IFN signature is present systemically either in all or subsets of patients suffering from these diseases. On the other hand, patients suffering from these autoimmune diseases often develop insulin resistance and metabolic syndrome. For example, association with obesity, type 2 diabetes and other components of metabolic syndrome is established in SLE and psoriasis. Interestingly, several recent studies have discovered a critical role of type I interferon induction in obesity-associated insulin resistance and hepatic steatosis in both animal models and human patients. We have discovered that in situ type I IFN induction in response to toll-like receptor 9 (TLR9) triggering from pDCs recruited into visceral adipose tissue in obese individuals drives adipose tissue inflammation and insulin resistance. In animal models of the disease, deficiency of TLR9 was found to ameliorate insulin resistance, adipose tissue inflammation and hepatic steatosis. Taking these recent studies into consideration, we proposed a pathogenetic link between induction of type I interferon response in varied clinical contexts and development of metabolic syndrome. We also proposed a syndromic description encompassing all the relevant clinical contexts that share a key pathogenetic event as well as targets for potential new generation therapies. To target this shared pathogenetic event, we have developed a family of novel TLR9 antagonists and started pharamacologic characterization. A novel biologic therapy is also being explored in preclinical models of metabolic syndrome. DNA-bending non-histone proteins can make regular 30- nm chromatin structure unviable Ranjith Padinhateeri Indian Institute of Technology, Mumabi, India Why most of the in vivo experiments do not find the theoretically expected 30-nm chromatin fiber is a puzzle. Two basic physical inputs that are crucial for understanding the structure of the 30-nm fiber are the bendability of the linker DNA and the relative orientations of the DNA entering/exiting nucleosomes. Based on these inputs we simulate chromatin structure and show that the presence of non- histone proteins, which bind and locally bend linker DNA, destroys any regular higher order structures (e.g., zig-zag). Our theory predicts a phase-diagram for the chromatin structure as a function of DNA-bending non-histone protein density and mean linker DNA length. For a wide range of linker lengths, we show that as we vary one parameter, that is, the fraction of bent linker region due to non-histone proteins, the steady-state structure will show a transition from zig-zag to an irregular structure-a structure that is reminiscent of what is observed in experiments recently. Our theory can explain the recent in vivo observation of irregular chromatin having co- existence of finite fraction of the next-neighbor i+2 and neighbor i+1 nucleosome interactions. MMP-cytoskeletal crosstalk in cancer invasion Shamik Sen Indian Institute of Technology, Mumabi, India Cancer invasion through dense extracellular matrices (ECMs) is mediated by matrix metalloproteinases (MMPs) which degrade the ECM thereby creating paths for migration. However, how this degradation influences the phenotype of cancer cells is not fully clear. Here we address this question by probing the function of MMPs in regulating biophysical properties of cancer cells relevant to invasion. We show that MMP catalytic activity regulates cell spreading, motility, contractility and cortical stiffness of highly invasive cancer cells by stabilizing integrins at the membrane and activating focal adhesion kinase. We further show that MMP inhibition causes a transition to a rounded amoeboidal phenotype through a combination of cytoskeletal disassembly-induced cell softening, and nuclear softening through increased phosphorylation of lamin A/C. Suppression of amoeboidal migration by cation-induced cell and nuclear stiffening establishes the importance of cell and nuclear softening in sustaining amoeboidal migration. Together, our results reveal an integrin mediated crosstalk between MMPs and the actomyosin cytoskeleton that enables invasion plasticity, i.e., switch from protease-dependent mesenchymal mode of migration to protease-independent amoeboidal mode of migration by altering cell and nuclear properties. .
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