Characterization of nPIST Mediated Actin Cytoskeleton Regulation

Das, Swagata (2022) Characterization of nPIST Mediated Actin Cytoskeleton Regulation. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Swagata Das (12IP011)) 12IP011_DBS.pdf - Submitted Version Restricted to Repository staff only Download (5MB)

Abstract

Actin cytoskeleton plays crucial role in cell shape maintenance and several cellular activities like muscle contraction, cell motility, cytokinesis, endocytosis, exocytosis, intracellular trafficking etc. Multidirectional vesicular trafficking pathway requires rapid turnover of actin network structure at distinct stages of vesicle biogenesis, transport and fusion. Several classes of actin binding proteins regulate actin cytoskeleton architecture at different compartments of trafficking pathway. Actin and its interacting proteins have also been implicated in the organization and integrity maintenance of Golgi network which serves as the prime vesicle sorting machinery of the cell. Trans-Golgi associated protein PIST has two coiled-coil domains at the N-terminal and PDZ domain at the C-terminal containing protein. It interacts with several plasma membrane proteins and regulates their cell surface targeting. Its neuronal isoform nPIST is exclusively expressed in neuronal tissues and contains eight extra amino acid residues within the second coiled-coil domain. Till date, the underlying mechanism of PIST/nPIST mediated trafficking of several cell surface proteins remains to be elucidated. In this study, we have characterized nPIST as an actin binding protein. Our in-silico modeling of nPIST suggests presence of a putative WH2-like (WASP homology 2) domain lying between the second coiled-coil domain and the PDZ domain. In vitro experimental analysis shows that apart from the predicted WH2-like region, nPIST contains another three actin binding regions. Multiple actin binding region promotes actin filament stabilization activity of nPIST in vitro. When overexpressed inside cell, nPIST causes blotched colocalized accumulation of cellular actin in perinuclear region. Moreover, overexpression of nPIST also alters cellular Golgi distribution pattern. Overexpression studies with actin binding altered mutant nPIST constructs gives evidence of possible linkage between nPIST mediated actin cytoskeleton regulation and Golgi integrity maintenance. Furthermore, siRNA mediated depletion of endogenous PIST causes fragmentation of Golgi network, signifying its crucial role in Golgi architecture preservation. Taken together, our study unveils new insights into functional aspect of nPIST as well as PIST; for their indispensable role in Golgi integrity maintenance and actin cytoskeleton regulation inside the cell.

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