Role of Cortical Cytoskeleton Mesh in the Regulation of Endocytosis and Exocytosis Near the Plasma Membrane in Normal and Under Stressed Conditions

Biswas, Anilisa (2014) Role of Cortical Cytoskeleton Mesh in the Regulation of Endocytosis and Exocytosis Near the Plasma Membrane in Normal and Under Stressed Conditions. Masters thesis, Indian Institute of Science Education Research Kolkata.

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Abstract

The dynamics of the intracellular endomembrane compartments during trafficking and the various mechanisms for the modulation of membrane surface area are not so well understood. One of our major focuses is to understand how the membrane undulations are regulated in a cell. The highly dynamic cortical actin affects the fluctuations of the undulations of the membrane by pinning the membrane, actively pushing and pulling the membrane and altering the membrane tension to regulate the surface area and thereby also regulating the interaction of vesicles with the plasma membrane. We have addressed the question of how the cortical actin could regulate the trafficking of vesicles across it-, making vesicles available for fusion, either allowing an increase or decrease in membrane surface area or hence modulating membrane fluctuations. It is important to note that vesicles are small and deformable and hence it is important to estimate the vesicle size distributions near the actin cortex and away from the plasma membrane. The actin mesh also has a size distribution and the size being estimable to the diffraction limit makes it challenging further to directly get to numbers. The important fact is that the actin meshwork can act as a kinetic barrier to the movement of vesicles because the sizes are in the same order of magnitudes. Active cytoskeletal rearrangements accompany vesicular transport and fusion. (J. Exp. Biol. 139 (1988) 253 – 266).The objective of this project is to closely monitor the movement of vesicles by Single Particle Tracking and Particle Image Velocimetry and try to calculate the actual numbers for the size distribution of vesicles and then correlate the effect of the presence of actin dynamics on it. Insights into these processes would allow us to elucidate the roles of the cortical actin mesh in the regulation of the rates of endocytosis and exocytosis during vesicular trafficking and surface area regulation. Thus, it is clearly understood that vesicle labeling and actin labeling has to be standardized and also novel methods have to be thought of which would enable us to quantify the actin and tracking of vesicles. In order to elucidate the roles of actin cytoskeleton disrupting drugs on the actin meshwork and vesicular movements we took the stable transfected cell lines of CAAX-GFP and LifeAct-mcherry and incubated them for 30 minutes with 2μM of Cytochalasin D, 80μM of Dynasore and 100μM of ATP Depleting agent. The effects of the drugs on the processes of trafficking were quantified by Particle Image Velocimetry.

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