Cytoskeletal stresses - Driving force for focal adhesion and adherens junction remodeling in epithelial cells under flow
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Renal epithelial cells experience wide range of shear stress due to urine flow within the kidney tubules and adapt to it by altering various cellular processes including remodeling of actin cytoskeleton and cell adhesions. It is known that actin cytoskeleton plays a crucial role in transmitting the forces to various mechanotransducers in the cell, which may trigger the downstream adaptation processes including actin remodeling. How the shear stress is distributed in the cytoskeleton and how cytoskeletal dynamics alters cellular adhesion adaptation has not been explored. In this thesis, using stress sensitive FRET probes we measured the forces in cytoskeletal linking proteins in live cells under precisely controlled shear stresses in a microfluidic chip. We hypothesize that the flow can cause spatial and temporal distribution of cytoskeletal stress during actin reorganization and these actin dynamics can be transmitted to focal adhesions and adherens junctions through cross-linking proteins and regulate their remodeling under flow. We tested this hypothesis in three steps. 1) We measured real time actin cytoskeletal stresses correlated with actin reorganization by using actinin-sstFRET sensor in epithelial cells subjected to a fluid shear stress of 0.74 dyn/cm 2 ; 2) we correlated actin dynamics with focal adhesion; and 3) we correlated actin dynamics with adherens junction remodeling under flow by co-transfection of the stress sensor with fluorescence labeled junction proteins. Our results show that flow induces dynamic actin stress variations in epithelial cells, consisting of three different phases, leading to a net reduction in cellular stress and formation of peripheral actin bundles. The actin dynamics play a crucial role in driving focal adhesion and adherens junction remodeling under flow. The fate of focal adhesions under flow is determined by their interactions with linked stress fibers that provide the forces to the binding proteins at the adhesion complex. Long term exposure to shear stress leads to reduction of cell-ECM adhesion (focal adhesions diminish in cell interior), whereas an enhancement of cell-cell adhesions (adherens junctions stabilize along cell periphery). We integrate findings of this study and propose a model for the process of actin cytoskeletal reorganization and simultaneous remodeling of focal adhesions and adherens junctions in epithelial cells under flow.