Adhesion receptor-cytoskeleton interactions in migrating tissue cells

Cell migration is a phenomenon requiring dynamic adhesive interactions between the internal cell motile machinery and the external substratum, with adhesion receptors, such as integrins, serving as the transmembrane link. The goal of this dissertation was to investigate the mechanism by which integrin interacts with cytoskeletal elements to give rise to cell movement. We have employed an available fibroblast system in which the cytoskeleton-binding domain of integrin was altered by site-directed mutagenesis. Three distinct approaches were taken: (1) laser optical trapping and nanometer-precision tracking were utilized to investigate the dynamics of integrin-cytoskeleton coupling; (2) computational simulations were performed to estimate the kinetics of these associations; and (3) cell migration parameters were examined to relate integrin-cytoskeleton binding with net cell movement.Nanometer-precision tracking and laser optical trapping were used to show that integrin is supplied by a cytoskeleton-dependent rapid transport mechanism to the cell's leading edge where nascent adhesions are formed. In contrast, large aggregates of integrin formed stable linkages to rearward-moving cortical actin, with stronger linkages existing at the cell front compared to the rear. This asymmetry in integrin-cytoskeleton interactions should yield a net transmission of force at the front, allowing the cell to move forward. We also conducted Brownian dynamics simulations with an added probability for linkage to a moving cytoskeletal element to mimic the directed transport of integrin. These studies reveal relative association and dissociation rate constants for integrin's interaction with the cytoskeletal element that gives rise to this transport. Finally, we used video microscopy and image analysis to measure cell migration speeds for the same fibroblasts containing site-directed mutations in integrin. We found that cells transfected with integrins that interact strongly with the cytoskeleton (as determined from laser trapping experiments) migrate significantly slower than cells transfected with weakly interacting integrins. These results confirm integrin's crucial role in migration and suggest that cellular regulation of locomotion may occur at the level of integrin-cytoskeleton associations. Furthermore, alteration of integrin-cytoskeleton coupling could be a potential candidate for targeted manipulation of cell migration in medical applications.U of I OnlyETDs are only available to UIUC Users without author permissio