| Description |
Adhesion and migration are required for proper development and for the maintenance of adult tissues. Cellular adhesion is regulated by signaling, adaptor, and actin associated proteins that assemble at the cell cortex in response to integrin activation. PINCH, ILK, and RSU-1 are three such adaptor proteins that form a physical complex downstream of integrins and have evolutionarily conserved overlapping, yet not identical, roles in integrin-mediated adhesion. The 5 LIM domain scaffolding protein PINCH is the central molecule in this complex, making contacts with ILK via LIM1 and RSU-1 via LIM5. In Drosophila, PINCH and ILK are required for maintaining actin-membrane linkages in the embryonic muscle, and null mutant clones in the wing display a loss of adhesion resulting in blisters. RSU-1 is not required for viability, but like PINCH and ILK, is required for maintaining adhesion of the wing epithelia. To further understand the contributions of these proteins to integrin function in vivo, a molecular analysis of PINCH was conducted using mutations that disrupt the interaction with ILK (PINCHQ38A and PINCHΔLIM1) or RSU-1 (PINCHD303V and PINCHΔLIM5). We find that PINCHQ38A transgenes rescue the stck (PINCH) null mutant, and rescued flies display no defects in integrin function. However, a dramatic reduction in viability is observed in PINCHQ38A rescued flies upon loss of RSU-1. Disruption of the PINCH-ILK interaction does not affect the levels of either protein, in contrast to results observed with complete loss of either PINCH or ILK, suggesting that PINCH and ILK mediate other interactions iv that contribute to their function and stability. PINCHΔLIM1 transgenes rescue the late embryonic/early larval lethality of the stck null mutant, but rescued animals only survive to larval and pupal stages, indicating that LIM1 function is required later in development. PINCHD303V transgenes rescue the stck null mutant, although rescued adult flies are not healthy. PINCHD303V rescued flies exhibit reduced levels of RSU-1, which is mislocalized, indicating that the PINCH-RSU-1 interaction is required to maintain levels of RSU-1 and to localize RSU-1 to adhesion sites. PINCHΔLIM5 transgenic flies were also generated, but the transgenes fail to rescue the stck null mutant due to instability of the transgenic protein. Taken together the data presented in this dissertation demonstrate functional consequences of disrupting the PINCH-ILK-RSU-1 complex, and suggest that multiple contacts made by each complex member contribute to protein localization, stability, and function downstream of integrin. |
