While considerable attention has been paid to the biochemical regulation of apoptosis, less effort has been directed towards elucidating the mechanisms that account for the morphology of apoptosis. This proposal will utilize fluorescent analogs of key cytoskeletal proteins to examine apoptosis-associated changes in cellular architecture in real time in living cells. Regulation by Rho family GTPases and cleavage of cytoplasmic components by caspases will be investigated. Particular attention will be paid to the role of D4GDI cleavage by CPP32 and how this alters Rho GTPase activity. Novel fluorescent indicators of protein cleavage will be developed to examine the kinetics and location of D4GDI and actin during apoptosis. Cytoskeletal control of specific Rho GTPases and their interactions in regulatory cascades will be determined by functional assays employing dominant negative and constitutively active mutants.