The long-term goal of these studies is to understand mechanisms controlling neural apoptosis, with the expectation that this information will improve therapeutic approaches to decrease neural death following trauma, pathophysiological insults and neurodegenerative disease. The immediate goal of the study is to identify key issues concerning regulation and function of cytoplasmic execution with particular emphasis on biochemical and functional characterization of MRLC-P/membrane blebbing. Several hypotheses will be tested: (1) Apoptotic membrane blebbing is regulated by phosphorylation of MRLC (MRLC-P) through signaling pathways activating myosin light chain kinase and Rho kinase.
Aim 1 proposes to characterize signaling pathways controlling membrane blebbing/MRLC-P during the execution phase. A cell free system has been developed in which cellular extracts from apoptotic and control cells can be treated with various pharmacological inhibitors or co-factors, or be immunodepleted of regulators. (2) MRLC-P/membrane blebbing functions to package the cell and prepare the corpse for phagocytosis by helping to generate apoptotic bodies.
Aim 2 proposes to investigate the role of membrane blebbing/MRLC-P in cellular packaging, formation of apoptotic bodies and phagocytosis. A combination of pharmacological and molecular manipulation will be used to modulate membrane blebbing/MRLC-P and the effects on cellular packaging, formation of apoptotic bodies and phagocytosis noted. (3) Disassembly of microtubules at the onset of execution acts as an initiating event to regulate other downstream events in the execution phase.
Aim 3 proposes to investigate the regulatory role of microtubule disassembly occurring at the onset of execution in subsequent events in execution and determine if extracts from apoptotic cells increase microtubule assembly. The effects of either stabilizing or dissembling microtubules on downstream apoptotic events will be assessed. (4) The Rho family of small GTPases are key regulators of many of the cytoplasmic events occurring in the execution phase.
Aim I V proposes to characterize the role of the Rho family of GTPases (Rok Rac, and Cdc42) in events occurring in the execution phase. Dominant-negative and constitutively-active forms of Rho, Rac, Cdc42, Pak and Rok will be introduced into cells and the effects on the events in cytoplasmic execution noted.
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