Wednesday, January 15, 2025
1/15/2025
PROJECT SUMMARY The actin cytoskeleton impacts virtually every function of a human cell. Sophisticated regulatory mechanisms ensure that actin polymerizes when and where it is needed, and the assembly of actin filaments de novo is driven by proteins called nucleation factors. While the cytoskeleton is known to provide structure and generate forces in cellular processes that maintain viability, how it affects programmed cell death in response to stress is not well understood. To address this question, we are investigating the mechanisms by which actin nucleation factors from the mammalian Wiskott-Aldrich Syndrome Protein (WASP) family control apoptosis following DNA damage. Our recent studies demonstrate that the WASP-family members JMY and WHAMM activate the Arp2/3 complex to assemble a juxtanuclear F-actin-rich territory that regulates apoptotic signaling. The cytoskeletal territory (I) serves as a compartment for coupling the biogenesis of apoptosomes to the localized processing of caspases, and (II) sequesters the active caspases to temporarily protect the rest of the cell from proteolytic cleavage. However, the molecular interactions between the apoptotic components and cytoskeletal factors are not known. The specific goals of this project are to (1) define how apoptosome proteins and the actin nucleation machinery interact in early apoptosis, and (2) determine how caspases disrupt F-actin-rich territories to execute terminal apoptosis. To accomplish the first goal, we will (a) deplete the core apoptosome components cytochrome c and/or Apaf-1 and define the localization patterns of JMY, WHAMM, and their functional domains in fixed and live apoptotic cells; (b) characterize the physical interactions between apoptosomal proteins and actin nucleation factors in vitro; (c) systematically quantify the abilities of purified JMY, WHAMM, and Arp2/3 complex to polymerize actin in isolation, in the presence of each other, and in a cytochrome c-regulated manner in vitro; and (d) measure the effects of precise JMY and WHAMM mutants on actin assembly, apoptosome organization, and caspase activation in cells. To accomplish the second goal, we will (a) deplete or mutate several caspases to identify the ones needed for dismantling F-actin-rich territories in cells; (b) determine if caspases can cleave F-actin, G-actin, and actin-binding proteins in vitro; (c) define the roles of actin crosslinking, bundling, capping, severing, and depolymerizing proteins on territory stability and localization of a caspase inhibitor in cells; and (d) ascertain whether disease-associated actin mutants are resistant to caspase cleavage and affect terminal apoptotic signaling in cells. Collectively, our results will provide new insights into the molecular mechanisms of communication between key components of the intrinsic apoptotic signaling cascade and the actin cytoskeleton.
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