? ? The broad aim of the studies described in this proposal is to gain an insight on the poorly understood processes regulating the functional localization of myosin II filaments in a nonmuscle cell context. Critical cellular events, such as cell division and cell locomotion, rely on the correct organization and localization of myosin II within the cell; however, the signaling pathways regulating myosin II during these cellular events are poorly understood. Studies in the simple amoeba Dictyostelium have established that the dynamic process of myosin II bipolar filament assembly and disassembly influences the temporal and spatial localization of myosin II in a cell. More specifically, the phosphorylation of sites in the """"""""tail"""""""" segment of myosin heavy chain (MHC) results in the disassembly of cytoskeleton-associated myosin filaments into a soluble, cytoplasmic pool of monomers. MHC Kinase A (MHCK A) is a key enzyme driving myosin II filament disassembly through MHC phosphorylation in Dictyostelium and is organized into three functional domains: a) an N-terminal coiled-coil region, b) a novel kinase domain unrelated to any conventional protein kinases, and c) a WD-repeat domain at its C-terminus. MHCK A is rapidly recruited to the cell cortex upon chemoattractant stimulation, and is preferentially recruited to sites of pseudopod formation. This recruitment, which is actin-dependent, is meditated by the coiled-coil domain of MHCK A and is proposed to play an important role in regulating localized disassembly of myosin II filaments at cortical sites. The experiments described here will explore the mechanism and functional consequence of MHCK A recruitment to the actin-rich cortex of the cell. To this end, the following questions will be addressed: 1) What part of the coiled -coil domain of MHCK A mediates recruitment to the cell cortex? MHCK A coiled-coil domain truncations will be expressed as GFP fusion proteins in Dictyostelium cells and then tested for chemoattractant-stimulated translocation to the actin-rich cortex. 2) Is the coiled-coil domain alone able to interact directly with actin filaments? Truncations of MHCK A containing the entire coiled-coil domain will be tested in co-sedimentation assays for actin-binding. 3) Is the activity of MHCK A regulated by its interaction with actin filaments? Several assays of MHCK A activity will be used to determine if MHCKA translocation to actin filaments affects the activity of the kinase. Results from these studies will contribute to our understanding of the mechanisms controlling localized myosin II assembly in a nonmuscle cell context. This information is critical for understanding the molecular factors regulating cellular contractile processes such as cytokinesis and cell migration and ultimately contributes to our understanding of how these processes go awry in cancer cells exhibiting uncontrolled cell division and metastasis. In another context, these studies are important for understanding how acto-myosin reorganization is regulated during the process of chemotaxis in immune system cells such as neutrophils. ? ? ?
