Cell motility contributes to both normal (e.g. wound healing) and pathological (e.g. metastasis) cell behavior. Underlying the migratory abilities of cells are the dynamics of actin at the leading edge, regulated in response to Rho GTPases. One such Rac GTPase-initiated pathway crucial to directed cell migration involves the precise spatial regulation of cofilin-dependent actin depolymerizing/severing activity through a phosphoregulatory cycle. During the past grant period, our laboratory identified and characterized a novel cofilin/ADF phosphatase which we termed chronophin (CIN). Our data show CIN to be an important regulator of cofilin phosphocycling, thereby modulating actin dynamics during cell division and motility. The regulation of CIN activity by upstream signals modulating Rac-dependent actin remodeling and cell motility will be investigated. We will use biochemical and molecular biological approaches, along with microscopy-based cell imaging, to determine the signaling pathways, proteins, and molecular complexes that influence cofilin regulation by CIN. The role(s) of CIN in coordinating Rac-dependent actin dynamics and cell motility will be studied. We will use quantitative fluorescent speckle microscopy (qFSM) to analyze the regulation of actin dynamics through CIN-modulated cofilin phosphocycling at the leading edge. In addition, we have shown for the first time that we can apply FSM to directly investigate growth factor (EGF)- regulated leading edge actin dynamics in the carcinoma cell line MTLn3. Cytoskeletal rearrangements induced by heat shock and/or ischemia/ATP depletion are a major cause of injury to cardiovascular cells, neuronal cells, and renal proximal tubule cells. These cytoskeletal responses may have both adaptive (survival) and injurious consequences. We have found that Hsp90 is an endogenous binding partner of CIN and negatively regulates CIN activity. We will examine the participation of Rho GTPase-Hsp90/CIN-cofilin signaling in ATP-depletion-mediated cytoskeletal remodeling. We will investigate the biochemical role of Hsp90 as a direct regulator of CIN activity under these conditions, and assess how the CIN-dependent cofilin cycle participates in stress-mediated cytoskeletal responses and cell survival. Relevance to public health: Cell motility plays important roles in normal physiology and in numerous disease states. Our studies will investigate a novel regulator of this important process. In addition, this protein is likely to play important roles in ischemic disorders and other stress responses. ? ? ?
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