Microtubules (MTs) and filamentous actin (f-actin), are required for directed tissue cell motility. Spatial control of f-actin polymerization and actomyosin contraction is essential to generate a self perpetuating asymmetry of lamellipodial protrusion in the front and retraction in the rear to drive cell motility. MTs provide spatial and temporal orchestration of these f-actin-based events, however the molecular basis is poorly understood. MTs and f-actin exhibit two mechanistic categories of interactions in migrating cells. """"""""Structural Interactions"""""""" in which f-actin and MTs are physically cross linked, and thus directly affect each other's organization and dynamics. """"""""Regulatory Interactions"""""""" are those in which the activities of Rho-family small GTPase signaling cascades are spatiotemporally controlled by the assembly and disassembly of MTs, and at the same time, Rho GTPases regionally co-regulate the dynamics of MTs and f-actin. It is critical for our understanding of the mechanisms of polarized cell motility to discover the molecular linkages between the microtubules and actin cytoskeletons and the molecular mechanisms that regulate these linkages and the feedback between the dynamics of the actin and MT cytoskeletal arrays.
Specific Aims :A.1. Test the hypothesis that specific classes of structural interactions between MTs and f-actin are required to mediate directed cell motility.A.2. Dissect the molecular mechanism of Rac1 GTPase-induced MT growth and test the hypothesis that Rac1-mediated promotion of microtubule growth is required for cell motility.A.3. Test the hypothesis that the adenomatous polyposis coli protein (ARC) mediates MT growth- activation of lamellipodial protrusion and Rac1 GTPase. A.4. Develop automated image analysis tools to quantitate the assembly, disassembly and movement of large populations of MTs in living cells. The experiments proposed in this grant will provide significant advances in our understanding of the basic mechanism of cell motility and provide important new technology that will aid in the advancement of other areas of biological science if they are achieved.
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