Microtubules (MTs) are ubiquitous elements of the cytoskeleton that are essential for the generation of cellular asymmetry during development and differentiation. In undifferentiated cells, most MTs are highly dynamic, whereas in polarizing or differentiated cells, certain MTs become stabilized and organized into specific arrays that are necessary for supporting polarized cell function. In many cases, alterations in proteins that regulate the formation of these MT arrays result in disease. Thus, lissencephaly is caused by alterations in the Lisl protein, which affects dynein functions in cell division and polarity. The nonsyndromic deafness syndrome, DFNA1, is cause by mutations in human Dia, a protein we have found regulates microtubule stabilization in fibroblasts. Our overall goal in this project is to determine the molecular mechanisms controlling MT stabilization and polarization and to explore their significance for cell function using the simple model system of in vitro wound healing. We have found that there are two distinct molecular pathways that regulate MTs during cell polarization and migration into wounds. These pathways resemble those described in yeast, where they function to position the nucleus and spindle. This suggests that MT-based polarization pathways have been conserved from yeast to mammals. One pathway involves the stabilization of MT oriented toward the wound and is regulated by the small GTPase Rho and its downstream effector mDia. We will explore how mDia regulates MT stabilization during in vitro wound healing by identifying the domain(s) of mDia important for MT stabilization and by identifying downstream effectors of mDia. Human Dia is mutated in DFNA1, and we will test the possibility that the mutation in DFNA1 alters MT stabilization. The second -pathway involves the reorientation of the MT organizing center (MTOC) toward the leading edge of cells adjacent to the wound margin. We have found that MTOC reorientation is regulated by CDC42 and is controlled independently of MT stabilization. We will study dynein and other potential downstream effectors to understand how CDC42 regulates MTOC reorientation. Lisl protein interferes with dynein function when overexpressed and we will test whether Lis1 overexpression effects MTOC orientation. We will study whether inhibition of MT stabilization or MTOC reorientation interferes with the polarization of cells, the migration of the cells into the wound and other wound responses. These studies should establish the molecular pathways by which MTs are regulated in cells and will contribute to our understanding of how cell polarity is established by MTs in all eukaryotic cells. These studies will also contribute to our understand of the basic molecular pathways involved in wound responses and will help explore the basic cell biological consequences of mutations in human Dia that causes deafness.
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