One of the central themes in the development and differentiation of all multicellular organisms, is the elaboration of new cellular morphologies. Alterations in normal cellular morphologies are frequently associated with, and in some cases thought to cause, pathological states. Filamentous elements within the cell, especially microtubules (MT), are thought to be principle determinants of cellular morphology. The long-range goal of this project is to determine how the complex arrays of MTs within cells are coordinated and ultimately utilized to generate cellular asymmetries. To examine these questions, we will use the onset of cell motility of cultured fibroblasts as a model system. The MTs in these cells are highly dynamic; however, we previously found that when fibroblasts are triggered to undergo motility (by experimentally wounding cell monolayers), selected MTs in the leading edge of the cell are removed from the dynamic pool of MTs and are stabilized. To further establish the relationship between MT stabilization and the onset of motility, we will develop additional systems in which fibroblast motility can be triggered from a nonmotile state. We will then characterize the properties of the oriented and stable MTs using ultrastructural assays and MT growth assays to determine if the unusual stability of these MTs is due to a novel regulatory activity that caps the MTs. We will also develop an in vitro assay for MT capping that will be used to identify and purify the putative capping factor. By using growth factors and pharmacological agents, we will determine the nature of the trans-membrane signal that triggers the selective stabilization of MTs in the leading edge of motile cells. Finally, we will test the hypothesis that the stable MTs in the oriented array contribute to the establishment of cellular asymmetry by acting as specialized tracks for organelle movements. Our proposed experiments on the selective stabilization of MTs in motile cells will identify novel regulatory factors that modulate MT function and will lead to a better understanding of the role of MTs in morphogenetic events.
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