The overall goal of this project is to understand how small GTPases of the Rho family regulate the stability and organization of microtubules (MTs) during polarization of cells for migration. The dynamics of MTs allows them to response to external signals during cell polarization and Rho GTPase signaling pathways are known to transduce these signals to MTs to bring about changes in their stability and organization. Cell migration into an in vitro wound is a model system for studying the signals regulating MT as the contribution of soluble, matrix and cell-associated factors can be dissected. The serum factor lysophosphatidic acid (LPA) stimulates separate Rho and Cdc42 signaling pathways that regulate two characteristic rearrangements of MTs in wound edge migrating fibroblasts: formation of a subset of unusually stable MTs and reorientation of the centrosome. In previous grant periods, we identified many of the factors in the pathways that are activated by these GTPases and have found that the pathways alter the dynamics of MTs near the cell cortex, a process termed MT capture. Yet, it is unknown how the proteins activated by these signaling pathways mediate MT capture or how the captured MTs influence cellular behavior. The current aims focus on the mechanism by which MT capture leads to long-lived MTs and the role of these stabilized MTs in motile processes in migrating cells. In addition to their well-characterized roles in regulating the actin cytoskeleton, formins have emerged as important regulators of MTs. We will explore the role of the formin mDia1 in generating stabilized MTs by defining its sites of interaction with MTs, determining its intrinsic ability to regulate MT dynamics, examining how interacting proteins, such as the MT +TIPs, alter its activity toward MTs and testing whether its interaction with MTs alters its actin polymerizing activity. We will explore the relationship between mDia1 and a kinesin motor protein that we have identified as a novel regulator of MT stabilization in cells. Approaches will be developed to specifically interfere with the generation of stable MTs in migrating cells so that their contribution to motile processes in cells can be determined. Understanding how Rho GTPase signaling pathways act to regulate MTs will provide new information about the fundamental ways cells transduce signals to cytoskeletal systems during cell migration, a process of importance for development, wound healing and metastasis.
Microtubules are dynamic cytoskeletal elements that contribute to cellular responses to external cues, such as growth factor stimulation. In migrating cells, microtubules contribute to the polarization of cellular activities that allow the cell to migrate in a directional fashion but the molecular mechanisms involved are unclear. Understanding the molecular mechanism controlling microtubules in migrating cells will contribute to fundamental knowledge of how cells migrate during development, wound healing and immune response and may lead to the identification of novel therapeutic targets for combating diseases involving abnormal cell migration, such as cancer metastasis and inflammation.
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