Aspects of cell motility play fundamental roles in cell cycling, division, migration, membrane transport and signal transduction, all of which are essential for normal cell growth, development and differentiation. Cell motility is mediated by a host of structural and motor proteins known collectively as the """"""""cytoskeleton"""""""". Retrograde flow is a form of cell motility that is mediated by the actin protein cytoskeleton and is widespread in eukaryotic cells. The flow process involves the continual movement of the lamellipodial plasma membrane, membrane proteins and underlying actin cytoskeleton from the cell periphery towards the cell center. This phenomenon has been postulated as functioning in cellular translocation, in the targeting of cellular migrations, and in aspects of morphogenesis. Retrograde flow has been extensively studied in only a few cell types (neurons, fish keratocytes and mammalian fibroblasts) and, despite these efforts, the exact mechanism and regulation of this fundamental process is still largely unknown. This proposal outlines a series of experiments aimed at elucidating the mechanism and regulation of retrograde flow using a unique experimental model, the sea urchin coelomocyte. These cells display a highly exaggerated form of flow and possess a number of properties which make them well suited for this study, including their optical properties, the availability of immunological probes for cytoskeletal proteins, and the readiness with which flow can be started and stopped and the cytoskeletal organization can be altered. Light and electron microscopic methods, combined with pharmacological and micromanipulation approaches will be used to address the following Specific Aims: 1. To determine the structural and functional relationships between actin filaments, between actin filaments and actin-binding and motor proteins, and between actin filaments and microtubules in cells undergoing retrograde flow. 2. To determine the regulatory roles of intracellular second messengers, protein kinase/phosphatase activity, and the Rho family of monomeric GTPase proteins on the process of retrograde flow. 3. To analyze the role of retrograde flow and cytoskeletal dynamics in cell wound healing.
