Mechanism and Regulation of Actin-Based Retrograde Flow

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DESCRIPTION (provided by applicant): Retrograde flow is a form of cell motility that is mediated by the actin 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 indicated as being important in cellular translocation, in the targeting of cellular migrations, in cell-substrate interactions, and in modulating the crosstalk between actin and microtubules. Retrograde flow has been extensively studied in only a few cell types (particularly neurons and mammalian and amphibian tissue culture cells) and, despite these efforts, the exact mechanism and regulation of this fundamental process is still largely unknown. The objective of this proposal is to expand the knowledge of the mechanisms underlying retrograde flow using a unique experimental model, the sea urchin coelomocyte. These cells display a highly exaggerated form of retrograde flow and possess a number of attributes 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 and actin-binding and motor proteins in the context of coelomocyte retrograde flow 2. To elucidate the physical forces operating in the context of coelomocyte retrograde flow. 3. To analyze the structural and functional relationships between actin filaments and microtubules in the context of coelomocyte retrograde flow. 4. To study the inducible transformation of coelomocytes from a lamellipodial to a filopodial form.