DESCRIPTION (Verbatim from the applicant's abstract): The broad objective of this proposal is to understand the crawling motility of animal cells. The proposal is focused on one aspect of the crawling process, namely the molecular mechanism of protrusion. The conceptual framework underlying the proposal is that protrusive motility can be expressed in terms of a novel, recently formulated hypothesis termed the dendritic nucleation/array treadmilling model. This model stipulates that steady-state protrusion is the result of a cycle of branched nucleation of actin filaments, elongation, capping, debranching and depolymerization. The specific aims targeted for the grant period are designed to test elements of the dendritic nucleation/array treadmilling hypothesis and to evaluate the design of the motility machinery in terms of the velocity of motility and magnitude of pushing force. The aims are: (1) to establish a bead-based motility assay; (2) to evaluate whether the Arp2/3 complex is critical for the balance between nucleation and elongation of actin filaments; (3) to evaluate the concept that capping protein functions to cap actin filament barbed ends globally but not locally; (4) to investigate the basis for the privileged status of uncapped ends at the working interface; (5) to evaluate mechanisms by which ADF/cofilin accelerates actin filament turnover; and (6) to determine the contribution of cross-linking proteins to the persistence of motility. Our research strategy will emphasize a combination of supramolecular structural, dynamic, model system and functional approaches. Experiments are proposed in vitro with a bead-based model system and in vivo with favorable cell systems,--amphibian keratocytes and human melanoma cell lines. Novel methods and approaches include the use of high resolution platinum replica and immuno-electron microscopy, digital fluorescence speckle microscopy, bead-based motility assays and laser optical trapping. The results will contribute to an understanding of the mechanism of actin polymerization-driven protrusion and cell motility which are fundamental processes of normal and malignantly transformed cells.
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NIH ExPorter