My long-term research objective is aimed at verifying the structure and function of cytoskeletal systems in amoeboid cells in terms of the molecular basis of cellular behavior with regard to the dynamic reorganization of cytoskeletal elements during cellular motile processes. The specific goals of the present proposal are to extend our recent findings that myosin molecules form thick filaments in Dictyostelium and its organization is changed in a dynamic manner during the chemotactic response to c-AMP in population of cells. To obtain more direct evidence, we wish to perform a detailed analysis of actomyosin and microtubule systems in individual cells locally stimulated with chemotactic agents using micromanipulators. This line of studies will be also done on individually identified cells using a motion analysis system. We also wish to examine the coordinated changes in these two cytoskeletal systems in individual mitotic cells. Thirdly, we wish to analyze the cytoskeletal organization of mutant cells which include abnormal myosin. To assess the possible role of myosin-I, we also wish to study differential localization of myosin-I and II. Finally, we will attempt to activate permeabilized cells with ATP to test the sliding movement of individual myosin filaments. The reactivating model will be also applied to studying differential functions of myosin-I and II. This proposal also aims at an electron microscopic identification of the myosin filaments using the quick-freeze, deep-etched platinum replication method. The methods will include high-resolution, high-contrast, high-sensitive videomicroscopy coupled with image-processing system, the application of chemotactic agents using ionophoresis apparatus equipped with micromanipulator, analysis of the behavioral response using motion-analysis system, and platinum-replication transmission electron microscopy.
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