Actin and myosins in organelle motility

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DESCRIPTION (provided by applicant): The goals of the proposed research are to characterize the role of actin filaments and myosin motors in transport of melanosomes (pigment granules) within retinal pigment epithelial (RPE) cells from fish. Melanosomes undergo massive migrations in fish RPE cells in vivo in response to light condition. Melanosome motility persists in dissociated, cultured RPE cells in vitro in response to chemical signals. Recent investigations resulted in a model for melanosome motility in RPE cells. The model hypothesizes that centripetal aggregation of melanosomes in RPE projections occurs by passive association of melanosomes with actin filaments undergoing constitutive retrograde flow. Centrifugal dispersion of melanosomes is hypothesized to occur by transport of melanosomes along actin filaments via myosin motors. To test the predictions of this model, the specific aims of the proposed research are 1) To determine whether actin filaments within RPE apical projections undergo retrograde actin flow using fluorescent speckle microscopy (FSM), and to simultaneously view actin flow and melanosome motility during aggregation to determine whether the movement of melanosomes is coordinated with actin flow. 2) To characterize actin-based motors on RPE melanosomes using in vitro motility assays. To accomplish this, melanosomes will be purified using density gradient centrifugation, and proteins associated with melanosome membranes will be identified using SDS-PAGE and immunoblotting. Actin-dependent motility of melanosomes will be tested using in vitro motility assays. Two assays will be used: in the Nitella assay, isolated melanosomes are applied to actin cables of dissected cells of the Characean alga, Nitella, and viewed using time-lapse video. The second assay is the sliding filament assay, where fluorescently labeled actin filaments are observed moving over melanosomes attached to a substrate. These assays will help to characterize melanosome-associated myosin motors. The proposed research will contribute to a better understanding of actin-dependent organelle motility, a universal characteristic of eukaryotic cells.