Transport of organelles and macromolecular complexes through the cytoplasm is essential for survival of every eukaryotic cell. This process is performed by motor proteins that move their cargo along the cytoskeletal filaments, microtubules and actin filaments. Spatial and temporal control of organelle transport is critical for such processes as cell division, secretion and embryonic development. Our long term goal is to understand molecular mechanisms regulating organelle transport in the cytoplasm. Previous work by this laboratory established a permanent cell line of pigment cells (melanophores) from the frog Xenopus laevis as a model system to study the regulation of organelle movement at the molecular level. Melanophores move pigment organelles from the cell center to the periphery in response to high concentrations of cAMP, while at low cAMP levels pigment aggregates to the cell center. Movement of organelles in this system requires the coordinated activity of two cytoskeletal systems: microtubules/microtubule motors and actin filaments/myosin. We identified a myosin bound to pigment organelles in these cells as myosin V, a class of myosins known to be involved in organelle transport in other systems. The goal of this proposal is to analyze the role of myosin V in organelle movement in Xenopus melanophores and to study the mechanisms regulating its activity. We will obtain a cDNA clone encoding myosin V from Xenopus and will use dominant negative mutations and inhibitory antibodies to examine the role of myosin V in the movement of pigment along actin filaments in melanophores. In order to study how organelle transport by myosin V is regulated, we will analyze phosphorylation of this motor during pigment aggregation and dispersion. Phosphorylation sites will be mapped and changed by site-directed mutagenesis. DNA encoding these mutant proteins will be transfected into melanophores and the effects of the mutations on the ability of cells to aggregate and disperse pigment will be examined. Using similar techniques we will also study how activity of myosin V is regulated during cell division. In addition, we will identify and characterize proteins on the surface of organelles that are involved in myosin V binding. We will extend our findings to other cell types, such as neurons, in order to understand general mechanisms controlling actin and myosin-dependent organelle transport and the role of myosin V in nerve cells.
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