As a model system to study organelle transport, pigment granule aggregation and dispersion will be analyzed in lysed melanophores from the teleost fish, Tilapia mossambica and Fundulus heteroclitus, and the movements of isolated granules along microtubules will be assessed in vitro. The aim of the project is to determine whether or not dynein and kinesin have opposing roles in the generation of bidirectional pigment granule transport in melanophores and to understand the mechanism by which the two directions of transport are regulated. This laboratory has previously determined that dispersion requires protein phosphorylation via cAMP dependent protein kinase while aggregation requires protein dephosphorylation via calcineurin. Experiments will be carried out to determine if dynein, kinesin, or other components of the pigment granule transport system in the melanophores are substrates for the kinase and phosphatase. If these substrates as well as the motors generating opposing movements are identified, then it may be possible to elucidate the mechanism by which the phosphorylation and dephosphorylation signal is translated into transport in a given direction. The orderly transport of membrane-enclosed subcellular compartments from one region of the cytoplasm to another is a very important problem in cell biology. Such transport phenomena are responsible for a wide variety of cellular functions, including endocytosis and secretion. In the case of melanophores of fish, the transport of the pigment granules radially from an "aggregated" central location to a "dispersed" situation results in a change in the coloration of the skin of the fish, and is an important adaptive response to predation. This project will result in information that will be of use in not only understanding fish coloration, but also all other microtubule-mediated translocations of subcellular compartments.
