The proposed studies seek to elucidate the mechanisms by which bidirectional organelle movements are mediated and regulated in fish melanophore cells. The principal investigator has recently demonstrated that pigment granule dispersion and aggregation are regulated by protein phosphorylation and dephosphorylation, respectively. Moreover, these studies suggested either that pigment granule dispersion is mediated by protein phosphorylation alone or that dispersion is mediated by a unique force generating ATPase that can utilize ATP-gamma-S for motility. In the proposed studies, experiments are designed to reveal if an ATPase is involved in dispersion. Additional experiments will be conducted to determine if exogenous cAMP protein kinases can rescue dispersion in cells treated with 8- azido-ATP, which inhibits endogenous protein kinases and ATPases. Studies will also be done to identify the protein whose phosphorylation and dephosphorylation are responsible for controlling the direction of pigment transport, to determine whether it is localized on pigment granules or in the cytoskeleton, and to determine if its interaction with these organelles differs during granule aggregation and dispersion. The ability of isolated granules to move along purified microtubules or melanophore cytoskeletons will be assessed, and the transport proteins will be identified and isolated. Cell biologists have long recognized that particles, molecules and organelles are transported in a highly regulated fashion within the cell. Recently, progress has been made in identifying the molecular basis of particle transport. The results of the experiment proposed here will contribute materially to our understanding of the transporter system and the proteins that constitute it.
