The long-term objective of this research is to understand the regulation of microtubule-dependent organelle and vesicle transport (particle transport) using human embryonic fibroblasts as a cell model. The synthesis and incorporation of cellular material into organelles and vesicles, and their subsequent directed transport to specific sites in the cell, is a fundamental process of all cells. For example, some neuronal particles carry material from the synapse to the cell body. Some pathologies are believed to be correlated to a breakdown of this basic process. Very little is known about the mechanism and regulation of particle transport.
The specific aims of this proposal are the determine the signaling pathway(s) through which particle transport is inhibited by serum and vasopressin; to describe the particle subpopulations that are affected by the factors that inhibit transport; to describe the biochemical changes that occur on particle-associated proteins that are involved in particle transport. Video-enhanced contrast differential interference contrast and video intensification fluorescence microscopy will be used to identify specific particle subpopulations and to assay the effects of various bioactive factors and agents on particle transport. This will lead to an identification of the signaling pathway(s) through which serum and vasopressin inhibit particle transport and an identification of the particle subpopulations whose transport are affected by these factors. Density gradient centrifugation and two-dimensional polyacrylamide gel electrophoresis will be used to isolate the particles and analyze particle- associated proteins, respectively. Particle-associated proteins from cells treated with particle transport-inhibiting factors will be compared to those from untreated cells to determine the proteins involved in particle transport, and the biochemical changes (phosphorylation/dephosphorylation) these proteins undergo.
