This is a proposal to determine the mechanism of binding of kinesin to organelle membranes and to establish the role of kinesin in the intracellular transport of cell organelles. This application builds on our previous success in creating an organelle motility assay that is based on defined components (chromaffin granules, microtubules (MTs), and kinesin) and gives efficient motility on Mts in vitro (greater than 30% simultaneous granule movement). It has also been observed that a significant portion of cellular kinesin is bound to organelle surfaces, particularly secretory granules and light microsomal components containing the Golgi apparatus. The goals are to identify the molecules of the organelle surface that are responsible for binding and to describe the binding mechanism, and study the function and regulation of the molecule.
The specific aims are to: (1) Use the organelle motility assay based on video-enhanced differential interference contrast (DIC) microscopy to determine the effects of solution conditions and soluble proteins on kinesin-dependent organelle transport and analyze the equilibrium binding of kinesin to chromaffin granule surfaces. (2) Identify the proteins and lipids that are required for kinesin-organelle interactions and determine if there is a specific membrane receptor for kinesin. (3) Identify the organelle-binding domain of the kinesin heavy chain and determine if it is located at the carboxy- terminal, tail-end of the protein. (4) Test the in vivo significance of kinesin for organelle transport, particularly with respect to the active migration of tubular membrane processes from the Golgi to other nearby organelles, by disrupting the kinesin-Golgi interactions with inhibitory antibodies to kinesin and kinesin-receptor-proteins. (5) Determine if the binding of kinesin to organelles or organelle motility are regulated by the phosphorylation of kinesin or kinesin-receptor-proteins. These studies will advance our understanding of the molecular basis of organelle transport and provide important new insights for future studies on the mechanism and regulation of kinesin-dependent motility in cells.
