The general aim of this proposal is to develop a more fundamental understanding of the molecular interactions which serve to direct organelle traffic along microtubules in eukaryotic cells. Our studies will continue to focus on axoplasm extruded from the squid giant axon because this preparation is highly enriched for vesicular organelles that are transported along microtubules. One long-term aim is to elucidate the biochemical mechanisms that target kinesin and dynein, two motor proteins for movement in opposite directions on microtubules, to specific populations of organelles programmed to move toward either the plus- or minus-ends of microtubules. In pursuit of this question, we propose two approaches to identify vesicular membrane proteins that interact with these motor proteins. In one approach, biotinylated organelles, attached by rigor to microtubules, will be detergent extracted, leaving the motor- receptor complex attached to the microtubule. The motor-receptor complex will be eluted with ATP and the components further purified; biotinylated proteins will be analyzed by an avidin-chemiluminescent procedure after gel electrophoresis. In a second approach toward identifying a kinesin receptor, the C-terminal tail domain of the kinesin heavy chain, which is believed to interact with organelles, will be expressed as a glutathione S- transferase fusion protein, coupled to glutathione sepharose, and used to screen detergent extracts of highly purified vesicles for proteins that interact with the tail domain. We also propose experiments aimed at identifying mechanisms that regulate the direction of organelle transport. In these studies, we will use an in vitro organelle motility assay and optical tweezers to screen for factors that cause individual organelles to change their direction of movement. A second long-term aim of our research is to develop a molecular model for kinesin and dynein-driven movement. In these studies, optical tweezers will be used to manipulate beads carrying single motor proteins onto microtubules; the motion of these beads will be tracked at nm-scale resolution with an image processing technique, with the aim of imaging the molecular mechanical events underlying motility. These studies will be executed with recombinant kinesin motor domains, expressed as fusion proteins with sites for specific coupling to beads, in an effort to relate elements of the primary structure of kinesin to the process of mechanochemical transduction. In collaborative studies, we will employ instrumentation for tracking the motion of beads with significantly higher temporal resolution than is possible with video.
The aim of these studies is to characterize motion during the power stroke in increasing detail.
