REGULATION OF CYTOPLASMIC DYNEIN BASED VESICLE TRANSPORT

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Microtubule-based motility powers a number of intracellular transport functions including the dynamic saltatory movements of many membrane- bound organelles and nuclear and chromosome movement in mitosis. Cytoplasmic dynein, a ubiquitous, soluble isoform of the well-known enzyme axonemal dynein, participates in many of these intracellular movements. In vitro, cytoplasmic dynein is a microtubule-stimulated ATPase that can power the sliding movement of plastic beads and glass coverslips relative to microtubules. Although cytoplasmic dynein transports membrane vesicles on microtubules in vivo, in vitro the enzyme requires the participation of other soluble factors, such as the 20S dynactin complex, to drive vesicle movement. Further evidence of a requirement for dynactin complex in cytoplasmic dynein-driven motile events has been provided by genetic studies. Dynactin complex contains ten distinct subunits, eight of which have been identified by peptide sequencing, antibody cross-reactivity and molecular cloning techniques. Rotary shadow EM imaging reveals the molecule to be composed of two distinct structural domains, a 37 nm filament that resembles f-actin and a projecting shoulder and fine sidearm. Antibody decoration experiments show the 37 nm filament to be composed of the actin-related protein Arp1, actin-capping protein and the p62 subunit, while the fine sidearm contains the p160/p150Glued subunit. In experiments detailed here, the biochemical properties of the Arp1 and p160/p150Glued subunits will be studied in vitro. Pathways for complex assembly will be explored in crosslinking, disruption and reassembly experiments. Little is known about the interaction of cytoplasmic dynein or the dynein complex with membranes, nor is the mechanism by which dynactin stimulates dynein activity understood. The interaction of these two macromolecular complexes with membranes will be examined, paying particular attention to the effects of dynactin (and its subunits) on dynein binding. The effects of dynactin complex and its specific subunits on dynein ATPase activity will also be studied. Membrane proteins required for membrane motility will be isolated by reconstitution into proteoliposomes and their potential interactions with dynein and dynactin complex studied further.