The long term goal is to understand microtubule-dependent, intracellular transport processes such as: chromosome movement, vesicle transport, pronuclear migration and ciliary/flagellar motility. These processes depend on microtubules to serve as """"""""roadways"""""""" for a variety of microtubule-dependent motors. Intracellular transport processes are essential for cell and therefore organism survival. Defects in these processes may lead to serious health problems including cancer, aneuploidy, neuronal developmental defects, sterility and respiratory impairment. The focus of this study is to understand how microtubule motors interact with microtubules: l) to generate movement coupled to ATP hydrolysis: and 2) to transport other microtubules as cargo. The means by which motors use the energy produced from ATP hydrolysis to generate force is not clear. but one essential component of the translocation process is the motor's continual binding and release of microtubule as it moves along the microtubule lattice. To date, the sites of contact between motors and microtubules are poorly characterized and are, at best, described as domains of approximately 50-200 amino acids. Almost nothing is known concerning the interaction at tee molecular level, i.e., what amino acid groups are involved and what types of chemical associations occur. In addition to binding to microtubules to generate movement, some motors bind microtubules at a second site independent of ATP. These motors have cargo- binding sites that recognize microtubules as cargo, and thus transport cargo microtubules relative to the microtubules upon which the motor is moving. Again, sites on motors and on microtubules that are responsible for this interaction are poorly characterized. The goals of this study are to identify domains and amino acids on both motors and microtubules that are involved in ATP-dependent and ATP- independent interactions and to characterize the functional role(s) of these groups in the binding site(s). This knowledge will contribute to understanding the complex mechanisms involved in both movement and cargo recognition. A combination of classic biochemical techniques and modern molecular methods will be used to accomplish these goals. Several different native and bacterial-expressed motors will be studied to develop a general picture of motor-microtubule binding as well as to identify differences that may be related to a specific motor's function and/or properties. Binding sites will be identified principally by: l) crosslinking of motormicrotubule complexes; 2) protein modification of motors and microtubules: 3) peptide-binding screens; and 4) the two-hybrid system. Domains and amino acids identified by these methods will be functionally characterized using: 1) synthetic peptides; 2) in vitro mutagenesis: and 3) the two-hybrid system.
