Proper rearrangement of the cell's microtubule cytoskeleton is an essential component of successful development. Important constituents of the cytoskeleton are its associated motor proteins which provide the force necessary for many microtubule-based movements. The long-term goal of this work is to understand how the activity of motor proteins is modulated during the complex changes in the cytoskeleton that occur during development. This proposal makes use of a small group of motor proteins related to the KIFC1 motor, in order to examine how relatively small changes in motor protein structure can alter function. KIFC1 isoforms differ in the domains responsible for cargo attachment and are found in several important microtubule complexes during mammalian spermatogenesis. The importance of the divergent cargo domains of KIFC1 isoforms to motor function will be investigated in the mitotic and meiotic spindles and the spermatid manchette. These microtuble-based structures perform very different functions during germ cell maturation but both contain KIFC1 isoforms suggesting a common function for these motors. The function of the spindles is well known: they and their associated motors act to segregate the genetic material during cell division. Less well characterized is the manchette that forms around the nucleus of elongating spermatids. Composed of about 1,000 microtubules, this structure is essential for the formation of viable sperm. The primary hypothesis to be tested in this study is that KIFC1 isoforms are targeted to different microtuble complexes where they act to stabilize these structures. Proposed experiments will identify the KIFC1 isoform composition of the spindle and manchette. The importance of the specific domains of these motors in directing these motors to the spindle and in microtuble bundling within the structure will be examined using in vivo and in vitro assays. Finally, the role of KIFC1 isoforms in the manchette will be examined by biochemical characterization and with in vitro motility assays. These studies will increase our understanding of how the interaction between motor and cargo is determined. Furthermore, the involvement of KIFC1 isoforms in multiple microtubule complexes during spermatogenesis has broad implications for regulation of the cytoskeleton in many other developmental systems. Given the involvement of KIFC1 homologs in the mitotic spindle, these motors are prime candidates for proper regulation of cell division and for misregulation during oncogenesis.
