Cytoplasmic motility processes that direct the movement of specific molecules, supramolecular structures, and organelles to particular locations in the cell are essential in eukaryotes. Defining the mechanisms behind these processes is important in understanding both how cells function normally, and how they malfunction in medical conditions such as cancer, aneuploidy, infertility, and paralysis. It has become evident that many cellular components move by being pulled along microtubules by forceproducing """"""""motor"""""""" proteins. My research focuses on kinesin, a recently discovered motor protein that is capable of moving plastic beads along microtubules in vitro. My long term goals are 1) to define kinesin's biological functions, and 2) to learn the molecular details of how kinesin accomplishes those functions in the cell. To achieve these goals, we are studying kinesin in Drosophila using genetics, molecular genetics, biochemistry, and cytology. With collaborators, I have characterized Drosophila kinesin, prepared antibodies against the force-producing element, the kinesin heavy chain, and cloned and characterized the Drosophila kinesin heavy chain gene. Recently, I have isolated 13 lethal mutations in the kinesin heavy chain gene (khc). To determine how the heavy chain works in vivo, we will determine what structural features of the heavy chain are required for viability. The genetic lesions in each of the 13 mutations in hand and in another 40 that we propose to isolate will be located by DNA heteroduplex mismatch cleavage, and identified by partial sequence analysis. I expect this work to define the location of important functional sites in the kinesin heavy chain, and to illuminate some of the biochemistry operative at those sites. The information gained in this study will allow development of refined molecular models of kinesin structure/function and subsequent tests of those models by in vitro mutagenesis. We have begun to identify what kinesin's biological functions are by analysis of the effects of loss of kinesin function caused by heavy chain mutations. The data suggest that kinesin function is critical in neuronal tissue, and that its primary role there may be in axonal transport. This hypothesis will be tested by examining the structure and function of neurons in khc mutant and control larvae, using electron microscopy and electrophysiology. The kinesin heavy chain is a maternally loaded protein, so determining what kinesin's functions are in oocytes and embryos will be done by 1) germline clonal analysis of khc null alleles, and 2) studying dominant interactions of khc alleles with other mutations known to cause problems with microtubule-based motility processes. Preliminary results indicate that kinesin participates in meiotic chromosome segregation. I expect further analysis to identify other cellular processes that depend on kinesin function, and to define how kinesin participates in those processes.
Showing the most recent 10 out of 31 publications
