Most intracellular motility processes employ motor proteins to move organelles or other cargo along cytoskeletal filaments. There are many essential cargoes, many specific destinations for them, and a large number of different motors. The key questions are: How do motors create movement? and Which motors accomplish what tasks? The proposed research will focus on the microtubule-based motor kinesin. It will investigate how kinesin interacts with microtubules, how its two motor subunits interact with one another, and what its functions are in a developmentally complex organism (Drosophila). In addition, a genetic screen will be used to identify new proteins that interact with kinesin as cargo linkers or regulators. The specific goals of the proposed research are as follows: 1) To identify amino acids of the kinesin heavy chain (KHC) that mediate binding to microtubules. This will be done by: a) identifying the amino acid changes in four mutated Drosophila kinesin heavy chain genes (khc) that show allele specific genetic interaction with a mutation in the Beta2 tubulin gene and b) testing the effects of those changes and others on the in vitro microtubule binding activity of dimerized KHC motor domains. 2) To identify amino acids of KHC that are important for the exceptionally processive movement of kinesin on microtubules. This will be done by: a) screening a large collection of lethal khc mutations for amino acid changes in a small region that is thought to be responsible for coordinating the stepping activities of the two motor domains of a KHC dimer and b) testing the effects of those changes on processive movement in vitro. 3) To determine if kinesin is important for male or female germ cell development/function or for embryogenesis. This will be done by creating chimeric flies that have germlines with no KHC and analyzing gametogenesis. fertility, and embryogenesis. 4) To study newly discovered neuronal defects caused by a loss of kinesin function: distal axonal neuropathy and photoreceptor specific retinal degeneration. These experiments may identify some of kinesin's cargoes in axons and may provide a new understanding of the role of axonal transport motors in the pathology of vertebrate neuropathies. 5) To identify and study the functions of proteins that are involved in kinesin-cargo linkage and in kinesin regulation. This will be done using a fast genetic screen of the Drosophila genome for deficiencies that cause synthetic distal neuropathy when combined with khc mutations in double heterozygotes.
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