Our long-term goal is to understand the molecular basis and logic of intracellular transport, which is crucial for normal neuronal and other cellular functions, and may play important roles in neurodegenerative and other disease processes. Our major emphasis is on kinesins, which generate directed movements along microtubules in a variety of cellular contexts including mitosis, vesicle traffic, and axonal transport. Specifically, we want to answer two general questions: What is the logic of kinesin motor utilization? How are kinesin motors regulated and attached to intracellular cargoes? To answer these questions, we propose to focus primarily on the functions of conventional kinesin (kinesin-I), because many of the issues of interest that are playing out in the motor function field can be attacked in studies of this well-studied single motor and its components. Tactically, we will use both Drosophila and mice in our experiments because of their unique and complementary advantages. Thus, Drosophila will be used to identify new genes encoding potential regulatory or attachment proteins and to provide basic information about accessory component functions. Mice will be used for detailed physiological, cell biological, and biochemical analyses of genes first identified or analyzed in Drosophila. To achieve these general goals, we will attack three specific aims in the next project period: 1) To understand the range of functions of conventional kinesin (kinesin-I) by analyzing mutants in the three different kinesin heavy chain subunits in mice (KIF5A, KIF5B, and KIF5C). This work will be carried out by generating systemic and conditional knockout mutants using the lox-cre system. These mutants will be analyzed primarily in six different cell types including cultured embryonic fibroblasts, hepatocytes, photoreceptors, motor neurons, sensory neurons, and cultured hippocampal neurons. 2) To test the hypothesis that kinesin light chain (KLC) is required for the cargo-attachment or regulation of kinesin-I.
This aim will be achieved by analyzing the biochemical and cellular phenotype of systemic and conditional mouse mutants lacking each KLC subunit. 3) To identify and analyze new kinesin regulatory and cargo-attachment components. These new components will be identified and cloned in Drosophila and then characterized in depth using genetic, cytological, and biochemical methods.
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