Intracellular transport by molecular motor proteins is essential for many neuronal and other cellular processes. These processes appear to include positioning organelles and organizing secretion, as well as neuronal signaling, damage responses, and processes that fail in neurodegenerative and other diseases. Our focus is on kinesins, which generate directed movements along microtubules in many cellular contexts including mitosis, vesicle traffic, and axonal transport. Considerable work from our laboratory and others has provided much information about the basic properties of these systems, but has left unanswered several important questions, two of which we will attack in the coming funding period: 1) What is the logic of kinesin motor utilization? 2) How are kinesin motors regulated and attached to intracellular cargoes? To answer these questions, we propose to focus primarily on conventional kinesin, kinesin-I. Tactically, we use both mice and Drosophila because of their unique and complementary features. To achieve our goals, we will attack three specific aims in the next project period. Specifically, we propose: 1) To test the hypothesis that neuron specific forms of kinesin-I carry out neuron-specific functions such as slow and fast axonal transport of neuron-specific cargoes, while ubiquitous forms carry out the same general functions in both neural and non-neural cells. Null and conditional knockout mutants in the three kinesin heavy chain (KIF5A, KIF5B, and KIF5C), and the two major kinesin light chain (KLC1 and KLC2) genes in the mouse will be generated and analyzed using the Iox-cre system. We will focus on a few representative cell types including cultured embryonic fibroblasts, hepatocytes, photoreceptors, motor neurons, sensory neurons, and cultured hippocampal neurons. In addition, we will test the related hypothesis that kinesin-I plays a major role in the slow axonal transport of neurofilaments. 2) To test the hypothesis that kinesin light chain (KLC) is required for both cargo-attachment and regulation of kinesin-I. This hypothesis will be tested by analyzing the biochemical and cellular phenotype of mouse mutants lacking defined KLC subunits. 3) To elucidate the structure and function of two different proposed kinesin-I vesicular attachment complexes. We will determine the composition and role in kinesin-I transport of components of the syd/JIP-3 and the amyloid precursor protein complexes.
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