Intracellular transport along the microtubule cytoskeleton is essential for neuronal development and function, with defects in the microtubule transport system causing neurodegenerative and neurodevelopmental diseases. For example, mutations in the microtubule-based motor KIF1C are linked to spastic ataxia 2, an autosomal recessive motor neuron disease. In polarized neuronal cells, KIF1C, a member of the kinesin-3 family, transports cargos toward axon terminals. Cytoplasmic dynein-1 moves the same cargos in the opposite direction. Adaptor proteins link kinesins and dynein to their cargos. In the case of KIF1C, the full set of its cargos is unknown, as is what adaptors mediate these cargo interactions and the molecular mechanisms that control KIF1C motility. To identify new KIF1C cargos and adaptors, I performed proximity-dependent biotinylation in human HEK293 cells. This method, recently applied by the Reck-Peterson lab to identify the dynein interactome, proved to be a powerful approach for identifying dynein adaptors and cargos. The KIF1C interactome was highly enriched for stress granule proteins. Another top ?hit? was the dynein adaptor protein HOOK3. Using immunoprecipitation experiments I confirmed that KIF1C interacts with multiple stress granule proteins and HOOK3. Stress granules are protein and RNA assemblies that form during cellular stress and are important for apoptosis, stress signaling, and mRNA sorting. Their assembly and disassembly is a dynamic process that requires the microtubule cytoskeleton. Deregulated stress granule accumulation and mutations in stress granule proteins are associated with motor neuron degeneration.
In Aim 1 of this proposal I will expand my studies of the KIF1C interactome to motor neurons, the cell type affected by KIF1C mutations and defects in stress granule homeostasis.
In Aim 2 I will determine how KIF1C interacts with protein and RNA stress granule components and the role of KIF1C in stress granule assembly/disassembly.
In Aim 3 I will investigate the role of cargo adaptor proteins, such as HOOK3, in linking KIF1C to stress granules, and the role such adaptors play in regulating both KIF1C and dynein motility. I will accomplish these Aims through experiments that combine proteomics, biochemistry, cell biology, in vitro single-molecule studies, and advanced three-dimensional live-cell imaging using lattice light sheet microscopy. My analysis will unravel the role of KIF1C in stress granule formation and/or disassembly, ultimately contributing to our understanding of neurodegenerative disease pathogenesis.
In human cells, neurological diseases are caused by the improper function of components of the cytoskeletal system. The proposed research will study one of these cytoskeletal components, the kinesin-3 molecular motor KIF1C, and unravel its role and regulation of intracellular transport in neuronal cells.
