Molecular motors that drive cargo transport along cytoskeletal filaments are critical for processes such as cell division, cell motility, intracellulr trafficking and ciliary function. Kinesin- based transport along microtubule filaments is particularly important in neuronal cells due to the polarized nature of the cell and the long distances between the cell body and synaptic regions. Defects in motor-driven transport processes are known to contribute to neurodegenerative and other diseases. Yet defining the exact transport contribution of each kinesin motor during neurite outgrowth, cellular polarization, axon generation and pathfinding, and circuit formation has been hindered by a lack of methods to control transport in an acute and motor-specific manner. In this proposal, we will take an "engineered chemical-genetic approach" to generate kinesin motors that can be inhibited by small molecules. Using kinesin-1 as a prototype, we will genetically modify kinesin-1's motor domain with sequences that can be chemically targeted by cell-permeable inhibitors. We will first characterize the inhibitable motors using in vitro assays that yield quantitative measures of motor activity. Successful inhibitory strategies will then be characterized by live cell imaging in fibroblasts and primary hippocampal neurons to define the exact complement of kinesin-1 cargoes. This work will provide the basis for future work aimed at a) developing animal models for studying kinesin-1 transport and b) generating inhibitable motors of the kinesin-2 and kinesin-3 families. This work will provide exciting new insights into how kinesin motors give rise to coordinated transport of protein complexes in cells and will suggest therapeutic targets in human disease.
The work in this proposal is relevant to public health because many human pathologies, such as neurodegeneration, cancer and the ciliopathies are caused by the disregulation of kinesin transport events. The proposed study will identify and characterize key behaviors of kinesin motor proteins and will advance our understanding of motor transport in order to promote treatment of motor-based diseases.