Biophysical studies of kinesin-1 (conventional kinesin) in live cells.

Movement is one of the most characteristic features of life. While motion on a large biological scale is accomplished by the concerted activities of muscles, tendons and ligaments, motion on a nano-biological scale is accomplished by ingenious protein machines called molecular motors. Kinesin-1 is a molecular motor that uses the energy of ATP hydrolysis to carry cargoes along microtubule tracks in cells. Defects in Kinesin-1 transport have been linked to neurodegenerative diseases such as Alzheimer's and Huntington's diseases. A variety of biochemical and biophysical methods have been used to study Kinesin-1 in vitro, however, very little is known about the molecular mechanisms that control Kinesin-1 activity in vivo. Using a quantitative Fluorescence Resonance Energy Transfer (FRET) method, I determined the overall structure of Kinesin-1 in the inactive and active states, the conformational changes upon activation, and the specific regions of Kinesin-1 that contribute to autoinhibition. I showed that two cellular binding partners of Kinesin-1 are required for activation. Together, these results constitute the first discoveries about kinesin activation in living cells. To understand the mechanical properties of Kinesin-1 during transport in the crowded intracellular environment, I developed new methods for single molecule imaging in live-cells (SMILe). I determined that single Kinesin-1 motors that cannot bind cargo move in vivo with an average speed of 0.78 +/- 0.11 mum/s and an average run length of 1.17 +/- 0.38 mum, similar to in vitro. These results suggest that the motility of single motors is neither hindered in cells nor upregulated by unknown cellular factors. SMILe enables the study in live cells of a wide variety of cellular events (e.g. transcription, synaptic transmission, and membrane trafficking) that are driven by the action of a surprisingly low number of molecules. Collectively, these studies demonstrate the unique abilities of biophysical methods for studying complicated molecular mechanisms in the most physiological environment, the cell. Furthermore, the development of SMILe provides techniques to study rare events in cells. Pushing the development of ultra sensitive biophysical techniques and answering the most challenging cell biology questions are the major directions of my future research.PhDBiological SciencesBiophysicsCellular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126368/2/3253223.pd