Single molecule approaches in studying biological nano-motors

Kinesin motor proteins are biological machines that use ATP hydrolysis to take 8-nanometer steps along the microtubule tracks. They perform a range of diverse intracellular functions in vesicle and organelle motility, control of the cell shape and chromosome segregation during meiosis and mitosis. Majority of kinesin motors carry their catalytic domain in the amino-terminus and it was believed until recently, that all these N-terminal motors move exclusively towards the plus-end of microtubules. However, in contrast to this dogma, we have recently reported that the S. cerevisiae mitotic kinesin-5 motors, which are essential for the mitotic spindle function in eukaryote cells, move towards the minus-end of the microtubules and change directionality under various experimental conditions. An additional bi-directional kinesin-5 from S. pombe was recently reported, indicating that the bi-directional motility of kinesin-5 motors is not as unusual as was previously thought. However, the mechanism and regulation of this bi-directional motility remains unknown.

Motility of kinesin motors is studied in a range of biochemical and biophysical in vitro assays, including microtubule gliding, antiparallel sliding and single molecule motility assays. We use such assays, in combination of live cell imaging, in order to study the mechanism which enables the bi-directional motility of some motors. Principles of these methodologies, as well as their implications will be presented and discussed.