The goal of this new project is to understand how the motors which power fast axonal transport transduce the chemical energy associated with the hydrolysis of ATP into directed movement of organelles along microtubules. We are analyzing the sliding of microtubules along glass coated with kinesin, the protein which powers anterograde transport and analyzing the motion of plastic beads coated with either kinesin or the retrograde translocator in order to determine the sequence of chemical reactions which comprise the mechanochemical cycle; the different structural configurations which kinesin and the retrograde translocator undergo during the work cycle; and how the chemical cycle is coupled to the work cycle. The heart of this project involves analyzing microtubule based motility by video microscopy, using a digital processor to generate images with sufficient contrast to visualize single microtubules and to acquire and analyze motion as a function of manipulations of the chemical environment. This information complements biochemical measurements including binding of microtubules and nucleotides to the translocators and ATPase activities. Electron microscopy of rapidly frozen kinesin on glass or plastic beads is used to determine structural configurations of transient intermediates. These investigations define fundamental biophysical properties of kinesin and retrograde translocator-based movement which will serve as a baseline for assaying the effects of other proteins in cytoplasm which interact with and perhaps regulate the motors. Finally, our ability to combine, in a single experiment, biochemistry with motion analysis and electron microscopy using purified components promises to make a major contribution toward understanding the fundamentals of force transduction in motility systems.
