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 began by analyzing the sliding of microtubules along glass coated with kinesin, the protein which powers anterograde transport. In order to understand how kinesin works in this simple system, we addressed three problems: to define the sequence of chemical reactions which comprise the mechanochemical cycle; to define the different structural configurations which kinesin undergoes during the work cycle; to determine 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 both generate images with sufficient contrast to visualize single microtubules and to acquire and quantitatively analyze motion data. Motion is analyzed as a function of biochemical manipulations in a flow cell. This information complements conventional biochemical measurements characterizing the binding of kinesin to microtubules and of nucleotide substrates and products to kinesin. Electron microscopy of rapidly frozen kinesin on glass is used to determine structural configurations of transient intermediates. These investigations define fundamental biophysical properties of kinesin-based movement which will serve as a baseline for assaying the effects of other proteins in cytoplasm which interact with and perhaps regulate kinesin. 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.
