The goal of this project is to understand how the motors which power fast axonal transport promote movement. Metabolic-uncouplers (eg., DNP, CCCP, valinomycin) block organelle movements along microtubules in vitro, but do not block movements of latex beads induced by kinesin or dynein. It thus appears that an ionic gradient across the organelle membrane is responsible for programming an organelle to go in the anterograde or retrograde direction. The next question is how kinesin or dynein is organized on the organelle surface and microtubule substrate. A new scanning transmission electron microscope (STEM), as described in Project # Z01-NS-02610-07 LN, has allowed kinesin to be visualized directly on the surfaces of microtubules and organelles. This STEM was used to determine at 2nm resolution the shapes and mass distributions of purified squid brain kinesin molecules--both free and bound to microtubules. Maps of kinesin bound to purified, taxol-stabilized bovine microtubules provided the first direct evidence for cross-bridging of microtubules by single kinesins which suggests that kinesins in cells might also translocate microtubules and therefore have some role in slow as well as fast axonal transport. The dynamic properties of other biological motors are being studied for comparison with the axonal transport motors; the bacterial flagellar motor in E coli has also been shown to depend on interactions or the flagellar structure with membrane proteins though these motors are driven by, rather than controlled by, ionic gradients. This motor system, like the axonal motor system, can also switch direction. A newly discovered component of the flagellar motor has led us to propose a novel structure model. Molecular genetic analysis of the new structural components may lead to an understanding of directional switching.
