This chapter describes a biochemically defined organelle motility assay reduced to the most basic components of vesicles, kinesin, and a microtubule (MT) substrate suspended in a simple MgATP motility buffer. The chromaffin cells of the bovine adrenal medulla are used because they are one of the best characterized examples of vesicular secretion, while containing large amounts of secretory vesicles and kinesin. The exceptionally pure bovine adrenal or brain kinesin supports the ATP-dependent movements of purified chromaffin granule “ghosts” along MTs preattached to a substrate in the presence of a low-salt buffer, a condition that had been demonstrated previously to support high levels of MT-activated ATPase activity. Moreover, under these conditions kinesin supports the movements of granule ghosts. These movements are frequent in number and exhibit a velocity equal to that of kinesin-coated beads translocating along MTs. This suggests that kinesin alone in the presence of low salt can initiate and sustain the anterograde translocation of membranous organelles without the requirement of other soluble factors or proteins. Analysis of video microscopic images shows that vesicles in Brownian motion bind to MTs and translocate at rates of 0.4–0.6 μm/s in the presence of any of the three different kinesin preparations.
ASJC Scopus subject areas
- Cell Biology