David P. Lorch*, Charles B. Lindemann, Alan J. Hunt*
Flagellar dynein generates forces that produce relative shearing between doublet microtubules (MTs) in the axoneme, this drives propagated bending of flagella and cilia. To better understand axonemal dynein's role in this intricately coordinated flagellar/ciliary motion, we have set up in vitro assays of MTs gliding across doublet MTs from disintegrated bovine sperm flagella. This allows study of exposed, active dynein distributed in physiologic geometry along doublet MTs. In the presence of ATP, MTs exhibit variable behaviors during their interactions with dynein such as stops, starts, and occasionally complete dissociation from the doublet. Gliding velocities measured have ranged from ~40 nm/sec to ~740 nm/sec. We are pursuing further characterization of these movements using optical tweezers. Specifically, MTs attached by tau-protein linkages to 1 micron silica beads held by the optical trap will be brought into contact with active dynein on the immobilized doublet MTs, allowing for high resolution characterization of the force and displacement generated by axonemal dynein. This technique should lead to more accurate measurements of the mechanical properties of axonemal dynein and refine existing models of flagellar motion.