| Flagella and cilia play critical roles in mammalian and other eukaryotic life by providing propulsion for swimming cells and moving fluids across tissue surfaces. Flagellar/ciliary bending is caused by the sliding of doublet microtubules (MTs) past each other due to a molecular motor called dynein attached to one doublet MT (dMT) "walking" along an adjacent dMT. Due to dMTs being fixed at the same end, this translocation produces a bend in the whole structure. While it is clear how the dynein molecules cause a bending of the dMTs, the mechanism underlying the generation of propagated waves of flagellar/ciliary motion has yet to be fully understood, especially with regard to the magnitude and regulation of the forces produced by dynein. Several outside studies have shown that some of dynein's mechanical properties such as velocity of MT gliding and force generation seem to be regulated by its multiple nucleotide binding sites. To better understand dynein's role in coordinated flagellar motion, we developed two in situ assays: one in which polymerized MTs glide along dMTs extruded from disintegrated bovine sperm flagella and an optical tweezers assay which is identical in geometry and environment except the MT is held in an optical trap to measure displacements and forces rather than velocities. The exposed, active dynein in each assay remain attached to their respective dMTs, allowing translocation of single MTs to be observed in an environment with direct control of chemical conditions. In the gliding assay, translocation of MTs by dynein exhibits Michaelis-Menten type kinetics, with Vmax = 4.7 +/- 0.2 mum/sec and Km = 124 +/- 11 muM. The character of MT translocation is variable, including smooth gliding, stuttered motility, oscillations, buckling, complete dissociation from the dMT, and occasionally movements reversed from the physiologic direction. The gliding velocity is independent of the number of dynein motors present and shows no indication of increased activity due to ADP regulation. In the optical tweezers assay, average force was found to be independent of [ATP] and [ADP] and distances of dynein's excursions indicates non-processivity. These combined results reveal dynein's motor activity, individually and cooperatively, within mammalian flagella. |
