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We utilize optical tweezers to examine displacements produced by small numbers of dynein motors located in situ on doublet microtubules from disintegrated mammalian sperm axonemes. In contrast with cytoplasmic dynein, we find that axonemal dynein is not processive, and the duration of individual force-generating interactions with a microtubule are longer than predicted from the velocity of movements generated by large ensembles of motors. These findings suggest that tension is required for rapid release of dynein following a power stroke and may explain how axonemal dynein is adapted to work in arrays within an axoneme, where cyclical bending patterns require motors to function over a range of sliding velocities.

Flagellar dynein generates forces that produce relative shearing between doublet microtubules in the axoneme; this drives propagated bending of flagella and cilia. To better understand dynein’s role in coordinated flagellar and ciliary motion, we have developed an in situ assay in which polymerized single microtubules glide along doublet microtubules extruded from disintegrated bovine sperm flagella at a pH of 7.8. The exposed, active dynein remain attached to their respective doublet microtubules, allowing gliding of individual microtubules to be observed in an environment that allows direct control of chemical conditions. In the presence of ATP, translocation of microtubules by dynein exhibits Michaelis-Menten type kinetics, with Vmax 5 4.7 6 0.2 lm/s and Km 5 124 6 11 lM. The character of microtubule translocation is variable, including smooth gliding, stuttered motility, oscillations, buckling, complete dissociation from the doublet microtubule, and occasionally movements reversed from the physiologic direction. The gliding velocity is independent of the number of dynein motors present along the doublet microtubule, and shows no indication of increased activity due to ADP regulation. These results reveal fundamental properties underlying cooperative dynein activity in flagella, differences between mammalian and non-mammalian flagellar dynein, and establish the use of natural tracks of dynein arranged in situ on the doublet microtubules of bovine sperm as a system to explore the mechanics of the dynein-microtubule interactions in mammalian flagella. Cell Motil. Cytoskeleton 65: 487–494, 2008. ' 2008 Wiley-Liss, Inc.