ABSTRACT
Outer-arm dynein is the main engine providing the motive force in cilia. Using three-dimensional tracking microscopy, we found that contrary to previous reports Tetrahymena ciliary three-headed outer-arm dynein (αßγ) as well as proteolytically generated two-headed (ßγ) and one-headed (α) subparticles showed clockwise rotation of each sliding microtubule around its longitudinal axis in microtubule corkscrewing assays. By measuring the rotational pitch as a function of ATP concentration, we also found that the microtubule corkscrewing pitch is independent of ATP concentration, except at low ATP concentrations where the pitch generated by both three-headed αßγ and one-headed α exhibited significantly longer pitch. In contrast, the pitch driven by two-headed ßγ did not display this sensitivity. In the assays on lawns containing mixtures of α and ßγ at various ratios, the corkscrewing pitch increased dramatically in a nonlinear fashion as the ratio of α in the mixture increased. Even small proportions of α-subparticle could significantly increase the corkscrewing pitch of the mixture. Our data show that torque generation does not require the three-headed outer-arm dynein (αßγ) but is an intrinsic property of the subparticles of axonemal dyneins and also suggest that each subparticle may have distinct mechanical properties.