Analysis of microtubule sliding patterns in Chlamydomonas flagellar axonemes reveals dynein activity on specific doublet microtubules.

Generating the complex waveforms characteristic of beating eukaryotic cilia and flagella requires spatial regulation of dynein-driven microtubule sliding. To generate bending, one prediction is that dynein arms alternate between active and inactive forms on specific subsets of doublet microtubules. Using an in vitro microtubule sliding assay combined with a structural approach, we determined that ATP induces sliding between specific subsets of doublet microtubules, apparently capturing one phase of the beat cycle. These studies were also conducted using high Ca2+ conditions. In Chlamydomonas, high Ca2+ induces changes in waveform which are predicted to result from regulating dynein activity on specific microtubules. Our results demonstrate that microtubule sliding in high Ca2+ buffer is also induced by dynein arms on specific doublets. However, the pattern of microtubule sliding in high Ca2+ buffer significantly differs from that in low Ca2+. These results are consistent with a 'switching hypothesis' of axonemal bending and provide evidence to indicate that Ca2+ control of waveform includes modulation of the pattern of microtubule sliding between specific doublets. In addition, analysis of microtubule sliding in mutant axonemes reveals that the control mechanism is disrupted in some mutants.