Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonemes Available to Purchase

The movement of eukaryotic flagella and cilia is regulated by intracellular calcium. We have tested a model in which the central pair of microtubules mediate the effect of Ca²⁺ to modify the dynein activity. We used a novel microtubule sliding assay that allowed us to test the effect of Ca²⁺ in the presence or absence of the central-pair microtubules. When flagellar axonemes of sea-urchin sperm were exposed to ATP in the presence of elastase, they showed different types of sliding disintegration depending on the ATP concentration: at low concentrations of ATP (≤50μM), all the axonemes were disintegrated into individual doublets by microtubule sliding; by contrast, at high ATP concentrations (≥100 μM),a large proportion of the axonemes showed limited sliding and split lengthwise into a pair of two microtubule bundles, one of which was thicker than the other. The sliding behaviour of the axonemes was also influenced by Ca²⁺. Thus, at 1 mM ATP, the proportion of axonemes that split into two bundles increased from 25% at <10^–9 M Ca²⁺to 60% at 10^–4 M Ca²⁺, whereas the sliding velocity of doublets during the splitting did not change. Electron microscopy of split bundles showed that the thicker bundles contained five or six doublets and the central pair, whereas the thinner bundles contained three or four doublets but not the central pair. Closer examinations revealed that the thicker bundles were dominated by four patterns of doublet combinations:doublets 8-9-1-2-3-4, 8-9-1-2-3, 4-5-6-7-8 and 3-4-5-6-7-8. This indicates that the sliding occurred preferentially at one or two fixed interdoublet sites on either side of the central-pair microtubules, whereas the sliding at the remaining interdoublet sites was inhibited under these conditions. Ca²⁺ reduced the appearance of the 4-5-6-7-8 and 3-4-5-6-7-8 patterns and increased the 8-9-1-2-3-4 and 8-9-1-2-3 patterns. The splitting patterns are possibly related to the switching mechanism of the dynein activity underlying the cyclical flagellar bending. To investigate the role of the central pair in the regulation of the dynein activity by Ca²⁺,we studied the behaviour of singlet microtubules applied to the dynein arms exposed on the doublets of the split bundles that were either associated with the central pair or not. Microtubules moved along both the thicker and the thinner bundles but the frequency of microtubule sliding on the thinner (i.e. the central-pair-less) bundles was three to four times (at≤10^–5 M Ca²⁺) and ten times (at 10^–4 M Ca²⁺) as large as that on the thicker,central-pair-associated bundles. Furthermore, the velocity of microtubule sliding at 1 mM ATP on the thicker bundles were significantly reduced by 10^–7-10^–4 M Ca²⁺, whereas that on the thinner bundles was not changed by the concentration of Ca²⁺. These results indicate that Ca²⁺ inhibits the activity of dynein arms on the doublets through a regulatory mechanism that involves the central pair and the radial spoke complex. This mechanism might control the switching of the dynein activity within the axoneme to induce the oscillatory bending movement of the flagellum.