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

Abstract

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.

The radial spokes and central apparatus: Mechano‐chemical transducers that regulate flagellar motility

“…These results indicate that calcium regulation of flagellar motility in Chlamydomonas involves the regulation of dynein-driven microtubule sliding, that calmodulin and calmodulin-dependent kinase may mediate the calcium signal, and that the central apparatus and radial spokes are among the key components of the calcium signaling pathway [Smith, 2002a]. Similar microtubule sliding assays and reactivation studies using sea urchin sperm axonemes have also implicated the central apparatus in calcium-induced modulation of motility [Bannai et al, 2000;Nakano et al, 2003].…”

“…In these cell types, specific interactions of the radial spokes with the central apparatus may positively or negatively control dynein activity by a switching mechanism that does not involve central apparatus rotation. This notion is supported by the recent studies of isolated sea urchin sperm axonemes using a modified microtubule sliding assay [Yoshimura and Shingyoji, 1999;Nakano et al, 2003]. It is possible that rotation provides additional regulatory mechanisms that are primarily founded in the asymmetry of the central apparatus and that provide axonemes with versatility and an array of waveforms [for example, see Gray, 1928].…”

Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii

“…We have observed that lf1, lf2, and lf4 flagellar waveforms show serious deviations from the WT waveform. This points the way to further work involving an in vitro microtubule-sliding assay with isolated axonemes so as to examine the possible role of the central pair in modulating dynein activity on specific doublet microtubules [38][39][40]. A closer look at mutant cell shapes is, therefore, warranted.…”

Sperm dysfunction and ciliopathy

“…2 axonemes (Fig. 1b, c) and are involved in the activation or inactivation of dyneins on a specific doublet microtubule, resulting in the determination of the ciliary beating plane [14,15,164,165]. Mutations in the genes for RS/CP leading to ciliopathy include RS proteins (RSPH1, RSPH4A, and RSPH9) and those in the CP (HYDIN and SPAG17) ( Table 1).…”