The changes in the bending pattern of flagella induced by an increased intracellular Ca2+ concentration are caused by changes in the pattern and velocity of microtubule sliding. However, the mechanism by which Ca2+ regulates microtubule sliding in flagella has been unclear. To elucidate it, we studied the effects of Ca2+ on microtubule sliding in reactivated sea urchin sperm flagella that were beating under imposed head vibration, We found that the maximum microtubule sliding velocity obtainable by imposed vibration, which was about 170-180 rad/second in the presence of 250 μM MgATP and < 10-9 M Ca2+, was decreased by 10-6-10-5 M Ca2+ by about 15-20%. Similar decrease of the sliding velocity was observed at 54 and 27 μM MgATP. The Ca2+-induced decrease of the sliding velocity was due mainly to a decrease in the reverse bend angle. When the plane of beat was artificially rotated by rotating the plane of vibration of the pipette that held the sperm head, the asymmetric bending pattern also rotated at 10-5 M Ca2+ as well as at < 10-9 M Ca2+. The rotation of the bending pattern was observed at MgATP higher than 54 μM (~ 100 μM ATP). These results indicate that the Ca2+-induced decrease of the sliding velocity is mediated by a rotatable component or components (probably the central pair) at high MgATP, but is not due to specific dynein arms on particular doublets. We further investigated the effects of a mild trypsin treatment and of trifluoperazine on the Ca2+-induced decrease in sliding velocity. Axonemes treated for 3 minutes with a low concentration (0.1 μg/ml) of trypsin beat with a more symmetrical waveform than before the treatment. Also, their microtubule sliding velocity and reverse bend angle were not affected by high Ca2+ concentrations. Trifluoperazine (25-50 μM) had no effect on the decrease of the sliding velocity in beating flagella at 10-5 M Ca2+. However, the flagella that had been 'quiescent' at 10-4 M Ca2+ resumed asymmetrical beating following an application of 10-50 μM trifluoperazine. In such beating flagella, both the sliding velocity and the reverse bend angle were close to their respective values at 10-5 M Ca2+. Trypsin treatment induced a similar recovery of beating in quiescent flagella at 10-4 M Ca2+, albeit with a more symmetrical waveform. These results provide first evidence that, at least at ATP concentrations higher than ~ 100 μM, 10-6-10-5 M Ca2+ decreases the maximum sliding velocity of microtubules in beating flagella through a trypsin-sensitive regulatory mechanism which possibly involves the central pair apparatus. They also suggest that calmodulin may be associated with the mechanism underlying flagellar quiescence induced by 10-4 M Ca2+.
|ジャーナル||Journal of Cell Science|
|出版ステータス||Published - 2000|
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