Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonernes
Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonernes
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DOI：
10.1242/jcs.00336
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发表时间：
2003-04-15
影响因子：
4
通讯作者：
Shingyoji, C
中科院分区：
文献类型：
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作者：
Nakano, I;Kobayashi, T;Shingyoji, C
真核生物鞭毛和纤毛的运动受细胞内钙的调节。我们测试了一个模型，其中中央微管对介导 Ca2+ 的作用来改变动力蛋白活性。我们使用了一种新颖的微管滑动测定，使我们能够测试 Ca2+ 在存在或不存在中央微管对的情况下的作用。当海胆精子的鞭毛轴丝在弹性蛋白酶存在下暴露于 ATP 时，根据 ATP 浓度的不同，它们表现出不同类型的滑动崩解：在低浓度 ATP（小于或等于 50 μM）下，所有轴丝通过微管滑动崩解成单独的双联体；相比之下，在高 ATP 浓度（大于或等于 100 μM）下，大部分轴丝表现出有限的滑动并纵向分裂成一对两个微管束，其中一个比另一个更粗。轴丝的滑动行为也受到 Ca2+ 的影响。因此，在 1 mM ATP 下，分裂成两束的轴丝比例从小于或等于 10(-9) M Ca2+ 时的 25% 增加到 10(-4) M Ca2+ 时的 60%，而分裂过程中双联体的滑动速度没有变化。分裂束的电子显微镜显示，较厚的束包含五个或六个双峰和中心对，而较薄的束包含三个或四个双峰，但不包含中心对。仔细检查发现，较厚的束主要由四种双联体组合模式主导：双联体 8-9-1-2-3-4、8-9-1-2-3、4-5-6-7-8 和 3-4-5-6-7-8。这表明滑动优先发生在中心对微管两侧的一个或两个固定双联体位点处，而在这些条件下其余双联体位点处的滑动受到抑制。 Ca2+减少了4-5-6-7-8和3-4-5-6-7-8图案的出现，并增加了8-9-1-2-3-4和8-9-1-2-3图案。分裂模式可能与周期性鞭毛弯曲背后的动力蛋白活性的切换机制有关。为了研究中心对在 Ca2+ 调节动力蛋白活性中的作用，我们研究了应用于动力蛋白臂的单线态微管的行为，该动力蛋白臂暴露在与中心对相关或不相关的分裂束的双联体上。微管沿着较粗和较细的束移动，但微管在较细的（即中心对相关的）束上滑动的频率是较厚的、中心对相关的束上的三到四倍（在小于或等于10（-5）M Ca2+时）和十倍（在10（-4）M Ca2+时）。此外，10(-7)-10(-4) M Ca2+ 显着降低了较粗管束上 1 mM ATP 下的微管滑动速度，而较细管束上的微管滑动速度并未因 Ca2+ 浓度而改变。这些结果表明，Ca2+ 通过涉及中心对和径向辐条复合物的调节机制抑制双联体上动力蛋白臂的活性。这种机制可能控制轴丝内动力蛋白活性的转换，以诱导鞭毛的振荡弯曲运动。
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 (less than or equal to50 muM), all the axonemes were disintegrated into individual doublets by microtubule sliding; by contrast, at high ATP concentrations ( greater than or equal to100 muM), 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 less than or equal to10(-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-pairless) bundles was three to four times (at less than or equal to10(-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.