Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella.
Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella.
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DOI：
10.1091/mbc.e11-08-0692
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发表时间：
2012-01
影响因子：
3.3
通讯作者：
Nicastro D
中科院分区：
文献类型：
--
作者：
Barber CF;Heuser T;Carbajal-González BI;Botchkarev VV Jr;Nicastro D
衣藻鞭毛的冷冻电子断层扫描揭示了径向辐条以前未表征的特征，包括结构异质性以及与动力蛋白以及辐条头之间的直接相互作用。 “径向辐条 3 替代”占据了具有辐条三元组的生物体中第三条辐条的位置。径向辐条（RS）在轴丝动力蛋白活性的调节以及纤毛和鞭毛运动的调节中发挥着重要作用。然而，有关所涉及的复合物的细节知之甚少。使用冷冻电子断层扫描和亚断层扫描平均，我们以前所未有的细节可视化了衣藻鞭毛径向辐条的三维结构。与许多其他物种不同，衣藻的每个轴丝重复只有两个辐条，RS1 和 RS2。我们的数据揭示了以前未表征的特征，包括有助于对接双联微管的双管辐条底座，以及内部动力蛋白直接连接到辐条。对野生型和无头辐条突变体 pf17 的结构进行比较，以确定头部的形态和边界，包括直接的 RS1 与 RS2 相互作用。尽管辐条的整体结构非常相似，但我们也观察到了一些差异，证实了最近关于 RS1 和 RS2 对接异质性的发现。我们发现了一种未表征的、较短的电子密度代替了第三个径向辐条，称为“径向辐条 3 替代品”，其结构与 RS1 和 RS2 没有相似之处，并且在 pf17 突变体中没有改变。这些发现表明，径向辐条在结构上是异质的，并且可能在轴丝调节中发挥功能上不同的作用。
Cryo–electron tomography of Chlamydomonas flagella reveals previously uncharacterized features of the radial spokes, including structural heterogeneity and direct interactions with dyneins and between the spoke heads. A “radial spoke 3 stand-in” occupies what would be the site of a third spoke in organisms with spoke triplets. Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo–electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named “radial spoke 3 stand-in,” which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.