Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex
Scaffold subunits support associated subunit assembly in the Chlamydomonas ciliary nexin-dynein regulatory complex
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DOI：
10.1073/pnas.1910960116
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发表时间：
2019-11-12
影响因子：
11.1
通讯作者：
Nicastro, Daniela
中科院分区：
文献类型：
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作者：
Gui, Long;Song, Kangkang;Nicastro, Daniela
运动纤毛和鞭毛中的Nexin-dynein调节复合体(N-DRC)作为相邻的双微管之间的连接物，起到稳定轴丝核心结构的作用，也是调节纤毛运动的中枢。虽然N-DRC已经通过遗传学、生物化学和结构方法进行了广泛的研究，但11个(或更多)N-DRC亚基的准确排列仍然未知。在这里，我们使用冷冻电子断层扫描，比较了衣藻野生型鞭毛的结构与特定DRC亚基缺失的菌株和带有标记的DRC亚基的挽救菌株的结构。我们的结果表明，DRC7是一个中央连接亚基，帮助连接N-DRC和外动力蛋白臂。DRC8的组装需要DRC11，而DRC8/11在连接器域的近端叶形成一个亚复合体，这是形成与邻近B管稳定接触所必需的。标记亚基的金标记法确定了DRC4先前不明确的N末端和DRC5的C末端的准确位置。DRC4现在被证明对N-DRC的核心支架做出了贡献。我们的结果揭示了N-DRC的总体结构，3个亚基DRC1/2/4形成了一个核心复合体，作为“功能亚基”组装的支架，即DRC3/5-8/11。这些发现揭示了N-DRC的组装及其在调节鞭毛跳动中的作用。
The nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the "functional subunits," namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.