Bertiaux Eloïse, Louvel Vincent, McCafferty Caitlyn L, van den Hoek Hugo, Batman Umut, Mukherjee Souradip, Bournonville Lorène, Mercey Olivier, Mean Isabelle, Müller Adrian, Van der Stappen Philippe, Buss Garrison, Daraspe Jean, Genoud Christel, Stearns Tim, Engel Benjamin D, Hamel Virginie, Guichard Paul
University of Geneva, Department of Molecular and Cellular Biology, Sciences III, Geneva, Switzerland.
Biozentrum, University of Basel, Basel, Switzerland.
bioRxiv. 2025 Jun 18:2025.06.17.660204. doi: 10.1101/2025.06.17.660204.
Centrioles are polarized microtubule-based structures with appendages at their distal end that are essential for cilia formation and function. The protein C2CD3 is critical for distal appendage assembly, with mutations linked to orofaciodigital syndrome and other ciliopathies. However, its precise molecular role in appendage recruitment remains unclear. Using Ultrastructure Expansion Microscopy (U-ExM), iterative U-ExM, and cryo-electron tomography (cryo-ET), we reveal that C2CD3 adopts a radially symmetric 9-fold organization within the centriole's distal lumen. We show that the C-terminal region of C2CD3 localizes close to a ~100 nm luminal ring structure consisting of ~27 nodes, while its N-terminal region localizes close to a hook-like structure that attaches to the A-microtubule as it extends from the centriole interior to exterior. This hook structure is adjacent to the DISCO complex (MNR/CEP90/OFD1), which marks future appendage sites. C2CD3 depletion disrupts not only the recruitment of the DISCO complex via direct interaction with MNR but also destabilizes the luminal ring network composed of C2CD3/SFI1/centrin-2/CEP135/NA14, as well as the distal microtubule tip protein CEP162. This reveals an intricate "in-to-out" molecular hub connecting the centriolar lumen, distal microtubule cap, and appendages. Although C2CD3 loss results in shorter centrioles and appendage defects, key structural elements remain intact, permitting continued centriole duplication. We propose that C2CD3 forms the luminal ring structure and extends radially to the space between triplet microtubules, functioning as an architectural hub that scaffolds the distal end of the centriole, orchestrating its assembly and directing appendage formation.
中心粒是基于微管的极化结构，其远端有附属物，对纤毛的形成和功能至关重要。蛋白质C2CD3对远端附属物的组装至关重要，其突变与口面指综合征和其他纤毛病有关。然而，其在附属物募集的确切分子作用仍不清楚。使用超微结构扩展显微镜（U-ExM）、迭代U-ExM和冷冻电子断层扫描（cryo-ET），我们发现C2CD3在中心粒的远端管腔内呈径向对称的九倍体组织。我们表明，C2CD3的C末端区域定位于靠近由约27个节点组成的约100nm管腔环结构，而其N末端区域定位于靠近一个钩状结构，该钩状结构在从中心粒内部延伸到外部时附着于A微管。这个钩状结构与DISCO复合体（MNR/CEP90/OFD1）相邻，后者标记了未来的附属物位点。C2CD3的缺失不仅通过与MNR的直接相互作用破坏了DISCO复合体的募集，还破坏了由C2CD3/SFI1/中心蛋白-2/CEP135/NA14组成的管腔环网络以及远端微管末端蛋白CEP162的稳定性。这揭示了一个复杂的“由内而外”分子枢纽，连接中心粒管腔、远端微管帽和附属物。尽管C2CD3的缺失导致中心粒变短和附属物缺陷，但关键结构元件仍然完整，允许中心粒继续复制。我们提出，C2CD3形成管腔环结构并径向延伸到三联微管之间的空间，作为一个构建枢纽，支撑中心粒的远端，协调其组装并指导附属物的形成。
