biostudies-literatureBertiaux EEuropean Molecular Biology OrganizationSwiss National Science FoundationSchweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungStaatssekretariat für Bildung, Forschung und InnovationPro Visu FoundationNIHUniversitätsspital BaselNIGMS NIH HHSe3003519https://www.ebi.ac.uk/biostudies/studies/S-EPMC12707624biostudies-literatureUnknown23(12)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) and iterative U-ExM on human cells, together with in situ cryo-electron tomography (cryo-ET) on mouse tissues, 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-liPLoS biologyThe luminal ring protein C2CD3 acts as a radial in-to-out organizer of the distal centriole and appendages.PMC12707624bridging postdoctoral fundsTMPFP3_224900MB22.00075205087310030_205087R35 GM130286PP00P3_187198310030284–2019187198224900R35GM130286Genoud CLouvel VVan der Stappen PStearns TBertiaux EHamel Vvan den Hoek HBournonville LMean IGuichard PRighetto RDDaraspe JBatman UMuller AMercey OMukherjee SBuss GMcCafferty CLEngel BDfalseThe luminal ring protein C2CD3 acts as a radial in-to-out organizer of the distal centriole and appendages.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) and iterative U-ExM on human cells, together with in situ cryo-electron tomography (cryo-ET) on mouse tissues, 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-li2025-01-01T00:00:00Z2025 Dec2026-06-06T05:36:34.026Z2026-05-26T03:12:13.94ZS-EPMC127076244136471910.1371/journal.pbio.3003519