Cep57 directly interacts with Cep63, but is dispensable for centriolar anchoring of the Cep63–Cep152 complex. (A) Mapping the Cep63-interacting regions of Cep57. The numbers indicate amino acid positions. The N-terminus of Cep57 is required for the interaction with Cep63 and the C-terminus for its centrosomal localization. (B) Domain mapping of Cep63. The numbers indicate amino acid positions. The N-terminus of Cep63 is required for the interaction with Cep57 and its centrosomal localization, whereas the C-terminus interacts with Cep152 (Zhao et al., 2013). (C) GST pulldown assay. Bacterial lysates expressing the indicated proteins were mixed and subjected to GST pulldown assays. Cep152M contains the fragment of mouse Cep152 from positions 1075 to 1383 aa (Zhao et al., 2013). (D) Schematics showing the interaction order of Cep57, Cep63 and Cep152. Cent represents the centrosomal-targeting region. (E) Confirmation of the KO cell lines. Cep57 or Cep63 knockout (KO) U2OS cells were lysed and subjected to immunoblotting. β-actin served as the loading control. (F,G) The effects of knockout of Cep63 on the centrosomal localization of Cep152 (F) or Cep57 (G) in U2OS cells. Note that knockout of Cep63 significantly reduced the centrosomal localization of Cep152, but not Cep57. (H,I) The effects of knockout of Cep57 on the centrosomal localization of Cep63 (H) or Cep152 (I) in U2OS cells. Note that both Cep63 and Cep152 still localized to the centrioles upon the Cep57 depletion. Quantification results (F–I) were from three independent experiments, and 75 cells at the G1 phase were scored per condition. Mean±s.d. values are presented in the plots. **P<0.01, ***P<0.001 (Student's t-test).

Cep57l1, the paralog of Cep57, forms a complex with Cep63 and Cep152 at the proximal end of centrioles. (A) Schematic diagram of mouse Cep57 and its paralog Cep57l1. (B) Schematic diagram of Cep57l1 and deletion mutants showing the ability to interact with Cep63 and to localize to the centriole. (C) Mapping the Cep63-interacting domain of Cep57l1. GFP-tagged Cep57l1 or its mutants were co-expressed with Flag–Cep63 in HEK 293T cells. Co-immunoprecipitation was then performed with GFP beads. Luc , luciferase control. (D) The requirement of full-length Cep57l1 for its centriolar localization. U2OS cells expressing GFP-tagged Cep57 or deletion mutants for 48 h were pre-extracted with 0.5% Triton X-100 for 40 s and co-stained with Cep152 (red) and Cetn (blue). Note that the centrosomal localization of both N- and C-terminal deletion mutants is much weaker than that of the full-length Cep57l1. (E) Schematic diagram of Cep63 and deletion mutants showing the ability to interact with Cep57l1 and Cep152 and to localize to the centriole. (F) Mapping of the Cep57l1-interacting regions of Cep63. GFP-tagged Cep57l1 was co-expressed with Flag-tagged Cep63 or mutants in HEK 293T cells. Co-Immunoprecipitation was then performed with GFP beads. Luc, luciferase control. (G) Schematics showing the interaction relationship of Cep57l1, Cep63 and Cep152. Cent represents the centriolar-targeting region. (H) GFP–Cep57l1 colocalizes with Cep63 and Cep152 at the proximal end of centrioles in U2OS cells. GFP–Cep57l1 was transiently expressed in human U2OS cells, and cells were also co-stained for Cetn and Cep63 (left) or Cep152 (right). (I) The subcellular localization of GFP–Cep57l1 during centriole amplification in mTECs. Representative 3D-SIM images were acquired from mTECs at day 3 after inducing multiciliation with the air-liquid interface system (ALI d3). Magnified areas show details for MCD (above; indicated by arrowheads) and DD (below; indicated by arrows) pathway centriole amplification.