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Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function.

Lee YL, Santé J, Comerci CJ, Cyge B, Menezes LF, Li FQ, Germino GG, Moerner WE, Takemaru K, Stearns T - Mol. Biol. Cell (2014)

Bottom Line: Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies.Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium.This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1(-/-) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford School of Medicine, Stanford University, Stanford, CA 94305.

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Cby1 facilitates primary cilia formation. (A) Cby1+/+ or Cby1−/− MEFs were serum starved, fixed, and stained for polyglutamylated tubulin (Glu-tub, green), Cby1 (red), and DNA (DAPI, blue). Scale bars, 10 μm. (B) Cby1+/+ or Cby1−/− MEFs were fixed after 0, 24, 48, or 72 h of serum starvation, stained for polyglutamylated tubulin, and scored for primary cilia. Results shown are the mean of three independent experiments ± SEM (100 cells/experiment; p values shown above each time point). (C, D) Cby1−/− MEFs were transfected with GFP or GFP-Cby1, serum-starved for 24 h, fixed, and stained for GFP (green), polyglutamylated tubulin (Glu-tub, red) and DNA (DAPI, blue). A representative image of Cby1−/− MEFs transfected with GFP-Cby1 is shown in C. Insets, enlarged images of centrosomal/ciliary regions. Note in C that the cell expressing GFP-Cby1 is ciliated, whereas the neighboring cell not expressing GFP-Cby1 is not ciliated. Scale bar, 10 μm. These data are quantified in D. Results shown are the mean of four experiments ± SEM (>100 cells/experiment; ***p < 0.001). (E) Ofd1+/+ or Ofd1−/− mouse embryonic stem cells were fixed and stained for γ-tubulin (γ-tub, red), Ofd1 (cyan), Cby1 (green), and DNA (DAPI, blue). Scale bar, 5 μm.
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Figure 2: Cby1 facilitates primary cilia formation. (A) Cby1+/+ or Cby1−/− MEFs were serum starved, fixed, and stained for polyglutamylated tubulin (Glu-tub, green), Cby1 (red), and DNA (DAPI, blue). Scale bars, 10 μm. (B) Cby1+/+ or Cby1−/− MEFs were fixed after 0, 24, 48, or 72 h of serum starvation, stained for polyglutamylated tubulin, and scored for primary cilia. Results shown are the mean of three independent experiments ± SEM (100 cells/experiment; p values shown above each time point). (C, D) Cby1−/− MEFs were transfected with GFP or GFP-Cby1, serum-starved for 24 h, fixed, and stained for GFP (green), polyglutamylated tubulin (Glu-tub, red) and DNA (DAPI, blue). A representative image of Cby1−/− MEFs transfected with GFP-Cby1 is shown in C. Insets, enlarged images of centrosomal/ciliary regions. Note in C that the cell expressing GFP-Cby1 is ciliated, whereas the neighboring cell not expressing GFP-Cby1 is not ciliated. Scale bar, 10 μm. These data are quantified in D. Results shown are the mean of four experiments ± SEM (>100 cells/experiment; ***p < 0.001). (E) Ofd1+/+ or Ofd1−/− mouse embryonic stem cells were fixed and stained for γ-tubulin (γ-tub, red), Ofd1 (cyan), Cby1 (green), and DNA (DAPI, blue). Scale bar, 5 μm.

Mentions: To test directly whether loss of Cby1 compromises cilium formation, we examined primary cilium formation in mouse embryonic fibroblasts (MEFs) from Cby1+/+ and Cby1−/− embryos. Cells were induced to form a cilium by serum starvation and assayed over a 72-h time course. Cby1−/− MEFs exhibited a delay in primary cilia formation relative to wild type (Figure 2, A and B). After 24 h of serum starvation, >40% of Cby1+/+ MEFs had a cilium, whereas <5% of Cby1−/− MEFs had a cilium (Figure 2B). The fraction of Cby1−/− MEFs with a cilium increased over time of serum starvation, although it remained lower than that for Cby1+/+ MEFs (Figure 2B). Complementation of the Cby1−/− deletion with a human GFP-Cby1 construct (Figure 2, C and D) rescued the defect in primary cilium formation, assessed at 24 h of serum starvation. Unlike in previous RNAi experiments (Steere et al., 2012), these data demonstrate that Cby1 is not essential for forming a primary cilium but is important for facilitating the process.


Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function.

Lee YL, Santé J, Comerci CJ, Cyge B, Menezes LF, Li FQ, Germino GG, Moerner WE, Takemaru K, Stearns T - Mol. Biol. Cell (2014)

Cby1 facilitates primary cilia formation. (A) Cby1+/+ or Cby1−/− MEFs were serum starved, fixed, and stained for polyglutamylated tubulin (Glu-tub, green), Cby1 (red), and DNA (DAPI, blue). Scale bars, 10 μm. (B) Cby1+/+ or Cby1−/− MEFs were fixed after 0, 24, 48, or 72 h of serum starvation, stained for polyglutamylated tubulin, and scored for primary cilia. Results shown are the mean of three independent experiments ± SEM (100 cells/experiment; p values shown above each time point). (C, D) Cby1−/− MEFs were transfected with GFP or GFP-Cby1, serum-starved for 24 h, fixed, and stained for GFP (green), polyglutamylated tubulin (Glu-tub, red) and DNA (DAPI, blue). A representative image of Cby1−/− MEFs transfected with GFP-Cby1 is shown in C. Insets, enlarged images of centrosomal/ciliary regions. Note in C that the cell expressing GFP-Cby1 is ciliated, whereas the neighboring cell not expressing GFP-Cby1 is not ciliated. Scale bar, 10 μm. These data are quantified in D. Results shown are the mean of four experiments ± SEM (>100 cells/experiment; ***p < 0.001). (E) Ofd1+/+ or Ofd1−/− mouse embryonic stem cells were fixed and stained for γ-tubulin (γ-tub, red), Ofd1 (cyan), Cby1 (green), and DNA (DAPI, blue). Scale bar, 5 μm.
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Figure 2: Cby1 facilitates primary cilia formation. (A) Cby1+/+ or Cby1−/− MEFs were serum starved, fixed, and stained for polyglutamylated tubulin (Glu-tub, green), Cby1 (red), and DNA (DAPI, blue). Scale bars, 10 μm. (B) Cby1+/+ or Cby1−/− MEFs were fixed after 0, 24, 48, or 72 h of serum starvation, stained for polyglutamylated tubulin, and scored for primary cilia. Results shown are the mean of three independent experiments ± SEM (100 cells/experiment; p values shown above each time point). (C, D) Cby1−/− MEFs were transfected with GFP or GFP-Cby1, serum-starved for 24 h, fixed, and stained for GFP (green), polyglutamylated tubulin (Glu-tub, red) and DNA (DAPI, blue). A representative image of Cby1−/− MEFs transfected with GFP-Cby1 is shown in C. Insets, enlarged images of centrosomal/ciliary regions. Note in C that the cell expressing GFP-Cby1 is ciliated, whereas the neighboring cell not expressing GFP-Cby1 is not ciliated. Scale bar, 10 μm. These data are quantified in D. Results shown are the mean of four experiments ± SEM (>100 cells/experiment; ***p < 0.001). (E) Ofd1+/+ or Ofd1−/− mouse embryonic stem cells were fixed and stained for γ-tubulin (γ-tub, red), Ofd1 (cyan), Cby1 (green), and DNA (DAPI, blue). Scale bar, 5 μm.
Mentions: To test directly whether loss of Cby1 compromises cilium formation, we examined primary cilium formation in mouse embryonic fibroblasts (MEFs) from Cby1+/+ and Cby1−/− embryos. Cells were induced to form a cilium by serum starvation and assayed over a 72-h time course. Cby1−/− MEFs exhibited a delay in primary cilia formation relative to wild type (Figure 2, A and B). After 24 h of serum starvation, >40% of Cby1+/+ MEFs had a cilium, whereas <5% of Cby1−/− MEFs had a cilium (Figure 2B). The fraction of Cby1−/− MEFs with a cilium increased over time of serum starvation, although it remained lower than that for Cby1+/+ MEFs (Figure 2B). Complementation of the Cby1−/− deletion with a human GFP-Cby1 construct (Figure 2, C and D) rescued the defect in primary cilium formation, assessed at 24 h of serum starvation. Unlike in previous RNAi experiments (Steere et al., 2012), these data demonstrate that Cby1 is not essential for forming a primary cilium but is important for facilitating the process.

Bottom Line: Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies.Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium.This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1(-/-) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford School of Medicine, Stanford University, Stanford, CA 94305.

Show MeSH
Related in: MedlinePlus