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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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Regulation of Ahi1 by Cby1. (A) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1, serum starved for 48 h, fixed, and immunostained for GFP (green), Cep164 (blue), and Ahi1 (red). Scale bar, 2.5 μm. (B) Centrosomal levels of Ahi1 in Cby1−/− MEFs expressing GFP or GFP-Cby1 quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >50 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (C) Cby1+/+ or Cby1−/− MEFs were fixed after 48 h of serum starvation and stained for Cep164 (red), Ahi1 (green), and acetylated tubulin (cyan). Scale bar, 5 μm. (D) Centrosomal levels of Ahi1 in MEFs with or without acetylated tubulin-labeled cilia quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >60 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (E) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1 or GFP-Ahi1, serum starved for 48 h, fixed, stained for GFP (green), polyglutamylated tubulin (glu-tub, red), and DNA (blue), and (F) scored for frequency of primary cilium formation. Results shown are the mean of three independent experiments ± SEM (>100 cells/experiment; *p < 0.05, **p < 0.01). Scale bar, 5 μm. (G) Cby1+/+ or Cby1−/− MEFs were transduced with lentivirus expressing GFP-Ahi1, serum starved for 24 h, fixed, and stained for GFP (green) and Cep164 (red). Scale bar, 5 μm. (H) Centrosomal levels of GFP quantified and displayed as a Tukey boxplot. Results from three independent experiments, >100 observations/experiment, ***p < 0.001 by Mann–Whitney U test.
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Figure 6: Regulation of Ahi1 by Cby1. (A) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1, serum starved for 48 h, fixed, and immunostained for GFP (green), Cep164 (blue), and Ahi1 (red). Scale bar, 2.5 μm. (B) Centrosomal levels of Ahi1 in Cby1−/− MEFs expressing GFP or GFP-Cby1 quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >50 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (C) Cby1+/+ or Cby1−/− MEFs were fixed after 48 h of serum starvation and stained for Cep164 (red), Ahi1 (green), and acetylated tubulin (cyan). Scale bar, 5 μm. (D) Centrosomal levels of Ahi1 in MEFs with or without acetylated tubulin-labeled cilia quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >60 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (E) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1 or GFP-Ahi1, serum starved for 48 h, fixed, stained for GFP (green), polyglutamylated tubulin (glu-tub, red), and DNA (blue), and (F) scored for frequency of primary cilium formation. Results shown are the mean of three independent experiments ± SEM (>100 cells/experiment; *p < 0.05, **p < 0.01). Scale bar, 5 μm. (G) Cby1+/+ or Cby1−/− MEFs were transduced with lentivirus expressing GFP-Ahi1, serum starved for 24 h, fixed, and stained for GFP (green) and Cep164 (red). Scale bar, 5 μm. (H) Centrosomal levels of GFP quantified and displayed as a Tukey boxplot. Results from three independent experiments, >100 observations/experiment, ***p < 0.001 by Mann–Whitney U test.

Mentions: Given the close spatial relationship between Cby1, Ahi1, and Ofd1, we tested whether association of either Ahi1 or Ofd1 with the centrosome depends on Cby1. The fluorescence signal for each protein was determined in Cby1−/− MEFs and Cby1+/+ controls. The amount of Ahi1 at the centrosomes of serum-starved Cby1−/− MEFs was more than twofold lower than in Cby1+/+ controls (Figure 5, A and C), and this could be complemented with a human GFP-Cby1 construct (Figure 6, A and B). This was likely due to a defect in recruitment of Ahi1 to the centrosome rather than a reduction of total Ahi1 level, because centrosomal Ahi1 was similar in Cby1−/− and Cby1+/+ cycling cells before starvation-induced recruitment of Ahi1 (Figure 5, A and C), and total Ahi1 assessed by Western blotting of cycling cells was unchanged (Figure 5G). The reduction in centrosomal Ahi1 was apparent in both ciliated and nonciliated Cby1−/− cells (Figure 6, C and D) and thus is not due to failure of the centrioles in Cby1−/− cells to undergo the transition to forming a cilium. In contrast to Ahi1, the amounts of centrosomal Cep164 (Figure 5B), Ofd1 (Figure 5, D and F), Tmem237, and Sdccag8 (Supplemental Figure S2, A–E) were unchanged in Cby1+/+ and Cby1−/− cells, regardless of time in serum starvation. Loss of CP110 from the mother centriole precedes cilia formation (Tsang et al., 2008); this loss still occurred in Cby1−/− MEFs (Supplemental Figure S2, F and G). These data demonstrate that Cby1 is required, directly or indirectly, for efficient recruitment of the transition zone protein Ahi1 and that this defect is manifested after initiation of the centriole-basal body transition marked by CP110 loss.


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)

Regulation of Ahi1 by Cby1. (A) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1, serum starved for 48 h, fixed, and immunostained for GFP (green), Cep164 (blue), and Ahi1 (red). Scale bar, 2.5 μm. (B) Centrosomal levels of Ahi1 in Cby1−/− MEFs expressing GFP or GFP-Cby1 quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >50 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (C) Cby1+/+ or Cby1−/− MEFs were fixed after 48 h of serum starvation and stained for Cep164 (red), Ahi1 (green), and acetylated tubulin (cyan). Scale bar, 5 μm. (D) Centrosomal levels of Ahi1 in MEFs with or without acetylated tubulin-labeled cilia quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >60 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (E) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1 or GFP-Ahi1, serum starved for 48 h, fixed, stained for GFP (green), polyglutamylated tubulin (glu-tub, red), and DNA (blue), and (F) scored for frequency of primary cilium formation. Results shown are the mean of three independent experiments ± SEM (>100 cells/experiment; *p < 0.05, **p < 0.01). Scale bar, 5 μm. (G) Cby1+/+ or Cby1−/− MEFs were transduced with lentivirus expressing GFP-Ahi1, serum starved for 24 h, fixed, and stained for GFP (green) and Cep164 (red). Scale bar, 5 μm. (H) Centrosomal levels of GFP quantified and displayed as a Tukey boxplot. Results from three independent experiments, >100 observations/experiment, ***p < 0.001 by Mann–Whitney U test.
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Figure 6: Regulation of Ahi1 by Cby1. (A) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1, serum starved for 48 h, fixed, and immunostained for GFP (green), Cep164 (blue), and Ahi1 (red). Scale bar, 2.5 μm. (B) Centrosomal levels of Ahi1 in Cby1−/− MEFs expressing GFP or GFP-Cby1 quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >50 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (C) Cby1+/+ or Cby1−/− MEFs were fixed after 48 h of serum starvation and stained for Cep164 (red), Ahi1 (green), and acetylated tubulin (cyan). Scale bar, 5 μm. (D) Centrosomal levels of Ahi1 in MEFs with or without acetylated tubulin-labeled cilia quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >60 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (E) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1 or GFP-Ahi1, serum starved for 48 h, fixed, stained for GFP (green), polyglutamylated tubulin (glu-tub, red), and DNA (blue), and (F) scored for frequency of primary cilium formation. Results shown are the mean of three independent experiments ± SEM (>100 cells/experiment; *p < 0.05, **p < 0.01). Scale bar, 5 μm. (G) Cby1+/+ or Cby1−/− MEFs were transduced with lentivirus expressing GFP-Ahi1, serum starved for 24 h, fixed, and stained for GFP (green) and Cep164 (red). Scale bar, 5 μm. (H) Centrosomal levels of GFP quantified and displayed as a Tukey boxplot. Results from three independent experiments, >100 observations/experiment, ***p < 0.001 by Mann–Whitney U test.
Mentions: Given the close spatial relationship between Cby1, Ahi1, and Ofd1, we tested whether association of either Ahi1 or Ofd1 with the centrosome depends on Cby1. The fluorescence signal for each protein was determined in Cby1−/− MEFs and Cby1+/+ controls. The amount of Ahi1 at the centrosomes of serum-starved Cby1−/− MEFs was more than twofold lower than in Cby1+/+ controls (Figure 5, A and C), and this could be complemented with a human GFP-Cby1 construct (Figure 6, A and B). This was likely due to a defect in recruitment of Ahi1 to the centrosome rather than a reduction of total Ahi1 level, because centrosomal Ahi1 was similar in Cby1−/− and Cby1+/+ cycling cells before starvation-induced recruitment of Ahi1 (Figure 5, A and C), and total Ahi1 assessed by Western blotting of cycling cells was unchanged (Figure 5G). The reduction in centrosomal Ahi1 was apparent in both ciliated and nonciliated Cby1−/− cells (Figure 6, C and D) and thus is not due to failure of the centrioles in Cby1−/− cells to undergo the transition to forming a cilium. In contrast to Ahi1, the amounts of centrosomal Cep164 (Figure 5B), Ofd1 (Figure 5, D and F), Tmem237, and Sdccag8 (Supplemental Figure S2, A–E) were unchanged in Cby1+/+ and Cby1−/− cells, regardless of time in serum starvation. Loss of CP110 from the mother centriole precedes cilia formation (Tsang et al., 2008); this loss still occurred in Cby1−/− MEFs (Supplemental Figure S2, F and G). These data demonstrate that Cby1 is required, directly or indirectly, for efficient recruitment of the transition zone protein Ahi1 and that this defect is manifested after initiation of the centriole-basal body transition marked by CP110 loss.

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