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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.

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

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Cby1 is required for efficient recruitment of Ahi1 to the centrosome. (A) Cby1+/+ or Cby1−/− MEFs were fixed after 0 or 48 h of serum starvation and stained for Cep164 (red), Ahi1 (green), and DNA (DAPI, blue). Scale bar, 5 μm. (B, C) Centrosomal levels of Cep164 (B) or Ahi1 (C) were quantified and normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment, *p < 0.05). (D) Cby1+/+ or Cby1−/− MEFs were fixed after 0 or 48 h of serum starvation and stained for γ-tubulin (γ-tub, red), Ofd1 (green), and DNA (DAPI, blue). Scale bar, 5 μm. (E) Centrosomal levels of γ-tubulin were quantified and normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment). (F) Centrosomal levels of Ofd1 were quantified as fluorescence intensity within the region defined by γ-tubulin labeling and then normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment). (G) Western blot analysis of extracts from Cby1+/+ or Cby1−/−MEFs probed with anti-Ahi1 or anti–α-tubulin as a loading control. For each sample, Ahi1 signal intensity was quantified and divided by tubulin signal intensity. Values shown are normalized to Cby1+/+ signal intensity.
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Figure 5: Cby1 is required for efficient recruitment of Ahi1 to the centrosome. (A) Cby1+/+ or Cby1−/− MEFs were fixed after 0 or 48 h of serum starvation and stained for Cep164 (red), Ahi1 (green), and DNA (DAPI, blue). Scale bar, 5 μm. (B, C) Centrosomal levels of Cep164 (B) or Ahi1 (C) were quantified and normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment, *p < 0.05). (D) Cby1+/+ or Cby1−/− MEFs were fixed after 0 or 48 h of serum starvation and stained for γ-tubulin (γ-tub, red), Ofd1 (green), and DNA (DAPI, blue). Scale bar, 5 μm. (E) Centrosomal levels of γ-tubulin were quantified and normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment). (F) Centrosomal levels of Ofd1 were quantified as fluorescence intensity within the region defined by γ-tubulin labeling and then normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment). (G) Western blot analysis of extracts from Cby1+/+ or Cby1−/−MEFs probed with anti-Ahi1 or anti–α-tubulin as a loading control. For each sample, Ahi1 signal intensity was quantified and divided by tubulin signal intensity. Values shown are normalized to Cby1+/+ signal intensity.

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)

Cby1 is required for efficient recruitment of Ahi1 to the centrosome. (A) Cby1+/+ or Cby1−/− MEFs were fixed after 0 or 48 h of serum starvation and stained for Cep164 (red), Ahi1 (green), and DNA (DAPI, blue). Scale bar, 5 μm. (B, C) Centrosomal levels of Cep164 (B) or Ahi1 (C) were quantified and normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment, *p < 0.05). (D) Cby1+/+ or Cby1−/− MEFs were fixed after 0 or 48 h of serum starvation and stained for γ-tubulin (γ-tub, red), Ofd1 (green), and DNA (DAPI, blue). Scale bar, 5 μm. (E) Centrosomal levels of γ-tubulin were quantified and normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment). (F) Centrosomal levels of Ofd1 were quantified as fluorescence intensity within the region defined by γ-tubulin labeling and then normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment). (G) Western blot analysis of extracts from Cby1+/+ or Cby1−/−MEFs probed with anti-Ahi1 or anti–α-tubulin as a loading control. For each sample, Ahi1 signal intensity was quantified and divided by tubulin signal intensity. Values shown are normalized to Cby1+/+ signal intensity.
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Figure 5: Cby1 is required for efficient recruitment of Ahi1 to the centrosome. (A) Cby1+/+ or Cby1−/− MEFs were fixed after 0 or 48 h of serum starvation and stained for Cep164 (red), Ahi1 (green), and DNA (DAPI, blue). Scale bar, 5 μm. (B, C) Centrosomal levels of Cep164 (B) or Ahi1 (C) were quantified and normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment, *p < 0.05). (D) Cby1+/+ or Cby1−/− MEFs were fixed after 0 or 48 h of serum starvation and stained for γ-tubulin (γ-tub, red), Ofd1 (green), and DNA (DAPI, blue). Scale bar, 5 μm. (E) Centrosomal levels of γ-tubulin were quantified and normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment). (F) Centrosomal levels of Ofd1 were quantified as fluorescence intensity within the region defined by γ-tubulin labeling and then normalized to Cby1+/+ 0-h serum starvation condition. Results shown are the mean of three independent experiments ± SEM (>100 centrosomes/experiment). (G) Western blot analysis of extracts from Cby1+/+ or Cby1−/−MEFs probed with anti-Ahi1 or anti–α-tubulin as a loading control. For each sample, Ahi1 signal intensity was quantified and divided by tubulin signal intensity. Values shown are normalized to Cby1+/+ signal intensity.
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