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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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Effects of depleting Ahi1. (A) Cby1+/+ MEFs transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control were serum starved for 24 h, fixed, and stained for Cep164 and Ahi1. Scale bar, 5 μm. (B) Centrosomal levels of Cep164 or Ahi1 quantified and displayed as Tukey boxplots. Results from duplicate experiments, >100 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (C) Western blot analysis of extracts from Cby1+/+ MEFs stably transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control and probed with anti-Ahi1 or anti–α-tubulin as a loading control. For each sample, Ahi1 signal intensity, shown at bottom, was quantified and divided by tubulin signal intensity. Values shown are normalized to control shRNA signal intensity. (D) MEFs transduced with lentivirus expressing short hairpin RNA against Ahi1 or a scrambled control or with shRNA against Ahi1 followed by GFP-human-Ahi1 transgene, serum starved for 24 h, fixed, and stained for Arl13b. Scale bar, 5 μm. (E) Ciliary Arl13b intensity quantified and displayed as a Tukey boxplot. Results from three independent experiments, >40 observations/experiment. ***p < 0.001; *p < 0.05; n.s., not statistically significant; by Mann–Whitney U test. (F) Cby1+/+ MEFs transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control were fixed and stained for Cep164 and Cby1. Scale bar, 5 μm. (G) Centrosomal levels of Cby1 quantified and displayed as Tukey boxplots. Results from duplicate experiments, >100 observations/experiment.
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Figure 7: Effects of depleting Ahi1. (A) Cby1+/+ MEFs transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control were serum starved for 24 h, fixed, and stained for Cep164 and Ahi1. Scale bar, 5 μm. (B) Centrosomal levels of Cep164 or Ahi1 quantified and displayed as Tukey boxplots. Results from duplicate experiments, >100 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (C) Western blot analysis of extracts from Cby1+/+ MEFs stably transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control and probed with anti-Ahi1 or anti–α-tubulin as a loading control. For each sample, Ahi1 signal intensity, shown at bottom, was quantified and divided by tubulin signal intensity. Values shown are normalized to control shRNA signal intensity. (D) MEFs transduced with lentivirus expressing short hairpin RNA against Ahi1 or a scrambled control or with shRNA against Ahi1 followed by GFP-human-Ahi1 transgene, serum starved for 24 h, fixed, and stained for Arl13b. Scale bar, 5 μm. (E) Ciliary Arl13b intensity quantified and displayed as a Tukey boxplot. Results from three independent experiments, >40 observations/experiment. ***p < 0.001; *p < 0.05; n.s., not statistically significant; by Mann–Whitney U test. (F) Cby1+/+ MEFs transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control were fixed and stained for Cep164 and Cby1. Scale bar, 5 μm. (G) Centrosomal levels of Cby1 quantified and displayed as Tukey boxplots. Results from duplicate experiments, >100 observations/experiment.

Mentions: To determine whether Ahi1, like Cby1, also regulates ciliary transport, we stably transduced Cby1+/+ MEFs with lentivirus expressing a short hairpin interfering RNA (shRNA) targeting Ahi1 or a scrambled nontargeting control. MEFs were serum starved for 24 h and stained for Ahi1, with the markers indicated in Figure 7. Fluorescence intensity measurements indicated that centrosomal Ahi1 levels in cells depleted of Ahi1 were reduced to approximately half of the levels in control cells (Figure 7, A and B). Western blots of whole-cell lysates indicated similar depletion of total Ahi1 (Figure 7C). Fluorescence intensity measurements of Arl13b indicated that ciliary Arl13b in Ahi1-depleted cells was reduced to ∼75% of the levels in control cells (Figure 7, D and E), consistent with a report that Arl13b was lost from some primary cilia of Ahi1-mutant patient fibroblasts (Tuz et al., 2013). Overexpression of depletion-resistant GFP-tagged human Ahi1 compensated for the observed defect in Arl13b, confirming that the shRNA used specifically depleted Ahi1. Cby1 levels were similar in both control cells and cells depleted of Ahi1 (Figure 7, F and G). These data indicate that partial depletion of Ahi1 results in a phenotype that is similar to, but less severe than, the reduction of centrosomal Ahi1 caused by deletion of Cby1 and that Ahi1 acts downstream of Cby1. These experiments, together with previous work linking Ahi1 to cilium formation and function (Hsiao et al., 2009; Lancaster et al., 2009; Tuz et al., 2013), suggest that defective recruitment of Ahi1, and probably other transition zone proteins, in Cby1−/− MEFs might provide a molecular explanation for the observed ciliogenesis defect.


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)

Effects of depleting Ahi1. (A) Cby1+/+ MEFs transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control were serum starved for 24 h, fixed, and stained for Cep164 and Ahi1. Scale bar, 5 μm. (B) Centrosomal levels of Cep164 or Ahi1 quantified and displayed as Tukey boxplots. Results from duplicate experiments, >100 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (C) Western blot analysis of extracts from Cby1+/+ MEFs stably transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control and probed with anti-Ahi1 or anti–α-tubulin as a loading control. For each sample, Ahi1 signal intensity, shown at bottom, was quantified and divided by tubulin signal intensity. Values shown are normalized to control shRNA signal intensity. (D) MEFs transduced with lentivirus expressing short hairpin RNA against Ahi1 or a scrambled control or with shRNA against Ahi1 followed by GFP-human-Ahi1 transgene, serum starved for 24 h, fixed, and stained for Arl13b. Scale bar, 5 μm. (E) Ciliary Arl13b intensity quantified and displayed as a Tukey boxplot. Results from three independent experiments, >40 observations/experiment. ***p < 0.001; *p < 0.05; n.s., not statistically significant; by Mann–Whitney U test. (F) Cby1+/+ MEFs transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control were fixed and stained for Cep164 and Cby1. Scale bar, 5 μm. (G) Centrosomal levels of Cby1 quantified and displayed as Tukey boxplots. Results from duplicate experiments, >100 observations/experiment.
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Figure 7: Effects of depleting Ahi1. (A) Cby1+/+ MEFs transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control were serum starved for 24 h, fixed, and stained for Cep164 and Ahi1. Scale bar, 5 μm. (B) Centrosomal levels of Cep164 or Ahi1 quantified and displayed as Tukey boxplots. Results from duplicate experiments, >100 observations/experiment, ***p < 0.001 by Mann–Whitney U test. (C) Western blot analysis of extracts from Cby1+/+ MEFs stably transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control and probed with anti-Ahi1 or anti–α-tubulin as a loading control. For each sample, Ahi1 signal intensity, shown at bottom, was quantified and divided by tubulin signal intensity. Values shown are normalized to control shRNA signal intensity. (D) MEFs transduced with lentivirus expressing short hairpin RNA against Ahi1 or a scrambled control or with shRNA against Ahi1 followed by GFP-human-Ahi1 transgene, serum starved for 24 h, fixed, and stained for Arl13b. Scale bar, 5 μm. (E) Ciliary Arl13b intensity quantified and displayed as a Tukey boxplot. Results from three independent experiments, >40 observations/experiment. ***p < 0.001; *p < 0.05; n.s., not statistically significant; by Mann–Whitney U test. (F) Cby1+/+ MEFs transduced with lentivirus expressing shRNA targeting Ahi1 or scrambled control were fixed and stained for Cep164 and Cby1. Scale bar, 5 μm. (G) Centrosomal levels of Cby1 quantified and displayed as Tukey boxplots. Results from duplicate experiments, >100 observations/experiment.
Mentions: To determine whether Ahi1, like Cby1, also regulates ciliary transport, we stably transduced Cby1+/+ MEFs with lentivirus expressing a short hairpin interfering RNA (shRNA) targeting Ahi1 or a scrambled nontargeting control. MEFs were serum starved for 24 h and stained for Ahi1, with the markers indicated in Figure 7. Fluorescence intensity measurements indicated that centrosomal Ahi1 levels in cells depleted of Ahi1 were reduced to approximately half of the levels in control cells (Figure 7, A and B). Western blots of whole-cell lysates indicated similar depletion of total Ahi1 (Figure 7C). Fluorescence intensity measurements of Arl13b indicated that ciliary Arl13b in Ahi1-depleted cells was reduced to ∼75% of the levels in control cells (Figure 7, D and E), consistent with a report that Arl13b was lost from some primary cilia of Ahi1-mutant patient fibroblasts (Tuz et al., 2013). Overexpression of depletion-resistant GFP-tagged human Ahi1 compensated for the observed defect in Arl13b, confirming that the shRNA used specifically depleted Ahi1. Cby1 levels were similar in both control cells and cells depleted of Ahi1 (Figure 7, F and G). These data indicate that partial depletion of Ahi1 results in a phenotype that is similar to, but less severe than, the reduction of centrosomal Ahi1 caused by deletion of Cby1 and that Ahi1 acts downstream of Cby1. These experiments, together with previous work linking Ahi1 to cilium formation and function (Hsiao et al., 2009; Lancaster et al., 2009; Tuz et al., 2013), suggest that defective recruitment of Ahi1, and probably other transition zone proteins, in Cby1−/− MEFs might provide a molecular explanation for the observed ciliogenesis defect.

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