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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 facilitates ciliary recruitment of Arl13b. (A) Cby1+/+ or Cby1−/− MEFs were serum starved for 48 h, fixed, and stained for acetylated α-tubulin (Ac-tub, red), Arl13b (green), and DNA (DAPI, blue). Scale bar, 5 μm. (B, C) Quantification of ciliary Arl13b and acetylated tubulin levels in Cby1+/+ or Cby1−/− MEFs. Results shown are the mean of three independent experiments ± SEM (>100 cells/experiment, *p < 0.05). (D) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1, serum-starved for 48 h, fixed, and stained for GFP (green), Arl13b (red), and DNA (DAPI, blue). Scale bar, 5 μm. (E) Ciliary Arl13b intensity was quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >30 observations/experiment, ***p < 0.001 by Mann–Whitney U test.
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Figure 3: Cby1 facilitates ciliary recruitment of Arl13b. (A) Cby1+/+ or Cby1−/− MEFs were serum starved for 48 h, fixed, and stained for acetylated α-tubulin (Ac-tub, red), Arl13b (green), and DNA (DAPI, blue). Scale bar, 5 μm. (B, C) Quantification of ciliary Arl13b and acetylated tubulin levels in Cby1+/+ or Cby1−/− MEFs. Results shown are the mean of three independent experiments ± SEM (>100 cells/experiment, *p < 0.05). (D) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1, serum-starved for 48 h, fixed, and stained for GFP (green), Arl13b (red), and DNA (DAPI, blue). Scale bar, 5 μm. (E) Ciliary Arl13b intensity was quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >30 observations/experiment, ***p < 0.001 by Mann–Whitney U test.

Mentions: The primary cilium is a signaling center for several pathways, and concentration of signaling proteins in the cilium compartment is essential to proper signaling (Berbari et al., 2009; Mahjoub and Stearns, 2012). Arl13b is a cilium-localized small GTPase that regulates cilium morphology and ciliary trafficking (Caspary et al., 2007; Duldulao et al., 2009; Cevik et al., 2010; Larkins et al., 2011). Loss of Arl13b in the zebrafish scorpion mutant results in cystic kidneys (Sun et al., 2004; Cantagrel et al., 2008), and mutations in ARL13B are causative for the ciliopathy Joubert syndrome (Cantagrel et al., 2008). We examined the localization of Arl13b to cilia in Cby1+/+ and Cby1−/− MEFs. The amount of ciliary Arl13b in Cby1−/− MEFs was reduced twofold compared with Cby1+/+ MEFs (Figure 3, A and B), and this reduction could be complemented by a human GFP-Cby1 construct (Figure 3, D and E). The amount of ciliary acetylated α-tubulin was similar in Cby1+/+ and Cby1−/− MEFs (Figure 3, A and C). Despite the reduction of ciliary Arl13b in Cby1−/− MEFs, these cells retained the ability to control protein access to the ciliary compartment, as assessed by recruitment of the transmembrane protein Smoothened (Smo) in response to treatment with Smo agonist (SAG), an activator of the pathway (Supplemental Figure S1).


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 ciliary recruitment of Arl13b. (A) Cby1+/+ or Cby1−/− MEFs were serum starved for 48 h, fixed, and stained for acetylated α-tubulin (Ac-tub, red), Arl13b (green), and DNA (DAPI, blue). Scale bar, 5 μm. (B, C) Quantification of ciliary Arl13b and acetylated tubulin levels in Cby1+/+ or Cby1−/− MEFs. Results shown are the mean of three independent experiments ± SEM (>100 cells/experiment, *p < 0.05). (D) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1, serum-starved for 48 h, fixed, and stained for GFP (green), Arl13b (red), and DNA (DAPI, blue). Scale bar, 5 μm. (E) Ciliary Arl13b intensity was quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >30 observations/experiment, ***p < 0.001 by Mann–Whitney U test.
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Figure 3: Cby1 facilitates ciliary recruitment of Arl13b. (A) Cby1+/+ or Cby1−/− MEFs were serum starved for 48 h, fixed, and stained for acetylated α-tubulin (Ac-tub, red), Arl13b (green), and DNA (DAPI, blue). Scale bar, 5 μm. (B, C) Quantification of ciliary Arl13b and acetylated tubulin levels in Cby1+/+ or Cby1−/− MEFs. Results shown are the mean of three independent experiments ± SEM (>100 cells/experiment, *p < 0.05). (D) Cby1−/− MEFs were transduced with lentivirus expressing GFP or GFP-Cby1, serum-starved for 48 h, fixed, and stained for GFP (green), Arl13b (red), and DNA (DAPI, blue). Scale bar, 5 μm. (E) Ciliary Arl13b intensity was quantified and displayed as a Tukey boxplot. Results from duplicate independent experiments, >30 observations/experiment, ***p < 0.001 by Mann–Whitney U test.
Mentions: The primary cilium is a signaling center for several pathways, and concentration of signaling proteins in the cilium compartment is essential to proper signaling (Berbari et al., 2009; Mahjoub and Stearns, 2012). Arl13b is a cilium-localized small GTPase that regulates cilium morphology and ciliary trafficking (Caspary et al., 2007; Duldulao et al., 2009; Cevik et al., 2010; Larkins et al., 2011). Loss of Arl13b in the zebrafish scorpion mutant results in cystic kidneys (Sun et al., 2004; Cantagrel et al., 2008), and mutations in ARL13B are causative for the ciliopathy Joubert syndrome (Cantagrel et al., 2008). We examined the localization of Arl13b to cilia in Cby1+/+ and Cby1−/− MEFs. The amount of ciliary Arl13b in Cby1−/− MEFs was reduced twofold compared with Cby1+/+ MEFs (Figure 3, A and B), and this reduction could be complemented by a human GFP-Cby1 construct (Figure 3, D and E). The amount of ciliary acetylated α-tubulin was similar in Cby1+/+ and Cby1−/− MEFs (Figure 3, A and C). Despite the reduction of ciliary Arl13b in Cby1−/− MEFs, these cells retained the ability to control protein access to the ciliary compartment, as assessed by recruitment of the transmembrane protein Smoothened (Smo) in response to treatment with Smo agonist (SAG), an activator of the pathway (Supplemental Figure S1).

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