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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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Superresolution imaging of Cby1 and centriole proteins. (A–C) 3D-SIM imaging. (A) MEFs or MTECs were fixed and stained for Ahi1 (green), Cby1 (red), and Cep164 (cyan). (B) MEFs or MTECs were fixed and stained for Ofd1 (green), Cby1 (red), and acetylated α-tubulin (Ac-tub, cyan). (C) MEFs or MTECs were fixed and stained for Sdccag8 (green), Cby1 (red), and polyglutamylated tubulin (Glu-tub, cyan). For A–C, MEF and MTEC top rows are maximum Z-projections; MTEC bottom rows are XZ-projections. Scale bars, 500 nm. (D) MTECs were fixed and stained for Tmem237, Ahi1, Cby1, Ofd1, or Sdccag8 and imaged using STED microscopy. Insets, enlarged centriolar regions. Mean diameters and SDs of centriolar rings for each protein (n ≥ 45) are indicated to the right. Mean diameters were derived from maximum intensity points of radial intensity profiles (Supplemental Figure S3). Ring diameters of each protein are statistically different from all other proteins with p < 0.01 (Student's two-tailed t test). Scale bars, 500 nm.
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Figure 4: Superresolution imaging of Cby1 and centriole proteins. (A–C) 3D-SIM imaging. (A) MEFs or MTECs were fixed and stained for Ahi1 (green), Cby1 (red), and Cep164 (cyan). (B) MEFs or MTECs were fixed and stained for Ofd1 (green), Cby1 (red), and acetylated α-tubulin (Ac-tub, cyan). (C) MEFs or MTECs were fixed and stained for Sdccag8 (green), Cby1 (red), and polyglutamylated tubulin (Glu-tub, cyan). For A–C, MEF and MTEC top rows are maximum Z-projections; MTEC bottom rows are XZ-projections. Scale bars, 500 nm. (D) MTECs were fixed and stained for Tmem237, Ahi1, Cby1, Ofd1, or Sdccag8 and imaged using STED microscopy. Insets, enlarged centriolar regions. Mean diameters and SDs of centriolar rings for each protein (n ≥ 45) are indicated to the right. Mean diameters were derived from maximum intensity points of radial intensity profiles (Supplemental Figure S3). Ring diameters of each protein are statistically different from all other proteins with p < 0.01 (Student's two-tailed t test). Scale bars, 500 nm.

Mentions: When visualized by 3D-SIM, all of the proteins appeared to be organized as rings of ∼250–300 nm, consistent with being in close apposition to the centriole microtubules, in both cell types (Figure 4, A–C). For this characterization we used MTECs, which, in contrast to MEFs, have hundreds of centrioles docked in the same orientation and plane at the apical surface, allowing for determination of the relative localization of the proteins along the longitudinal axis of the centriole. XZ-projections of 3D-SIM images of MTECs demonstrated that Cby1 is located at a similar position along the longitudinal axis as Ahi1 and Ofd1 (Figure 4, A and B). In contrast, Cep164 and Sdccag8 are located closer to the proximal end of the centriole than Cby1 (farther from the cilium; Figure 4, A and C). It was not possible to image Tmem237 under the 3D-SIM conditions.


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)

Superresolution imaging of Cby1 and centriole proteins. (A–C) 3D-SIM imaging. (A) MEFs or MTECs were fixed and stained for Ahi1 (green), Cby1 (red), and Cep164 (cyan). (B) MEFs or MTECs were fixed and stained for Ofd1 (green), Cby1 (red), and acetylated α-tubulin (Ac-tub, cyan). (C) MEFs or MTECs were fixed and stained for Sdccag8 (green), Cby1 (red), and polyglutamylated tubulin (Glu-tub, cyan). For A–C, MEF and MTEC top rows are maximum Z-projections; MTEC bottom rows are XZ-projections. Scale bars, 500 nm. (D) MTECs were fixed and stained for Tmem237, Ahi1, Cby1, Ofd1, or Sdccag8 and imaged using STED microscopy. Insets, enlarged centriolar regions. Mean diameters and SDs of centriolar rings for each protein (n ≥ 45) are indicated to the right. Mean diameters were derived from maximum intensity points of radial intensity profiles (Supplemental Figure S3). Ring diameters of each protein are statistically different from all other proteins with p < 0.01 (Student's two-tailed t test). Scale bars, 500 nm.
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Figure 4: Superresolution imaging of Cby1 and centriole proteins. (A–C) 3D-SIM imaging. (A) MEFs or MTECs were fixed and stained for Ahi1 (green), Cby1 (red), and Cep164 (cyan). (B) MEFs or MTECs were fixed and stained for Ofd1 (green), Cby1 (red), and acetylated α-tubulin (Ac-tub, cyan). (C) MEFs or MTECs were fixed and stained for Sdccag8 (green), Cby1 (red), and polyglutamylated tubulin (Glu-tub, cyan). For A–C, MEF and MTEC top rows are maximum Z-projections; MTEC bottom rows are XZ-projections. Scale bars, 500 nm. (D) MTECs were fixed and stained for Tmem237, Ahi1, Cby1, Ofd1, or Sdccag8 and imaged using STED microscopy. Insets, enlarged centriolar regions. Mean diameters and SDs of centriolar rings for each protein (n ≥ 45) are indicated to the right. Mean diameters were derived from maximum intensity points of radial intensity profiles (Supplemental Figure S3). Ring diameters of each protein are statistically different from all other proteins with p < 0.01 (Student's two-tailed t test). Scale bars, 500 nm.
Mentions: When visualized by 3D-SIM, all of the proteins appeared to be organized as rings of ∼250–300 nm, consistent with being in close apposition to the centriole microtubules, in both cell types (Figure 4, A–C). For this characterization we used MTECs, which, in contrast to MEFs, have hundreds of centrioles docked in the same orientation and plane at the apical surface, allowing for determination of the relative localization of the proteins along the longitudinal axis of the centriole. XZ-projections of 3D-SIM images of MTECs demonstrated that Cby1 is located at a similar position along the longitudinal axis as Ahi1 and Ofd1 (Figure 4, A and B). In contrast, Cep164 and Sdccag8 are located closer to the proximal end of the centriole than Cby1 (farther from the cilium; Figure 4, A and C). It was not possible to image Tmem237 under the 3D-SIM conditions.

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