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The oral-facial-digital syndrome gene C2CD3 encodes a positive regulator of centriole elongation.

Thauvin-Robinet C, Lee JS, Lopez E, Herranz-Pérez V, Shida T, Franco B, Jego L, Ye F, Pasquier L, Loget P, Gigot N, Aral B, Lopes CA, St-Onge J, Bruel AL, Thevenon J, González-Granero S, Alby C, Munnich A, Vekemans M, Huet F, Fry AM, Saunier S, Rivière JB, Attié-Bitach T, Garcia-Verdugo JM, Faivre L, Mégarbané A, Nachury MV - Nat. Genet. (2014)

Bottom Line: How centriole length is precisely set remains elusive.Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene.Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.

View Article: PubMed Central - PubMed

Affiliation: 1] Equipe d'Accueil 4271 Génétique des Anomalies du Développement, Fédération Hospitalo-Universitaire, Université de Bourgogne, Dijon, France. [2] Centre de Référence Maladies Rares "Anomalies du Développement et Syndromes Malformatifs" de l'Est, Centre de Génétique et Pédiatrie 1, Hôpital d'Enfants, Centre Hospitalier Universitaire Dijon, Dijon, France. [3].

ABSTRACT
Centrioles are microtubule-based, barrel-shaped structures that initiate the assembly of centrosomes and cilia. How centriole length is precisely set remains elusive. The microcephaly protein CPAP (also known as MCPH6) promotes procentriole growth, whereas the oral-facial-digital (OFD) syndrome protein OFD1 represses centriole elongation. Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene. Concordant with the clinical overlap, C2CD3 colocalizes with OFD1 at the distal end of centrioles, and C2CD3 physically associates with OFD1. However, whereas OFD1 deletion leads to centriole hyperelongation, loss of C2CD3 results in short centrioles without subdistal and distal appendages. Because C2CD3 overexpression triggers centriole hyperelongation and OFD1 antagonizes this activity, we propose that C2CD3 directly promotes centriole elongation and that OFD1 acts as a negative regulator of C2CD3. Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.

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C2cd3 co-localizes with Ofd1 at the distal end of centrioles and procentrioles and physically interacts with Ofd1IMCD3-[GFP-C2cd3] cells stained for:(a) The centriolar marker branched-glutamylated tubulin (GT335, pink).(b) The distal centriole and procentriole marker Centrin (red).(c) The procentriole marker Sass6 (red).(d) Ninein (red, marks the subdistal appendages and the proximal end of the centrioles and procentrioles) and glutamylated tubulin (GT335, pink, marks the ciliary axoneme).(e) Cep164 (red, marks the distal appendages) and glutamylated tubulin (GT335, pink).(f) Cep290 (red, marks the transition zone) and glutamylated tubulin (GT335, pink).(g) Ofd1 (red) and γ-tubulin (pink, marks the pericentriolar material near the proximal part of the centriole).(h) The distal cap marker CP110 (red) and γ-tubulin (pink).All cells were treated with nocodazole before processing for immunofluorescence. All scale bars are 1 µm.(i) Immunoprecipitation of OFD1 from RPE cell extract recovers C2CD3. For the C2CD3 immunoblot, 160 equivalent of eluates and 1 equivalent of lysate were ran on the gel. For the OFD1 immunoblot, 4 equivalent of eluates and 1 equivalent of lysate were ran on the gel. The eluate and lysate panels are cropped from the same film exposure.(j) GST capture assays. GFP-C2cd3 and MBP-GFP-FKBP (negative control) expressed in HEK cells were captured by bacterially expressed GST fusions to Ofd1 fragments. Captured proteins were detected by immunoblotting for GFP. 80 equivalent of eluates and 1 equivalent of lysate were ran on the gel.
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Figure 2: C2cd3 co-localizes with Ofd1 at the distal end of centrioles and procentrioles and physically interacts with Ofd1IMCD3-[GFP-C2cd3] cells stained for:(a) The centriolar marker branched-glutamylated tubulin (GT335, pink).(b) The distal centriole and procentriole marker Centrin (red).(c) The procentriole marker Sass6 (red).(d) Ninein (red, marks the subdistal appendages and the proximal end of the centrioles and procentrioles) and glutamylated tubulin (GT335, pink, marks the ciliary axoneme).(e) Cep164 (red, marks the distal appendages) and glutamylated tubulin (GT335, pink).(f) Cep290 (red, marks the transition zone) and glutamylated tubulin (GT335, pink).(g) Ofd1 (red) and γ-tubulin (pink, marks the pericentriolar material near the proximal part of the centriole).(h) The distal cap marker CP110 (red) and γ-tubulin (pink).All cells were treated with nocodazole before processing for immunofluorescence. All scale bars are 1 µm.(i) Immunoprecipitation of OFD1 from RPE cell extract recovers C2CD3. For the C2CD3 immunoblot, 160 equivalent of eluates and 1 equivalent of lysate were ran on the gel. For the OFD1 immunoblot, 4 equivalent of eluates and 1 equivalent of lysate were ran on the gel. The eluate and lysate panels are cropped from the same film exposure.(j) GST capture assays. GFP-C2cd3 and MBP-GFP-FKBP (negative control) expressed in HEK cells were captured by bacterially expressed GST fusions to Ofd1 fragments. Captured proteins were detected by immunoblotting for GFP. 80 equivalent of eluates and 1 equivalent of lysate were ran on the gel.

Mentions: We then mapped the precise location of C2cd3 within centrioles. We frequently observed two juxtaposed dots of C2cd3 associated with a single spot of glutamylated tubulin (Fig. 2a). Since the two juxtaposed foci of C2cd3 were each positive for centrin, which marks the distal lumen of mature and pro- centrioles (Fig. 2b and Supplementary Fig. 4a) and since procentriole microtubules are not polyglutamylated22, the glutamylated tubulin-negative spot of C2cd3 likely represented the procentriole. Staining cells for Sas-6, a marker of procentrioles but not mature centrioles23, confirmed that C2cd3 is present at procentrioles (Fig. 2c). In mature centrioles, we found C2cd3 slightly distal to the Ninein-or Odf2-marked subdistal appendages (Fig. 2d and Supplementary Fig. 4b). C2cd3 was precisely located between the Cep164-marked distal appendages (Fig. 2e). Finally, the transition zone25 marker Cep290 was clearly distal to C2cd3 (Fig. 2f), leading us to conclude that C2cd3 is localized near the distal tip of centrioles. CP110 and Ofd1 have also been localized near the distal end of centrioles, with CP110 marking a slightly more distal location than Ofd1 (Ref. 7), namely the centriole cap whose removal allows for elongation of the ciliary axoneme26. C2cd3 perfectly co-localized with Ofd1 (Fig. 2g) and was slightly proximal to CP110 (Fig. 2h).


The oral-facial-digital syndrome gene C2CD3 encodes a positive regulator of centriole elongation.

Thauvin-Robinet C, Lee JS, Lopez E, Herranz-Pérez V, Shida T, Franco B, Jego L, Ye F, Pasquier L, Loget P, Gigot N, Aral B, Lopes CA, St-Onge J, Bruel AL, Thevenon J, González-Granero S, Alby C, Munnich A, Vekemans M, Huet F, Fry AM, Saunier S, Rivière JB, Attié-Bitach T, Garcia-Verdugo JM, Faivre L, Mégarbané A, Nachury MV - Nat. Genet. (2014)

C2cd3 co-localizes with Ofd1 at the distal end of centrioles and procentrioles and physically interacts with Ofd1IMCD3-[GFP-C2cd3] cells stained for:(a) The centriolar marker branched-glutamylated tubulin (GT335, pink).(b) The distal centriole and procentriole marker Centrin (red).(c) The procentriole marker Sass6 (red).(d) Ninein (red, marks the subdistal appendages and the proximal end of the centrioles and procentrioles) and glutamylated tubulin (GT335, pink, marks the ciliary axoneme).(e) Cep164 (red, marks the distal appendages) and glutamylated tubulin (GT335, pink).(f) Cep290 (red, marks the transition zone) and glutamylated tubulin (GT335, pink).(g) Ofd1 (red) and γ-tubulin (pink, marks the pericentriolar material near the proximal part of the centriole).(h) The distal cap marker CP110 (red) and γ-tubulin (pink).All cells were treated with nocodazole before processing for immunofluorescence. All scale bars are 1 µm.(i) Immunoprecipitation of OFD1 from RPE cell extract recovers C2CD3. For the C2CD3 immunoblot, 160 equivalent of eluates and 1 equivalent of lysate were ran on the gel. For the OFD1 immunoblot, 4 equivalent of eluates and 1 equivalent of lysate were ran on the gel. The eluate and lysate panels are cropped from the same film exposure.(j) GST capture assays. GFP-C2cd3 and MBP-GFP-FKBP (negative control) expressed in HEK cells were captured by bacterially expressed GST fusions to Ofd1 fragments. Captured proteins were detected by immunoblotting for GFP. 80 equivalent of eluates and 1 equivalent of lysate were ran on the gel.
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Figure 2: C2cd3 co-localizes with Ofd1 at the distal end of centrioles and procentrioles and physically interacts with Ofd1IMCD3-[GFP-C2cd3] cells stained for:(a) The centriolar marker branched-glutamylated tubulin (GT335, pink).(b) The distal centriole and procentriole marker Centrin (red).(c) The procentriole marker Sass6 (red).(d) Ninein (red, marks the subdistal appendages and the proximal end of the centrioles and procentrioles) and glutamylated tubulin (GT335, pink, marks the ciliary axoneme).(e) Cep164 (red, marks the distal appendages) and glutamylated tubulin (GT335, pink).(f) Cep290 (red, marks the transition zone) and glutamylated tubulin (GT335, pink).(g) Ofd1 (red) and γ-tubulin (pink, marks the pericentriolar material near the proximal part of the centriole).(h) The distal cap marker CP110 (red) and γ-tubulin (pink).All cells were treated with nocodazole before processing for immunofluorescence. All scale bars are 1 µm.(i) Immunoprecipitation of OFD1 from RPE cell extract recovers C2CD3. For the C2CD3 immunoblot, 160 equivalent of eluates and 1 equivalent of lysate were ran on the gel. For the OFD1 immunoblot, 4 equivalent of eluates and 1 equivalent of lysate were ran on the gel. The eluate and lysate panels are cropped from the same film exposure.(j) GST capture assays. GFP-C2cd3 and MBP-GFP-FKBP (negative control) expressed in HEK cells were captured by bacterially expressed GST fusions to Ofd1 fragments. Captured proteins were detected by immunoblotting for GFP. 80 equivalent of eluates and 1 equivalent of lysate were ran on the gel.
Mentions: We then mapped the precise location of C2cd3 within centrioles. We frequently observed two juxtaposed dots of C2cd3 associated with a single spot of glutamylated tubulin (Fig. 2a). Since the two juxtaposed foci of C2cd3 were each positive for centrin, which marks the distal lumen of mature and pro- centrioles (Fig. 2b and Supplementary Fig. 4a) and since procentriole microtubules are not polyglutamylated22, the glutamylated tubulin-negative spot of C2cd3 likely represented the procentriole. Staining cells for Sas-6, a marker of procentrioles but not mature centrioles23, confirmed that C2cd3 is present at procentrioles (Fig. 2c). In mature centrioles, we found C2cd3 slightly distal to the Ninein-or Odf2-marked subdistal appendages (Fig. 2d and Supplementary Fig. 4b). C2cd3 was precisely located between the Cep164-marked distal appendages (Fig. 2e). Finally, the transition zone25 marker Cep290 was clearly distal to C2cd3 (Fig. 2f), leading us to conclude that C2cd3 is localized near the distal tip of centrioles. CP110 and Ofd1 have also been localized near the distal end of centrioles, with CP110 marking a slightly more distal location than Ofd1 (Ref. 7), namely the centriole cap whose removal allows for elongation of the ciliary axoneme26. C2cd3 perfectly co-localized with Ofd1 (Fig. 2g) and was slightly proximal to CP110 (Fig. 2h).

Bottom Line: How centriole length is precisely set remains elusive.Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene.Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.

View Article: PubMed Central - PubMed

Affiliation: 1] Equipe d'Accueil 4271 Génétique des Anomalies du Développement, Fédération Hospitalo-Universitaire, Université de Bourgogne, Dijon, France. [2] Centre de Référence Maladies Rares "Anomalies du Développement et Syndromes Malformatifs" de l'Est, Centre de Génétique et Pédiatrie 1, Hôpital d'Enfants, Centre Hospitalier Universitaire Dijon, Dijon, France. [3].

ABSTRACT
Centrioles are microtubule-based, barrel-shaped structures that initiate the assembly of centrosomes and cilia. How centriole length is precisely set remains elusive. The microcephaly protein CPAP (also known as MCPH6) promotes procentriole growth, whereas the oral-facial-digital (OFD) syndrome protein OFD1 represses centriole elongation. Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene. Concordant with the clinical overlap, C2CD3 colocalizes with OFD1 at the distal end of centrioles, and C2CD3 physically associates with OFD1. However, whereas OFD1 deletion leads to centriole hyperelongation, loss of C2CD3 results in short centrioles without subdistal and distal appendages. Because C2CD3 overexpression triggers centriole hyperelongation and OFD1 antagonizes this activity, we propose that C2CD3 directly promotes centriole elongation and that OFD1 acts as a negative regulator of C2CD3. Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.

Show MeSH
Related in: MedlinePlus