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Drosophila Ana2 is a conserved centriole duplication factor.

Stevens NR, Dobbelaere J, Brunk K, Franz A, Raff JW - J. Cell Biol. (2010)

Bottom Line: Functional orthologues of all but SAS-5 have been found in other species.Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms.We propose that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Gurdon Institute, Cambridge, England CB2 1QN, UK.

ABSTRACT
In Caenorhabditis elegans, five proteins are required for centriole duplication: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. Functional orthologues of all but SAS-5 have been found in other species. In Drosophila melanogaster and humans, Sak/Plk4, DSas-6/hSas-6, and DSas-4/CPAP-orthologues of ZYG-1, SAS-6, and SAS-4, respectively-are required for centriole duplication. Strikingly, all three fly proteins can induce the de novo formation of centriole-like structures when overexpressed in unfertilized eggs. Here, we find that of eight candidate duplication factors identified in cultured fly cells, only two, Ana2 and Asterless (Asl), share this ability. Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms. We show that Ana2 is the likely functional orthologue of SAS-5 and that it is also related to the vertebrate STIL/SIL protein family that has been linked to microcephaly in humans. We propose that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery.

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Overexpression of Ana2 drives centriole overduplication in spermatocytes. (A and B) Centriole number (A) and conformation (B) in G2 primary spermatocytes expressing either the centriole marker RFP-PACT alone or both RFP-PACT and GFP-Ana2. Centrioles were counted in a total of 109 RFP-PACT cells and 138 GFP-Ana2 RFP-PACT cells from seven testes per condition. (C and D) G2 primary spermatocytes expressing either RFP-PACT (red) alone (C) or both RFP-PACT and GFP-Ana2 (D). DNA is in blue. The cell in C has the normal two centriole pairs. Overexpression of GFP-Ana2 induces centriole triplets and quadruplets (D). (E–G) Magnified images of RFP-PACT–labeled doublet (E), triplet (F), and quadruplet (G) centriole groups. (H and I) Secondary spermatocytes in meiosis II expressing either RFP-PACT (red) alone (H) or both RFP-PACT and GFP-Ana2 (I). Tubulin is in green and DNA in blue. The cell in H has the normal two centrioles whereas the one in I has three centrioles forming a tripolar spindle. Bars: (C and D) 10 µm; (H and I) 5 µm.
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fig2: Overexpression of Ana2 drives centriole overduplication in spermatocytes. (A and B) Centriole number (A) and conformation (B) in G2 primary spermatocytes expressing either the centriole marker RFP-PACT alone or both RFP-PACT and GFP-Ana2. Centrioles were counted in a total of 109 RFP-PACT cells and 138 GFP-Ana2 RFP-PACT cells from seven testes per condition. (C and D) G2 primary spermatocytes expressing either RFP-PACT (red) alone (C) or both RFP-PACT and GFP-Ana2 (D). DNA is in blue. The cell in C has the normal two centriole pairs. Overexpression of GFP-Ana2 induces centriole triplets and quadruplets (D). (E–G) Magnified images of RFP-PACT–labeled doublet (E), triplet (F), and quadruplet (G) centriole groups. (H and I) Secondary spermatocytes in meiosis II expressing either RFP-PACT (red) alone (H) or both RFP-PACT and GFP-Ana2 (I). Tubulin is in green and DNA in blue. The cell in H has the normal two centrioles whereas the one in I has three centrioles forming a tripolar spindle. Bars: (C and D) 10 µm; (H and I) 5 µm.

Mentions: Ana2 can drive de novo formation of centriole-like structures as efficiently as DSas-6 and Sak (Peel et al., 2007; Rodrigues-Martins et al., 2007b). We wanted to verify, however, that it also has a role in canonical centriole duplication. Overexpressing GFP-Sak or GFP–DSas-6 from the ubiquitin (Ubq) promoter induces centriole overduplication in brains and embryos, respectively (Peel et al., 2007). Surprisingly, however, overexpression of Sak, DSas-6, or DSas-4 cannot drive centriole overduplication in primary spermatocytes (Peel et al., 2007), which suggests that another duplication protein is limiting. To test if Ana2 might be this limiting factor, we generated Ubq-GFP-Ana2 transgenic lines. Strikingly, we found that in spermatocytes expressing Ubq-GFP-Ana2, in addition to the normal centriole pairs (doublets), we observed centriole triplets, quadruplets, and even quintets (Fig. 2, A–G). The extra centrioles in these clusters appeared to be fully functional; they separated from one another by the end of meiosis I (as centriole doublets normally do), and the extra centrioles inherited by secondary spermatocytes recruited PCM and nucleated MT asters, and so formed multipolar spindles during meiosis II (Fig. 2, H and I).


Drosophila Ana2 is a conserved centriole duplication factor.

Stevens NR, Dobbelaere J, Brunk K, Franz A, Raff JW - J. Cell Biol. (2010)

Overexpression of Ana2 drives centriole overduplication in spermatocytes. (A and B) Centriole number (A) and conformation (B) in G2 primary spermatocytes expressing either the centriole marker RFP-PACT alone or both RFP-PACT and GFP-Ana2. Centrioles were counted in a total of 109 RFP-PACT cells and 138 GFP-Ana2 RFP-PACT cells from seven testes per condition. (C and D) G2 primary spermatocytes expressing either RFP-PACT (red) alone (C) or both RFP-PACT and GFP-Ana2 (D). DNA is in blue. The cell in C has the normal two centriole pairs. Overexpression of GFP-Ana2 induces centriole triplets and quadruplets (D). (E–G) Magnified images of RFP-PACT–labeled doublet (E), triplet (F), and quadruplet (G) centriole groups. (H and I) Secondary spermatocytes in meiosis II expressing either RFP-PACT (red) alone (H) or both RFP-PACT and GFP-Ana2 (I). Tubulin is in green and DNA in blue. The cell in H has the normal two centrioles whereas the one in I has three centrioles forming a tripolar spindle. Bars: (C and D) 10 µm; (H and I) 5 µm.
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Related In: Results  -  Collection

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fig2: Overexpression of Ana2 drives centriole overduplication in spermatocytes. (A and B) Centriole number (A) and conformation (B) in G2 primary spermatocytes expressing either the centriole marker RFP-PACT alone or both RFP-PACT and GFP-Ana2. Centrioles were counted in a total of 109 RFP-PACT cells and 138 GFP-Ana2 RFP-PACT cells from seven testes per condition. (C and D) G2 primary spermatocytes expressing either RFP-PACT (red) alone (C) or both RFP-PACT and GFP-Ana2 (D). DNA is in blue. The cell in C has the normal two centriole pairs. Overexpression of GFP-Ana2 induces centriole triplets and quadruplets (D). (E–G) Magnified images of RFP-PACT–labeled doublet (E), triplet (F), and quadruplet (G) centriole groups. (H and I) Secondary spermatocytes in meiosis II expressing either RFP-PACT (red) alone (H) or both RFP-PACT and GFP-Ana2 (I). Tubulin is in green and DNA in blue. The cell in H has the normal two centrioles whereas the one in I has three centrioles forming a tripolar spindle. Bars: (C and D) 10 µm; (H and I) 5 µm.
Mentions: Ana2 can drive de novo formation of centriole-like structures as efficiently as DSas-6 and Sak (Peel et al., 2007; Rodrigues-Martins et al., 2007b). We wanted to verify, however, that it also has a role in canonical centriole duplication. Overexpressing GFP-Sak or GFP–DSas-6 from the ubiquitin (Ubq) promoter induces centriole overduplication in brains and embryos, respectively (Peel et al., 2007). Surprisingly, however, overexpression of Sak, DSas-6, or DSas-4 cannot drive centriole overduplication in primary spermatocytes (Peel et al., 2007), which suggests that another duplication protein is limiting. To test if Ana2 might be this limiting factor, we generated Ubq-GFP-Ana2 transgenic lines. Strikingly, we found that in spermatocytes expressing Ubq-GFP-Ana2, in addition to the normal centriole pairs (doublets), we observed centriole triplets, quadruplets, and even quintets (Fig. 2, A–G). The extra centrioles in these clusters appeared to be fully functional; they separated from one another by the end of meiosis I (as centriole doublets normally do), and the extra centrioles inherited by secondary spermatocytes recruited PCM and nucleated MT asters, and so formed multipolar spindles during meiosis II (Fig. 2, H and I).

Bottom Line: Functional orthologues of all but SAS-5 have been found in other species.Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms.We propose that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Gurdon Institute, Cambridge, England CB2 1QN, UK.

ABSTRACT
In Caenorhabditis elegans, five proteins are required for centriole duplication: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4. Functional orthologues of all but SAS-5 have been found in other species. In Drosophila melanogaster and humans, Sak/Plk4, DSas-6/hSas-6, and DSas-4/CPAP-orthologues of ZYG-1, SAS-6, and SAS-4, respectively-are required for centriole duplication. Strikingly, all three fly proteins can induce the de novo formation of centriole-like structures when overexpressed in unfertilized eggs. Here, we find that of eight candidate duplication factors identified in cultured fly cells, only two, Ana2 and Asterless (Asl), share this ability. Asl is now known to be essential for centriole duplication in flies, but no equivalent protein has been found in worms. We show that Ana2 is the likely functional orthologue of SAS-5 and that it is also related to the vertebrate STIL/SIL protein family that has been linked to microcephaly in humans. We propose that members of the SAS-5/Ana2/STIL family of proteins are key conserved components of the centriole duplication machinery.

Show MeSH
Related in: MedlinePlus