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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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Ana2 and DSas-6 functionally and physically interact. (A) Percentage of unfertilized eggs laid by mothers of the given genotypes that contained MT asters. All transgenes were GFP fusions with a Ubq promoter. Eggs from mothers expressing one or two copies of Ubq-GFP–DSas-6 and Ubq-Ana2-GFP were analyzed; all combinations expressed one copy of each transgene. n > 80 eggs per genotype (for values, see Materials and methods). (B and C) Almost all unfertilized eggs from mothers expressing one copy of Ubq-GFP–DSas-6 and one copy of Ubq-Ana2-GFP assemble large numbers of asters (stained for tubulin). The arrow indicates polar bodies. (D) Single aster from an egg laid by a Ubq-GFP–DSas-6/Ubq-Ana2-GFP mother stained for tubulin (blue) and DSas-4 (red). (E) Very similar structures are found in eggs from mothers expressing very high levels of GFP–DSas-6 alone from the stronger UAS promoter. (F) Schematic of our Y2H analysis of Ana2 and DSas-6 and comparison with Y2H analyses of C. elegans SAS-5 and SAS-6. Brackets indicate the protein fragments tested, and interactions are shown with arrows. In Drosophila, the C-terminal region of Ana2 interacts with the N-terminal region of DSas-6. In C. elegans, the C-terminal region of SAS-5 interacts with the coiled-coil region of SAS-6 (Leidel et al., 2005; Boxem et al., 2008). Both Ana2 and SAS-5 (Leidel et al., 2005; Boxem et al., 2008) also interact with themselves. (G) Western blot of an immunoprecipitation experiment from S2 cells overexpressing Ana2-GFP; performed with random rabbit IgG (RRb), Ana2, or DSas-6 antibodies; and probed with Ana2 antibodies. DSas-6 antibodies, but not RRb, coimmunoprecipitated Ana2-GFP (endogenous Ana2 was undetectable). Bars: (B) 100 µm; (C) 20 µm; (D and E) 2 µm.
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fig4: Ana2 and DSas-6 functionally and physically interact. (A) Percentage of unfertilized eggs laid by mothers of the given genotypes that contained MT asters. All transgenes were GFP fusions with a Ubq promoter. Eggs from mothers expressing one or two copies of Ubq-GFP–DSas-6 and Ubq-Ana2-GFP were analyzed; all combinations expressed one copy of each transgene. n > 80 eggs per genotype (for values, see Materials and methods). (B and C) Almost all unfertilized eggs from mothers expressing one copy of Ubq-GFP–DSas-6 and one copy of Ubq-Ana2-GFP assemble large numbers of asters (stained for tubulin). The arrow indicates polar bodies. (D) Single aster from an egg laid by a Ubq-GFP–DSas-6/Ubq-Ana2-GFP mother stained for tubulin (blue) and DSas-4 (red). (E) Very similar structures are found in eggs from mothers expressing very high levels of GFP–DSas-6 alone from the stronger UAS promoter. (F) Schematic of our Y2H analysis of Ana2 and DSas-6 and comparison with Y2H analyses of C. elegans SAS-5 and SAS-6. Brackets indicate the protein fragments tested, and interactions are shown with arrows. In Drosophila, the C-terminal region of Ana2 interacts with the N-terminal region of DSas-6. In C. elegans, the C-terminal region of SAS-5 interacts with the coiled-coil region of SAS-6 (Leidel et al., 2005; Boxem et al., 2008). Both Ana2 and SAS-5 (Leidel et al., 2005; Boxem et al., 2008) also interact with themselves. (G) Western blot of an immunoprecipitation experiment from S2 cells overexpressing Ana2-GFP; performed with random rabbit IgG (RRb), Ana2, or DSas-6 antibodies; and probed with Ana2 antibodies. DSas-6 antibodies, but not RRb, coimmunoprecipitated Ana2-GFP (endogenous Ana2 was undetectable). Bars: (B) 100 µm; (C) 20 µm; (D and E) 2 µm.

Mentions: A small percentage of eggs laid by mothers carrying two copies of a Ubq-GFP–DSas-6 transgene (as opposed to the much stronger UASp-GFP–DSas-6 discussed above) assemble centriole-like structures (Peel et al., 2007). To see if we could enhance this effect, we generated flies carrying one copy of Ubq-GFP–DSas-6 and one copy of Ubq-Ana2-GFP, neither of which alone (as a single copy) induces the assembly of centriole-like structures (Fig. 4 A). Strikingly, almost all the unfertilized eggs laid by these females contained hundreds of large structures that stained for centriole markers, recruited PCM, and nucleated asters (Fig. 4, A–D; and Fig. S2 A). Importantly this interaction was specific to Ana2 and DSas-6. In eggs from mothers carrying one copy of either Ubq-Ana2-GFP or Ubq-GFP–DSas-6 together with one copy of either Ubq-GFP-Sak, Ubq-Asl-GFP, or Ubq–DSas-4–GFP, we observed at most a very small number of asters in very few eggs (Fig. 4 A).


Drosophila Ana2 is a conserved centriole duplication factor.

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

Ana2 and DSas-6 functionally and physically interact. (A) Percentage of unfertilized eggs laid by mothers of the given genotypes that contained MT asters. All transgenes were GFP fusions with a Ubq promoter. Eggs from mothers expressing one or two copies of Ubq-GFP–DSas-6 and Ubq-Ana2-GFP were analyzed; all combinations expressed one copy of each transgene. n > 80 eggs per genotype (for values, see Materials and methods). (B and C) Almost all unfertilized eggs from mothers expressing one copy of Ubq-GFP–DSas-6 and one copy of Ubq-Ana2-GFP assemble large numbers of asters (stained for tubulin). The arrow indicates polar bodies. (D) Single aster from an egg laid by a Ubq-GFP–DSas-6/Ubq-Ana2-GFP mother stained for tubulin (blue) and DSas-4 (red). (E) Very similar structures are found in eggs from mothers expressing very high levels of GFP–DSas-6 alone from the stronger UAS promoter. (F) Schematic of our Y2H analysis of Ana2 and DSas-6 and comparison with Y2H analyses of C. elegans SAS-5 and SAS-6. Brackets indicate the protein fragments tested, and interactions are shown with arrows. In Drosophila, the C-terminal region of Ana2 interacts with the N-terminal region of DSas-6. In C. elegans, the C-terminal region of SAS-5 interacts with the coiled-coil region of SAS-6 (Leidel et al., 2005; Boxem et al., 2008). Both Ana2 and SAS-5 (Leidel et al., 2005; Boxem et al., 2008) also interact with themselves. (G) Western blot of an immunoprecipitation experiment from S2 cells overexpressing Ana2-GFP; performed with random rabbit IgG (RRb), Ana2, or DSas-6 antibodies; and probed with Ana2 antibodies. DSas-6 antibodies, but not RRb, coimmunoprecipitated Ana2-GFP (endogenous Ana2 was undetectable). Bars: (B) 100 µm; (C) 20 µm; (D and E) 2 µm.
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fig4: Ana2 and DSas-6 functionally and physically interact. (A) Percentage of unfertilized eggs laid by mothers of the given genotypes that contained MT asters. All transgenes were GFP fusions with a Ubq promoter. Eggs from mothers expressing one or two copies of Ubq-GFP–DSas-6 and Ubq-Ana2-GFP were analyzed; all combinations expressed one copy of each transgene. n > 80 eggs per genotype (for values, see Materials and methods). (B and C) Almost all unfertilized eggs from mothers expressing one copy of Ubq-GFP–DSas-6 and one copy of Ubq-Ana2-GFP assemble large numbers of asters (stained for tubulin). The arrow indicates polar bodies. (D) Single aster from an egg laid by a Ubq-GFP–DSas-6/Ubq-Ana2-GFP mother stained for tubulin (blue) and DSas-4 (red). (E) Very similar structures are found in eggs from mothers expressing very high levels of GFP–DSas-6 alone from the stronger UAS promoter. (F) Schematic of our Y2H analysis of Ana2 and DSas-6 and comparison with Y2H analyses of C. elegans SAS-5 and SAS-6. Brackets indicate the protein fragments tested, and interactions are shown with arrows. In Drosophila, the C-terminal region of Ana2 interacts with the N-terminal region of DSas-6. In C. elegans, the C-terminal region of SAS-5 interacts with the coiled-coil region of SAS-6 (Leidel et al., 2005; Boxem et al., 2008). Both Ana2 and SAS-5 (Leidel et al., 2005; Boxem et al., 2008) also interact with themselves. (G) Western blot of an immunoprecipitation experiment from S2 cells overexpressing Ana2-GFP; performed with random rabbit IgG (RRb), Ana2, or DSas-6 antibodies; and probed with Ana2 antibodies. DSas-6 antibodies, but not RRb, coimmunoprecipitated Ana2-GFP (endogenous Ana2 was undetectable). Bars: (B) 100 µm; (C) 20 µm; (D and E) 2 µm.
Mentions: A small percentage of eggs laid by mothers carrying two copies of a Ubq-GFP–DSas-6 transgene (as opposed to the much stronger UASp-GFP–DSas-6 discussed above) assemble centriole-like structures (Peel et al., 2007). To see if we could enhance this effect, we generated flies carrying one copy of Ubq-GFP–DSas-6 and one copy of Ubq-Ana2-GFP, neither of which alone (as a single copy) induces the assembly of centriole-like structures (Fig. 4 A). Strikingly, almost all the unfertilized eggs laid by these females contained hundreds of large structures that stained for centriole markers, recruited PCM, and nucleated asters (Fig. 4, A–D; and Fig. S2 A). Importantly this interaction was specific to Ana2 and DSas-6. In eggs from mothers carrying one copy of either Ubq-Ana2-GFP or Ubq-GFP–DSas-6 together with one copy of either Ubq-GFP-Sak, Ubq-Asl-GFP, or Ubq–DSas-4–GFP, we observed at most a very small number of asters in very few eggs (Fig. 4 A).

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