Drosophila Ana2 is a conserved centriole duplication factor

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.

Figure 1.

Overexpression of Ana2 or Asl drives de novo formation of centriole-like structures. (A and B) Unfertilized eggs laid by UAS-Ana2-GFP (A) or UAS-Asl-GFP (B) mothers containing numerous MT asters (stained for tubulin). Arrows indicate the polar bodies. (C and D) Single asters from UAS-Ana2-GFP (C) or UAS-Asl-GFP (D) eggs stained for tubulin (blue) and DSas-4 (red). GFP is in green. Each aster contains several structures that stain for centriole markers. Bars: (A and B) 20 µm; (C and D) 2 µm.

Figure 2.

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.

Figure 3.

Ana2 is a centriole component with a unique asymmetric localization. (A) Centriole pair from a G2 primary spermatocyte expressing Ana2-GFP (green) stained for the centriole marker GTU88* (red). Ana2-GFP localizes to the proximal and distal centriole ends and also exhibits a unique asymmetric distribution, localizing preferentially along one centriole barrel, which can be identified as the daughter from the GTU88* staining (see main text). (B) Centriole pair from a G2 primary spermatocyte expressing GFP-Ana2 (green) and RFP-PACT (red). GFP-Ana2 localization is indistinguishable from Ana2-GFP. (C) Centriole pair from a primary spermatocyte at anaphase of meiosis I: the centrioles are beginning to separate. The cell is expressing RFP-PACT (red) and GFP-Ana2 (green), which is no longer obviously asymmetric. (D) Two basal bodies from spermatids expressing RFP-PACT (blue) and GFP–DSas-6 (green), and stained for Ana2 (red). GFP–DSas-6 and Ana2 colocalize at the proximal centriole-like structure, a nodule adjacent to the basal body marked by RFP-PACT. Bars, 2 µm.

Figure 4.

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.

Figure 5.

Ana2 is related to vertebrate STIL. Schematic of human STIL Drosophila Ana2, and C. elegans SAS-5. All three proteins have a central, coiled-coil domain (green) and a conserved region near the C terminus (blue): the STAN motif. An alignment of the STAN motif is shown in full, with an alignment including SAS-5 below. Both are colored according to the Blosum62 coloring scheme, where dark blue indicates a match to the consensus sequence and light blue indicates a positive Blosum62 score. Asterisks indicate residues are identical in all aligned sequences, colons indicate conserved substitutions, and periods indicate semiconserved substitutions.