(a) The anti-Sas-4 antibody labels embryonic centrosomes. Centrosomes are marked by Asl-GFP. Blue, DAPI. Scale bar, 2 μm. (b) Sas-4’s location in sperm centrosomes varies over development. Ana1-GFP, a centriolar marker, labels centrosomes. Scale bar, 1 μm. (c) Sas-4 labels a ‘toroid-shaped’ structure around the centriole core. 3D-structured illumination microscopy of Sas-4-labelled (green) mitotic centrosomes in Drosophila S2 cells (). Tubulin (red) and CP-190 (blue) mark the PCM. Scale bars are 1 μm.

(a) Immunoprecipitations using the anti-Sas-4 antibody of HSLs reveal associations between Sas-4 and other proteins. Embryonic extracts were used as the positive control; uncoated beads were used as the negative control (Control). (b) CNN, Asl, D-PLP, CP-190 and β-tubulin co-purify with Sas-4 in tandem affinity purification (TAP) of HSL from Sas-4–TAP-expressing embryos (Sas-4–TAP). The centriolar protein Sas-6 was only detected in embryonic extracts. (c) CNN (CNN-IP) or Asl (Asl-IP) co-immunoprecipitates Sas-4, D-PLP and tubulin from the TAP-purified Sas-4-containing complexes (Sas-4–TAP). A portion of the TAP-purified Sas-4-containing complexes was not precipitated by the antibody-coated beads (Unbound), which contains Sas-4 and tubulin. None of the above proteins is detected from uncoated beads (Control). (d, e) TAP-purified Sas-4-containing complexes fractionate around 7 S in a 15–60% sucrose gradient. (d) Whereas Sas-4, β-tubulin and γ-tubulin are widely distributed, CNN, D-PLP and CP-190, were restricted to only a narrow range of fractions (~7 S). (e) Asl was also detected as a narrow distribution in experiments that included nocodozole (100 ng ml^{− 1}) which inhibits microtubule polymerization.

(a) In control testes, developing and elongated centrioles are labelled by Ana1-GFP throughout the testis. The elongated centrioles within the dashed box are enlarged in the inset. In Sas-4^s2214-null mutants, Ana1-GFP only labels nascent centrioles present in spermatogonia; Ana1-GFP is absent from differentiating spermatocyte cells. Dotted lines mark a testis’ boundaries. In this analysis, we excluded the maternally contributed centrioles that are restricted to the stem cells, identified by Fas III (magenta) labelling^,,. Scale bar, 10 and 2 μm for higher and lower magnification, respectively. (b–f) In early differentiating sperm cells, centriolar structures in control testes and Sas-4^s2214-null mutant testes are labelled by Sas-6-GFP. CNN (b), Asl (c), CP-190 (d) and γ-tubulin (e) are present in control centrioles but not in Sas-4^s2214 nascent procentrioles. Dashed boxes mark the enlarged areas shown in the lower panels. Scale bar, 2 μm. (f) Charts showing the fraction of centriolar structures (CSs) that are positive for each PCM protein in the control (grey filling) and in Sas-4^s2214 (white filling); for each chart, Sas-6-GFP-labelled CSs were counted within a 20 μm² area that is 10 μm from the stem cell region or ~25 μm from the tip of a testis (dotted lines). The mean ± s.e.m. of six independent testes are shown.

(a–d) Sas-4-GFP overexpression (OE) in S2 cells induces foci that contain Asl (a), D-PLP (b) and CP-190 (c) but not the centriolar protein Ana1 (d). In (d), yellow arrows point out the Sas-4- and Ana1-positive centrioles; the white arrow points out a Sas-4-positive but Ana1-negative focus. This is an example of a focus that was induced by Sas-4 OE. In (a), the nuclear Asl staining is nonspecific. Scale bar, 1 μm. (e) Quantification of control (grey filling) and Sas-4-GFP-induced (white filling) foci shown in panels (a–d) (n = 50 except for Ana1, where n = 20). Note that ~30% of the S2 cells normally possess three to four centrioles per cell. (f) Velocity sedimentation of Sas-4-induced foci. Extracts of untransfected control cells and Sas-4-transfected cells were separated on a 15–60% sucrose gradient. The intermediate density fractions induced by Sas-4 OE are not detected in control cells. The Sas-4-induced foci co-fractionate with CNN, Asl and CP-190. The fractionation pattern of the centriole core-specific protein Sas-6 remained unchanged in both control cells and Sas-4-transfected cells.

(a) Schematics of the Sas-4-containing, GFP-tagged constructs: Sas-4, Sas-4Δ150, and Sas-4-N. (b) Co-immunoprecipitation experiments using S2 cells that express Sas-4, Sas-4Δ150, or Sas-4-N. HSL, high-speed cell lysate; IP, immunoprecipitation; IgG, beads coated with a nonspecific mouse IgG; and Embryonic: embryonic extract. The asterisk marks a band that may be an isoform of Sas-4. (c) Sas-4-N190 (which includes only the first 190 amino acids of Sas-4) pulls down CNN, Asl, D-PLP and β-tubulin but not the centriolar-specific protein Sas-6 from embryonic HSL. The purified recombinant proteins used in this figure are shown in . (d) Sas-4 binds directly to CNN via the N-terminal half of CNN. Anti-Sas-4 antibody-coated beads immobilized Sas-4-N190, which was used to bind CNN or its truncated variants. Sas-4-N190 binds purified full-length CNN (CNN) and CNN’s N-terminal half. The C-terminal half of CNN (CNN-C) does not bind Sas-4-N190. B, bound; U, unbound. (e) The N-terminal domain of Sas-4 is essential in the formation of S-CAP complexes and Sas-4-induced foci. Comparison of fractionation patterns for S2 cell extracts transfected with either Sas-4 or Sas-4Δ150. (f) The N-terminal domain of Sas-4 is required for the formation of functional centrosomes. Analysed are testes of homozygous Sas-4^S2214-null mutants that have been transfected with Sas-4, heterozygous Sas-4^S2214 mutants transfected with Sas-4Δ150, and homozygous Sas-4^S2214 mutants transfected with Sas-4Δ150. The tip of a testis is marked with dotted lines. Scale bar, 10 and 2 μm for higher (left) and lower (right) magnification, respectively.

(a–d) In vivo, the PN2-3 domain is necessary for Sas-4’s recruitment of S-CAP complex components to a centriole. (a) The number of centrioles observed at the tip of a testis ( < 60 μm from the hub) and the fractions of centrioles that are positive for CNN or Asl. The mean ± s.e.m. of six independent testes are shown. (b, c) CNN and Asl are recruited to centrosomes of Sas-4^s2214-null mutants that express Sas-4 or Sas-4Δ90. (d) In contrast, these proteins are not recruited to centrioles when Sas-4ΔPN2-3 is expressed. Dashed boxes mark the enlarged areas. Scale bar, 2 μm. (e) In vitro, the PN2-3 domain is necessary for Sas-4’s binding to S-CAP complex components and their recruitment to a centriole. Co-immunoprecipitation experiments using extracts of S2 cells transfected with Sas-4 or Sas-4ΔPN2-3. In cells that overexpress Sas-4, CNN, Asl, CP-190, β-tubulin and γ-tubulin are immunoprecipitated (IP) from high-speed cell lysates (HSLs). In contrast, CP-190 and γ-tubulin but not CNN, Asl and β-tubulin are immunoprecipitated from HSLs of S2 cells that overexpress Sas-4ΔPN2-3. IgG: beads coated with a mouse IgG. (f) The PN2-3 domain of Sas-4 is required to pull down Asl, CNN or β-tubulin from embryonic HSLs. The purified recombinant proteins used are shown in Coomassie-stained gels. Sas-4-N–GST (which only includes the first 190 amino acids of Sas-4) pulls down Asl, CNN, β-tubulin, CP-190 and γ-tubulin from embryonic lysates. However, a Sas-4-N–GST variant, which lacks its PN2-3 domain (‘NΔPN2-3’), can only pull down CP-190 and γ-tubulin.

(a) Centrosomes purified from wild-type embryonic extracts contain Sas-4, CNN, Asl, CP-190, tubulin and Sas-6, while stripped centrosomes contain only Sas-6 and tubulin. Purified Sas-4 complexes contain the Sas-4, CNN, Asl, CP-190 and tubulin. (b) Binding assays of purified Sas-4 complexes, Sas-4-N190 or Sas-4-Δ90 to stripped centrosomes and analyses by discontinuous sucrose gradient. Low-density (LD) fractions contain unbound proteins and high-density (HD) fractions contain stripped centrosomes and the proteins bound to them. In the absence of stripped centrosomes, Sas-4 complexes, Sas-4-N190 or Sas-4-Δ90 remain only in low-density fractions. When mixed with stripped centrosomes, Sas-4 complexes and Sas-4-Δ90 but not Sas-4-N190 are found in the HD fractions. Excess complexes and proteins remain in the unbound fractions. (c) Binding assays of Sas-4-Δ90, CNN or a combination of Sas-4-Δ90 and CNN to stripped centrosomes. LD fractions contain unbound proteins, whereas HD fractions contain stripped centrosomes and proteins bound to them. CNN or Asl alone cannot bind stripped centrosomes. In the presence of Sas-4-Δ90 and stripped centrosomes, CNN and Asl are bound to stripped centrosomes. (d) Schematic of the Sas-4 protein domains. Tethering: as Sas-4Δ90 binds to stripped centrosomes while Sas-4-N190 does not, the centrosome-binding domain of Sas-4 appears to be between residues 191 and 901. CNN, Asl, β-tubulin binding depends on the presence of the PN2-3 domain. D-PLP and γ-tubulin binding: as Sas-4-N190 binds to D-PLP and γ-tubulin, these proteins’ binding domain in Sas-4 appears to be between residues 1 and 190. Green box: conserved segment in PN2-3 domain. CP-190 binding: as Sas-4-N190 and Sas-4Δ150 bind to CP-190, the CP-190 domain of Sas-4 appears to be between residues 151 and 190. (e) A model for the role of Sas-4 in PCM formation. Centrosome biogenesis begins with the formation of a nascent procentriole and the assembly of centriole microtubules. S-CAP complexes form in the cytoplasm and are then tethered to a procentriole via Sas-4. The centriole elongates and matures to a functional centrosome. Sas-4 (blue); microtubules (green); PCM (grey); appendages (orange).