Plasmids and flies
Transgenic flies and Sas-4 constructs were previously described14,21
. P[lacW]l(3)s2214 (Sas-4s2214) was obtained from the Bloomington Stock Center. GST-CNN was constructed by insertion of a Kpn
I fragment of cnn-RA cDNA into the Kpn
I site of pGEX2 (Stratagene). The resulting plasmid expresses amino acids 17–1,148 of CNN-PA with GST at its amino terminus. GST-CNN-N and GST-CNN-C were PCR amplified and cloned into pGEX2 to produce plasmids that express CNN’s amino acids 17–558 and 517–1,048, respectively. Sas-4 cDNA (GM21734) was obtained from the Berkeley Drosophila
Genome Project. For Sas-4-C90, amino acids 90–900 was amplified from Sas-4 cDNA and cloned into pET24B that express Sas-4-C90 as HIS-tagged fusion protein. Sas-4-N190 was a generous gift from the Tang lab. For Sas-4-N190ΔPN2-3, the PN2-3 domain was deleted from Sas-4-N190 and cloned into pGEX2. For Sas-4ΔPN2-3, the PN2-3 domain was deleted from Sas-4 cDNA by using Quick change exchange kit (Stratagene).
All Sas-4 constructs for in vivo expression were subcloned into pUAS vectors in a manner that the cloned proteins were expressed using a Sas-4 promoter rather than overexpressed using the GAL4 system.
Drosophila embryonic and S2 cell extracts
A 0–8-h Drosophila
embryo extracts were prepared as described4
. Briefly, embryos were homogenized in extract buffer containing 80 mM K-PIPES, pH 6.8, 1 mM MgCl2
, 1 mM Na3
EGTA, 14% sucrose, 100 mM KCl, 1 mM PMSF, protease inhibitor cocktail and EDTA-free Roche complete tablets (Roche). The crude extract was centrifuged for 20 min at 1,500 g
at 4°C to obtain a low-speed lysate, which contained centrosomes and cytoplasmic complexes. A HSL was prepared similarly but was instead centrifuged for 30 min at 200,000 g
. Similar procedures were used for Drosophila
S2 cells. Stable Drosophila
S2 cell lines that express variants of Sas-4 were established by co-transfecting plasmids containing a Sas-4 construct with a hygromycin-resistant plasmid (Invitrogen).
Purification of native Sas-4 complexes
Protein G beads were coated with an anti-Sas-4 or anti-GFP (Roche) antibody overnight at 4°C. The antibody-coated beads and embryonic/S2 extracts were mixed and incubated at 4°C for 4 h, twice washed with extract buffer containing 0.1% Triton X-100, and then twice washed with extract buffer. The beads were eluted by boiling in Laemmli sample buffer. Recombinant Sas-4-N190 (35 μM) was used to purify native Sas-4 complexes. For TAP purification, HSL was incubated with IgG-coated beads, the bound product was TEV cleaved, and the Sas-4 complexes were bound to calmodulin beads. The complexes were eluted in buffer containing 10 mM EGTA. For GST pull downs, the bait was recombinant Sas-4-N-GST or Sas-4-N190ΔPN2-3 bound to GST agarose and the source of Sas-4-N190 was embryonic HSL; 20 mM glutathione was used for the elution. When required, an anti-GFP antibody (Roche) was used for the co-immunoprecipitation experiments.
Sucrose gradient velocity sedimentation
Sucrose was dissolved in extract buffer including either 100 or 500 mM KCl. Continuous gradients of 15–60% were generated with a Gradient Master (Biocomp). Centrifugation at 243,000 g for 13 h at 4 °C was performed using an SW-40 rotor (Beckman Coulter).
Liquid chromatography MS/MS
Protein samples were in-solution trypsinized. Tryptic peptides were analysed by liquid chromatography MS/MS. Peptides were separated across a 45-min gradient ranging from 10 to 35% (v/v) acetonitrile in 0.1% (v/v) formic acid in a microcapillary (125 μm×18 cm) column packed with C18 reverse-phase material: Magic C18AQ, 5 μm particles, 200 Å pore size (Michrom Bioresources). The peptides were analysed using an LTQ XL linear ion trap mass spectrometer (ThermoFisher). For each cycle, one full MS scan was followed by ten MS/MS spectra from the ten most abundant ions. MS/MS spectra were searched using the Sequest algorithm against a Drosophila protein database. All peptide matches were filtered based on tryptic state, Xcorr and dCorr.
Samples were resolved in 8% acrylamide gels or 3–12% gradient gels for D-PLP. Proteins were transferred to nitrocellulose membranes and incubated with primary antibodies overnight at 4 °C followed by secondary antibodies at room temperature for 1 h. Super Signal West Pico or Femto Chemilluminescent substrate (Pierce) detected peroxidase activity. Molecular masses were determined by comparison with ProSieve molecular standards (Lonza). The apparent molecular weights of proteins detected by specific antibodies were Sas-6: 54 kDa, Asl: 115 kDa, CNN: 150 kDa, Sas-4: 100 kDa, CP-190: 190 kDa and tubulin: 50 kDa.
A cDNA fragment containing amino acids 2–150 of Drosophila Sas-4 was cloned into a pET vector (Invitrogen) and expressed in Escherichia coli. Inclusion bodies were prepared and resolved using SDS–polyacrylamide gel electrophoresis. Protein was extracted from the gel and injected into mice. Hybridomas were prepared and the monoclonal antibody was collected using standard protocols.
For western blots, the monoclonal mouse anti-Sas-4 (1:500), rabbit anti-CNN (1:5,000, courtesy of C. Kaufman), mouse anti-γ-tubulin (1:5,000, Sigma-Aldrich), mouse anti-GFP (1:5,000, Roche), mouse or rabbit anti-β-tubulin (1:5,000, Sigma), rabbit anti-Sas-6 (1:5,000, Gopalakrishnan et al.36
), rabbit anti-Asl (1:5,000, Blachon et al.14
), rabbit anti-D-PLP (1:1,000, courtesy of J. Raff), rabbit anti-CP-190 (1:5,000, courtesy of V. Corces) and rabbit anti-Plk4 (1:5,000, courtesy of M. Bettencourt). Peroxidase-conjugated secondary antibodies were used at 1:5,000 (Vector Labs).
For immunofluorescence, rat anti-β-tubulin (1:200, Chemicon), mouse anti-γ-tubulin (1:200), the above-described monoclonal mouse anti-Sas-4 (1:100), rabbit anti-CNN (1:200), rabbit anti-D-PLP (1:200), rabbit anti-Asl (1:200), and rabbit anti-CP-190 (1:200) were used. Secondary antibodies Cy5 goat anti-mouse and rhodamine donkey anti-rat were used at 1:200 (ImmunoResearch). 4,6-diamidino-2-phenylindole (DAPI) (1 μg ml−1, Sigma) stained DNA.
S2 cells were grown on concanavalin A-coated cover slips for 1 h at 25 °C and then fixed with formaldehyde (3.7% in PBS) for 5 min. Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 1% BSA, 0.1% Triton X-100 in PBS for 45 min. Antibody labelling was performed for 1 h at room temperature followed by three washes in PBS. A similar protocol was used for testes labelling. Confocal images were collected using a Leica TCS SP5 scanning confocal microscope; 3D-SIM images were collected using the DeltaVision|OMX microscope33
. Images were processed using Adobe Photoshop. Immunoelectron microscopy was performed as described14,36
Sas-4-N190, Sas-4-N190ΔPN2-3, Sas-4Δ-90, CNN and Asl proteins were expressed in Escherichia coli strain BL21(DE3)pLysE via isopropylthiogalactoside induction. The soluble fraction of Sas-4-N190 or Sas-4-N190ΔPN2-3 was purified using GST-agarose affinity beads. For CNN and Sas-4-Δ90, cells were sonicated and inclusion bodies were pelleted by centrifugation for 30 min at 18,500 g. Inclusion bodies were twice homogenized in 1% Triton X-100, 1 M urea, 100 mM Tris buffer pH 7, 5 mM EDTA and centrifuged at 18,500 g. The homogenized inclusion bodies were washed in 100 mM Tris pH 7, 5 mM EDTA, suspended in 20 mM Tris pH 7.5, 100 mM NaCl, 8 M urea, 10 mM 2-mercaptoethanol, and dialysed against progressively decreasing urea, in 1 M steps, with, at least, 1 h between steps. The refolded proteins were dialysed against 20 mM Tris pH 7.5, 100 mM NaCl, 30% glycerol. Sas-4-Δ90 was purified similarly yet with the step involving 8 M urea omitted. The affinity-purified protein was then dialysed against 20 mM Tris pH 7.5. Recombinant proteins were then subjected to size exclusion using Superose 12 column, and the purified peak fractions were collected.
Centrosome binding assay
To assay the ability of a Sas-4 complex or recombinant Sas-4-N190, Sas-4-Δ90, CNN or Asl to bind to centrosomes or to stripped centrioles, discontinuous sucrose gradient sedimentation was used. SW-55 tubes (Beckman) were cushioned with 300 μl 70% sucrose followed by 40% sucrose, both in extract buffer having 100 mM KCl (described above). Sas-4 complexes or recombinant Sas-4 proteins were mixed with centrosomes or stripped centrosomes to a volume of 4 ml in the presence of 1 mM GTP and mixed for 4 h at 4 °C. This mixture was layered on the 40% sucrose cushion and was spun in an SW-55 rotor at 300,000 g for 1 h. Fractions were collected from the bottom of the gradient by puncturing the tube with an 18.5-gauge needle. The first three fractions (250 μl each), the next five fractions (500 μl) and a final fraction (the remaining volume) were collected. The first two fractions, that is, the high-density fractions, contained centrosomes with bound proteins; the final fraction, that is,, the low-density fraction, contained only unbound proteins.