Genome-wide RNAi screen
Logarithmically growing Kc167 cells were trypsinized, washed, and resuspended in serum-free medium at 1 × 106 cells/ml, and 10 μl of cells was plated to each well of the 384-well plate containing dsRNA. Cells were incubated for 1 h at room temperature before 35 μl of Schneider's medium (Invitrogen) with 1× antibiotics (Invitrogen), and 10% FCS (Omega) was added to each well and incubated for an additional 4 d at 25°C. Cells were fixed for 5 min at room temperature in 100% methanol and washed twice in 1× TBS with 0.1% Triton X-100 (TBST). Cells were treated for 30 min with blocking solution (TBST containing 2% BSA), which was replaced by 10 μl of blocking solution containing chicken anti-CID antibody (1:100 dilution) and mouse anti-HP1 (1:400 dilution) and incubated overnight at 4°C. Cells were washed twice for 5 min with blocking solution, and 10 μl of secondary antibodies (Alexa 568 anti–chicken and Alexa 488 anti–mouse antibody at 1:400 dilutions) was added and incubated for 1 h at room temperature. Secondary antibodies were washed away three times with TBST. DNA was stained with 2 μg/ml Hoechst 33342 in TBS for 10 min at room temperature and washed with TBS. Plates were imaged using a 40× objective using an automated plate-imaging microscope (ImageXpress; MDS Analytical Technologies).
A MetaMorph (available at http://straightlab.stanford.edu/analysis
) journal was written to segment nuclei based on DNA staining. Cell area was estimated by a 10-pixel dilation from the nuclei. Centromere staining was identified within the cells through a morphological top hat filter with a 5-pixel diameter. The integrated intensity of the centromere and cell body staining was collected. Each well was assigned a score equal to the sum of the cell body intensity divided by the sum of the centromere intensity. Positives were verified by manual image analysis and retesting (Fig. S1).
Indirect IF on fixed cells
S2 cells were settled on glass slides and fixed with either 4% PFA, 4% formalin, or 100% methanol for 10 min. After three washes with PBS for 10 min each, cells were blocked in either 5% milk or 2% BSA in PBST (PBS with 0.2% Triton X-100) for 10 min before primary antibody incubation overnight at 4°C followed by three washes of 10 min in PBST. To detect the kinetochore localization of GFP-CAL1, a stable line expressing GFP-CAL1 was treated with 0.5% sodium citrate for 7 min followed by centrifugation on a slide using cytospin (Shandon) at 2,900 rpm for 10 min; cells were then fixed, washed, blocked in PBST with 5% milk, and incubated overnight at 4°C with anti-CID and anti-Rod antibodies. After washes and incubation with anti–chicken and anti–rabbit secondaries, cells were fixed again for 5 min with 4% formalin, washed, and incubated overnight with Alexa 488–conjugated anti-GFP antibody (Invitrogen). All of the secondary antibodies used were Alexa 488, Alexa 546, or Alexa 647 (Invitrogen) conjugates, and they were incubated at 1:500 dilutions for 45 min at room temperature. After three 10-min washes in PBS, cells were mounted in 2.5% 1,4-diazabicyclo[2.2.2]octane and 1 μg/ml DAPI in 50% glycerol. The dilutions and antibodies used were 1:300 chicken anti-CID (Blower and Karpen, 2001
), 1:300 guinea pig anti–CENP-C, 1:500 rabbit anti-GFP (Invitrogen), 1:10 mouse anti-CYCA (Developmental Studies Hybridoma Bank), 1:500 mouse antitubulin (Sigma-Aldrich), 1:500 rabbit anti-PH3 (H3S10ph; Millipore), 1:500 mouse antifibrillarin (Cytoskeleton, Inc.), and 1:500 rabbit anti-Rod (Scaerou et al., 1999
). All images were taken on a microscope (Deltavision Spectris; Applied Precision, LLC) and deconvolved using softWoRx (Applied Precision, LLC). Images were taken as z stacks of 0.2- or 0.3-μm increments using a 100× oil-immersion objective.
Embryos were collected either overnight or for 1 h, aged, and dechorionized with 50% bleach for 2 min. Embryos extensively washed in 100 mM NaCl + 0.5% Triton X-100 were fixed with formaldehyde saturated with heptane for 20 min and hand devitillinized on double sticky tape with a 35-gauge needle in PBTA (1% BSA + 0.2% Triton X-100 and 0.05% NaN3 in PBS). Antibodies were diluted in PBTA and incubated overnight. After three 10-min washes in PBTA, secondary antibodies were incubated for 2 h. After three washes in PBS, embryos were mounted in Vectashield containing DAPI and imaged on a Deltavision microscope as stated in the previous paragraph. The mutant embryos used for this study were y, ry (control), cycAC8LR1, cenpCprl-41 (C. Lehner, University of Zurich, Zurich, Switzerland), rca1IX, and pBacCG5148.
CENP-C antibodies were generated by cloning the first 2,196 bp of the DmCENP-C gene into the pET100/D-TOPO vector (Invitrogen). Protein was expressed in BL21-star cells and purified using the pETQIA expressionist kit (QIAGEN). Polyclonal antibodies were produced in guinea pigs by Covance, and crude serum was used for immunostaining. The first 176 amino acids of CAL1 and the first 124 amino acids of CID were fused to GST using a modified pGEX-6P vector (EMD). Fusion proteins were expressed in BL21 (DE3) pLysS cells and purified with glutathione agarose (Sigma-Aldrich). Polyclonal antibodies were produced in rabbits, and antisera were affinity purified against the antigen after removal of the GST tag with PreScission Protease (EMD).
Immunoprecipitation from Kc167 cells
Approximately 8 × 1010 cells stably expressing GFP-tagged CLD proteins at levels equal to or lower than the endogenous levels were harvested and washed in PBS. Nuclei were isolated by lysing the cells in 25 ml of nuclear extraction buffer (20 mM Hepes, pH 7.7, 50 mM KCl, 2 mM MgCl2, 5 mM β-mercaptoethanol [β-ME], 1% Triton X-100, 1 mM PMSF, 2 mM benzamidine-HCl, and 10 μg/ml LPC [leupeptin, pepstatin, and chymostatin]) followed by a wash in 25 ml of nuclear wash buffer (20 mM Hepes, pH 7.7, 50 mM KCl, 2 mM MgCl2, 5 mM β-ME, 1 mM PMSF, 2 mM benzamidine-HCl, and 10 μg/ml LPC). Nuclei were resuspended in 3 ml of micrococcal nuclease buffer (20 mM Hepes, pH 7.7, 50 mM KCl, 2 mM MgCl2, 5 mM β-ME, 3 mM CaCl2, 1 mM PMSF, 2 mM benzamidine-HCl, and 10 μg/ml LPC), and chromatin was digested with 0.1 U/μl micrococcal nuclease (Worthington Biochemical) for 30 min at 25°C. 3 ml of 2× extraction buffer (20 mM Hepes, pH 7.7, 575 mM KCl, 5 mM EGTA, 5 mM EDTA, 5 mM β-ME, 20% glycerol, 0.1% Igepal-CA630, 1 mM PMSF, 2 mM benzamidine-HCl, and 10 μg/ml LPC) was added to stop the reactions. The lysate was sonicated twice for 30 s and cleared by centrifugation at 10,000 g for 15 min. Cleared lysate was added to 30 μl of anti-GFP resin (0.5 mg/ml polyclonal rabbit anti-GFP antibody coupled to protein A–Sepharose beads) and incubated for 2 h at 4°C. The anti-GFP resin was washed three times in 20 mM Hepes, pH 7.7, 300 mM KCl, 2.5 mM EGTA, 2.5 mM EDTA, 5 mM β-ME, 10% glycerol, 0.05% Igepal, 1 mM PMSF, 2 mM benzamidine-HCl, and 10 μg/ml LPC, boiled in 70 μl of SDS sample buffer, and analyzed by Western blotting.
Approximately 4 × 107 cells were harvested and washed once in PBS. The cells were lysed in 20 mM Hepes, pH 7.7, 500 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.5% Igepal, 1 mM PMSF, 2 mM benzamidine-HCL, and 10 μg/ml LPC. Protein concentrations were equalized by Bradford assays, and 30 μg of total protein was loaded per sample. Proteins were transferred to a nitrocellulose membrane in 10 mM 3-(cyclohexylamino)-1-propane sulfonic acid, 0.1% SDS, and 20% methanol for 45 min for CID and CYCA or for 90 min for CENP-C and CAL1. Affinity-purified antibodies were used at a concentration of 1 μg/ml.
Kc167 cells were grown in 6-well dishes and harvested at 24, 48, 72, and 96 h after RNAi treatment. Cells were washed once in PBS and fixed by dropwise addition into 70% ethanol while mixing. Cells were washed twice in PBS. The cells were stained by the addition of 100 μl of 100 μg/ml RNaseA and 400 μl of 50 μg/ml propidium iodide. DNA content was measured using a FACS analyzer (FACScan; BD). FACS data were analyzed with FlowJo software (Tree Star, Inc.).
dsRNA was prepared using a kit (MegaSCRIPT T7; Applied Biosystems) according to the manufacturer's procedures. Templates were generated by PCR from genomic DNA using the following primers: CYCA reverse, 5′-GCCAAGAAATCGAATGTGGT-3′; CYCA forward, 5′-ATTTCACGTCATGGTTCTCTT-3′; RCA1 reverse, 5′-TTTCAATCGCCACACAGTAG-3′; RCA1 forward, 5′-GCCTCGCTTATGAAAACCC-3′; CAL1 forward, 5′-TGGATGCCAGGAAAGTTAGT-3′; CAL1 reverse, 5′-CTATAGGGATTGTTGATATCAGC-3′; CENP-C forward, 5′-TGGTAAACTATTTGGGTCTCTC-3′; CENP-C reverse, 5′-GGTACCAGTTCGTTCTCCA-3′; CID forward, 5′-ACCGTGCAGCAGGAAAG-3′; and CID reverse, 5′-CCCCGGTCGCAGATGTA-3′. 106 logarithmically growing S2 cells were plated in 1 ml of serum-free medium, and 15 μg dsRNA was added to the culture. Control wells received water instead of dsRNA. After 1 h of incubation, 1.5 ml of serum-containing medium was added, and incubation proceeded for 4 d. Samples of 100 μl were taken every day and were subjected to indirect IF analysis.
Quantitation of defective mitoses and mitotic index
S2 cells were depleted of CID, CLD-2, CENP-C, RCA1, or CYCA by RNAi. After 4 d, cells were fixed for 10 min with 10% formalin in PBS and stained for DNA (DAPI), CID, CENP-C, tubulin, or H3S10Ph by indirect IF. Mitotic cells were scored as defective or normal based on chromosome and spindle morphology compared with control cells, and the significance of the differences was determined using the χ2 test.
SNAP tag labeling of newly synthesized CID
Drosophila Kc cells stably expressing SNAP-tagged CID (SNAP-CID) were transfected with 5 μg dsRNA against CENP-C or CAL1 using the 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) transfection reagent (Roche) or transfection reagent alone (control). SNAP-CID was quenched with BTP after 72 h of RNAi depletion. BTP was removed, and cells were allowed to grow for another 24 h to allow synthesis of unlabeled SNAP-CID protein followed by TMR* labeling. Cells were fixed with 4% formaldehyde in PBST for 5 min, and total SNAP-CID was detected by IF with a rabbit α-SNAP polyclonal antibody (Covalys) used at 1:500.
Time-lapse analysis and GFP constructs
Time-lapse videos were performed using a Deltavision Spectris microscope. Cells were mounted using the hanging drop method (Heun et al., 2006
). For the mitotic time-lapse videos, cells expressing mCherry-tubulin and H2B-GFP in prophase/prometaphase (gift of G. Goshima and R. Vale, University of California, San Francisco, San Francisco, CA) were imaged every 1 or 2 min until cytokinesis for a total of 30–45 min with the exception of CID RNAi, in which, in some severe cases, cells were imaged for 90–120 min and did not undergo cytokinesis. Cells were imaged 3–4 d after RNAi treatment except for CYCA and RCA1 RNAi, in which cells were imaged after 1–2 d of treatment. 5–10 videos of randomly selected prophase cells were imaged for each RNAi experiment. 80–100% of videos per RNAi experiment showed mitotic defects with the exception of CYCA and RCA1 RNAi, in which the percentage of defective mitoses was ~60%. Videos of GFP-CID, GFP-CDL2, GFP–CENP-C, and GFP-CYCA in mCherry-tubulin–expressing cells were imaged every 2 min until cytokinesis was completed. Videos were edited in Photoshop (Adobe) to reduce video size, and the time in minutes in the still images reflects the actual elapsed time during image acquisition.
GFP constructs were generated for all five CLD genes after PCR amplification with a template PCR system (Expand Long; Roche) from either Drosophila
Genomic Resource Center clones or from Drosophila
Kc167 cell poly-A RNA using the following primers flanked by an AscI site at the 5′ end and a PacI site at the 3′ end: CID forward, 5′-GCATCATATGCAGCACGCTGTTTCCGCTG-3′; CID reverse, 5′-GCATGCTAGCGCTTTTTTGGAACAGTGTGACCG-3′; CG5148 forward, 5′-ATGGCGAATGCGGTGGTG-3′; CG5148 reverse, 5′-TTACTTGTCACCGGAATTATTCTCG-3′; CENP-C forward, 5′-ATGTCGAAGCCCCAGAAC-3′; CENP-C reverse, 5′-CTAACTGCGTATACACATCAG-3′; RCA1 forward, 5′-ATGAGCGCCTATTATCGGCG-3′; RCA1 reverse, 5′-CTAAAAGCAGAGCCGCTTGAGCGAGTT-3′; CYCA forward, 5′-ATGGCCAGTTTCCAGATCCAC-3′; CYCA reverse, 5′-ATGTCCGTGACGGATGTTCAGTC-3′; and Δ55-CYCA forward, 5′-AACAATGTGCCGCGTCCG-3′. PCR products were cloned into pCopia–localization and purification (LAP) digested with AscI and PacI. pCopia-LAP was generated by replacing EGFP with the LAP tag (Cheeseman and Desai, 2005
) and modifying the polylinker such that AscI/PacI cloning would result in the N-terminal LAP (GFP) tagging of genes. The Copia promoter was PCR amplified from the pCoPuro plasmid (Iwaki et al., 2003
) with the primers forward (5′-GCATCATATGGGCAAATGGGTTTAGGATTGGG-3′) and reverse (5′-GCATGCTAGCGGAAGGTCGTCTCCTTGTGAGG-3′) and was cloned into the EGFP vector using the NdeI and NheI sites. The plasmid for nondegradable cyclin expression was generated by ligating Δ55-CYCA into the AscI and PacI sites of pCopia-LAP to generate pCopia-GFP–Δ55-CYCA.
S2 cells were transfected with Cellfectin (Invitrogen) according to the manufacturer's instructions. Stable lines were obtained by cotransfecting the LAP tag constructs with the plasmid pHygro (Invitrogen) and by selection in the presence of 100 μg/ml hygromycin B (Invitrogen).
Nondegradable CYCA rescue experiments
Kc167 cells were depleted of CYCA and RCA1 by treatment with dsRNA. Immediately after the incubation of cells with dsRNA, the cells were transfected with plasmids expressing GFP, pCopia–GFP-CYCA, or pCopia-GFP–Δ55-CYCA with FuGene 6 (Roche) according to the manufacturer's instructions. After 4 d, the cells were fixed for IF and stained for DNA, GFP, and CID. CID centromere intensities were measured in the GFP-positive and GFP-negative populations.
For the experiments to test nondegradable CYCA rescue of CAL1 depletion, cells were transfected with 5 μg pCopia-GFP–Δ55-CYCA alone or in combination with 5 μg dsRNAi against CAL1 using the DOTAP transfection reagent. 4 d after incubation, cells were fixed and stained with anti-GFP and anti-CID antibodies.
Online supplemental material
Fig. S1 depicts the workflow of the genome-wide siRNA screen for centromere propagation and the quantification of CID levels after RNAi depletion of the CLD genes. Fig. S2 shows the alignment of CAL1 homologues in several drosophilid species. Fig. S3 shows the localization of CLDs in interphase and mitosis. Fig. S4 shows the effect of cenpC
, and rca1
mutants on centromeres in Drosophila
embryos, the fragmentation of chromatin used in the LAP-CLD purifications, and the expression levels of LAP-CLD fusions in Kc167 cells. Fig. S5 shows the rescue of centromeric CID localization in RCA1-depleted cells by nondegradable CYCA. Videos 1–5 show time-lapse videos of Drosophila
S2 cells expressing mCherry-tubulin and GFP-CID, GFP–CENP-C, GFP-CAL1, GFP-CYCA, and GFP-RCA1, respectively. Videos 6–10 show time-lapse videos of Drosophila
S2 cells expressing GFP-H2B and mCherry-tubulin after dsRNA depletion with control, CID, CENP-C, CAL1, and CYCA RNA, respectively. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200806038/DC1