CDK-dependent phosphorylation and nuclear exclusion coordinately control kinetochore assembly state.

Cell culture and transfection

Human cell lines were maintained in DMEM supplemented with 100 U/ml streptomycin, 100 U/ml penicillin, 2 mM glutamine, and 10% (vol/vol) fetal calf serum. All cells were cultured at 37°C with 5% CO 2 . HeLa LacZeo/TO cells (a gift from S. Taylor, University of Manchester, Manchester, England, UK) were maintained in 2 µg/ml Blasticidin and 200 µg/ml Zeocin. Cells expressing tetracycline-inducible GFP LAP or mCherry LAP fusions were generated by Flp recombinase–mediated integration of a pCDNA5-FRT-TO–based plasmid (Invitrogen). Cells were then maintained in 2 µg/ml Blasticidin and 400 µg/ml Hygromycin B. Clonal cell lines constitutively expressing GFP LAP or mCherry LAP fusions were generated using retroviral infection of HeLa cells with a pBABE-blast– or pBABE-Puromycin–based vector. See Table S1 for a list of cell lines used in this study. Transient transfection of cells with plasmids encoding mCherry-Ran T24N and mCherry-Dsn1 K-R was performed with transfection reagent (Effectene; QIAGEN) according to the manufacturer’s instructions. Codon-optimized and RNAi-resistant CENP-T (Mr. Gene) was used in all experiments. RNAi-resistant Ndc80 was generated by site-directed mutation (Agilent Technologies) of siRNA-targeting nucleotides in the human coding sequence. siRNA transfections were performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Cells were analyzed 48 h after siRNA transfection unless stated otherwise. Nontargeting control siRNAs and gene-specific siRNAs were obtained from Thermo Fisher Scientific. Single siRNAs were used against CENP-T (5′-CGGAGAGCCCUGCUUGAAA-3′), Ndc80/Hec1 (5′-GAAGUUCAAAAGCUGGAUGAUCUU-3′), and PP2R2A–B55-α (5′-CUGCAGAUGAUUUGCGGAUUAUU-3′; Schmitz et al., 2010 ), and a pool of siRNAs was used against Skp1A (5′-CUACUUGCAUGUAAAGAAU-3′, 5′-GGAGAAAUGUAACUGGACA-3′, 5′-CUAGUAUGAUGGAAAGUUU-3′, and 5′-CGCAAGACCUUCAAUAUCA-3′).

Immunofluorescence and microscopy

Where indicated, cells were incubated with 20 µM MG132 (Sigma-Aldrich), 5 µM Flavopiridol (CDKi; Sigma-Aldrich), 10 µM BI2536 (Plk1 inhibitor [Plki]; Tocris Bioscience), 2 µM AZ3146 (Mps1i; Tocris Bioscience), 2 µM ZM447439 (Aurora B inhibitor; Tocris Bioscience), or 0.66 µM nocodazole before fixation or live-cell imaging. For immunofluorescence analysis, cells were grown on glass coverslips. Where indicated, cytoplasmic contents were preextracted before fixation by incubation for 2 min in PBS + 1% (vol/vol) Triton X-100. Cells were fixed by incubation for 15 min in PBS + 4% (vol/vol) formaldehyde before incubation for 30 min in TBS + 3% (wt/vol) BSA + 0.1% (vol/vol) Triton X-100. Antibodies used for staining are listed in Table S2 . Cy2-, Cy3-, and Cy5-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. All antibodies were diluted in TBS + 3% BSA and incubated with cells for 1 h at room temperature. DNA was visualized by a 5-min incubation with 10 µg/ml Hoechst in PBS. Images were acquired on a deconvolution microscope (DeltaVision Core; Applied Precision) equipped with a charge-coupled device camera (CoolSNAP HQ2; Photometrics). For fixed-cell analysis, 40 z sections were acquired at 0.2-µm steps using a 60×, 1.3 NA U-Plan Apochromat objective (Olympus). Images were acquired at room temperature through glycerol-based mounting media. Cy2, Cy3, Cy5, or Hoechst florescence was observed using appropriate filters. Images were deconvolved using 10 cycles of enhanced ratio deconvolution on DeltaVision software (Applied Precision). For time-lapse imaging, cells were imaged in CO 2 -independent media (Invitrogen) at 37°C. Images were acquired every 2 min using six z sections at 0.7-µm intervals using a 40× U-Plan Apochromat/340 NA objective (Olympus). GFP or mCherry fluorescence was observed using appropriate filters. To quantify fluorescent intensity, maximum intensity projections were generated, and individual kinetochores were analyzed at each frame using MetaMorph software (Molecular Devices). Integrated fluorescence intensity was measured in a 7 × 7–pixel region containing a kinetochore. Background was subtracted by subtracting from this the integrated intensity from a 7 × 7–pixel region of cytoplasm in the same cell. At least 10 kinetochores were analyzed per cell. To visualize chromosome movement during live-cell imaging, cells were incubated for 30 min in 1 µg/ml Hoechst. Media were replaced with CO 2 -independent media without Hoechst before imaging. Online supplemental material Fig. S1 assesses factors controlling kinetochore assembly kinetics. Fig. S2 shows that inhibition of CDK, but not other mitotic kinases, affects kinetochore assembly. Fig. S3 shows that inhibition of Ndc80 complex disassembly does not cause immediate mitotic defects.

Table

S1 shows cell lines used in this study.

Table

S2 shows antibodies used to visualize kinetochore proteins. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201301006/DC1 .

Online supplemental material Fig. S1 assesses factors controlling kinetochore assembly kinetics. Fig. S2 shows that inhibition of CDK, but not other mitotic kinases, affects kinetochore assembly. Fig. S3 shows that inhibition of Ndc80 complex disassembly does not cause immediate mitotic defects.

Table

S1 shows cell lines used in this study.

Table

S2 shows antibodies used to visualize kinetochore proteins. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201301006/DC1 .