Cell culture, RNAi, drug treatments, and Western blot analysis
S2 and Jupiter-GFP cells (provided by A. Debec, Institut Jacques Monod, Paris, France) were cultured in Schneider's (Sigma-Aldrich) or M3 media, respectively, containing 10% FBS (Invitrogen) in a 25°C incubator. The S2 GFP–α-tubulin cell line was provided by R. Vale (University of California, San Francisco, San Francisco, CA). HeLa cells were grown in DME supplemented with 10% FBS in a 37°C incubator with 5% CO2
and humidified atmosphere. RNAi depletion of Drosophila
Mtor was performed as previously described (Qi et al., 2004
) or using a 256-bp fragment specific for the 3′ untranslated region (UTR) of Mtor cDNA sequence using the following set of primers: 5′-TAATACGACTCACTATAGGGGGCGAGGAGTTCGGCGGACC-3′ and 5′-TAATACGACTCACTATAGGGATCGACAAAATTACACATAT-3′. For the rescue experiment, CuSO4
was added for induction of Mtor-mCherry expression from a metallothionein promoter (pMT) vector (Invitrogen) to a final concentration of 500 µM for 18 h before the analysis. mCherry cDNA was provided by R. Tsien (University of California, San Diego, La Jolla, CA). Mad2 RNAi in S2 cells was performed as previously described (Orr et al., 2007
). Depletion of human Tpr was performed using published siRNA oligonucleotides against the targeting sequence 5′-GGTGGAGAGCGAACAACAG-3′ and transfected using HiPerFect (QIAGEN). Phenotypes were analyzed and quantified after 72 h, and protein knockdown was monitored by Western blotting using the following antibodies and dilutions: mouse anti-Mtor 1:1,000, rabbit anti-Tpr 1:1,000 (provided by L. Gerace, The Scripps Research Institute, La Jolla, CA), rabbit anti-CLASP1 1:400 (provided by N. Galjart, Erasmus University Medical Center, Rotterdam, Netherlands), and mouse anti-Chromator 1:2,000. Appropriate secondary antibodies were visualized using the ECL system (GE Healthcare). MT depolymerization in S2 cells was induced by colchicine at 100 µM during live cell recordings or at 30 µM for 18 h for the quantification of mitotic index or immunofluorescence analysis of KT proteins. MG132 was used at 2 µM for 120 min followed by washout with fresh medium while recording. For HeLa cells, nocodazole was used at 1 µM for 16 h for the analysis of Mad1 and Mad2 KT accumulation and mitotic index quantification.
Immunofluorescence microscopy in Drosophila
S2 cells was performed as described previously (Maiato et al., 2006
). HeLa cells were grown on poly-l
-lysine–coated coverslips and processed for immunofluorescence as described previously (Maiato et al., 2006
). The following primary antibodies were used: mouse anti–α-tubulin clone B512 (1:2,000; Sigma-Aldrich), rat anti–α-tubulin YL1/2 1:10 (AbD Serotec), rabbit anti-DmBubR1 and anti-DmMad2 1:2,000 and 1:30, respectively, mouse anti-Mtor 1:10, mouse anti-DmMad1 1:50 (provided by R. Karess, Institut Jacques Monod, Paris, France), mouse anti–lamin B T40 1:200 (provided by P. Symmons, University of Tübingen, Tübingen, Germany), rabbit anti-CID 1:500 (provided by S. Henikoff, Fred Hutchinson Cancer Research Center, Seattle, WA), chicken anti-Ndc80 1:100 (provided by T. Maresca and T. Salmon, University of North Carolina, Chapel Hill, NC) rabbit anti-Rod 1:300 (provided by R. Karess), mouse anti–phosphohistone H3 1:100,000 (Abcam), human anticentromere antibodies (ACA) 1:5,000 (provided by B. Earnshaw, University of Edinburgh, Edinburgh, Scotland, UK), rabbit anti-Tpr 1:500, mouse anti-Tpr 1:500 (provided by V. Cordes, University of Heidelberg, Heidelberg, Germany), rabbit anti-HsMad1 1:500 (provided by P. Meraldi, ETH Zurich Institute of Biochemistry, Zurich, Switzerland), and sheep anti-HsMad2 1:500 (provided by S. Taylor, University of Manchester, Manchester, England, UK). Secondary antibodies used were Alexa Fluor 350, 488, 568, and 647 (1:2,000; Invitrogen) and 1 µg/ml DAPI. For mitotic index analysis in S2 and Hela cells, 1,000 or 500 cells, respectively, were scored in three independent experiments.
S2 stable cell lines were grown on concanavalin A–coated coverslips in modified Rose chambers with Schneider's medium containing 10% FBS. The Mtor-mCherry construct was obtained by PCR amplification of the coding region of Mtor cloned into a pMT-mCherry vector in which a blasticidin resistance cassette for stable selection had been previously inserted into SalI site. The mRFP-Mad2 fusion protein under the control of its endogenous promoter (Buffin et al., 2005
) was cloned into pAC-HisB vector (Invitrogen) in which a blasticidin resistance cassette had been previously inserted into SalI site. Four dimensional datasets were collected at 25°C with a spinning disc confocal system (Revolution; Andor) equipped with an electron multiplying charge-coupled device camera (iXonEM+; Andor) and a CSU-22 unit (Yokogawa) based on an inverted microscope (IX81; Olympus). Two laser lines (488 and 561 nm) were used for near-simultaneous excitation of GFP and mCherry/mRFP, and the system was driven by iQ software (Andor). Time-lapse imaging of z stacks with 1-µm steps covering the entire volume of the mitotic apparatus were collected every 30 or 5 s, according to the experiments.
We used a custom routine written in Matlab to compensate for rotation and translation of the spindle. The direct output is a whole-spindle kymograph resulting from conversion of each time point image matrix into a vector. Chromosome poleward velocity relative to the equator was measured following each CID-mCherry track obtained from kymographs. CID cDNA was provided by G. Karpen (University of California, Berkeley, Berkeley, CA). Velocity was determined measuring the slope of the fastest linear movement after anaphase onset within each half-spindle. Spindle elongation velocity was measured following spindle MT minus ends.
FRAP was performed with a spectral confocal (SP2; Leica) with a 63×/1.4 NA objective lens and an additional zoom of 6×. Images were acquired every 422 ms or 1 s. Bleaching was conducted for 1.7 s after two frames of prebleach imaging. GFP intensity of the bleached area was normalized using the intensity of a neighboring cell after background subtraction. Nonlinear (exponential) curve fit was applied to recovery curves (Microcal Origin; OriginLab Corporation) for half-time recovery calculation.
Mad2 and Mps1 accumulation at KTs was measured for individual KTs by quantification of the pixel gray levels of the focused z plane within a region of interest (ROI). Background was measured outside ROI and was subtracted to the measured fluorescent intensity inside ROI. Results were normalized against a constitutive KT marker (CID for S2 cells or ACA in HeLa cells) using a custom routine written in Matlab (Mathworks). For quantification of mRFP-Mad2, pixel gray levels of the focused z plane within ROIs were defined for the spindle region and cytoplasm of prometaphase cells, and the respective ratio was determined after background subtraction. For quantification of GFP–α-tubulin, Jupiter-GFP, and Mtor-mCherry after MT depolymerization, ROIs were defined for the spindle region and cytoplasm. After background subtraction and bleaching correction, pixel gray levels of the focused z plane within ROIs were quantified over time.
For co-IP experiments in Drosophila, anti-Mtor or control antibodies were bound to protein G beads (Sigma-Aldrich) for 4 h at 4°C on a rotating wheel in IP buffer. Antibody-coupled beads or beads only were incubated overnight at 4°C with 1 ml of 0–3 h embryonic lysate on a rotating wheel. Beads were washed extensively with IP buffer. The resulting bead-bound immunocomplexes were analyzed by SDS-PAGE and Western blotting using dMad2 or dMad1 antibodies. mAb 12F10 was used to detect Mtor to monitor the IP efficiency. Co-IP experiments in human cells were performed with native protein extracts (5 mg of total protein in a total volume of 500 µl of IP buffer obtained from HeLa cells or a derivative clone stably expressing EGFP-Tpr, both enriched for mitotic cells by incubation with nocodazole). Extracts were incubated with the precipitating antibody (rabbit anti-GFP, unspecific rabbit–IgG, or rabbit anti-Mad2; Bethyl Laboratories, Inc.) and 40 µl of protein A–Sepharose slurry overnight at 4°C on a rotating platform. Samples were centrifuged, the supernatant was retained as unbound sample, and the pelleted beads (IP) were washed three times with washing buffer (IP buffer with 250 mM KCl). Precipitated proteins were removed from the beads by boiling 5 min in SDS sample buffer and analyzed by SDS-PAGE and Western blotting with the appropriate antibodies.
Generation of Mps1-GFP and Mps1KD-GFP constructs
coding region was PCR amplified from the cDNA LD08595, subcloned into pEGFP-N1 (Clontech Laboratories, Inc.), and the GFP-fusion was transferred to pMTV5-HisB (Invitrogen). Mutagenic PCR to introduce the point mutation for the kinase dead was performed with the following oligonucleotides: 5′-GATCGC
TTTTGGCATAGCCAGC-3′ and 5′-GCTGGCTATGCCAAAAG
CGATC-3′, corresponding to D478A in Mps1 protein (Tighe et al., 2008
). The underlined nucleotides were mutated from the original Mps1 sequence. The resulting fragment was substituted in the pMTV5-Mps1-GFP vector.
Soluble fractions from S2 cell extracts transiently expressing Mps1-GFP, Mps1KD-GFP, or GFP after 36 h of induction were obtained after centrifugation at 14,000 rpm for 5 min at 4°C. 10 µl of protein A beads (GE Healthcare) was preincubated with anti-GFP antibody (ab290; GE Healthcare) and used to immunoprecipitate 1 mg from the protein extracts obtained before a 2-h incubation at 4°C. Beads were washed with IP buffer supplemented with 300 mM NaCl and subsequently with the kinase buffer. For determination of kinase activity, the extracts were resuspended in kinase buffer supplemented with 3 µCi γ-[ATP32], 30 µM ATP, and 0.5 µg/µl dephosphorylated maltose-binding protein (Sigma-Aldrich). After a 60-min incubation at 25°C, samples were resolved by SDS-PAGE and subsequently exposed to an x-ray film (GE Healthcare).
Generation of mps1KD mutant stock
A point mutation was introduced in the coding region of a genomic clone containing the mps1
gene and promoter region by PCR mutagenesis as described for the generation of Mps1KD
-GFP construct. The resulting 4.7-kb fragment was cloned into pTV2 and subsequently microinjected into Drosophila
embryos. Gene targeting by homologous recombination was used to generate the mps1KD Drosophila
stock as described previously (Rong and Golic, 2000
Statistical analysis was performed using either parametric one-way analysis of variance or nonparametric analysis of variance (Kruskal-Wallis) for multiple group comparisons according to the normality of the distribution. All pairwise multiple comparisons were subsequently analyzed using either posthoc Student-Newman-Keuls (parametric) or Dunn's (nonparametric) tests. Parametric t test or nonparametric Mann-Whitney was used for two group comparisons. All statistical analyses were performed using SigmaStat 3.5 (Systat Software, Inc.).
Online supplemental material
Figs. S1 and S2 present additional characterization of Mtor RNAi phenotype and mRFP-Mad2, Mtor-mCherry, and Tpr localization and function. Fig. S3 provides a detailed characterization of the mps1KD
allele. Videos 1–6 show the colocalization of Mtor-mCherry or mRFP-Mad2 with MTs in living cells and their response to MT depolymerization. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200811012/DC1