Strains and plasmids
All yeast strains were derived from the S288C background, except for a protease-compromised strain (DDY1810), and were grown in yeast extract/peptone or synthetic medium supplemented with appropriate nutrients and 2% glucose at 25°C unless otherwise noted. All epitope- and fluorescently tagged genes were expressed at their endogenous loci under their native promoters as described previously (
Longtine et al., 1998). The mutations in
sli15-17A (S137A, S177A, S276A, T277A, S298A, S305A, S326A, S382A, S383A, S391A, T395A, S413A, S432A, S460A, T564A, S578A, and T589A) were generated by gene synthesis (GenScript) in combination with site-directed mutagenesis (QuikChange Mutagenesis kit; Agilent Technologies). Wild-type
SLI15 and
sli15-17A were cloned into pBSKS+ and then a nourseothricin resistance gene cassette (
Goldstein and McCusker, 1999) was inserted 100 bp 3′ of each ORF. These inserts were transformed into a diploid strain heterozygous for a
SLI15 deletion to target each construct to the endogenous locus. The 10-kb LacO array (
Straight et al., 1997) was inserted into an intergenic region 1.8 kb away from
CEN15 as described previously (
Goshima and Yanagida, 2000). The Cin8 expression plasmid was constructed by cloning the
CIN8 ORF (BamHI–NotI) into the pRS426 plasmid containing the GAL1-10 promoter, a TEV protease recognition site, and encoding seven repeats of the myc epitope (
Rodal et al., 2003). All yeast strains and plasmids used in this study are listed in
Table S1.
Yeast cell cycle synchronization
To create preanaphase spindle conditions, cells were treated with 0.1 M hydroxyurea (HU) for 2.5 h, which maintains spindle MT dynamicity and biorientation (
Kosco et al., 2001). To synchronize cells in metaphase by depletion of Cdc20, an activator of the anaphase-promoting complex (
Uhlmann et al., 2000), cells containing
CDC20 driven by
MET3 promoter were first arrested in G1 with 10 µg/ml α-factor for 3 h and then released into media containing 10 mM methionine for 3 h. To induce metaphase arrest by depolymerizing MTs, cells were treated with 15 µg/ml nocodazole for 2 h.
Statistics
Error bars show standard deviations except where otherwise indicated. Whiskers on box plots show the extreme values of the data. Top and bottom of box indicate the 75th and 25th percentiles. Dark bar at the waist of the box indicates the median (50th percentile). If the notches (indentations in the sides of the box) of two datasets do not overlap then this is considered strong evidence that the two medians are different (
Chambers et al., 1983).
Statistical analyses and box plots were performed using R (
http://www.r-project.org;
R Development Core Team, 2010). Unless otherwise noted, significances (P values) were determined using the two-tailed Student’s
t test (two-sample, unequal variance). Normality of data was evaluated using the Shapiro-Wilk test. For tests using the Mann-Whitney
U test (two-tailed test), similarity of the data distributions was evaluated using quantile–quantile plots. Also calculated were ρ values (=
U/(n
1*n
2), where n
is are the sample sizes) that give an estimate of the probability that a randomly chosen value from the first population will be larger than a randomly chosen value from the second population (
Hernstein et al., 1976). Unless otherwise noted, P values obtained from the Mann-Whitney
U test were for tests of the null hypothesis (H
0) that the medians were identical.
In vitro kinase assay
In vitro kinase assays were conducted as described previously (
Cheeseman et al., 2002) with the following modifications. BSA was added to the reaction to a final concentration of 67 µg/ml. 1 pmol of CPC (containing only wild-type subunits, ipl1-K133R, or sli15-17A) and 100 pmol of substrate (Dam1 complex or histone H3-H4 tetramer) were used for each reaction. The reaction was conducted at ambient temperature and samples were collected every 10 min for 30 min. The Dam1 complex was expressed and purified as described previously (
Westermann et al., 2005). In brief, BL21Rosetta cells (EMD) containing the plasmid pC43HSK3H (
Miranda et al., 2005) was grown at 37°C to OD
0.4, and then protein expression was induced in the presence of 1 mM IPTG for 4 h. After the cells were harvested and washed with phosphate-buffered saline (PBS) the cells were lysed in 20 mM sodium phosphate (pH 6.8), 500 mM NaCl, 1 mM EDTA, 20 mM imidazole, and 0.5% (vol/vol) Triton X-100 by sonication, and the lysate was cleared by centrifugation. Ni-NTA agarose beads (QIAGEN) were incubated with the cleared lysate for 2 h at 4°C and then were collected and washed with the lysis buffer. The proteins were eluted in 6 ml 20 mM sodium phosphate, 500 mM NaCl, 1 mM EDTA, and 200 mM imidazole. After overnight dialysis against 20 mM sodium phosphate, 150 mM NaCl, and 1 mM EDTA, the dialysate was loaded onto a 1-ml HiTrap SP Sepharose column (GE Healthcare), and proteins were eluted with a 10-ml linear gradient of 0.15 to 1 M NaCl in 20 mM sodium phosphate (pH 6.8) 1 mM EDTA buffer. The
Xenopus histone H3/H4 tetramer was a kind gift from P. Kaufmann (University of Massachusetts, Worcester, MA).
Chromatin immunoprecipitation
Chromatin immunoprecipitation from preanaphase cells was conducted based on previously described methods (
Braunstein et al., 1993;
Kang et al., 2001). The yeast cells were grown to early log phase (OD
600 0.2–0.4) and then treated with 0.1 M hydroxyurea for 2.5 h at 30°C. The protein–DNA complexes were then cross-linked with 1% formaldehyde (37% stock; Thermo Fisher Scientific) for 2 h at 25°C. The reaction was terminated by addition of glycine (Sigma-Aldrich) to a final concentration of 125 mM and incubating for 5 min. After washing twice with 50 ml of cold TBS, the cells were resuspended in 900 ml of the lysis buffer (all buffer compositions were as described in
Braunstein et al., 1993) containing proteinase inhibitor cocktail IV (EMD). The cells were lysed using a Mini-Beadbeater (Biospec Products) with an equal volume of glass beads at 4°C at the maximum speed. Cycles of 2 min of beating followed by 1 min on ice were performed until >80% cells were lysed. After collecting the lysate by centrifugation, the lysate was sonicated six times for 10 s on ice (at 20% duty cycle at 2.5 output setting; Sonicator W-385 [Misonix]). The lysates were then cleared by centrifugation and split into three tubes: input, mock treatment, and target sample. The target sample was incubated with anti-myc antibody (9E10) for 1 h before the addition of preblocked Protein G–Sepharose (GE Healthcare) and 4 µg/µL lambda DNA (sonicated to 200–1000 bp). The mock-treated sample was treated as the target sample except no antibody was added. The protein G–Sepharose was preblocked in a solution consisting of 1x TE 1.54 mM NaN
3 1 mg/ml BSA. After a 3-h incubation at 4°C, the beads were washed and resuspended in ChIP elution buffer containing proteinase K (Fermentas) to a final concentration of 250 µg/ml and incubated at 37°C for 12 h. The input DNA (the sonicated lysate) was also treated with proteinase K at this time. All samples were then incubated at 65°C for 6 h. DNA was recovered by phenol-chloroform extraction followed by ethanol precipitation. DNA was resuspended in TE and subjected to PCR analysis.
Western blotting
Yeast whole-cell extracts were prepared as described previously (
Keogh et al., 2006). Target proteins were detected using anti-myc antibody (9E10), anti-GFP antibody (Torrey Pines Biolabs), anti-Pgk1 antibody (Invitrogen), or as otherwise noted.
FRAP and live-cell microscopy
Cells suspended in minimal growth medium were mounted on a coverslip coated with concanavalin A (Sigma-Aldrich). FRAP was conducted using a confocal microscope (LSM710; Carl Zeiss) equipped with a 63x 1.4 NA Plan Apochromat oil objective. Pinhole size was set to two Airy units. After photobleaching, images were acquired using ZEN 2009 software (Carl Zeiss) at ambient temperature every 2.6 s as a stack of four images with a 700-nm axial separation. These images were converted to a series of 2D images by maximum intensity projection using ZEN 2009 software. Image processing and the intensity measurements were conducted using iVision (BD) and Imaris (Bitplane) software. Initial corrections to fluorescence values were conducted by established methods (
http://www.embl.de/eamnet/frap/FRAP6.html;
Phair et al., 2004). Half-time for fluorescence recovery (t
1/2) values were determined by plotting ln(y
max − y) vs. time, where y is the corrected fluorescence and y
max was determined from the binomial regression curve fit to the corrected fluorescence values. The slopes of these lines (k) were then converted to t
1/2 values: t
1/2 = ln(0.5)/k.
To display the averaged curves, fluorescence decay values were converted to a percentage of the prebleach fluorescence. Recovery values were converted to a percentage of the difference between the pre- and post-bleach fluorescence. The means and standard deviations for values falling in 5-s intervals were then determined and plotted; where necessary, curves were shifted vertically to align y-intercepts.
Other live-cell microscopy experiments were performed using a microscope (IX81-OMAC; Olympus) equipped with 100x 1.4 NA Plan Apochromat oil objective. Cells suspended in minimal growth medium were mounted on a coverslip coated with concanavalin A (Sigma-Aldrich). Images were acquired using MetaMorph software (Molecular Devices) at ambient temperature unless otherwise noted as a stack of seven images with 400-nm axial separation using an Orca II camera (Hamamatsu Photonics) with 2 × 2 binning (1 pixel, ~129 nm). Alexa 488–labeled CPC and TRITC rhodamine-labeled MTs were visualized using the same equipment described above with 1 × 1 binning (1 pixel, ~64.5 nm). Images were processed using Imaris (Bitplane) and ImageJ software (
http://rsb.info.nih.gov/ij).
CPC purification
CPC purification was conducted as described previously (
Nakajima et al., 2009), with the following modifications. Nbl1 was purified from bacteria and denatured in buffer containing 6 M guanidine HCl for 30 min on ice and then added to the cleared lysate from insect cells (ES-Sf9) expressing Ipl1, Sli15, and Bir1. For fluorescently labeled CPC, the complex was incubated with Alexa 488 C5 maleimide (Invitrogen) on ice in the dark for 2 h before gel filtration.
Size exclusion chromatography of the CPC
For analysis of the native yeast-CPC, a protease-compromised strain (DDY1810) expressing SLI15-13myc at the endogenous locus under the native promoter was grown to early log phase (OD600, 0.3–0.35) before addition of either 15 µg/ml nocodazole or 0.1 M hydroxyurea. After being spheroplasted, the cells were lysed through homogenization into buffer supplemented with metabolic, protease, and phosphatase inhibitors (50 mM Bis-Tris propane, pH 7.4, 0.1 M KCl, 5 mM EGTA, 5 mM EDTA, 10 mM NEM, 20 mM β-glycerophosphate, 40 µM cantharidin, 20 mM Na pyrophosphate, 10 mM NaN3, 20 mM NaF, 0.8 mM Na orthovanadate, 1 mM PMSF [all from Sigma-Aldrich], and 1x protease inhibitor cocktail IV [EMD]) to preserve the stability and post-translational modifications. The cleared lysates were subject to gel filtration using a Superose 6 10/300 column (GE Healthcare) with a fraction size of 0.5 ml. The same gel filtration condition was used for molecular weight markers (HMW; GE Healthcare, Bio-Rad Laboratories). Detection of Sli15-13myc in gel filtration fractions was done by Western blot using anti-myc (9E10) antibody. Gel filtration of recombinant CPC and molecular weight markers (Bio-Rad Laboratories) was conducted using a Superose 6 10/300 column (GE Healthcare) with a fraction size of 0.5 ml.
Fluorescence-based MT association assay
To generate segmented MTs, unlabeled guanylyl-(a,b)-methylene-diphosphonate (GMPCPP)-MT seeds were polymerized by incubating 2 µM tubulin (purified from bovine brain) with 1 mM GMPCPP in BRB80 buffer (80 mM Pipes-KOH, pH 6.8, 1 mM MgCl2, and 1 mM EGTA) for 30 min at 37°C. These seeds were then used to elongate TRITC rhodamine-labeled GDP-MTs by diluting 10-fold and incubating with a 10-µM labeled/unlabeled tubulin mix (5% TRITC rhodamine-labeled porcine tubulin [Cytoskeleton] and 95% unlabeled tubulin) and 100 µM GTP in BRB80 buffer for 20 min at 37°C. To stabilize the MTs, taxol (Paclitaxel; Sigma-Aldrich) was then added to a final concentration of 20 µM, and the MTs were incubated for another 10 min at 37°C.
Alexa 488–labeled CPCs were diluted into CPC buffer (50 mM Bis-Tris propane, pH 6.8, 100 mM arginine, 100 mM glutamic acid, 300 mM NaCl, 2 µM Zn(CH
3CO
2)
2, 2 µM CaCl
2, 100 µM MgCl
2, 100 µM ATP, 1 mM DTT, and 20% glycerol) supplemented with 1 mg/ml BSA and dialyzed into CPC buffer containing 50 mM NaCl at 4°C for 2 h. After dialysis, taxol was added to the CPCs to a final concentration of 10 µM. Segmented MTs (0.2 µM tubulin) in CPC buffer were incubated with 2 nM Cin8 for 10 min at ambient temperature, then 2 nM final concentration of CPC was added to the reaction for 3 min before imaging in the presence of oxygen scavengers (0.2 mg/ml glucose oxidase, 35 µg/ml catalase, 0.25% β-mercaptoethanol, and 4.5 mg/ml glucose;
Desai et al., 1999).
Fluorescent spot detection and analysis were done with Imaris software (Bitplane) using default settings except for setting the minimum spot diameter to 100 nm. CPC spots on unlabeled seeds (in gaps aligned with TRITC MTs) were manually classified as MT bound. Frequency (µm−2) of Alexa 488 CPCs on MTs was determined for each MT bundle examined by dividing the number of Alexa 488–labeled CPC dots associated with the bundle by the total MT area in the field containing the MT bundle. A total of 14 microscope fields containing MT bundles were examined for both the WT and 17A CPCs (containing 17 and 16 separate bundles, respectively) and no fields containing bundles were ignored. The total MT areas that were analyzed for the WT and 17A CPCs were 69.7 × 109 µm2 and 54.109 µm2, respectively. Total Alexa 488 fluorescence intensity over the area of the spot was determined for each spot.
MT cosedimentation assay
The recombinant CPCs were supplemented with 1 mg/ml BSA and dialyzed into CPC buffer containing 50 mM NaCl for 2 h at 4°C. After dialysis, taxol was added to a final concentration of 20 µM. MTs in CPC buffer containing 100 mM NaCl and 20 µM taxol were added to a final concentration of 0.25 µM tubulin. CPCs and MTs were incubated at ambient temperature for 20 min before ultracentrifugation at 50 krpm at 25°C for 15 min. The supernatant and pellet were separated and analyzed by Western blot using anti-Bir1 antibody. Signal intensity was quantified using ImageJ software.
Cin8 purification and immunoprecipitation of CPC
Cin8-7myc was overexpressed and purified as described previously (
Rodal et al., 2003) with the following modifications. Cin8-7myc purification buffer consisted of 20 mM Hepes, pH 7.5, 1 mM EGTA, 5 mM MgCl
2, 1 mM ATP, and 100 mM KCl. Cin8-7myc–bound IgG Sepharose (GE Healthcare) was washed with buffer containing 500 mM KCl to remove contaminants before myc-tag removal by TEV protease. TEV protease (pRK793) was expressed and purified as described previously (
Kapust et al., 2001).
For immunoprecipitation of CPC with Cin8, myc antibody (9E10) was conjugated to Dynabeads (Invitrogen) following the company’s instructions. WT and 17A CPCs were prepared as described for the fluorescence-based MT association assay. The Dynabeads-bound Cin8 was incubated with CPC in CPC buffer (50 mM NaCl) containing 1 mg/ml BSA for 30 min at 4°C. The beads were washed three times with CPC buffer and the bound and unbound proteins were analyzed by Western blot using anti-Sli15, anti-Bir1 (generous gifts from A. Desai, University of California, San Diego, La Jolla, CA), and anti-myc (9E10) antibodies.
Online supplemental material
Fig. S1 shows that Ipl1 kinase activity is required for CPC exclusion from preanaphase spindles. Fig. S2 shows the time course of CPC kinase reactions with the Dam1 complex and histone H3 as substrates, the requirement for Ipl1-dependent phosphorylation of Sli15 for normal cell cycle progression, CPC targeting to the central spindle in
ipl1 and sli15 temperature-sensitive mutants, Bim1 protein level and electrophoretic band shift in wild-type and
sli15-17A cells, and electrophoretic mobility of Sli15- and sli15-17A-13myc throughout the cell cycle. Fig. S3 shows the low affinity of the Alexa 488–labeled CPC for rhodamine-labeled MTs. Video 1 shows MT-dependent nuclear localization of the CPC. Table S1 lists the yeast strains and plasmids used in this study. Online supplemental material is available at
http://www.jcb.org/cgi/content/full/jcb.201009137/DC1.
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