Article |
Address correspondence to Fred Hutchinson Cancer Research Center, P.O. Box 19024, 1100 Fairview Ave., N. (A2-168), Seattle, WA 98109-1024. Tel.: (206) 667-1351. Fax: (206) 667-6526. E-mail: sbiggins{at}fhcrc.org
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Abstract |
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Key Words: Ipl1/Aurora protein kinase; spindle; mitosis; microtubule; budding yeast
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Introduction |
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Mitotic spindles consist of two types of microtubules: those that bind to kinetochores (kinetochore microtubules) and those that interdigitate with each other (interpolar microtubules) (for review see Desai and Mitchison, 1997). The microtubule minus ends are bound to the microtubule organizing center, whereas the dynamic plus ends either capture kinetochores or interdigitate. In budding yeast, the mitotic spindle is formed after separation of the duplicated spindle pole bodies (SPBs)* (for review see Winey and O'Toole, 2001). During metaphase, cytoplasmic microtubules ensure that the nucleus migrates to the bud neck (nuclear migration) and the spindle aligns parallel to the mother-bud axis (spindle orientation) (for review see Segal and Bloom, 2001). Anaphase then occurs in two phases. In anaphase A, the sister chromatids move toward the SPBs, and in anaphase B a rapid phase of spindle elongation is followed by a second slow phase that drives the SPBs apart (Yeh et al., 1995; Straight et al., 1997). When the spindle has reached its maximal length of 10 µm, it disassembles exclusively from the plus ends at the midzone, the site of overlap between the interpolar microtubules (Maddox et al., 2000). Proper regulation of microtubule dynamics is critical for chromosome segregation and is performed in part by motor and nonmotor microtubule-associated proteins.
The fidelity of spindle assembly is monitored by the spindle checkpoint that prevents cells from separating their sister chromatids until kinetochore attachment is complete and kinetochores are under tension (for review see Millband et al., 2002). Spindle checkpoint arrest is achieved through inhibition of the anaphase-promoting complex (APC), a multiprotein ubiquitin ligase that catalyses proteolysis of the mitotic inhibitor proteins Pds1 and Clb2 (for review see Peters, 2002). When the APC targets Pds1p for degradation, the Esp1 protease is liberated to cleave the cohesin Mcd1/Scc1p, resulting in sister chromatid separation (for review see Nasmyth, 2002).
The Ipl1/Aurora protein kinases are key regulators of chromosome segregation and cytokinesis (for reviews see Shannon and Salmon, 2002; Stern, 2002). In mammals, the kinases can be subdivided into three families: Aurora A, B, and C (for review see Nigg, 2001). Aurora A localizes to centrosomes and is required to maintain the separation of centrosomes and to form a bipolar spindle (Glover et al., 1995). Aurora B exhibits a "chromosomal passenger" localization pattern, where it localizes to the chromosomes and kinetochores, transfers to the spindle, and eventually accumulates at the spindle midzone and midbody (Bischoff et al., 1998; Schumacher et al., 1998; Terada et al., 1998; Petersen et al., 2001; Murata-Hori et al., 2002). Aurora B is in a complex with the chromosomal passenger proteins INCENP (inner centromere protein) and Survivin/Bir1 (Kim et al., 1999; Adams et al., 2000; Kaitna et al., 2000; Speliotes et al., 2000; Morishita et al., 2001; Rajagopalan and Balasubramanian, 2002).
In budding yeast, there is a single essential Aurora protein kinase, Ipl1 (Chan and Botstein, 1993; Francisco et al., 1994). In ipl1 mutant cells, sister chromatids are pulled to the same spindle pole instead of opposite poles (Biggins et al., 1999; Kim et al., 1999). Experiments in vitro and in vivo suggest that this defect is due to an inability of ipl1 mutants to release monooriented kinetochore-microtubule attachments in order to make the correct bioriented attachments (Biggins et al., 1999; Tanaka et al., 2002). ipl1 mutants also fail to activate the spindle checkpoint when kinetochores are not under tension (Biggins and Murray, 2001). Like mammalian homologues, Ipl1p localizes to kinetochores and the mitotic spindle (Biggins et al., 1999; Biggins and Murray, 2001; He et al., 2001; Kang et al., 2001; Tanaka et al., 2002). Several Ipl1p/Aurora substrates have been identified in various organisms. CENP-A, the human histone centromeric H3 variant, is an Aurora B kinase substrate, and nonphosphorylatable CENP-A mutants have cytokinesis defects (Zeitlin et al., 2001). In budding yeast, three kinetochore proteins are substrates either in vivo and/or in vitro and are good candidates for essential Ipl1p targets: the INCENP homologue (Sli15p), the Dam1 protein, and the CBF3 component Ndc10p (Biggins et al., 1999; Kang et al., 2001; Cheeseman et al., 2002; Li et al., 2002).
Here we show that the Ipl1p kinase has a role in spindle disassembly that is independent from its previously identified functions. There is a dynamic relocalization of Ipl1p in anaphase, and Ipl1p tracks the plus ends of depolymerizing microtubules. We propose that Ipl1p regulates microtubule plus ends to promote chromosome segregation and spindle disassembly.
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Results |
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The role of Ipl1p in spindle disassembly is an independent function
Since Ipl1p is required for chromosome segregation and the spindle checkpoint, we tested whether the spindle disassembly delay was a consequence of defects in these functions. We showed previously that if bipolar spindle assembly occurs before Ipl1p inactivation, chromosome segregation is normal (Biggins and Murray, 2001). Therefore, we arrested cells containing Tub1GFP in metaphase by depleting the Cdc20 protein, shifted the cells to the restrictive temperature to inactivate Ipl1p, and then released them into the cell cycle. Aliquots were taken every 5 min, and cells with a pole to pole distance corresponding to a late anaphase cell (equal or greater than 9 µm) were analyzed for the presence or absence of a spindle. Since tubulin is always at the SPB, the pole to pole distance can be measured regardless of whether a spindle is present. Spindle disassembly occurred in 68% of Cdc20-depleted cells compared with only 36% of the Cdc20-depleted ipl1321 double mutant cells (Fig. 4 A). In addition, there was no defect in spindle elongation in either strain. This result suggests that the spindle breakdown defect in ipl1321 mutants is independent from Ipl1p's role in chromosome segregation.
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Since mutants defective in the mitotic exit network exhibit a spindle breakdown delay (Stegmeier et al., 2002), we tested whether ipl1321 mutant cells delayed mitotic exit by monitoring the destruction of Clb2p, the major mitotic B-type cyclin. Cells from the experiment described in Fig. 4 A were collected every 5 min and then immunoblotted with anti-Clb2p antibodies (Fig. 4 C). Clb2p degradation occurred with similar kinetics in wild-type and ipl1321 mutant cells, indicating Ipl1p is not required for mitotic exit.
Ipl1p also has a function in the spindle checkpoint when kinetochore tension is not generated (Biggins and Murray, 2001) so we tested whether other spindle checkpoint genes are also required for spindle disassembly. We found that the dynamics of spindle elongation and breakdown in wild-type, mad1, and mad2
strains containing Tub1GFP were similar (Fig. S2 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1). Therefore, a function in spindle disassembly is not a general property of all checkpoint proteins. Together, these data suggest that Ipl1p's role in spindle disassembly is independent from its roles in chromosome segregation and the spindle checkpoint and identifies a previously unknown function for this protein kinase.
ipl1321 mutant cells can stabilize fragile spindles
Since ipl1321 mutants are defective in spindle breakdown, we tested whether they could stabilize fragile spindles that result from loss of sister chromatid cohesion in metaphase. When sister chromatid cohesion is released in the absence of APC function by a mutation in the Mcd1/Scc1 cohesion protein, spindle elongation occurs in the presence of high levels of the Pds1 and Clb2 proteins (Michaelis et al., 1997). This leads to fragile spindles where the spindle elongates but breaks down abnormally fast, creating an "anaphase-like prometaphase" (Severin et al., 2001b). Therefore, we tested whether the addition of an ipl1321 mutation could stabilize the fragile spindles. We used a cdc26 strain, which leads to temperature-sensitive inactivation of the APC, in combination with the cohesin mutation mcd11 to create fragile spindles. cdc26
mcd11 and cdc26
mcd11 ipl1321 mutant cells containing Tub1GFP were arrested in G1 using
-factor, released to the restrictive temperature, and monitored for budding, spindle formation, and spindle breakdown. As reported previously, 93% of cdc26
mcd11 mutants underwent spindle breakdown within 150 min of release from G1 (Fig. 4 D) (Severin et al., 2001b). However, only 48% of cdc26
mcd11 ipl1321 mutant cells had undergone spindle breakdown at this time, indicating that the ipl1321 mutation stabilizes the fragile spindles.
Ipl1p kinase activity increases when spindles disassemble
To analyze Ipl1p kinase activity when spindles disassemble, we developed a kinase assay using antibodies generated against a recombinant GST-Ipl1 fusion protein. The affinity-purified antibodies specifically recognize a single major band in yeast lysates that migrates just above 45 kD (Fig. S3 available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1). The antibodies were used to immunoprecipitate wild-type Ipl1p and the Ipl1321 protein that has reduced kinase activity at high temperatures (Biggins et al., 1999). The majority of Ipl1p present in the yeast lysates (Fig. 5 A, pre) was depleted by the antibody (Fig. 5 A, post). The immunoprecipitates (Fig. 5 A, IP) were then incubated with the histone-fold domain of the kinetochore protein Cse4 in a kinase reaction in vitro. Cse4p was radiolabeled in the presence of wild-type Ipl1p but not the kinase inactive Ipl1321 protein (Fig. 5 A, right), showing that the assay specifically reflects an Ipl1p-associated kinase activity.
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Ipl1p's substrates localize to the spindle midzone, and Ipl1p follows the plus ends of the depolymerizing spindle microtubules
The novel midzone localization pattern for Ipl1p led us to test whether proteins that Ipl1p regulates also localize to the spindle midzone. First, we tested Cse4p which we have shown here is an Ipl1p substrate in vitro. Localization of a Cse4GFP fusion showed that it does not transfer to the spindle (unpublished data). We next analyzed the localization of COOH-terminal GFP fusions to the Ndc10, Sli15, and Dam1 proteins (Fig. 6 A). Ndc10GFP localized to the midzone in late anaphase cells in addition to the previously reported spindle and kinetochore localization (Lechner and Carbon, 1991; Goh and Kilmartin, 1993). We found that Sli15GFP also accumulates at the spindle midzone and exhibits the same localization pattern as Ipl1p throughout the entire cell cycle (Fig. 6 A; unpublished data). Various labs have reported that Dam1p localizes to kinetochores throughout the cell cycle and to the mitotic spindle (Hofmann et al., 1998; He et al., 2001; Jones et al., 2001). Here we show that Dam1GFP also localizes to the spindle midzone in anaphase cells. Therefore, the majority of known Ipl1p substrates localize to the spindle midzone, providing several potential candidates for Ipl1p regulation of spindle disassembly.
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Since Ipl1p localizes to the spindle midzone very late in anaphase and then travels back to the poles, we tested whether it was following the plus ends of the depolymerizing spindle microtubules. Live microscopy was performed on cells coexpressing Tub1CFP (tubulin) and Ipl1GFP. Although CFP and GFP have overlapping spectrums, the Ipl1GFP and Tub1CFP signals were easily discernible (Fig. 6 B). It was not possible to use the nonoverlapping spectrum of YFP because the Ipl1YFP signal was not strong enough to perform time-lapse imaging of cells. We started imaging a cell when Ip1lp (Fig. 6 B, green) localized to the midzone of a long spindle (Fig. 6 B, red, 0'). After 1 min, the Ipl1p signal split and there was no longer any tubulin signal in the center of the spindle, indicating that the spindle is starting to break down. As the spindle depolymerized toward the poles, the Ipl1p signal always localized near the plus end of the microtubules (Fig. 6 B, 2'). At the end of spindle disassembly, the remaining tubulin at the pole colocalized with Ipl1p (Fig. 6 B, 3.5'). Therefore, Ipl1p accumulates at the spindle midzone in late anaphase and then follows the plus end of the depolymerizing microtubules back to the poles, suggesting it may specifically regulate the microtubule plus ends.
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Discussion |
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Ipl1p leaves the kinetochores after tension is established
We show here that Ipl1p exhibits a localization pattern similar to chromosomal passenger proteins, making it likely that Ipl1p is an Aurora B homologue. The dynamic localization of Ipl1p reflects its various mitotic functions. During prometaphase, Ipl1p monitors kinetochore tension and promotes microtubule release of monooriented kinetochore attachments, thus ensuring that sister kinetochores establish biorientation before chromosome segregation (Biggins and Murray, 2001; Tanaka et al., 2002). The proposal that Ipl1p might delocalize from kinetochores at metaphase provided an attractive mechanism for inactivating the kinase when tension and biorientation are established (Tanaka et al., 2002). However, we found that both Ipl1GFP and endogenous Ipl1p localize to kinetochores in metaphase. Our data agrees with the observation that a mammalian Aurora BGFP fusion protein left kinetochores 0.5 min after the initiation of anaphase, well after tension was established (Murata-Hori et al., 2002). K. Tanaka and T.U. Tanaka recently found that Ipl1p is on metaphase kinetochores by chromatin immunoprecipitation, thus reconciling our data (personal communication). To understand how tension regulates Ipl1p, we are actively trying to elucidate the mechanisms that control Ipl1 protein stability and kinase activity, since these may be alternative modes of regulation at metaphase.
Functions of the Ipl1/Aurora B protein kinase family
We show that the localization of Ipl1p to the mitotic spindle is correlated with spindle disassembly in budding yeast. Since Aurora B localizes to the spindle midzone in all organisms, this may be another conserved function of the Ipl1/Aurora B protein kinase family. Alternatively, this may reflect similarity to the role of Aurora A, since it regulates spindle function and phosphorylates the Eg5 motor protein in frog egg extracts (Giet et al., 1999; Giet and Prigent, 2000). In other organisms, Aurora B is required for cytokinesis, and this may be coupled to defects in spindle microtubule depolymerization that have not been noticed previously. In human cells, cytokinesis is regulated by the phosphorylation of CENP-A by Aurora B (Zeitlin et al., 2001). We report here that the budding yeast histone variant Cse4p is also a good substrate for Ipl1p in vitro. Further analysis of the effects of Cse4p phosphorylation by Ipl1p may reveal more details about spindle disassembly and/or cytokinesis in budding yeast. We show that the dynamics of spindle elongation are not altered in ipl1 mutant cells, unlike mutants in the AuroraINCENPSurvivin complex in Schizosaccharomyces pombe (Morishita et al., 2001; Rajagopalan and Balasubramanian, 2002). This difference may be due to the number of kinetochore microtubule-binding sites in each organism. In S. pombe, there are multiple binding sites, which results in lagging chromosomes if biorientation is not achieved. In budding yeast where there is a single microtubule binding site, defects in biorientation cannot generate lagging chromosomes. However, when a conditional dicentric chromosome is activated in budding yeast, thus creating a lagging chromosome, spindle elongation is delayed (Yang et al., 1997). Our study in budding yeast had the advantage that defects in biorientation do not interfere with spindle dynamics. To determine whether the spindle disassembly function of Ip1lp is conserved, spindle dynamics will need to be analyzed in situations where chromosome segregation is normal.
Consistent with a role in spindle disassembly, we found that Ipl1p kinase activity increases just before spindle breakdown. Few studies have looked at the regulation of Ipl1p homologues. In Drosophila and rat tissues, Aurora B protein levels and kinase activity peak during mitosis (Bischoff et al., 1998; Terada et al., 1998). However, the time points were not close enough in those studies to determine whether the peak of kinase activity corresponds to spindle breakdown. In fission yeast, Aurora B is not cell cycle regulated (Petersen et al., 2001; Leverson et al., 2002), making it unclear whether there are conserved mechanisms that regulate Ipl1/Aurora B protein levels and kinase activity. Our study also revealed that Ipl1p kinase activity is low when cells are arrested in metaphase with kinetochores under tension. This may reflect an active mechanism that regulates Ipl1p stability and/or activity once tension is established. Future work will be needed to elucidate the mechanisms that lead to changes in Ipl1p kinase activity during mitosis.
How does Ipl1p regulate spindle disassembly?
In support of a role for Ipl1p in direct regulation of microtubules, we found that ipl1 mutants are able to alleviate the spindle fragility of apc mcd1 mutant cells. However, since the mechanism that leads to fragile spindles is not known, it is not clear how Ipl1p stabilizes spindles. Although mutations in Ipl1p affect spindle breakdown, they do not do this by grossly altering the structure of the spindle midzone, since all the midzone proteins we tested still localized in an ipl1 mutant.
Our studies using live microscopy revealed a previously unidentified localization pattern for Ipl1p. At anaphase, Ipl1p is transported along the spindle to the midzone and then tracks the plus ends of the depolymerizing spindle microtubules back to the poles. To our knowledge, the only other protein in budding yeast that exhibits this localization pattern is the Ipl1p substrate Ndc10p and suggests regulated transport of these proteins on microtubules in anaphase, possibly by motor proteins (D. Bouck and K. Bloom, personal communication). The localization to the plus ends may indicate that Ipl1p directly destabilizes microtubules like catastrophe factors, such as the KINI family of motor proteins (Desai et al., 1999). However, although Ipl1p binds microtubules in vitro (Kang et al., 2001), we have not been able to induce microtubule depolymerization with bacterial Ipl1p in vitro (unpublished data). Therefore, Ipl1p may directly promote microtubule depolymerization in a manner that we have not yet detected, or it may instead control a microtubule-binding protein.
There are two nonessential proteins known to be involved in spindle microtubule disassembly in budding yeast: the motor protein Kip3 and the microtubule-associated protein Ase1 (Juang et al., 1997; Straight et al., 1998). Ipl1p and Kip3p may act in the same spindle disassembly pathway because the double mutant exhibits the same spindle breakdown defect as each single mutant. However, we have yet to obtain evidence that Ipl1p regulates Kip3p (unpublished data). There are other potential candidates for Ipl1p regulation that will need to be investigated, such as the midzone protein Stu2 that opposes the Kip3 protein and the Esp1p/Pds1p cell cycle regulation complex that is also found at the midzone and has a function stabilizing spindles during anaphase (Uhlmann et al., 2000; Jensen et al., 2001; Severin et al., 2001a). We also found that three known Ipl1p substrates are at the spindle midzone: Ndc10p, Sli15p, and Dam1p. It is interesting to note that Dam1p was originally identified for its role in regulating spindle dynamics (Hofmann et al., 1998; Jones et al., 1999). Therefore, several potential Ipl1p substrates localize to the spindle midzone, and it will need to be determined whether any of these candidates also promote spindle disassembly.
The spindle midzone: a kinetochore-like structure?
Several kinetochore proteins are now known to localize to the spindle midzone in anaphase, including Stu2p, Slk19p, and the motor protein Cin8 (Hoyt et al., 1992; Zeng et al., 1999; Kosco et al., 2001). Here we show four additional kinetochore proteins localizing to the midzone: the Ipl1/Aurora protein kinase, the INCENP homologue Sli15p, Dam1p, and Ndc10p. Since midzone staining is difficult to detect, it may have been overlooked in several other localization studies and many more kinetochore proteins may be present at the midzone. Most of the spindle midzone proteins have been implicated in the regulation of spindle dynamics in anaphase by either promoting spindle elongation or spindle disassembly. An intriguing possibility is that the microtubule plus ends at the spindle midzone are regulated in anaphase similarly to the kinetochore-microtubule attachments in prometaphase. Future work will determine whether the Ipl1/Aurora protein kinase and other spindle midzone proteins are global regulators of microtubule plus ends.
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Materials and methods |
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Yeast strain construction
Yeast strains are listed in Table I and were constructed by standard genetic techniques. Diploids were isolated on selective medium and subsequently sporulated at 23°C. All strains containing the pGAL-CDC20 construct were obtained through crosses with SLJ577, a gift from S. Jaspersen and M. Winey (University of Colorado, Boulder, Colorado). SBY1036 was created by integration of TUB1CFP:URA3 (a gift from K. Bloom, University of North Carolina at Chapel Hill, Chapel Hill, NC) with StuI at the URA3 locus. Strains containing CSE4-myc12 were obtained by integrating pSB246 cut with ClaI at the CSE4 locus. Strains containing TUB1GFP:LEU2 were obtained by integrating plasmid pSB340 cut with AgeI at the LEU2 locus and TUB1GFP:URA3 by integrating plasmid pMAS27 (a gift from M. Shonn, TUFTS University, Boston MA) cut with StuI at the URA3 locus. SBY554 was obtained by integrating pSB164 (pGAL-IPL1:URA3 [Biggins et al., 1999]) cut with StuI at the URA3 locus. SBY736 was obtained by integrating pSB257 (pGAL-myc12-IPL1:URA3) cut with StuI at the URA3 locus. Deletions in yeast genes and GFP, CFP, and myc epitope tags were made using the PCR-based integration system (Longtine et al., 1998). YFP epitope tags were made using pDH5, a gift from T. Davis (University of Washington, Seattle, WA). Specific primer sequences are available upon request. All deletions and epitope tags were confirmed by PCR.
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Protein and immunological techniques
Protein extracts were made and immunoblotted as described (Minshull et al., 1996). Anti-Tub1p antibodies were obtained from Accurate Chemical and Scientific and used at a 1:1,000 dilution, anti-Clb2p antibodies (a gift from A. Rudner, Harvard Medical School, Boston, MA) were used as described (Rudner et al., 2000), and anti-Ipl1p antibodies were used at 1:1,000. To generate anti-Ipl1p antibodies, GST-Ipl1p was injected into rabbits at Cocalico Biological Inc. For the fourth and fifth boosts, boiled protein was injected. The antibodies were affinity purified by coupling GST-Ipl1p to SulfoLink Coupling gel (Pierce Chemical Co.).
Microscopy
For live microscopy to analyze GFP fusion proteins, cells were grown in yeast meda, pH 7.0, washed, and resuspended in 1/10 volume minimal medium with casamino acids. For live microscopy at RT, 1.5 µl of cell preparation was put on a 2% agarose pad containing minimal medium with casamino acids. The slide was then sealed with VALAP (1:1:1, vaseline: lanolin: paraffin) and imaged. For live microscopy at 35°C, prepared cells were mounted directly onto a heated stage (Bioptechs). Images were collected through an Olympus 1X17 60x objective with a CH350 CCD camera (Roper Scientific) using the Softwox 2.5 (Applied Precision) software. The same software was used for deconvolution. At least 10 cells were analyzed for all reported experiments.
Chromosome spreads were performed as described (Loidl et al., 1991; Michaelis et al., 1997). Lipsol was obtained from Lip Ltd. 9E10 antibodies that recognize myc tag were used at a 1:500 dilution and obtained from Covance. Anti-Tub1p antibodies (Accurate Chemical and Scientific) were used at a 1:500 dilution. Anti-Ipl1p antibodies were used at a 1:250 dilution. Alexafluor-594 and Alexafluor-488 secondary antibodies were obtained from Molecular Probes and used at a 1:250 dilution.
Cse4 histone fold domain purification
The histone fold domain of CSE4 (encoding aa 121229) was PCR amplified from Saccharomyces cerevisiae genomic DNA and cloned into a T7 expression vector of the pCRT7/CT TOPO TA cloning kit (Invitrogen). The resulting expression plasmid (pT7Cse4c) was transformed into BL21-CodonPlus (DE3)-RIL cells (Stratagene), and 2 liters were induced with ITPG to 0.2 mM for 2.5 h at 37°C, and Cse4 was purified under denaturing conditions as described (Gelbart et al., 2001). The Cse4 histone fold domain purity was verified by SDS-PAGE and dialyzed against water. A substantial portion of the protein was not soluble in water, but solubility was adequate for kinase assays in vitro.
Ipl1p kinase assays
40-ml cultures of mid-log cells were collected and resuspended in 500 µl lysis buffer (100 mM NaCl, 50 mM Tris, pH 7.5, 50 mM NaF, 50 mM ß-glycerophosphate, pH 7.4, 2 mM EDTA, 2 mM EGTA, 0.1% Triton X-100). 2 mM NaVO4, 2 mM PMSF, 10 µg/ml LPC (leupeptin, pepstatin, and chymostatin; Chemicon), 1 mM DTT, 0.1 µg/ml microcystin (Calbiochem) were added fresh. All subsequent steps were performed at 4°C. Cells were lysed with glass beads in a beater (Biospec Products Inc.) for 30 s and then centrifuged for 10 min. 400 µl supernatant was added to 5 µl magnetic protein G beads (Dynal Biotech Inc.) and 4 µl anti-Ipl1p antibodies for 2 h. The beads were washed three times with 400 µl lysis buffer and once with 100 µl kinase buffer without ATP (50 mM Tris, pH 7.4, 1 mM DTT, 25 mM ß-glycerophosphate, 5 mM MgCl2) and then resuspended in buffer with 10 µM ATP, 5 µCi [32P]ATP and 5 µg Cse4p for 30 min at 30°C. 2x sample buffer was added, and reactions were separated on SDS-PAGE and subjected to autoradiography using a Phosphorimager Screen (Molecular Dynamics). Kinase assays were quantified using ImageQuant (Molecular Dynamics) software.
Online supplemental material
Videos 13 and Figs. S1S3 are available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1. Video 1 displays wild-type cells expressing Tub1-GFP from metaphase to anaphase. Video 2 shows ipl1-321 cells expressing Tub1GFP that are delayed in spindle disassembly. Video 3 shows Ipl1GFP localization during late anaphase. Fig. S1 shows endogenous Ipl1 localization on chromosome spreads. Fig. S2 demonstrates that the spindle checkpoint mutants mad1 and mad2
do not have spindle disassembly defects. Fig. S3 shows the Ipl1p antibody specificity.
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Footnotes |
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* Abbreviations used in this paper: APC, anaphase-promoting complex; INCENP, inner centromere protein; SPB, spindle pole body.
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Acknowledgments |
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This work was supported by grants from the Roche Research Foundation and the Department of Defense of the Army to S. Buvelot, a Damon Runyon Cancer Research Foundation fellowship (DRG-1554) to D. Vermaak, and a Sidney Kimmel Scholar award and National Institutes of Health grant to S. Biggins.
Submitted: 4 September 2002
Revised: 2 December 2002
Accepted: 24 December 2002
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References |
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