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First published online 9 January 2007
doi: 10.1242/jcs.03342
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Research Article |
Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK
Author for correspondence (e-mail: ms433{at}cam.ac.uk)
Accepted 14 November 2006
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Summary |
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13, encoding a truncation of the spindle pole body component Spc110p (the intranuclear receptor for the 
-tubulin complex). We found that Clb5p-GFP was localised to the spindle poles and intranuclear microtubules and that Clb5p-dependent kinase promoted cell cycle dependent phosphorylation of Spc110p contributing to spindle integrity. Two cyclin-dependent kinase consensus sites were required for this phosphorylation and were critical for the activity of spc11013 as a suppressor. Together, our results point to the function of cyclin-dependent kinase phosphorylation of Spc110p and provide, in addition, support to a model for Clb5p control of spindle polarity at the level of astral microtubule organisation.
Key words: Spindle, Cell cycle, Cyclin-dependent kinase, Microtubule organisation
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Introduction |
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; Nasmyth, 1993; Morgan, 2006) in combination with individual B-type cyclin gene disruptions. The use of the hypomorphic cdc28-4 sensitised cells to the loss of individual cyclins, thus circumventing the problem of functional redundancy (Segal et al., 1998). These studies revealed the specific requirement of the S-phase cyclin Clb5p for correct spindle polarity and dynamics (Segal et al., 1998; Segal et al., 2000b).
Spindle assembly and polarity establishment are tightly coupled processes in the yeast cell cycle. Both aspects of spindle morphogenesis are controlled by the spindle pole body (SPB; the yeast microtubule-organising centre) (Byers, 1981). SPBs are inserted in the nuclear envelope to organise intranuclear microtubules (MTs) that assemble into a spindle. The cytoplasmic face of the SPB organises astral (cytoplasmic) MTs that interact with the cell cortex to position the spindle (Carminati and Stearns, 1997; Shaw et al., 1997). Following conservative duplication at the G1-S transition (Lew et al., 1997; Adams and Kilmartin, 2000; Jaspersen and Winey, 2004), old and new SPBs separate to generate a short spindle. Polarity is apparent during SPB separation as each pole engages in asymmetrically oriented astral MT-cortex interactions. The old SPB reaches for the bud cortex, while the new SPB is confined to the mother cell. This asymmetric behaviour targets each pole to opposite compartments of the dividing cell. The ensuing alignment of the preanaphase spindle along the mother-bud axis, permits the translocation of the old SPB into the bud during spindle elongation across the bud neck (Shaw et al., 1997; Segal et al., 2000b; Pereira et al., 2001).
Spindle polarity is further revealed by the timing of astral MT-mediated labelling of SPBs by a dynein-heavy-chain-GFP fusion (Dyn1p-GFP), as observed by live cell imaging microscopy (Shaw et al., 1997). Initially, the fusion marks the old SPB. Following pole separation, label is progressively acquired by the new SPB. This `lag' in Dyn1p-GFP accumulation reflects a delay in astral MT organisation relative to SPB separation that prevents the new SPB from interacting with the bud cortex (Huisman and Segal, 2005). Loss of the S-phase cyclin Clb5p under conditions of limited CDK activity (cdc28-4 clb5 cells) abolishes this temporal control and results in diploid-specific lethality. In this mutant, both poles display Dyn1p-GFP label during separation (Segal et al., 2000b). The ensuing loss of polarity causes the terminal translocation of the preanaphase spindle into the bud, with high penetrance in cdc28-4 clb5 homozygous diploid cells (Segal et al., 1998; Segal et al., 2000a; Cross and Jacobson, 2000).
Here we have addressed possible roles of Clb5p-dependent kinase in control of spindle morphogenesis. First, we found that Clb5p is localised to the SPBs and mitotic spindle. Second, we undertook a genetic screen for high dosage suppressors of cdc28-4 clb5 diploid lethality. This screen identified a genomic sequence encoding a truncation of the SPB component Spc110p (Rout and Kilmartin, 1990; Kilmartin et al., 1993). Spc110p is a phosphoprotein with a cell-cycle-dependent profile of phosphorylation (Friedman et al., 1996; Stirling and Stark, 1996) attributed to the kinase Mps1p (Friedman et al., 2001). Here we present genetic analysis indicating that, in addition, CDK consensus sites in Spc110p contribute to its pattern of cell-cycle-dependent phosphorylation. In agreement with the in vivo analysis presented here, the involvement of Clb5p in selective targeting of Spc110p for phosphorylation has been demonstrated recently in vitro in proteomic-based studies (Ubersax et al., 2003; Loog and Morgan, 2005). We further show that these phosphorylation events may be important for spindle integrity and correct spindle dynamics. Finally, we address the mechanism of rescue of cdc28-4 clb5 lethality by the truncated SPC110. The mode of action of this dosage suppressor validates a model in which CDK may provide temporal control of astral MT organisation by the SPB, in coordination with spindle assembly, to promote correct spindle polarity (Segal and Bloom, 2001).
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; Liakopoulos et al., 2003). In light of the genetic data supporting the direct involvement of Clb5p in mitotic spindle morphogenesis (Segal et al., 1998; Segal et al., 2000b) we undertook analysis of Clb5p-GFP localisation by live-cell imaging microscopy. As shown in Fig. 1, Clb5p-GFP accumulated in the nucleus following bud emergence. In addition, a prominent dot-like label (Fig. 1A, 0 minutes, right cell, arrow) appeared to split and eventually give rise to a rod bisecting the nucleus (Fig. 1A, 2 minutes, right cell, arrowheads). The position of this rod suggested labelling of the short mitotic spindle. Following alignment along the mother-bud axis (Fig. 1A, 27.5 minutes, left cell, arrowheads), Clb5p-GFP label progressively disappeared, consistent with Clb5p proteolysis at anaphase onset. To confirm that Clb5p-GFP accumulated at the mitotic spindle, time lapse analysis was performed in wild-type cells coexpressing Clb5p-GFP and the SPB component Spc29p fused to CFP (Fig. 1B,C). Clb5p-GFP label began to accumulate in the nucleus coincident with bud emergence (Fig. 1B, 0-10 minutes) and appeared as a dot that colocalised with the SPB shortly after (Fig. 1B, 10-23 minutes, arrows). Following SPB separation, Clb5p-GFP label appeared as a short line between the two SPBs (Fig. 1C, arrowheads). Label disappeared progressively from the spindle and nucleus before initiation of spindle elongation (Fig. 1C, right cell, 5-11 minutes). Labelling of the spindle and SPBs (but not nuclear localisation) was lost upon a shift of a cdc28-13 mutant to the semipermissive temperature of 35°C (data not shown). Localisation of Clb5p-GFP to the SPBs and spindle was still apparent in a cnm67 mutant in spite of disrupting the outer plaque (Schaerer et al., 2001), although it was not possible to determine conclusively whether label at the SPBs was reduced compared with that in wild-type cells (Fig. 1D). The significance of this observation is addressed later. Taken together, these results indicate that Clb5p is localised to the SPB and mitotic spindle, consistent with its roles in spindle morphogenesis.
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lethality diploid lethality. We hypothesised that the screen might yield putative targets of Clb5p-associated kinase or gene products, which, in high dosage, can bypass the defect of cdc28-4 clb5 cells.
Based on the highly penetrant failure of cdc28-4 clb5 haploid cells to give rise to viable homozygous diploids following mating (Segal et al., 1998; Cross and Jacobson, 2000), a yeast genomic library on a multicopy (2-µm-based) vector was screened for clones increasing diploid formation between a tester 
cdc28-4 clb5 strain and a cdc28-4 clb5 cells transformed with the library (see Materials and Methods). The recovered plasmids were then retested for their ability to support growth of a diploid cdc28-4 clb5 GAL1:CLB5 strain upon transfer to glucose containing medium (not shown). Among the dosage suppressors recovered, plasmid pDLS13 improved cdc28-4 clb5 diploid viability but did not suppress temperature sensitivity of cdc28 clb5 haploids (Segal et al., 1998). Standard analysis indicated that the suppressor mapped to the 3' end of the library insert predicted to encode a truncation spanning amino acids 1-437 of the SPB component Spc110p here referred to as Spc11013p. Spc110p is a component of the inner plaque that serves as a target for the -tubulin complex responsible for intranuclear MT nucleation (Rout and Kilmartin, 1990; Knop and Schiebel, 1997; Schiebel, 2000). However, as shown in Fig. 2A, Spc11013p did not contain the crucial domains necessary for insertion at the central plaque of the SPB (Sundberg et al., 1996). Indeed, contrary to a full-length Spc110p-GFP fusion that localised to the SPBs (Rout and Kilmartin, 1990; Adams and Kilmartin, 1999), Spc11013p-GFP accumulated in the nucleus but did not label the SPBs even upon overexpression under the control of the GAL1 promoter (Fig. 2B). In contrast to the behaviour of spc11013, however, full-length SPC110 could not suppress cdc28-4 clb5 diploid lethality (not shown).
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; Stirling and Stark, 1996). Spc110p is subjected to cell-cycle-dependent phosphorylation (Friedman et al., 1996; Stirling and Stark, 1996), beginning at bud emergence as cells proceed into S phase (Fig. 3A). Phosphorylation is absent in cdc28ts or cdc4ts-induced blocks, whereas it is present in a clb1-4 strain (Friedman et al., 1996), implicating Clb5p in Spc110p phosphorylation. However, in this early analysis a link between CDK-consensus phosphorylation sites and cell-cycle-dependent modification was not observed. Instead, the pattern of cell-cycle-dependent phosphorylation was attributed to the kinase Mps1p (Friedman et al., 2001). More recently, Spc110p was identified as a putative target of CDKs by proteomics approaches (Ubersax et al., 2003). Consistent with those findings, Spc110p phosphorylation levels were sensitive to overexpression of Cdc14p, a phosphatase with specificity toward CDK-phosphorylated sites (Visintin et al., 1998). Extracts from cells arrested at metaphase by a nocodazole-induced block (Fig. 3B) or in S-phase by a hydroxyurea-induced block (not shown) exhibited reduced levels of phosphorylation upon induction of Cdc14p expression under the GAL1 promoter. Spc110p bulk phosphorylation was enhanced by overexpression of Clb5p. Indeed, following a nocodazole-induced metaphase block in raffinose-containing medium, a clb3 clb4 clb5 GAL1:CLB5 strain exhibited markedly reduced levels of Spc110p phosphorylation upon release and repression of GAL1:CLB5 by dextrose. By contrast, GAL1:CLB5 re-induction by releasing cells in galactose-containing medium resulted in overt phosphorylation of Spc110p (Fig. 3C). The effect of Clb5p overexpression on Spc110p phosphorylation was also prominent in cdc28-4 clb5 cells, but less obvious in a clb5 mutant (Fig. 3C). Indeed, it is important to point out that deletion of CLB5 in combination with cdc28-4 greatly reduced bulk levels of phosphorylation of Spc110p. By contrast, single cdc28-4 mutant cells exhibited comparable levels of Spc110p phosphorylation relative to wild-type cells at the permissive temperature (supplementary material Fig. S1). These results support the notion that functional cyclin redundancy is less apparent in a cdc28-4 background (Segal et al., 1998).
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Serine to alanine substitutions were introduced by site-directed mutagenesis to generate strains expressing Spc110p mutant variants lacking predicted CDK phosphorylation sites at positions 36 and 91. The resulting single or double mutants were viable (see also Friedman et al., 1996). Analysis of extracts from asynchronous cells showed that bulk phosphorylation was particularly reduced by a substitution at position 91. Substitution of Ser36 had a slight additive effect (Fig. 3D,E). To assess the contribution of these residues to the profile of cell-cycle-dependent phosphorylation of Spc110p, extracts from synchronised cell populations generated by release from an -factor-induced block were analysed (Fig. 3E and supplementary material Fig. S2). Compared with the pattern observed for wild-type Spc110p, phosphorylation of Spc11091Ap was delayed during S phase and reached relatively lower levels at later times. By contrast, initial phosphorylation of Spc11036Ap was apparent, however, phosphorylation levels were reproducibly lower by 60-75 minutes with the low mobility band under-represented, even at 90 minutes. Spc11036A91Ap combined the behaviour of both single mutants to give rise to a close-mobility doublet after 75 minutes.
These results indicated that CDK might contribute both to bulk level and the cell-cycle-dependent pattern of phosphorylation of Spc110p. The timing of phosphorylation inferred from these genetic data, together with previous findings (Friedman et al., 1996; Loog and Morgan, 2005) support the involvement of Clb5p, although other B-type cyclins can also direct CDK-dependent phosphorylation of Spc110p in a clb5 background (Fig. 3C).
As indicated above, Mps1p promotes cell-cycle-dependent phosphorylation of Spc110p. To assess the combined contribution of CDKs and Mps1p to the pattern of phosphorylation, spc11036A, spc11091A or spc11036A91A were introduced in an mps1-1 mutant (Schutz and Winey, 1998). As shown in Fig. 4, inactivation of Mps1p was not sufficient to abolish the cell-cycle-dependent pattern (Fig. 4A) and the additive effects (Fig. 4B) on Spc110p phosphorylation dependent upon the two CDK consensus sites. Conversely, substitutions cancelling three Mps1 phosphorylation sites at positions 60, 64 and 68 (Friedman et al., 2001) validated this observation and confirmed that the cell-cycle-dependent pattern of phosphorylation remaining in Spc11036A91Ap, was dependent on Mps1p (Fig. 4C and supplementary material Fig. S3). However, the combined substitutions at Mps1p and CDK phosphorylation sites did not appear to impair cell viability (not shown).
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In conclusion, the wild-type pattern of cell-cycle-dependent phosphorylation of Spc110p might require both CDK consensus sites as well as the previously identified sites for Mps1p-dependent phosphorylation.
Effect of mutations cancelling CDK phosphorylation sites in Spc110p
Mutations cancelling putative CDK phosphorylation sites in Spc110p did not appear to compromise cell viability but enhanced the temperature sensitivity of a mps1-1 strain (not shown). In order to investigate the functional significance of phosphorylation of Spc110p at the two CDK consensus sites, SPC110, spc11036A, spc11091A or spc11036A91A strains expressing GFP-Tub1p were used to determine the cell-cycle distribution of MT-based structures in asynchronous cell populations. Although spc11036A behaved like SPC110 cells, the spc11091A mutant exhibited a mild increase in the proportion of preanaphase spindles at the expense of elongated spindles (Fig. 5A). In addition, preanaphase spindles appeared to be longer. Analysis of spc11036A91A cells, however, showed that these two substitutions were not additive regarding this phenotype.
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factor arrest to generate histograms for spindle length distribution at onset of spindle elongation (Fig. 5B). In general, after SPB separation, spindle length increases from approximately 1 µm to reach a stable length of 1.6-2.0 µm before the onset of the fast phase of spindle elongation in which spindle length increases at a rate of approximately 1 µm/minute (Yeh et al., 1995; Straight et al., 1998; Segal et al., 2000b). As expected, wild-type cells exhibited a characteristic distribution of spindle lengths at a time-point corresponding to onset of spindle elongation, with the majority of spindles shorter than 2 µm. Substitutions at positions 36 and 91 resulted in a displaced distribution with a significant `shoulder' for spindle lengths between 2 and 3.2 µm, particularly in the case of spc11091A (Fig. 5B middle panel), consistent with a perturbation in spindle dynamics and a rate-limiting step at onset of anaphase B. This observation was further validated by time-lapse analysis of the spc11091A mutant to determine the kinetics for early spindle elongation. Fig. 6 shows representative time-lapse sequences for the behaviour of wild-type or spc11091A cells. After maintaining a stable preanaphase spindle length, wild-type cells initiated the fast phase of spindle elongation in a single transition (94%, n=17 cells recorded; Fig. 6A,B). By contrast, spc11091A cells did not reach a stable spindle length. Instead, length increased progressively until the fast phase of spindle elongation started (69%, n=19 cells recorded; Fig. 6C-E). Cells also exhibited a variable pause in early anaphase B (Fig. 6D). Following this delay, the rate of elongation was comparable in both strains (Fig. 6E). This kinetic delay was dependent on the spindle integrity checkpoint (Lew and Burke, 2003), as indicated by the impact of a mad2 on the distribution of spindle length of a spc11091A mutant. Indeed, the profile for spc11091A mad2 cells was shifted back to the left, reflecting the absence of a slow point in early spindle elongation (Fig. 7A). This was confirmed by the distribution of MT-based structures in asynchronous cell populations of the double mutant that did not exhibit an excess of preanaphase cells (Fig. 7B). Accordingly, spc11091A mad2 cells started spindle elongation in a single fast transition (91%, n=11 cells recorded; Fig. 7C). These data suggest that at least a substitution that cancels phosphorylation at Ser91 within a CDK site, compromised spindle integrity to cause a kinetic delay in spindle elongation by transient activation of the Mad2p-dependent checkpoint. Yet, elimination of this mild kinetic delay by mad2 did not significantly impair viability of spc11091A cells (not shown).
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Effect of Spc11013p expression on spindle polarity in cdc28-4 clb5 cells
As indicated above, phosphorylation of full-length Spc110p was significantly decreased, but not absent, in cdc28-4 clb5 cells (Fig. 3C). Moreover, dependency on CDK consensus sites was also observed for Spc11013p expressed in cdc28-4 clb5 cells (Fig. 8A). We therefore proceeded to determine whether phosphorylation at positions 36 and 91 was required for this truncation to rescue cdc28-4 clb5 mutant phenotypes.
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mutants expressing either Spc11013p or Spc11013-36A91Ap. A cross between an a cdc28-4 clb5 HIS3:spc11013 and a tester cdc28-4 clb5 resulted in 73.5% diploid viability (compared with 100% in a cdc28-4 CLB5+ control cross). By contrast, a cross using an a cdc28-4 clb5 HIS3:spc11013-36A91A yielded 16.5% viable diploid microcolonies as did the control cross between two cdc28-4 clb5 strains.
Spindle polarity can be assessed by the timing of MT-mediated labelling of SPBs by a Dyn1p-GFP fusion (Shaw et al., 1997). Cdc28p-Clb5p kinase is required for the characteristic delay in Dyn1p-GFP acquisition at the new SPB marking its commitment to mother-bound fate (Huisman and Segal, 2005). Indeed, disruption of SPB intrinsic asymmetry in cdc28-4 clb5 cells is accompanied by the absence of any `lag' in Dyn1p-GFP acquisition relative to SPB separation (Shaw et al., 1997; Segal et al., 2000b). To assess whether dosage suppression of the cdc28-4 clb5 mutant by spc11013 could be due to rescue of the spindle polarity defect, cdc28-4 clb5 HIS3:spc11013 cells expressing the Dyn1p-GFP fusion previously described (Shaw et al., 1997), were subjected to time-lapse analysis during spindle assembly. Fig. 8 shows a representative time-lapse sequence of this strain in which asymmetric Dyn1p-GFP acquisition was coupled to the correct establishment of spindle polarity (60% exhibited a lag in Dyn1p-GFP acquisition, n=15 cells recorded; Fig. 8B). By contrast, cdc28-4 clb5 HIS3:spc11013-36A91A exhibited mainly a symmetrical pattern of Dyn1p-GFP localisation coincident with SPB separation (5% showing a lag, n=19 cells recorded; Fig. 8C) as did the parental cdc28-4 clb5 strain (not shown) (Segal et al., 2000b).
Thus, spc11013 function as a dosage suppressor of cdc28-4 clb5 diploid lethality correlated with its ability to partially restore intrinsic SPB asymmetry and spindle polarity. Suppression, however, was critically dependent on the two CDK phosphorylation sites of Spc110p.
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Discussion |
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cells (Fig. 1D), and we could not measure a decrease in Clb5p-GFP label in this mutant that might reflect selective loss of association with the outer plaque.
Thus, Clb5p might be partly localised to the inner plaque of the SPB and to intranuclear MTs. In addition, Clb5p may be present at the bridge or half bridge to account for the persistent label at spindle poles in a cnm67 mutant. In contrast to this observation, association of Clb2p with SPBs is disrupted by a cnm67 mutation (Grava et al., 2006). We were unable, however, to detect Clb5p-GFP along astral MTs under any conditions. Nevertheless, our findings collectively support a direct role for Clb5p in the progression of the spindle pathway and the existence of Clb5p-dependent kinase substrates at the nuclear face of the SPB, in addition to supporting previously proposed targets at the outer plaque or astral MTs that mediate Cdc28p-Clb5p control of spindle polarity (Segal et al., 2000b; Liakopoulos et al., 2003; Maekawa et al., 2003; Moore et al., 2006).
Cell-cycle-dependent phosphorylation of Spc110p by CDKs
Loss of intrinsic spindle polarity renders cdc28-4 clb5 cells unable to generate viable homozygous diploids (Segal et al., 1998; Cross and Jacobson, 2000). We exploited this phenotype in a screen for high-dosage suppressors that might uncover putative targets of Clb5p-associated kinase. Alternatively, suppressors may bypass the perturbation in cdc28-4 clb5 cells thus providing mechanistic insight into the function of Clb5p in the establishment of spindle polarity. Surprisingly, the screen yielded a clone encoding a truncation of a previously identified component of the nuclear face of the SPB, Spc110p. Yet, in agreement with the premise of the screen, wild-type Spc110p might prove a substrate for Clb5p-dependent kinase. This provides further validation of our findings regarding Clb5p localisation. Moreover, the analysis of the mode of rescue of cdc28-4 clb5 by the dosage suppressor spc11013, as discussed below, hints to the actual role of Clb5p-dependent phosphorylation of Spc110p.
Previous studies implicated the kinase Mps1p in phosphorylation of Spc110p (Friedman et al., 2001). Here we show that, in addition, Spc110p phosphorylation levels were greatly diminished by overexpression of the phosphatase Cdc14p (Fig. 3B). Conversely, phosphorylation was enhanced by overexpression of Clb5p. This was apparent in a clb5 strain and more pronounced in clb3 clb4 clb5 or cdc28-4 clb5 mutants (Fig. 3C and supplementary material Fig. S1), supporting the notion of functional redundancy among B-type cyclins for phosphorylation of Spc110p in vivo. Two residues within CDK consensus sites, Ser36 and Ser91, contributed to bulk levels and cell-cycle-dependent phosphorylation of Spc110p starting in early S phase, even in the absence of Mps1p activity (Fig. 4). These data, together with a recent report (Loog and Morgan, 2005), favour the involvement of Cdc28p-Clb5p kinase in cell-cycle-dependent phosphorylation of Spc110p, independently of Mps1p activity. However, it remains possible that CDK also acts indirectly via Mps1p, as previously demonstrated for the SPB component Spc42p (Jaspersen et al., 2004).
We addressed the significance of CDK-mediated phosphorylation by mutational analysis. The most noticeable effect on cell-cycle distribution was observed in spc11091A cells, which exhibited a metaphase delay. We pursued the analysis of this mutant to assess spindle dynamics by digital imaging analysis. Profiles of spindle length distribution in synchronous populations as well as detailed time-lapse analysis pointed to a perturbation in spindle dynamics during early anaphase of spc11091A cells (Figs 5 and 6). The dynamic delay in early spindle elongation was abolished by deletion of MAD2 (Fig. 7). These findings indicated that phosphorylation at position 91 may be important for spindle integrity and normal progression of the spindle pathway. Previous mutational analysis targeting the N-terminal domain of Spc110p identified spc110-221 as a temperature-sensitive allele allowing for spindle assembly, yet, resulting in a MAD1-dependent arrest at the restrictive temperature (Sundberg and Davis, 1997). The two CDK sites characterised here are within the region mutated by this allele (coding multiple substitutions from positions 15 to 163 of the Spc110p sequence).
Mechanism of rescue of cdc28-4 clb5 diploid lethality by spc11013
The dosage suppressor spc11013 encoded a truncation that lacks the domain docking Spc110p at the central plaque of the SPB (Sundberg et al., 1996). Indeed, Spc11013p-GFP was localised to the nucleus without marking the SPBs or producing any aggregates (Fig. 2). Interestingly, neither a multicopy plasmid carrying wild-type SPC110 nor its overexpression under the GAL1 promoter suppressed cdc28-4 clb5 diploid lethality (not shown).
To address the mode of rescue, we examined intrinsic spindle polarity in cdc28-4 clb5 cells expressing Spc11013p, using the temporally regulated recruitment of a Dyn1p-GFP fusion at SPBs as readout. In principle, two classes of suppressors could be expected – those restoring spindle asymmetry (the `lag' in Dyn1p-GFP acquisition at the new SPB), and those enabling symmetric spindles to orient efficiently. spc11013 belonged to the first class (Fig. 8).
We have previously proposed that temporal control of astral MT organisation by the old and new SPBs in tight coordination with spindle assembly dictates spindle polarity (Segal and Bloom, 2001). Central to both processes is cell-cycle control of MT nucleation by the SPB. MT nucleation is initiated in vivo by -tubulin complexes in association with MT organising centres (Schiebel, 2000). The core components of this complex are conserved from yeast to mammals. In yeast, a Tub4p (-tubulin)-Spc98p-Spc97p complex is targeted to the outer plaque and (half) bridge via Spc72p and to the inner plaque via Spc110p (Knop and Schiebel, 1997; Knop and Schiebel, 1998). The truncation characterised here spans the Spc110p domain responsible for binding the -tubulin complex (Knop and Schiebel, 1998; Nguyen et al., 1998) and has been shown to interact with components of this complex even if unable to localise to the SPB (Sundberg et al., 1996). Thus, rescue of polarity in cdc28-4 clb5 cells could be explained by titration effects within the nucleus that restored the kinetic delay in MT organisation at the outer plaque. The analogous truncation encoding the binding domain for the -tubulin complex of Spc72p did not rescue cdc28-4 clb5 diploid lethality. It is possible, however, that titration can only be efficient from within the nucleus because truncated Spc72p was cytoplasmic (data not shown).
Importantly, the ability of spc11013 to act as a dosage suppressor of cdc28-4 clb5 diploid lethality was abolished by mutations cancelling the two CDK sites in Spc110p characterised here. Accordingly, the spc11013-S36A S91A mutated truncation did not restore SPB asymmetry in the Dyn1p-GFP fusion assay (Fig. 8). This is in contrast to the mild effects of the same substitutions for the functionality of full-length Spc110p. Even a spc110mps1-36A91A strain did not exhibit major spindle phenotypes or overt sensitivity to MT-depolymerising agents (not shown). Perhaps, in the context of a large structure with many phosphorylated components, further substitutions to abolish phosphorylation of multiple SPB substrates (of both CDKs and Mps1p) are necessary before fully penetrant phenotypes are observed. This has been the case, for example, in the genetic analysis of CDK-mediated control of the APC/CCdc20p (Cross, 2003).
Here we show that Spc11013p functions away from the SPB, a situation in which efficient binding of the -tubulin complex accounting for titration might be more reliant on CDK-dependent phosphorylation. By contrast, any effects on binding by full-length Spc11036A91Ap may only translate into slightly compromised spindle integrity. Finally, the rate of SPB separation and average spindle length at metaphase are increased in cdc28-4 clb5 cells (Segal et al., 2000b). High dosage of spc11013 in this strain slowed down SPB separation and decreased metaphase spindle length (data not shown) indicating that this construct also had the potential to interfere with organisation of nuclear MTs.
Our findings demonstrate that Clb5p-dependent kinase activity might enforce polarity by controlling an aspect of MT organisation imparting the correct delay at the new SPB, yet, the identity of the crucial target in this function at the outer plaque remains an open question. Nevertheless, the system described here provides a valuable tool for further dissecting the cell-cycle mechanisms controlling MT organisation at the SPB.
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Materials and Methods |
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; Segal et al., 1998). Strains carrying a cdc28-4 allele (Lörincz and Reed, 1986) and cyclin gene disruptions (Epstein and Cross, 1992; Richardson et al., 1992) were previously described (Segal et al., 1998). Deletion of SPC110, MAD2 and CNM67 was performed by replacing the open reading frame using KANR cassettes amplified by PCR (Wach et al., 1994). Strains expressing a Spc29p-CFP fusion were obtained by transformation with a SPC29:CFP:KANR cassette generated by PCR (Wach et al., 1997). Relevant genotypes of the strains used in this study are listed in supplementary material Table S1. Standard yeast genetic procedures were used (Sherman et al., 1986). Synthetic medium with supplements, or rich medium (YEP) contained 2% w/v dextrose or 3% w/v raffinose. Induction of GAL1-driven constructs was achieved by adding 3% w/v galactose. Yeast cultures were grown at 25°C unless stated. Quantitative yeast mating assays were performed as previously described (Segal et al., 1998), except that formation of viable diploids was assessed by scoring microcolonies under low magnification. Results were normalised to the frequency of zygote formation.
Screen for high-dosage suppressors of cdc28-4 clb5
Isolation of high-dosage suppressors of cdc28-4 clb5 lethality was carried out by screening a YEp24 yeast genomic DNA library (Carlson and Botstein, 1982) for plasmids improving diploid colony formation following mating as follows. After transformation of strain MY127 (a cdc28-4 clb5::ARG4 LEU2) with the library, colonies were replica-plated onto a lawn of strain MY128 ( cdc28-4 clb5::ARG4 TRP1) (Segal et al., 1998) prepared by spreading a suspension of the cells in a small volume of YEPD on plates of synthetic medium with relevant drop-outs for diploid selection at 23°C. Plasmids were recovered from colonies that confirmed plasmid linkage of the suppression. Clones containing CDC28, CLB5 and TRP1 were repeatedly isolated according to genetic analysis confirmed by restriction mapping. In addition, several partial suppressors were identified. In particular, pDLS13, improved diploid growth without otherwise suppressing temperature sensitivity of haploid cdc28-4 clb5 cells. Deletion analysis showed that the suppressing activity was dependent on a truncated SPC110 (encoding amino acids 1-437) at the 3' end of the library insert. This truncation is here referred to as Spc11013p.
Plasmids
Strains expressing a GFP-Tub1p or a Dyn1p-GFP fusion were obtained by transformation with pAFS91 linearised at the unique StuI site (Straight et al., 1997) or pKBY701 (Shaw et al., 1997; Segal et al., 2000b), respectively. The integrative plasmids pMST57 and pMSL42 were used for galactose-inducible expression of CLB5 (Segal et al., 1998).
pSMH1 was obtained by introducing a CLB5:GFP fusion under the control of the HIS3 promoter as a 2.5 kb EcoRI-SalI fragment into YIplac211 (Gietz and Sugino, 1988). The plasmid was linearised with EcoRV within URA3 in the vector for integration. pRS426-SPC110 carried a 3.1 kb BamHI-XhoI fragment generated by PCR encoding SPC110 in pRS426 (Sikorski and Hieter, 1989). pDLS13 was the original YEp24-based library clone identified as a high-dosage suppressor of cdc28-4 clb5 diploid lethality. This plasmid contained a 7790 bp insert ending after 1311 bases of the SPC110 ORF. Plasmid pCDC14-EMBLYex4 encoded a HIS6-tagged Cdc14p under the control of the inducible GAL1 promoter (Jensen et al., 2002).
Tagging constructs for this study were derived from pKGFP, pKHA3 or pKCFP (Jensen et al., 2002) by subcloning the tag cassettes into the indicated vectors of the plac (Gietz and Sugino, 1988) or pRS (Sikorski and Hieter, 1989) series. Plasmids YIplac211-SPC110tGFP and YIplac211-SPC110tHA3 contained a 1900 bp EcoRI-NotI fragment generated by PCR for 3' in-frame fusion to GFP or HA3 tags, respectively. Linearisation with SacI targeted these constructs for integration at the endogenous SPC110. pMFSG1 was a YIplac211-derivative in which a BamHI-SalI fragment spanning spc11013 fused to GFP was placed under the inducible GAL1 promoter. pMFSH1 was as pMFSG1 except that it expressed Spc11013p fused to HA3 under the control of the GAL1 promoter. These plasmids were linearised with StuI within URA3 in the vector for integration. Constructs for N-terminal tagging of Spc110p were generated as follows. pMFS1 was constructed by introducing a 3235bp KpnI-NotI fragment containing an HA3:SPC110 fusion under the control of the HIS3 promoter into pRS306. The HA3 tag was replaced by a XhoI-BamHI fragment encoding GFP to generate pMFS2. These constructs were linearised with StuI prior to transformation. pSMH8 was obtained by subcloning the KpnI-NotI insert of pMFS1 into pRS404. For integration, the plasmid was linearised with Bsu36I. pSMH24 was as pSMH8 except that the N-terminal HA3 tag was fused to the truncated spc11013.
Plasmids expressing Spc110p variants lacking CDK or Mps1p phosphorylation sites were generated by a two-round PCR-based strategy using the mutagenic oligonucleotides listed in supplementary material Table S2. Briefly, in a first round of PCR, the 3' end primer introduced the necessary mutations to encode serine to alanine substitutions at positions 36 or 91 of Spc110p. This product was extended and digested to produce a 793 bp BamHI-BglII fragment and used to replace the corresponding BamHI-BglII fragment of the wild-type sequence in pSMH8. spc11036A91A was generated by a second round of mutagenesis using the spc11036A sequence as template to introduce the mutation at position 91. pSMH25 was derived from pSMH24 to carry the HA3:spc110(1-1311) fusion mutated at positions 36 and 91. The allele spc110mps1 was created by introducing mutations cancelling Mps1 phosphorylation sites – S60A, T64A and T68A (Friedman et al., 2001) using the same strategy. The final constructs were linearised with Bsu36I within TRP1 in the vector prior to transformation of a spc110 strain carrying pRS426-SPC110. Plasmid shuffling was monitored by scoring for spontaneous loss of the URA3-containing pRS426-SPC110 to select for integrants carrying solely the mutated SPC110. All mutant constructs supported viability in the absence of SPC110 (not shown).
pRS406mps1-1t was used to introduce a mps1-1 allele in 15DaubA. The plasmid contained a 1291 bp SacI-XhoI fragment of the 3' end of MPS1 open reading frame spanning the site of the mutation in mps1-1 (Schutz and Winey, 1998) and 3' UTR amplified by PCR using yeast genomic DNA from strain 1381 W303 mps1-1 (kindly provided by M. Winey, University of Colorado, Boulder, CO). The construct was linearised with BamHI before transformation of 15DaubA cells.
Microscopy methods
Time-lapse recordings of cells mounted in selective medium containing 25% gelatin were carried out as previously described (Maddox et al., 1999; Huisman et al., 2004) using a Nikon Eclipse E800 with a CFI Plan Apochromat 100x, N.A. 1.4 objective, Chroma Technology filter sets and a Coolsnap-HQ CCD camera (Roper Scientific). Images were acquired using 2x2 binning. For cells expressing GFP fusions, five fluorescence images were acquired at a Z-distance of 0.8 µm between planes. A single differential interference contrast (DIC) image was taken in the middle focal plane. This acquisition regime was repeated at 30- or 60-second intervals. Images were processed using Metamorph software (Universal Imaging) (Maddox et al., 1999). Cells expressing Spc29p-CFP and Clb5p-GFP fusions were recorded according to a protocol that discriminates between CFP and GFP using a CFP/YFP filter set (Huisman et al., 2004). Three fluorescence images were acquired at a Z-distance of 0.8 µm at 30- or 60-second intervals. Recordings of cells expressing the Dyn1p-GFP fusion were performed by mounting the cells in synthetic glucose-containing medium, after a 2-hour incubation in galactose-containing medium, to limit the induction of the fusion (Segal et al., 2000b).
Spindle length profiles in synchronised cell populations were generated by measuring spindle length in digital images acquired as five Z stacks using Metamorph software. Cells were monitored during the time course to select the time-point closest to onset of spindle elongation (samples containing less than 10% of spindles longer than 3.5 µm).
Cell-cycle synchronisation
Time-course experiments following release from G1 arrest were carried out as follows. Cells in mid-log phase were diluted to 8x106 cells/ml and arrested in the presence of 300 ng/ml factor for 3 hours. After arrest was verified by microscopy, cells were rinsed and resuspended in prewarmed YEPD at the incubation temperatures indicated for each experiment. Aliquots were taken for preparation of cell extracts, as well as for microscopy and FACScan analysis at the indicated intervals.
Cultures of mps1-1 strains were instead grown at 23°C and synchronised at the same temperature. Cells were then preincubated at 34°C for 1 hour before release in fresh YEPD prewarmed to 34°C. Nocodazole arrest was induced by incubation of mid-log-phase cultures with 15 µg/ml nocodazole for 2 hours. In all experiments involving synchronisation, cell cycle progression was evaluated by FACScan analysis, quantification of spindle morphologies in cells fixed by incubation in 3.7% formaldehyde for 30 minutes or cells stained by DAPI as previously described (Clarke et al., 2001).
Lysate preparation and immunoblotting
Protein extracts were obtained using NP-40 buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 10 mM sodium pyrophosphate, 0.1 mM orthovanadate, 1 mM PMSF, 2 mg/ml aprotinin, leupeptin and pepstatin A) as previously described (Clarke et al., 2001). Protein samples were separated on 7.5% SDS-polyacrylamide gels. Western blots were probed with monoclonal antibody 16B12 (Babco) at 1:1000 dilution to detect Clb5p-HA3, with monoclonal antibody 12CA5 (Roche) at 1:1000 dilution to detect HA3-tagged Spc110p. His6-tagged proteins were detected using monoclonal antibody anti-HIS (Amersham) at 1:3000 dilution. GFP-tagged proteins were detected with a mixture of two monoclonal antibodies 7.1 and 13.1 (Roche) at 1:1000 dilution. -tubulin was detected with monoclonal antibody B-5-1-2 (Sigma) at 1:1000 dilution.
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* Present address: Haematology and Leukaemia Unit, St Vincent's Institute for Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia 
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45 minutes after the release from G1). Phosphorylation peaked at metaphase (see Friedman et al., 1996
