Correspondence to Jeff Bachant: jeffbach{at}citrus.ucr.edu
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Introduction |
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In an unperturbed cell cycle, regulation of preanaphase spindle length is achieved through two processes. First, the outward force of Cin8 and Kip1 is balanced by motors that promote spindle contraction (Saunders et al., 1997). Second, cohesion between replicated chromatids allows the pulling of bi-oriented kinetochores (KTs) toward opposite spindle poles to generate a form of traction that limits spindle extension. Yeast centromeres (CENs) replicate early in S phase, and DNA synthesis is completed before SPBs separate (McCarroll and Fangman, 1988). This timing ensures that, similar to the situation in other eukaryotes, chromatid pairs engage the spindle in a bipolar fashion during spindle assembly. Once all chromatids align, an inhibitory signal from the spindle checkpoint that monitors bi-orientation is relieved. The anaphase-promoting complex (APC) then targets Pds1/securin for ubiquitin-mediated degradation (Cohen-Fix et al., 1996). Pds1 proteolysis allows Esp1/separin to cleave the Mcd1/Scc1 cohesin subunit, triggering chromatid disjunction and spindle elongation (Ciosk et al., 1998).
Additional mechanisms are necessary to couple spindle extension to anaphase onset when the relative timing of S phase and spindle assembly is perturbed. After treatment with the ribonucleotide reductase inhibitor hydroxyurea (HU), for example, two protein kinases, Mec1 (homologue of vertebrate ATM/ATR) and Rad53 (homologue of vertebrate Chk2), control S phase checkpoint responses that coordinate chromosome replication and segregation. One facet of the S phase checkpoint promotes DNA synthesis in the face of nucleotide depletion by preventing fork collapse and by delaying the firing of origins of replication that are activated in mid to late S phase (Santocanale and Diffley, 1998; Lopes et al., 2001). Another facet controls a cell cycle arrest mechanism that prevents spindle extension (Allen et al., 1994; Weinert et al., 1994). Due to the apparent similarity of spindle extension in HU-treated mec1 and rad53 mutants to anaphase spindle elongation, it has been thought that the S phase checkpoint prevents spindle extension by preventing anaphase entry. In response to various forms of DNA damage that do not overtly stall DNA replication, Mec1 has in fact been shown to block anaphase through two pathways that make Pds1 resistant to APC proteolysis. One pathway, regulated by Rad53, prevents Pds1 recognition by the APC specificity factor Cdc20 (Agarwal et al., 2003). The other pathway, controlled by the Chk1 kinase, phosphorylates Pds1 to block ubiquitination (Wang et al., 2001). However, cell cycle arrest after HU treatment is independent of Pds1, suggesting an alternative mechanism to Pds1 stabilization restrains spindle extension during early S phase (Yamamoto et al., 1996; Clarke et al., 1999, 2003). The nature of this mechanism remains an unresolved issue in yeast cell cycle control.
Here, we present evidence that the S phase checkpoint controls cell cycle arrest not by inhibiting anaphase but by allowing the bi-orientation of replicating chromosomes to generate an inward force within the spindle that prevents extension during S phase distress. Cell imaging reveals that HU-treated rad53 mutants undergo cycles of spindle extension and collapse that are dissimilar to anaphase spindle elongation. In addition, S phase checkpoint arrest is independent of cohesin, indicating restraint of spindle extension during S phase is distinct from inhibition of anaphase. Three observations suggest that bipolar chromosome attachments provide a force to prevent defective extension during HU arrest and that Rad53 plays a fundamental role in promoting these connections. First, mutants defective for the KT proteins Ask1, Mif2, Ndc10, and Ndc80 and the KT regulator Ipl1 exhibit spindle extension after HU treatment. Second, a dicentric chromosome rescues spindle extension in HU-treated rad53 cells. Third, introducing as few as three minichromosomes where an origin of replication is placed in close proximity to a CEN is sufficient to block the rad53 spindle extension phenotype. These findings suggest a model in which the early replication timing of yeast CENs is a functionally significant aspect of cell cycle control. According to this view, the conserved role of checkpoint signaling in stabilizing DNA replication complexes ensures CEN duplication during S phase distress. Replicating chromosomes can then bi-orient and generate the traction necessary to restrain untimely spindle extension.
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Results |
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rad53 mutants treated with HU undergo dynamic alterations in spindle length
We continued our analysis of the rad53 S phase checkpoint defect by visualizing spindle extension using live cell imaging (Videos 15, available at http://www.jcb.org/cgi/content/full/jcb.200412076/DC1). WT and rad53-21 SPC42-GFP strains were released from G1 arrest into 200 mM HU media. Filming was initiated once spindle extension was occurring in 25% of the population, allowing visualization of the initial extension of the spindle in some cells and subsequent changes once spindle extension had occurred in others. In HU-arrested WT cells, spindle length remained relatively constant at
2 µm, although translocations of the spindle within the cell were observed (Fig. 2 A). Spindle translocations were also observed in rad53 mutants, but were accompanied by SPB separation at rates comparable to the 0.5 µm/min observed during the rapid phase of anaphase B (Straight et al., 1997; Fig. 2, BD). Unexpectedly, spindle extension was in fact reversible, with SPBs contracting at rates similar to the rate of extension. This cycle was variable, with seemingly stochastic conversions between extension and collapse. These dynamics account for the continuum of spindle lengths in HU-treated rad53 cells and are distinct from unidirectional spindle elongation during anaphase.
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Many KT-defective mutants exhibit perturbations to preanaphase spindle integrity characterized by defective extension (Jones et al., 1999; Goshima and Yanagida, 2000; Enquist-Newman et al., 2001; Janke et al., 2002; Nekrasov et al., 2003). However, there is considerable variability in this phenotype. For example, a recent paper examined a collection of dam1 mutants and found that different alleles exhibited phenotypes ranging from cell cycle arrest with typical preanaphase spindle morphology to an aberrant arrest accompanied by spindle extension (Cheeseman et al., 2001). This same paper found that dam1 mutants do not exhibit a loss of spindle integrity during HU arrest. However, based on our results with ask1-3 and mif2-2, we hypothesized that KT mutants exhibiting spindle extension when cell cycle progression was delayed in metaphase might also display extension during HU arrest. To test this hypothesis, we constructed a panel of cdc23-1 KT-defective double mutants. After release from G1 at a cdc23 nonpermissive temperature, we could then compare the effect of a KT-defective mutation on spindle length during either S phase arrest (by adding HU) or metaphase arrest (by withholding HU). It has been proposed that the KT assembles from protein complexes defining inner, medial, and outer KT regions (Cheeseman et al., 2002). Therefore, we examined mutations affecting the inner CBF3 complex (ndc10-1, ndc10-2, and ctf13-30), the medial Ndc80 complex (ndc80-1), and the outer Dam1/DDD/DASH complex (dam1-1 and duo1-2).
In the absence of HU, cdc23-1 mutants arrested with 24-µm metaphase spindles (average 2.9 ± 0.6 µm), whereas cdc23-1 strains released in the presence of HU displayed the shorter spindles typical of S phase checkpoint arrest (average 2.0 ± 0.5 µm; Fig. 7). Both with and without HU treatment, cdc23-1duo1-2, cdc23-1ctf13-30, and cdc23-1ndc10-2 strains arrested similarly to cdc23-1 controls (unpublished data). Thus, these mutants fall in the anticipated class that do not perturb preanaphase spindle integrity. In contrast, cdc23-1ndc10-1, cdc23-1ndc80-1, and cdc23-1dam1-1 mutants all displayed spindle extension at the cdc23 block. Of these, cdc23-1ndc10-1 and cdc23-1ndc80-1 mutants also displayed untimely extension after HU treatment. A relatively small population (12%) of cdc23-1dam1-1 cells treated with HU did in fact exhibit 3-µm spindles. However, there was much greater requirement for Dam1 after completion of S phase, with 92% of cdc23dam1-1 cells exhibiting spindle extension.
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Discussion |
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Chromosome bi-orientation generates S phase spindle traction
Chromatid cohesion is normally an important determinant of spindle length because it allows KT bi-orientation to offset the outward forces driving spindle extension. However, because CENs replicate early in S phase in Saccharomyces cerevisiae (McCarroll and Fangman, 1988), it is possible that KTs attach to the spindle in a bipolar fashion even before establishing cohesion during DNA replication distress. In this case, properties of a CEN-spanning replication bubble (such as intertwining of DNA strands) would presumably substitute for cohesion in tolerating tension on the chromosome fiber. KT bi-orientation normally provides the signal to induce anaphase entry. During early S phase, bi-orientation would not necessarily lead to the same outcome because it is unlikely that all chromosomes would be able to bi-orient (see the following section), and the machinery involved in triggering anaphase does not yet appear to be operational (Yamamoto et al., 1996; Clarke et al., 2003). Starting with our Esh screen, we in fact identified mutations affecting four KT proteins, Ask1, Mif2, Ndc10, and Ndc80, that engender spindle extension after HU treatment. These proteins participate in interactions that link CEN DNA to spindle MTs (Cheeseman et al., 2002), suggesting a mechanical requirement for KTspindle attachment during HU arrest. The observation that Ipl1 is also required to prevent S phase spindle extension further suggests KTs must connect to the spindle in a bipolar fashion. Thus, the simplest interpretation is that replicating chromosomes generate bipolar spindle traction in a cohesin-independent fashion, and this force is required to appropriately regulate spindle length in HU-arrested cells.
We note that there is considerable variability in whether or not KT-defective mutants exhibit spindle extension during HU arrest. One explanation for this variability is that increasingly severe alleles compromise KTMT interactions to a point where insufficient traction is generated to restrain extension. In support of this idea, KT-defective mutants exhibiting spindle extension after HU treatment also tend to exhibit extension when arrested in metaphase. Thus, the KT appears to be fulfilling similar roles in both the cohesin-independent and -dependent pathways restraining spindle extension. One apparent exception to this tendency is Dam1. In agreement with a previous paper, we find that Dam1 plays a more prominent role in restricting spindle extension after completion of DNA replication (Cheeseman et al., 2001), suggesting KTspindle connections are regulated differently or subjected to different forces once cohesion is established.
Spindle length is controlled not only by KT bi-orientation but also by modulating spindle dynamics. Indeed, while this manuscript was under consideration, an independent paper provided evidence that Cin8 and Stu2, a regulator of MT dynamics that promotes spindle extension, accumulate 25-fold in mec1 mutants after HU treatment (Krishnan et al., 2004). Inactivation of Cin8 and Stu2 reduced mec1 spindle extension, and overproduction of Cin8 forced spindle extension during HU arrest. Using a GFP tag adjacent to CEN5, the separation of CEN-proximal regions that accompanies KT bi-orientation during metaphase was not observed during HU arrest, prompting a model in which the S phase checkpoint compensates for an inability to bi-orient replicating chromosomes by down-regulating effectors of spindle extension. In an effort to reconcile these findings with our own conclusions, we note that CENs on different chromosomes do not replicate uniformly in HU (Yabuki et al., 2002), and variation in CEN bi-orientation between chromosomes might be expected. For example, it has been reported that 29% of CEN15-GFP cells exhibit bi-orientation after HU treatment (Goshima and Yanagida, 2000). Furthermore, KT bi-orientation during S phase might be difficult to visualize using GFP chromosome tagging if topological intertwining interfered with CEN separation. A second consideration is that inhibiting outward force production within the spindle is limited by the requirement to offset spindle collapse (Saunders and Hoyt, 1992). Therefore, we suggest KTspindle attachments restrict spindle extension during S phase checkpoint arrest, but the inward force afforded by these connections is likely to be reduced compared with metaphase cells. Therefore, it may be important to simultaneously reduce the force driving spindle extension; such an uncoupling of spindle dynamics and chromosome bi-orientation may underlie the cycles of spindle extension and collapse in HU-treated rad53 mutants documented in this paper.
CEN replication as an effector of S phase checkpoint arrest
Although our data suggests chromosome attachments to the spindle prevent extension during early S phase, several observations indicate checkpoint signaling is unlikely to play a direct role in KTspindle connections. In particular, the chromatin stretching that accompanies dicentric activation in HU-treated rad53 mutants argues that KTMT attachments form and withstand tension. Dicentric activation actually reduces rad53 spindle extension, suggesting chromosome bridging can provide a surrogate source of traction that compensates for the rad53 defect. If this interpretation is correct, it may be surprising that we see a >50% rescue of the rad53 phenotype. As dicentric KTs presumably connect equally to the same or opposite pole, 50% suppression should be maximal. Because Ipl1 is active in HU-treated rad53 mutants and spindles cycle between extension and collapse, each cell may experience multiple opportunities to bridge the dicentric.
If Rad53 is not required for KTspindle attachment, how could the S phase checkpoint be involved in generating spindle traction? Our observations suggest similarities between spindle extension in HU-treated rad53 mutants and the reductional anaphase of DNA replication mutants (Piatti et al., 1995). A recent paper has shown that the origins closest to all 16 CENs do in fact fire after treatment with 200 mM HU and that most CENs have a variable probability of replicating before fork stalling (Yabuki et al., 2002). Therefore, at the time HU-treated rad53 mutants initiate spindle extension, S phase checkpoint proficient cells would have replicated some, but not all, CEN sequences. Because a critical and conserved aspect of Rad53 function is to prevent convergent replication fork collapse (Lopes et al., 2001), a simple model for S phase checkpoint cell cycle arrest is that Rad53 promotes CEN replication while spindle assembly is ongoing. After KT assembly, replicating chromosomes could then bi-orient and restrict extension beyond appropriate preanaphase spindle length. This contingency plan would be supplanted as cells overcame the replication interference, established cohesion, and progressed toward metaphase. Our finding that multiple CENARS minichromosomes suppress spindle extension in HU-treated rad53 mutants, and that this suppression requires both initiation of DNA replication and KT function, provides strong support for a model in which Rad53 promotes cell cycle arrest by controlling CEN replication. If this model is correct, a key prediction is that the Rad53 substrates involved in preventing spindle extension will prove to be identical to the substrates controlling replication fork stabilization during HU treatment.
It has been known for some time that mammalian cells induced to enter mitosis without completing S phase generate KT fragments that align and segregate on the spindle (Brinkley et al., 1988). Similarly, recent evidence from yeast suggests that the primary role of cohesin in facilitating KT bi-orientation is to allow proper chromosome tensioning; in experimental situations, this function can be supplied by other forms of chromatid association such as topological intertwining (Dewar et al., 2004). These observations suggest that there is actually significant latitude in how KT bi-orientation is achieved. One implication of the work presented here is that budding yeast may have exploited this flexibility to accommodate a cell cycle in which spindle assembly and DNA replication are initiated simultaneously. Such a mode of mitotic control would necessitate a robust mechanism to ensure CEN replication when replication fork progression was impeded, providing a selective advantage to a chromosome architecture in which CENs were closely flanked by early firing origins of replication.
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Materials and methods |
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Microscopy and cell analysis
Ethanol-fixed cells were stained for flow cytometry with propidium iodide. Cells were processed for -tubulin immunofluorescence using YOL1/34 (Accurate Scientific) and FITC-conjugated antirat secondary antibodies (Sigma-Aldrich). 1 µg/ml DAPI staining was performed in mounting media (Vecta-Shield; Vector Laboratories). SPC42-GFP cells were fixed in 4% formaldehyde for 5 min. The distance between SPC42-GFP foci was quantified using MetaMorph software. Time-lapse imaging was performed by transferring cells to 2% agarose media pads in a depression chamber slide. Cells were visualized at ambient temperature on an upright microscope (model E800; Nikon) using a 100x Plan Apo 1.4 NA objective. Epifluorescence excitation was performed using a 100-W Hg bulb and a standard FITC bandpass filter cube; a number 8 neutral density filter was used to minimize photobleaching and toxicity. Samples were simultaneously visualized with low levels of bright-field illumination. Stacks of images were acquired at 2-min intervals using a CCD camera (model CoolSNAP fx; Photometrics) and exposure times of
200 ms/frame. The focus drive and camera shutters were controlled using the acquisition features of MetaMorph. The distance between SPC42-GFP foci was measured within image stacks using the MetaMorph XYZ application. Maximum projections were processed using the "flatten background" algorithm.
Protein techniques
For analysis of Clb2-HA Cdk1, 10 ml of cell cultures at OD600 0.8 were removed for each time point and washed into 1 mM NaN3, 10 mM EDTA, and 0.9% NaCl. Protein extracts were prepared as described previously (Bachant et al., 2002). 100 µg of the extract was incubated with a 1:100 dilution of 12CA5 -HA antibody for 12 h at 4°C. Immune complexes were collected using protein Aconjugated Sepharose beads and washed twice in lysis buffer and once in kinase buffer (20 mM Tris 7.5 and 7.5 mM MgCl2). Kinase reactions were assembled by resuspending the immunoprecipitates in 20 µl of kinase buffer, 2 µl of 1 mg/ml histone HI (Sigma-Aldrich), and 1 µl of
-[32P]ATP (NEN Life Science Products). After incubation at 37°C for 1 h, 4 µl of 6x SDS sample buffer was added and proteins were resolved by SDS-PAGE. Radioactivity incorporated into histone HI was quantified by PhosphoImager (Molecular Dynamics) analysis.
Online supplemental material
Videos 15 contain time-lapse sequences of spindle pole dynamics in WT and rad53-21 mutants exposed to HU. Fig. S1 depicts the kinetics of spindle extension and sister chromatid separation in mif2 and mif2mad2 mutants. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200412076/DC1.
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Acknowledgments |
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This work was supported by a grant from the National Institute of General Medical Sciences (GM-66190) to J. Bachant.
Submitted: 13 December 2004
Accepted: 22 February 2005
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