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First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02757
Research Article |
Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673
Author for correspondence (e-mail: mcbucs{at}imcb.a-star.edu.sg)
Accepted 24 October 2005
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cells is excessively dynamic and the nuclei move into mother-daughter junction prematurely. As a result, the chromatin mass undergoes partial division that requires neither anaphase promoting complex (APC) activity nor Scc1 cleavage. Partial division of the chromatin mass is accompanied by the loss of the centromeric region's ability to resist pole-ward pull by the spindle. Slk19 physically associates with Scc1 and this association appears necessary for efficient cleavage of Slk19 by separase. Our results suggest that Slk19 participates in regulating nuclear migration and, in conjunction with cohesin complex, may be involved in the maintenance of centromeric tensile strength to resist the pole-ward pull.
Key words: Slk19, Mitosis, Chromosome segregation, Kinetochore, Yeast
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).
Following DNA replication in S phase, duplicated chromosomes are prevented from premature segregation by the cohesin complex, which holds sister chromatids together until anaphase (Ciosk et al., 1998; Uhlmann et al., 1999). The cohesion between sister chromatids is also important for proper attachment of spindle microtubules to the chromosomes (Biggins and Walczak, 2003; Tanaka et al., 2000). In budding yeast Saccharomyces cerevisiae, the cohesin complex is comprised of Smc1, Smc3, Scc1 and Scc3 proteins (Michaelis et al., 1997). At the onset of anaphase, a cysteine protease called separase (encoded by the ESP1 gene) cleaves Scc1 and destroys cohesion between sister chromatids to initiate their separation (Uhlmann et al., 1999). However, the separase is restrained from action through most of the cell cycle by its binding to securin Pds1 (Ciosk et al., 1998). As cells transit from metaphase to anaphase, APCCdc20, a multi-subunit E3 ligase, targets securin for ubiquitylation-dependent degradation and sets the separase free to cleave Scc1 (Uhlmann et al., 1999). The initiation of anaphase is also under the control of the spindle checkpoint. In the event of spindle assembly defects or improper attachment of microtubules to the kinetochores, the checkpoint prevents activation of APCCdc20 and inhibits chromosome segregation (Lew and Burke, 2003).
Proper alignment and segregation of sister chromatids during mitosis depends on the amphitelic (bipolar) attachment of sister kinetochores to microtubules emanating from opposite centrosomes (spindle pole bodies in yeast). While multiple microtubules bind to each kinetochore in higher eukaryotes, a yeast kinetochore is estimated to attach to a single microtubule. Once bipolar attachment is established in mid to late S phase, sister kinetochores experience a pole-ward pull causing transient separation of centromeric chromatin termed `elastic deformation' of chromosomes (He et al., 2000; Tanaka et al., 1999). This force is presumably resisted by the cohesin complex, which holds sister chromatids together until the onset of anaphase. Consistent with this, cohesin binding sites have been mapped to pericentric DNA flanking the conserved centromeric DNA and along the chromosome arms (Blat and Kleckner, 1999; Megee et al., 1999; Tanaka et al., 1999; Weber et al., 2004). Transient separation of sister kinetochores prior to anaphase suggests that the cohesin complexes that reside proximal to the centromeric region are subjected to greater separating force (pole-ward pull) compared with the distal complexes in the chromosome arms. It is not clear whether cohesin complexes alone are sufficient to resist the pole-ward tug at centromeric region or auxiliary proteins are required to augment the resistance.
Kinetochores are large and dynamic protein assemblies on centromeric DNA whose composition varies considerably during the cell cycle (McAinsh et al., 2003). In budding yeast Saccharomyces cerevisiae, for example, Ipl1 (Aurora B kinase) and Sli15/INCENP, components of the central complex, localize initially to the kinetochores but relocate to the spindle mid-zone later in mitosis (Kang et al., 2001). Slk19 (the subject of this study) is also first localized to the kinetochore. At anaphase, it is cleaved by separase (at a site 77 amino acids from the N-terminus) and the larger of the cleaved products translocates to the spindle mid-zone (Sullivan et al., 2001). However, cleavage by separase does not appear to be essential for the translocation to the spindle. Though non-essential for viability, it has been suggested that Slk19 plays a role in the stabilization of the mitotic spindle (Zeng et al., 1999). Slk19 has also been identified as a component of the FEAR (Cdc Fourteen Early Anaphase Release) network, allegedly responsible for timely initiation of mitotic cyclin destruction (Stegmeier et al., 2002). However, the exact role of Slk19 in this pathway remains elusive. It also remains unclear what function, if any, Slk19 serves at the kinetochores.
Here we show that in Slk19-deficient cells, the chromatin mass becomes bi-lobed and stretched, and appears partially divided prior to the onset of anaphase (for convenience we refer to this phenotype as chromatin-mass deformation or CMD). This defect is greatly accentuated during G2-M delay/arrest and is accompanied by the loss of centromeric elasticity. Slk19 shows physical interaction with Scc1 both in vivo and in vitro. Interaction with Scc1 appears to be important for efficient cleavage of Slk19 by separase. We suggest that Slk19 plays an important role in the timing of nuclear migration. It may also participate, directly or indirectly, in the maintenance of centromeric tensile strength during mitotic stagnation (for instance, during activation of checkpoint controls) when cells need to preserve nuclear integrity until cell cycle progression can be resumed.
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cells, synchronized in G1 by 
factor treatment, were released at 24°C. While in wild-type cells the chromatin mass elongated briefly (110 minutes) and then rapidly underwent complete division, (Fig. 1), chromatin mass in slk19 cells elongated earlier (90 minutes) and lingered in elongated state (partial nuclear division) before resolving into two well-separated entities (Fig. 1, upper right panel and graph). To determine whether chromatin-mass deformation (CMD) in slk19 cells requires onset of anaphase, we examined the nuclei in slk19 cdc20 double mutant. Cdc20 is the activator of anaphase-promoting complex (APC) required for the initiation of nuclear division; hence CDC20-deficient cells are unable to trigger anaphase. When released into glucose medium from factor induced G1 arrest, cdc20 GAL-CDC20 mutant arrested with a single, round nucleus (Fig. 2A, top left panel). Surprisingly, 
90% of cdc20 slk19 GAL-CDC20 cells contained chromatin mass stretched out across the mother-daughter junction (Fig. 2A, upper right panel and graph). As expected, neither Scc1 cleavage nor Clb2 degradation was detected in either strain suggesting that neither anaphase nor mitotic exit had been initiated (Fig. 2A). Since Slk19 has been described as a kinetochore protein, it is possible that inactivation of other kinetochore proteins may also lead to CMD. We, therefore, have examined the nuclear division in a strain defective in Ndc10 function (a kinetochore component). cdc20 ndc10-1 GAL-CDC20 cells were subjected to experimental regime as described above. As expected, ndc10-1 cells assembled a bipolar spindle and fail to undergo nuclear division at the non-permissive temperature but did not exhibit chromatin mass deformation (Fig. 2A, bottom panel).
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These observations imply that chromatin mass deformation in slk19 cells occurs prior to anaphase initiation and does not require APC activity or Scc1 cleavage. Consistent with an earlier report (Zeng et al., 1999), the mitotic spindles in some cdc20 slk19 cells were abnormally short and in many cells took on an oblong dot-like appearance. However, staining with antibodies against SPB component Tub4 showed paired but clearly separated spots suggesting that these were short spindles (Fig. 2B). It is noteworthy that unlike cdc20 slk19 cells, cdc20 cells continue to enlarge while arrested at G2-M and eventually become almost 50% greater than the size of cdc20 slk19 cells.
Bipolar attachment in wild-type and slk19 cells
It is conceivable that the deformation of the chromatin mass in slk19 cells results from a splayed chromosomal arrangement at metaphase because shorter spindles in these cells are unable to establish bipolar attachment to kinetochores. A convenient indicator of bipolar attachment is the separation of sister chromatids at the centromeric region. We used a TetO/GFP-TetR system (Michaelis et al., 1997; Tanaka et al., 2000) to mark chromosome V centromeres (CENV proximal locus) with GFP; hence the appearance of two GFP dots is indicative of bipolar attachment. To compare the timing of sister centromeric separation in wild-type and Slk19-deficient cells, G1-arrested wild-type and slk19 strains carrying TetO/GFP-TetR constructs (US3437 and US3502) were allowed to resume cell cycle progression in fresh medium at 24°C. As shown in Fig. 3 (Graph), the timings of budding and centromeric separation are comparable in both strains suggesting that short spindles in slk19 cells are able to establish bipolar attachment. Two GFP dots are also seen in time points where chromatin mass is elongated in slk19 cells (Fig. 3, photomicrograph) implying that splaying of chromatin mass occurs despite bipolar attachment.
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cells arrested in G2-M using live cell imaging. We employed cdc20 to impose G2-M arrest since, unlike mutation in APC components such as Cdc16 or Cdc23, which also cause arrest in G2-M, cdc20 mutant cells do not exhibit any extra nuclear DNA movement and behave just as wild-type cells (Palmer et al., 1989). For live imaging, cdc20 GAL-CDC20 and slk19 cdc20 GAL-CDC20 cells expressing HTA2-EGFP (encoding histone H2A subtype) were used to allow visualization of the chromatin mass. G1-arrested cells were released into glucose medium at 24°C and subjected to time-lapse microscopy after the newly formed bud had grown to approximately half the size of the mother. As shown in Fig. 4, the nuclei in cdc20 cells maintained their round morphology and remained in the mother, as would be expected of cells arrested in G2-M. Throughout the recording, the nucleus remained locally mobile within the mother cell (as judged by its changing position with respect to the neck) but never entered the mother-daughter neck. The nuclei in cdc20 slk19 cells, conversely, transited rapidly between the round and elongated form but eventually entered the daughter compartment with loosely attached fragments of chromatin still remaining in the mother. As expected, both cdc20 and slk19 cdc20 cells remained firmly arrested in G2-M throughout the experiment and did not proceed to mitosis. These results suggest that in the absence of Slk19, the nucleus becomes more mobile and moves prematurely into the mother-daughter junction.
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cells is microtubule dependent cells prior to anaphase is caused by force exerted by the spindle. cdc20 GAL-CDC20 (US3398) and cdc20 slk19 GAL-CDC20 (US3399) cells were released from G1 arrest into glucose medium at 24°C with or without the microtubule depolymerizing agent nocodazole (Noc). While cdc20 cells arrested with single round nuclei irrespective of Noc treatment (Fig. 5A, panel a), cdc20 slk19 cells released in Noc-containing medium (precluding spindle formation) lost the CMD phenotype and arrested with undivided, round nuclei (Fig. 5A, middle section in panel b). In a parallel experiment, nocodazole was added after cdc20 slk19 GAL-CDC20 cells had arrested in G2-M (with short spindles and elongated nuclei) by growth in glucose medium. In this case the nuclei remained partially divided (Fig. 5A, bottom section in panel b). These observations suggest that partial nuclear division in slk19 cells results from the force exerted by the spindle. Moreover, the `chromatin deformation' is irreversible such that once introduced, it cannot be undone by removing the spindle. To determine where Slk19 is normally localized at the cell cycle arrest used in these experiments, cdc20 GAL-CDC20 cells expressing Slk19-GFP and Ndc10-ECFP (US4194) were arrested in G2-M by transferring them to glucose. Co-localization of Slk19 and the kinetochore component Ndc10 suggests that Slk19 is associated with kinetochores during Cdc20 depletion-induced arrest (Fig. 5A, panel c).
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To examine the spindle behavior more closely in the absence of Slk19, we determined the spindle length distribution in cdc20 GAL-CDC20 (US3398) and cdc20 slk19 GAL-CDC20 (US3399) strains. Cells were arrested at G2-M by growth in glucose, spindles were stained using anti-tubulin antibodies and spindle length was measured using Caliper Function (Metamorph). As shown in Fig. 5B, the spindle lengths show a wider distribution (0.8-2.2 µm) in slk19 cells compared with Slk19-proficient cells (2-3 µm). A wider distribution of spindle lengths may be a result of heightened mobility of spindles in Slk19-deficient cells. A small proportion of slk19 cells do contain spindles, which are of approximately the same length (or somewhat larger) as those in the Slk19-proficient cells. To observe spindle dynamics directly, we undertook live imaging of wild-type and slk19 cells expressing Spc42-GFP (US3786 and US4164). Spc42 is a SPB component; hence, the distance between the two Spc42-GFP spots can be taken as a measure of spindle length. Cells with closely spaced but visibly separated GFP spots were filmed until one of the spots entered the mother-daughter neck region and the distance between the GFP spots was measured every five minutes. Though spindles are generally shorter in slk19 cells (Fig. 5B) (Zeng et al., 1999), in this experiment we selected cells from both strains, which had approximately the same distance between Cdc42-GFP spots; this allowed us to measure spindle length with comparable accuracy. While spindle lengths in wild-type cells show relatively smaller oscillations around a mean length, spindles in slk19 cells show larger oscillations (Fig. 5C) suggesting that the spindle is relatively more dynamic in the absence of Slk19. This is consistent with the possibility of an involvement of Slk19 in spindle dynamics raised previously (Zeng et al., 1999). Although it is not immediately clear why absence of Slk19 at the kinetochore leads to an increasingly dynamic spindle (see Discussion), the increased `mobility' may contribute significantly to CMD observed in slk19 cells.
Loss of centromeric elasticity in Slk19-deficient cells
If the spindle is able to establish bipolar attachment to kinetochores and cohesion subunit Scc1 is not cleaved in G2-M arrested Slk19-deficient cells (Figs 2 and 3), what causes the nucleus to undergo partial division? One possibility is that cohesin complex assembly may be defective in Slk19-deficient cells; therefore centromeric cohesion is unable to resist pole-ward pull by the spindle. However, using a reciprocal immunoprecipitation regime we find that cohesin subunits Smc1 and Smc3 are physically associated with Scc1 in slk19 cells just as in wild-type cells (data not shown), suggesting that cohesin assembly is not grossly abnormal in slk19 cells. Alternatively, centromeric resistance to pole-ward pull prior to anaphase may require function of kinetochore proteins, such as Slk19, in addition to cohesin complex-mediated cohesion. As mentioned earlier, transient separation of centromeres (visualized by GFP-marked CEN proximal region) is a result of pole-ward pull experienced by them once bipolar attachment to the spindle microtubules has been established. The pre-anaphase separation of centromeres in wild-type cells is abolished the when spindle is disassembled by nocodazole (Haering and Nasmyth, 2003). This elastic behavior, which can be visualized as the merging of two distinct GFP signals into one upon nocodazole treatment, reflects the inherent tensile strength that prevents centromeric regions from irreversible deformation. To determine whether Slk19 deficiency causes loss of centromeric strength, cdc20 GAL-CDC20 and cdc20 slk19 GAL-CDC20 strains harboring TetO/GFP-Tet-R constructs (US3444 and US3448) were arrested in G2-M by growth in glucose. In both strains, 60% of cells show pre-anaphase separation of centromeres (Fig. 6A, left graph, –Noc). Upon nocodazole treatment, while centromere separation was seen in only 20% of cdc20 cells, centromeres remained separated in 60% of cdc20 slk19 cells implying that absence of Slk19 causes loss of elastic recoil of the centromeric region (Fig. 6A). Despite the loss of elasticity, the distance between the GFP marked centromeres in cdc20 slk19 cells is quite similar to that in cdc20 cells prior to nocodazole treatment (Fig. 6A, right graph).
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factor, hydroxyurea (HU) and nocodazole to arrest them in G1, early S phase and metaphase, respectively. Reciprocal immuno-precipitation (IP) experiments showed that Slk19 associates with the full-length Scc1 in asynchronous, G1, S and metaphase extracts (Fig. 6B, top left panel in section a). Likewise, both the full-length and the cleaved form of Slk19 can be immunoprecipitated with Scc1 (Fig. 6B, bottom left panel in section a). Controls for this experiment show that this interaction is specific (Fig. 6B, section b). Furthermore, truncation of the N-terminus 77 amino acids (1-77 amino acids cleaved by separase) appears to substantially reduce association of Slk19 with Scc1, implying that Scc1-Slk19 interaction is perhaps aided by the N-terminus but not exclusively dependent on it (Fig. 6B, section c). Consistent with the interaction between Scc1 and Slk19 in IP experiments, bacterially produced MBP-Slk19 fusion protein can efficiently bind Scc1-cmyc18 in cell extracts in a `MBP pull-down assay' (Fig. 6B, section d).
To determine the whether the N-terminus is important for Slk19's ability to provide tensile strength to the centromeres, cdc20 slk19 GAL-CDC20 cells harboring TetO/GFP-TetR constructs were transformed with a CEN plasmid expressing either wild-type Slk19 (US3986), separase-resistant Slk19 (R77E) (US4024) or Slk19(1-77) (US4025). Cells were arrested in G2-M by growth in glucose to allow spindle formation and transient separation of centromeres, followed by nocodazole treatment to destroy the spindle. Both wild-type Slk19 and non-cleavable Slk19 (R77E) restore the elastic behavior of centromeric regions to a significant extent (Fig. 7A). However, although Slk19 (1-77) is still able to localize to the kinetochores (data not included), it fails to alleviate the loss of elasticity (Fig. 7A). These experiments imply that the N-terminus of Slk19 is important for the maintenance of centromeric elasticity. We have noticed consistently that for some reason a CEN plasmid-borne wild-type Slk19 gene is not as efficient in restoring centromeric elasticity as the chromosomally integrated copy.
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slk19 mutant and were tested for their ability to restore centromeric elasticity. Only three of the four deletion versions [Slk19-(419-498), Slk19-(511-538), Slk19-(327-400)] were tested since we failed to obtain a KANMX version of slk19-(543-577). None of the three deletion constructs was able to efficiently restore the centromeric elasticity in Slk19-deficient cells (Fig. 7B, Table). Moreover, when GFP-fusions of these truncated versions of Slk19 were examined for their cellular location, none of them were able to localize to kinetochores (data not shown). Hence, these three coiled-coil domains in the C-terminus of Slk19 may be important for the interaction with Scc1 and this interaction is perhaps necessary for Slk19 to serve its function at kinetochores.
Scc1 binding to centromeric region and cleavage during cell cycle progression in the absence of Slk19
Keeping in view the physical interaction between Slk19 and Scc1, we asked whether absence of Slk19 alters the timing of Scc1 to cleavage by separase. To test this, cdc20 SCC1-cmyc18 GAL-CDC20 (US3910) and cdc20 slk19 SCC1-cmyc18 GAL-CDC20 (US3579) strains were arrested in G2-M by growth in raffinose at 25°C. Cells were then shifted to 16°C, galactose was added to induce expression of Cdc20, and Scc1 cleavage was monitored. We used a lower temperature for this experiment to monitor Scc1 cleavage over an extended period. As shown in Fig. 8A, the timing of the onset of Scc1 cleavage in both strains appears to be very similar. This experiment, of course, analyzes overall Scc1 cleavage and does not provide any specific information about the cleavage of CEN region-bound Scc1, where Slk19 is initially localized.
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cells because Scc1 binding to centromeric region is compromised in the absence of Slk19 function, resulting in weakening of centromeric resistance against pole-ward force. To test this we performed chromatin immunoprecipitation (ChIP) on extracts prepared from WT cells harboring SCC1-cmyc18 and SLK19-HA6 at their respective loci (US3779), and slk19 SCC1-cmyc18 cells (US3516). CEN3 and CEN16 were used as centromeric-target sequences and MET2 as target sequence in the chromosome arm (see Materials and Methods for sequence details). Exponentially growing cells were treated with 1% formaldehyde to crosslink Scc1 and Slk19 to the associated chromatin. Cell extracts were immunoprecipitated with anti-HA and anti-cmyc antibodies, and co-immunoprecipitated DNA was analyzed by PCR using centromeric- or chromosome arm-specific primers. Consistent with an earlier report (Zeng et al., 1999), PCR primers complementary to CEN3 and CEN16 gave clearly visible signals from co-immunoprecipitates obtained using anti-HA antibodies on extracts expressing Slk19-HA6 but no signal was seen with primers complementary to the arm-region (Fig. 8B, left panel). As expected, both sets of primers (centromeric and chromosome arm) gave good signals with co-immunoprecipitates obtained using anti-cmyc antibodies on extracts expressing Scc1-cmyc18 (the signal for Slk19 binding to CEN16 is somewhat weaker, but reproducible, compared with its binding to CEN3). However, absence of Slk19 did not significantly alter the cross-linking of Scc1 to the centromeric region (Fig. 8B, right panel), suggesting that Slk19 does not affect Scc1's binding to this region.
Effect of Scc1 inactivation on localization and cleavage of Slk19
Although Scc1 and Slk19 show physical interaction, the absence of Slk19 affects neither Scc1's binding to the centromeric region nor the timing of its cleavage by separase. In a reciprocal experiment, we tested whether inactivation of Scc1 influences Slk19 localization to the kinetochore or its cleavage by separase. Temperature sensitive scc1-73 and WT cells carrying SLK19-GFP (US4077, US2237) at its native locus were released from G1 arrest and allowed to resume cell cycle progression at 37°C. Slk19-GFP is seen at the kinetochores at 75 minutes in many cells and on the spindle at 90 minutes (Fig. 9A). By 90 minutes, approximately 50% of Slk19 is in the cleaved form (Fig. 9B). At 37°C, when Scc1 is inactivated, Slk19-GFP is at the kinetochore in many cells at 75 minutes; it translocates to the spindle in some cells by 90 minutes. However, the Slk19 cleavage is delayed substantially at 37°C in that noticeable cleavage is seen only after 150 minutes, although by this time the nuclei are already divided (Fig. 9). These results suggest that in the absence of functional Scc1, Slk19 cleavage is inefficient but its translocation to the spindle is not impaired. This is consistent with a previous report (Sullivan et al., 2001), which showed that Slk19 localization to the spindle does not require its cleavage by separase.
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Upon completion of DNA replication in S phase, the cohesion complex holds the sister chromatids together to ensure that chromosomes do not segregate prematurely under the influence of pole-ward force exerted by the spindle. The kinetochores (centromeric region) bear the brunt of the pole-ward pull and provide resistance to this force, presumably with the help of cohesins. However, it is not clear whether the cohesin complex alone is sufficient for this task or auxiliary proteins are required to augment the centromeric resistance. Pre-anaphase chromatin mass elongation (Fig. 1) and the loss of elastic recoil of the centromeric region in the absence of Slk19 (Fig. 6) may suggest its importance in providing tensile strength to the centromeric region (see below). Interaction of Slk19 with the cohesin subunit Scc1 (this study) and its genetic interaction with the components of alternative RFC complex (Mayer et al., 2004) involved in the establishment of sister chromatid cohesion are consistent with this notion. The N-terminal 77 amino acids (cleaved by separase at the onset of anaphase) seem necessary for the maintenance of centromeric strength and help in its interaction with Scc1 (Figs 6 and 7). Scc1-Slk19 interaction, though intriguing, is physiologically relevant since Slk19 cleavage by separase is significantly altered in the absence of Scc1 function (Fig. 9). Hence, it is tempting to imagine that separase action on Slk19 and Scc1 at anaphase deprives centromeres of both resistance to pole-ward pull and the cohesion leading to the smooth separation of sister chromatids. SLK19 deficiency causes gross chromatin mass deformation under the influence of the spindle, which shows increased mobility in the absence of Slk19 (Fig. 5). It is possible that the highly dynamic nature of the short spindle in SLK19-deficient cells is a result of weakened resistance at the kinetochores, eventually causing the nucleus to move untimely into the neck (also see below). Nevertheless, the cohesion complex is the main tethering agent holding the sister chromatids together prior to anaphase since inactivation of cohesion complex alone by growing cdc20 scc1-73 at 37°C leads to premature nuclear division (data not shown). Hence, we surmise that while the cohesion complex helps to fasten sister chromatids together, Slk19 may help to strengthen the `tether' at centromeric region, enabling it to counter the pole-ward pull by the spindle.
In vertebrates, cohesins dissociate from chromosome arms during mitotic prophase under the influence of Polo and Aurora B kinases but the centromeric cohesins remain bound, keeping the sister-chromatids together until anaphase onset (Losada et al., 1998; Haering and Nasmyth, 2003). Similarly in meiosis I, cohesion between arms of a homologous pair dissolves but centromeric cohesion between sister chromatids is protected from cleavage until anaphase of meiosis II (Kitajima et al., 2004). These observations imply that some aspects of cohesion at centromeres may be modulated differently from that along chromosome arms. Pre-anaphase deformation of chromatin due to pole-ward pull by kinetochore-microtubules may also exemplify atypical nature of cohesive force at centromeric region. Chromosome stretching leading to transient sister separation at centromeres has been correlated to the compaction of centromere proximal DNA, which is inferred to have a substantially lower packing ratio (with reference to B-form DNA) compared with bulk chromatin (He et al., 2000). Although it may be attractive to attribute the centromeric elasticity to the packing ratio of centromeric DNA and the cohesion complexes present in this region, contributions from other proteins present in this region cannot be discounted. The facts that loss of centromeric elasticity in slk19 cells occurs in the absence of Scc1 cleavage and that SCC1 binding to the CEN region is not significantly altered (at least as estimated by CHIP assays) implies that the cohesin complex, while tethering sister-chromatids to each other, may not be sufficient to endow centromeres with the tensile strength to resist pole-ward pull by the spindle microtubules. Since Slk19 binds centromeric DNA (Zeng et al., 1999) and also interacts with Scc1 (this study), it is possible that elastic behavior emerges from this unique interaction between centromeric DNA, the cohesin complex and Slk19. It is likely that other centromere-associated proteins also participate in varying degrees in rendering elasticity to centromeres. Recent studies using biophysical and biochemical techniques have identified new kinetochore complexes (MIND and COMA) that appear to be important for kinetochore assembly (Wulf et al., 2003). Some of the proteins in these assemblies are proposed to bridge the subunits directly in contact with DNA with those associated with microtubules. Slk19 exists in a tetramer and appears to interact with Nnf1p, a subunit of the MIND complex.
It should be noted that the CMD phenotype of slk19 cells reported here appears similar to the nuclear migration phenotype reported in FEAR pathway defective mutants in a previous study (Ross and Cohen-Fix, 2004). The tendency of the undivided nucleus to move into the daughter cell in FEAR mutants was considered to be an anaphase event by Ross and Cohen-Fix (Ross and Cohen-Fix, 2004). Since Slk19 is a component of the FEAR network (Stegmeier et al., 2002) and in our study the CMD phenotype of slk19 strain was observed in cells arrested in metaphase (due to Cdc20 depletion), it can be argued that chromatin mass deformation is the manifestation of the early stages of nuclear migration due to overall failure of the FEAR network (Ross and Cohen-Fix, 2004). However, we find that unlike slk19 strain, cells lacking Cdc14 or Spo12 function (both FEAR pathway components) do not show chromatin mass deformation even when held for extended periods at metaphase (data not shown). These observations together with the fact that CMD was observed in metaphase arrested cells (a stage when FEAR pathway is presumably inactive) imply that the CMD phenotype is most likely not due to the failure of FEAR network per se; instead they may hint at a new Slk19 function at the centromeric region.
What is the causal relationship between the chromatin mass elongation (partial nuclear division) and the loss of centromeric elasticity seen in slk19 cells stalled in G2-M? One possibility, as argued above, is that the loss of centromeric elasticity renders the kinetochore unable to resist pole-ward pull by the spindle, resulting in deformation of the chromatin mass. Alternatively, CMD may be caused primarily by the premature entry of the chromatin mass into the mother-bud neck in the absence of Slk19. This gross physical distortion of the chromatin could disrupt the normal `force distribution' (which involves kinetochores, the spindle, spindle poles and cytoplasmic microtubule), leading to the loss of centromeric elasticity. However, our results at present cannot distinguish between these possibilities. Since Slk19 is present at kinetochores and spindle mid-zone, albeit at different times in the cell cycle, it can perhaps influence the behavior of both the kinetochores and spindle. It is possible that the chromatin mass deformation we observe in the absence of Slk19 is not a result of altered behavior of only one of these structures but a combined effect of changed dynamics between the spindle, spindle poles, kinetochore and the movement of chromatin across the mother-daughter neck. Precisely how Slk19 deficiency may bring about these changes is an issue, which is at present experimentally difficult to address.
Although centromeric DNA loses its elasticity and the chromatin mass prematurely elongates in the absence of Slk19, slk19 cells remain viable. Is maintenance of centromeric resistance to spindle forces, then, at all necessary for cell survival? During unimpeded progression through the cell cycle, cellular events happen in fairly rapid succession once cells enter mitosis. Under these circumstances, partial loss of centromeric elasticity may not have severe consequences. However, during mitotic stagnation (such as checkpoint induced delays), loss of centromeric resistance to pole-ward pull leading to visible nuclear distortion (Fig. 1) may compromise the cells' fitness when they resume progression through the cell cycle. We have observed that cells recovering from mitotic arrest in the absence of Slk19 function do show a discernible loss of viability (data not shown). Despite its non-essentiality, our results suggest a novel function for Slk19 at the kinetochores. However, the precise nature of the interaction of Slk19 with the cohesin complex and the mechanism by which this interaction gives rise to the emergence of centromeric tensile strength remains to be elucidated. We suggest that the elastic nature of the centromeric region is important for its function in maintenance of the tension-ridden state of the metaphase spindle and envisage that protein(s) at kinetochores serving a function similar to that of Slk19 in yeast may exist in other organisms.
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Materials and Methods |
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Strains and plasmids
A combination of standard molecular biology and molecular genetic techniques such as gene transplacement, gene disruption, PCR-based tagging of endogenous genes (Knop et al., 1999) and tetrad dissection were used to construct plasmids and strains with various genotypes (shown in Table 1). Southern blot and/or PCR analysis was performed to confirm gene disruptions and transplacements.
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Synchronization by treatment with factor or nocodazole
For experiments requiring synchronous cultures, exponential phase cells were grown in medium at 24°C containing either 1 µg/ml factor (for bar1 cells) or 5 µg/ml factor (for BAR1 cells). After 3.0-3.5 hours of treatment, cells were filtered, washed and resuspended in fresh medium pre-incubated at the appropriate temperature. For nocodazole treatment, cells were treated with 15 µg/ml of nocodazole for 1.5 hours. Samples were then taken at specific intervals for western blot analysis, flow cytometry and immunofluorescent staining.
Cell extracts, immunoprecipitation and western blot analysis
Preparation of cell extracts for immunoprecipitations and western blot analysis, and precipitation of proteins by TCA were done as described before (Yeong et al., 2000). MBP and MBP-Slk19 fusion proteins were made in E. coli. Recombinant proteins were purified and bound to amylose resin beads according to protocols provided by New England Biolabs. For western blot analyses, immunodetection of Cdc28, Clb2 and tubulin was carried out using anti-Cdc28 polyclonal antibodies (1:1000 dilution), anti-Clb2 polyclonal antibodies (1:1000 dilution) and anti-tubulin polyclonal antibodies (YOL1/34 from Serotech, 1:1000 dilution), respectively. Enhanced chemiluminescence kit from Santa Cruz was used for all western blot analyses according to the manufacturer's instructions.
Chromatin immunoprecipitation
Yeast cells were grown to OD600=1.0, formaldehyde (final concentration 1%) was added and cells shaken for another 30 minutes. Glycine (125 mM) was added. After 5 minutes, cells were pelleted, washed twice in ice-cold PBS and were broken using glass beads. The lysate was briefly sonicated to give DNA fragments between 500 bp and 1 kb. Cross-linked chromatin was divided into two portions. One portion was used as input DNA while the other half was used for immunoprecipitation with anti-HA beads. A parallel set of cross-linked chromatin was divided into two portions. One portion served as a negative control (no antibody). The other half was immunoprecipitated with anti-myc beads. The samples were incubated at 65°C overnight and treated with RNase A. The input DNA and co-immunoprecipitated samples were analyzed with PCR (24 cycles) using pairs of primers corresponding to the following loci; CEN3: 5'-ATCAGCGCCAAACAATATGG-3' and 5'-GAGCAAAACTTCCACCAGTA-3', CEN16: 5'-TTGAAGCCGTTATGTTGTCG-3' and 5'-TACCATGGTGTGTCACTTCC-3', MET2: 5'-AGATCCCAACTACTTGGACG-3' and 5'-GGACACCACGCTTTGACCTT-3' (Zeng et al., 1999). PCR products were run on 2% agarose gels and visualized with ethidium bromide.
Flow cytometry, anti-tubulin staining and visualization of GFP signals
Flow cytometry was performed according to Lim et al. (Lim et al., 1996). To visualize signals of the GFP fusion proteins, cells collected at various time-points were frozen immediately on dry ice without fixation and stored until further use. Cells were later thawed and mounted on slides with Vectashield containing DAPI (Molecular Probes). The images were captured using a Leica DMRX microscope attached to a Hamamatsu charge-coupled device camera driven by the Metamorph software (Universal Imaging Corporation). Anti-tubulin staining was done according to Lim et al. (Lim et al., 1996).
For time-lapse imaging, the cells were mounted onto glass slides coated with gelatin containing 2% glucose in low immunofluorescence yeast nitrogen base with complete drop out medium supplemented with adenine. GFP signals were sampled every 4 minutes. For each time point, seven Z-sections (0.5 µm apart) were obtained. A time-stack of these projections was compiled from the Z projections of these planes using Metamorph software.
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