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Article |
Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase
Correspondence to Luis Aragón: luis.aragon{at}csc.mrc.ac.uk
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Mitotic cell division involves the equal segregation of all chromosomes during anaphase. The presence of ribosomal DNA (rDNA) repeats on the right arm of chromosome XII makes it the longest in the budding yeast genome. Previously, we identified a stage during yeast anaphase when rDNA is stretched across the mother and daughter cells. Here, we show that resolution of sister rDNAs is achieved by unzipping of the locus from its centromere-proximal to centromere-distal regions. We then demonstrate that during this stretched stage sister rDNA arrays are neither compacted nor segregated despite being largely resolved from each other. Surprisingly, we find that rDNA segregation after this period no longer requires spindles but instead involves Cdc14-dependent rDNA axial compaction. These results demonstrate that chromosome resolution is not simply a consequence of compacting chromosome arms and that overall rDNA compaction is necessary to mediate the segregation of the long arm of chromosome XII.
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Introduction |
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Chromosome XII is the largest chromosome, with 1 Mb plus the rDNA array (RDN1), which can vary in size, from 100 to 200 U of a 9.1-kb repeat (Petes, 1979), thus, reaching a total chromosome size of 2–3 Mb (Mortimer and Johnston, 1986). RDN1 is located on the right arm 
300 kb away from the centromere and 600 kb from the right telomere. The rDNA array not only organizes the nucleolus, the center for ribosomal RNA synthesis (Shaw and Jordan, 1995), but it also holds a key regulator of mitotic exit named the Cdc14 protein phosphatase (Garcia and Pillus, 1999; Shou et al., 1999; Visintin et al., 1999). Cdc14p is kept inactive in the nucleolus until mid-anaphase when it is released throughout the cell to reach its targets (Pereira et al., 2002; Stegmeier et al., 2002). Cdc14p participates in the segregation of rDNA (Granot and Snyder, 1991; Buonomo et al., 2003; D'Amours et al., 2004; Sullivan et al., 2004; Torres-Rosell et al., 2004) by targeting the condensin complex to rDNA and hence promoting both resolution and compaction of rDNA (Guacci et al., 1994; D'Amours et al., 2004; Sullivan et al., 2004). Besides its role in rDNA segregation, Cdc14 is also required to promote spindle stability during anaphase (Pereira and Schiebel, 2003).
An open question in the rDNA segregation process is how sister rDNA chromatids are partitioned during anaphase and whether rDNA compaction precedes resolution or whether, on the contrary, sister rDNAs resolve before overall compaction takes place. In the present report, we demonstrate that rDNA inheritance requires two distinct steps separated in time. First, at the anaphase onset, unzipping of sister rDNAs generates an intermediate stage, characterized by largely resolved sister rDNA chromatids across the mother and daughter cells. Second, the linear length of rDNA is reduced, by Cdc14-dependent rDNA axial compaction, to mediate segregation of sister rDNAs to opposite poles in late anaphase. Surprisingly, the second step does not require spindles, suggesting that the main biological function of rDNA compaction is to mediate segregation of this long chromosome arm. This two-stepped mode of inheritance allows us to propose that rDNA resolution and compaction are two functionally distinct processes separated in time, with the resolution of sister rDNA arrays preceding their overall compaction.
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Sister-chromatid rDNAs are resolved at centromere-proximal but not centromere-distal regions in rDNA bridges
To examine how sister chromatids are organized in the rDNA-containing arm of chromosome XII, we visualized chromosomal tags inserted at various positions along this arm using the tetO/tetR-YFP system (Michaelis et al., 1997) in cells that also expressed Net1p-CFP. The tag insertions chosen were: 45 kb right of CEN12 (tetO:194), the centromere-proximal edge of the rDNA array (tetO:450), the centromere-distal edge of the rDNA array (tetO:487), or 20 kb away from the right telomere of chromosome XII (tetO:1061). First, we looked at cells undergoing metaphase. The analysis revealed that DNA regions flanking the rDNA locus (tetO:450 and tetO:487) were always located at the base of the loop with the rest of the nuclear mass (Fig. S1). Chromosome tags inserted close to the centromere (tetO:194) or telomere (tetO:1061) were also located within the main nuclear mass. In contrast, chromosomal tags inserted in the middle of the rDNA array (RDN1:lacO) appeared clearly separated from the nuclear mass within the rDNA loop (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200408087/DC1). RDN1:lacO tags appeared as a single GFP dot within the loop which indicates that rDNA sister chromatids are fully cohesed at this stage. These results demonstrate that rDNA metaphase loops are restricted to rDNA sequences and sisters are fully cohesed throughout the locus.
We then extended our analysis to cells undergoing the "rDNA bridge" period, when rDNA arrays are stretched across the bud-neck (Fig. 1 B and Fig. 2). Centromere tags (tetO:194) and tags in the proximal flank of rDNA (tetO:450) were fully segregated in these cells (Fig. 2 A, tetO:194 and tetO:450 columns). Interestingly, tags in the distal flank of rDNA (tetO:487) were not separated and localized to the middle of the elongated rDNA (Fig. 2 A, tetO:487 column). Telomere tags (tetO:1061) were last to separate and always trailed sister rDNA arrays to the poles (Fig. 2 A, tetO:1061 column). Therefore, the 600-kb region between the distal flank of rDNA and the right telomere follows behind the rDNA arrays during segregation. Furthermore, we asked to what extent rDNA unzips in the bridge stage. To do so, we measured the average distance between separated sister tags in the centromere-proximal edge of the rDNA (tetO:450) during this stage and found the distance to be 5.2 µm (SD 1.4 µm; Fig. 2 B). This separation indicates that a large proportion of rDNA repeats within the array is resolved when cells reach the bridge. This is also in agreement with the observation that the distal edge of the rDNA lays around the middle of the bridge (Fig. 2 A, tetO:487). However, the fact that this distal tag appears as a single focus implies that not all the rDNA repeats in the array are resolved. We have determined the percentage of sister resolution in the rDNA array at the bridge stage by making use of a strain that bears tags in the middle of the rDNA (lacO:RDN1) (Torres-Rosell et al., 2004). The average distance separation between lacO:RDN1 tags in the bridge stage was 1.8 µm (SD 1.5 µm), thus, 77% of the array is already resolved at the bridge stage (Fig. 2 B, 2.6 µm from a total chromatid length of 3.4 µm). These results demonstrate that, during anaphase, sister rDNAs unzip from centromere-proximal to centromere-distal regions before they segregate to opposite poles. Furthermore, the directionality of the unzipping process demonstrates that the partitioning of sister telomeres is delayed compared with the rest of the chromosome arm.
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2.95 µm. Taking into account that 23% of the array is not seen as part of the bridge because they are not yet resolved (Fig. 2 B), the actual length of each rDNA chromatid at the anaphase bridge is 3.85 µm. Therefore, the transition from the metaphase loop to the rDNA bridge does not involve a significant shortening in the axial length of the rDNA (from 3.72 to 3.85 µm). In contrast, the transition from rDNA bridge to fully segregated rDNA shortens the axial length of each sister rDNA chromatid by a factor of 3.3 (from 3.85 to 1.17 µm).
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Pausing at and progressing out of the rDNA bridge stage
During our experiments with synchronous cultures, we noticed that as cells enter anaphase there is a rapid transition from loop to bridge followed by a several minute-long pause at the rDNA bridge (see Fig. 6, not depicted). We reasoned that this might suggest that progression out of bridges could be under specific cell cycle regulation. Therefore, we searched for mutants that would confer arrest specifically at the bridge stage.
Recently it has been shown that inactivation of FEAR network, Cdc14p or condensin causes nucleolar segregation defects (Freeman et al., 2000; Sullivan and Uhlmann, 2003; D'Amours et al., 2004; Sullivan et al., 2004; Torres-Rosell et al., 2004). We evaluated which mutants or combinations provided us with the closest arrest to the bridge stage observed in wild-type cells, with the rDNA stretched across the mother and daughter cells (Figs. 1 and 2). We found that FEAR and MEN double mutants (cdc15-2 spo12
and cdc15-2 slk19), despite their known impairments in full segregation of rDNA to the poles (D'Amours et al., 2004; Torres-Rosell et al., 2004), did not arrest with stretched rDNA. Instead, most of the cells with misegregated rDNAs showed separated rDNA arrays in the same cell body (Fig. 4 A). Polo kinase, Cdc5p, is the only common member of both FEAR and MEN networks (Stegmeier et al., 2002). Inactivation of polo through the cdc5-1 allele produced a severe impairment in both nuclear and nucleolar segregation (Fig. 4 A). A double mutant of condensin, smc2-8, and cdc15-2 also arrested before the anaphase bridge stage with severe impairment in the segregation of the main nuclear mass (Fig. 4 A). Cdc14p activity in anaphase triggers numerous events including segregation of rDNA (Granot and Snyder, 1991), targeting of Ipl1-Sli15 complex to spindle midzones (Pereira and Schiebel, 2003) and timely coordination of mitotic exit. We used the cdc14-1 mutant allele to test whether a cdc14-1 block resembles the bridge stage of wild-type cells since this allele has been reported to arrest with stretched nucleoli between mother and daughter cells (Granot and Snyder, 1991). We found that nucleoli did not segregate in >80% of cells, whereas nuclear masses appear fully separated in a similar percentage (Fig. 4 A). cdc14-1 blocked cells with unsegregated nucleoli could be divided into two categories, representing 50% of the cells each. These were: (a) cells with rDNA stretched across the mother and daughter bodies, similar to the bridge stage in wild-type cells (Fig. 4 A), and (b) cells with a single rDNA signal, in either mother or daughter cell body, connecting two separated nuclear masses in the same cell body (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200408087/DC1). In these cells, although the nucleolus is not stretched across the neck, it still seems to form a bridge between the DAPI-stained bulk DNAs.
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80% cells (Fig. 4, B and C) with an average distance between the tags of 3.9 µm (SD 1.7 µm; Fig. 4 D). Even in cdc14-1 blocked cells with a single rDNA signal on either mother or daughter cell bodies, sister tetO:450 tags were clearly separated (Fig. S3, arrow). We observed a reduced distance between sister tetO:450 tags in cdc14-1 bridges compared with wild type (Fig. 2 B and Fig. 4 D). However, we also found that many cdc14-1 arrested cells showed problems in the segregation of separated nuclear masses to the mother and daughter cells (Fig. S3, arrow). In such cells the distance between nuclear masses and separated tetO:450 tags is necessarily shorter that in bridged wild-type cells. Therefore, the reduced distance between sister tetO:450 tags in cdc14-1 bridges is partially due to nuclear segregation defects. We have also quantified the kinetics of the resolution and segregation of proximal and distal edges of rDNA in a synchronous culture of cdc14-1 relative to wild-type cells (Fig. 5). As expected, cdc14-1 progresses throughout G1/S phases as wild type, entering anaphase on schedule. However, cdc14-1 exhibited both a delay in the appearance of binucleated cells and a higher frequency of stretched nuclei (Fig. 5, second row of panels) suggesting nuclear segregation problems. These phenotypes can be attributed to the known role of Cdc14p in the segregation of telomeric regions (D'Amours et al., 2004). The resolution of centromere-proximal flanks of rDNA (tetO:450) was slightly delayed in cdc14-1 (5–10 min; Fig. 5, third row of panels). Segregation of the tags, measured as two foci located in different cell bodies, was clearly delayed for tetO:450. In contrast, both resolution and segregation of the centromere-distal flanks of rDNA (tetO:487) were severely delayed (Fig. 5, third row of panels). Similar separation kinetics for rDNA centromere-distal flanks have been recently reported for another mutant allele of CDC14, cdc14-3 (D'Amours et al., 2004). We conclude that the cdc14-1 arrest is similar to the anaphase bridge period in wild-type cells, with separated nuclear masses and sister rDNAs largely unzipped but not segregated, since the majority of tetO:450 tags and lacO:RDN1 tags (in the middle of rDNA), but not tetO:487 tags, are separated (Fig. 4, B and C; Fig. 5; Torres-Rosell et al., 2004). Together, these results demonstrate that cells can be genetically arrested in the bridge stage using the CDC14 mutant allele cdc14-1.
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To test whether spindles are required for the segregation of rDNA arrays after the bridge stage, we took advantage of the fact that a cdc14-1 block is similar to the anaphase bridge stage (Fig. 4, B and C). We exposed cdc14-1 cells to nonpermissive conditions and, after the block, the culture was separated in two and nocodazole was added to one half. After a further 10 min of incubation at 37°C, both cultures were returned to 23°C to reactivate Cdc14p and we took samples every 30 min for 2 h. The level of rDNA segregation (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200408087/DC1) and tetO:487 segregation (Fig. 7) followed identical kinetics in both experimental conditions. Therefore, the presence of nocodazole, i.e., absence of spindles, did not prevent the poleward segregation of rDNA arrays, demonstrating that the transition from the anaphase bridge stage to the full segregation of sister rDNAs does not require spindles. We conclude that after the anaphase bridge period Cdc14-dependent compaction of rDNA is required for the segregation of the long RDN1 locus.
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rDNA resolution and compaction are temporally separated during anaphase
The structure of the long arm of chromosome XII is different from most other chromosomes as it harbors the rDNA. We have analyzed the configuration of rDNA in metaphase and anaphase cells using a combination of chromosome tags along the arm and fluorescent rDNA-binding proteins. During metaphase, rDNA forms a loop as previously described by others (Guacci et al., 1994; Lavoie et al., 2004). The loop is restricted to rDNA repeats as chromosome tags at the proximal (tetO:450) and distal (tetO:487) edges of rDNA are located at the base of the rDNA loop in the nucleus (Fig. S1). A similar configuration of the rDNA in metaphase has been suggested in a recent report while writing of this manuscript was in progress (Fuchs and Loidl, 2004).
Previously, we identified a stage during mid-anaphase when the rDNA is stretched across the mother and daughter cells (Torres-Rosell et al., 2004). In the present report, we show that cells undergoing this "anaphase rDNA bridge" have sister rDNAs unzipped from each other and, thus, largely resolved (Fig. 2). Sister chromosome tags at the centromere-proximal edge (tetO:450) of rDNA are separated, with an average distance between the tags of 5.2 and 5.9 µm (Fig. 2 B and Fig. 3 A), and locate within the main nuclear masses which are already segregated. In contrast, sister chromosome tags in the centromere-distal edge (tetO:487) of rDNA are unresolved and locate to the middle of the nucleolar bridge (Fig. 2). The large distances between sister tetO:450 tags and the localization of tetO:487 tags to the middle of the bridge demonstrate that sister chromatids have resolved most of the rDNA array by the time they arrive at this anaphase rDNA bridge. Furthermore, we have also observed that a chromosome tag inserted in the middle of the RDN1 locus (RDN1:lacO) separates by 2 µm in bridged cells, demonstrating that around three fourths of the arrays are fully resolved at the bridge stage (Fig. 2 B). Interestingly, we have also shown that the rDNA compaction in bridged cells is not significantly different from that of the metaphase loops (Fig. 3 A) and that overall rDNA compaction occurs during late anaphase after the bridge stage (Fig. 3 A and Fig. 6). Therefore, we conclude that the rDNA resolution and compaction processes occur separately and consecutively during anaphase and that there is a narrow window in the anaphase timing (the "rDNA bridge stage") when sister rDNAs are largely resolved yet not compacted.
Hyper-compaction of rDNA is required during segregation because anaphase spindles are not sufficiently long
The presence of the rDNA arrays in the right arm of the chromosome XII makes this arm the longest of the genome (2–3 Mbp, which is at least 1 Mbp longer than the right arm of chromosome IV, the next in length). During mitosis, repetitive (rDNA) and unique regions in yeast chromosomes are 115–140 fold more compacted than B-form DNA, yielding an average compaction of 2.54 µm/Mb (Guacci et al., 1994). Based on this packaging, every chromosome arm in the genome except for the long arm of chromosome XII can be fully segregated with an 8 µm anaphase spindle (Fig. 8 A). However, the segregation of the long arm of chromosome XII would require a spindle length of between 9.8 and 14.9 µm depending on the size of the array (Fig. 8 A). Therefore, yeast cells would be faced with a problem if they were to execute cytokinesis with the chromosome folding levels of metaphase. Instead, they must require additional compaction during anaphase to ensure that the linear length of the right arm of chromosome XII is sufficiently reduced as to be segregated and avoid severing of this arm by cytokinesis. Interestingly, the coccidian parasite Aggregata eberthi, which also uses an intranuclear spindle during mitosis (like that of S. cerevisiae), has an abnormally reduced spindle size relative to the nucleus (Darlington, 1937). During metaphase A. eberthi chromosomes retain the length and appearance normally seen in prophase contracting further only in late anaphase (Darlington, 1937). Sometimes the anaphase compaction is delayed and the ends of longer chromosomes appear unseparated (Darlington, 1937). Therefore, hyper-compaction–mediated segregation might be a strategy to cope with long chromosome arms and a relative reduced spindle size. Here, we have presented strong evidence supporting the idea that rDNA compaction is the driving force behind the poleward segregation of the long arm of chromosome XII in late anaphase. First, live cell analysis revealed that rDNA segregation is accompanied by substantial axial compaction of sister rDNA but little increment in the spindle length (Fig. 3 A and Fig. 6). Second, cells arrested in the anaphase bridge can segregate rDNA in the absence of spindles when allowed to progress out of the arrest (Fig. 7).
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Recently, it has been reported that in a TEV-induced anaphase, the absence of Cdc14p activity generates a situation where tags in the proximal and distal edges of rDNA do not resolve, but tags in the centromeric and telomeric regions are not only resolved but also fully segregated to the poles (Sullivan et al., 2004). These findings raise the possibility that in wild-type cells the telomere of the long arm of chromosome XII might also segregate before and independent of the rest of arm itself. Nevertheless, in our investigation, we have never observed, neither in wild-type cells nor in the cdc14-1 mutant analyzed, that distal regions to rDNA (i.e., in particular, telomeric regions) segregate before the entire rDNA has been fully resolved (Figs. 2 and 4). The difference, with respect to tetO:450 separation, between a cdc14-1 block and the TEV-induced anaphase (i.e., resolution in cdc14-1 blocked cells but not in TEV-induced anaphases) could be due to problems in spindle stabilization in the TEV-anaphase due to the absence of separase activity (Sullivan et al., 2001). In contrast, anaphase spindles are fully stable in cdc14 mutants because of residual Slk19p function (Pereira and Schiebel, 2003). In cdc14-1 cells there was a minor delay in the separation of tetO:450 tags (Fig. 5), suggesting that Cdc14p activity is involved at the onset of rDNA resolution. However, Cdc14p is not essential for rDNA resolution since cells arrested by cdc14-1 can reach the bridge stage of wild-type cells with tetO:450 separated an average distance of 4 µm (Fig. 4 D).
We have shown that Cdc14-mediated compaction of rDNA is necessary for the resolution and segregation of tetO:487 tags independent of spindles (Fig. 7). Interestingly, the resolution of tetO:487 tags can also be induced in the absence of spindles by overexpressing Cdc14 in metaphase arrested cells (D'Amours et al., 2004). This demonstrates that rDNA resolution requires additional Cdc14-mediated mechanisms independent of spindles (D'Amours et al., 2004). Additionally, overexpression of Cdc14p in metaphase cells also has been shown to induce rDNA compaction (Sullivan et al., 2004). Our data and these studies support the idea that the separation and segregation of distal regions of chromosome XII require rDNA compaction. Together, the data demonstrate that progression out of the bridge stage is under specific cell cycle regulation, requiring Cdc14p activity. Furthermore, it strongly suggests that stable anaphase spindles are important during the unzipping of the rDNA array observed during the bridge period.
Conclusions
Here, we have shown that late anaphase compaction of rDNA is necessary for its segregation independent of spindles. The data demonstrate that rDNA unzipping/resolution and compaction occur at different times in wild-type cells, with the resolution of sister rDNAs preceding their compaction. Also we found that these two steps needed for rDNA segregation require distinct mechanisms. The first step occurs from metaphase to mid-anaphase and involves unzipping of sister rDNAs from centromere-proximal to centromere-distal regions. The second step takes place from mid- to late anaphase and involves the compaction of sister rDNAs (Fig. 8 B). These findings show that the biological function of rDNA compaction is to ensure full segregation and demonstrate that sister chromatid resolution does not simply result from the compaction of individual chromosome arms, at least for yeast rDNA.
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Materials and methods |
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-factor pheromone.
Microscopy
Yeast cells with CFP- or GFP-tagged proteins were analyzed by fluorescence microscopy after DAPI staining. Series of z-focal plane images were collected on a microscope (model IRB; Leica) using a C4742-95 digital camera (Hamamatsu) and OpenLab software (Improvision). A tunable light source (Polychrome IV) with a Xenon lamp was used. Images in different z-axis planes were flattened into a two-dimensional projection and processed in Openlab. Live cell imaging (Fig. 6) was done using 63x/1.4 or 100x/1.35 lenses and a Polychrome IV device, tunable light source optimized for live imaging that minimizes cell exposure. The viability of cells after imaging was comparable to nonimaged cells under the same conditions. DNA was stained using DAPI (Molecular Probes) at 1 µg/ml final concentration after short treatment of the cells with 1% Triton X-100. Imaging was done in antifade/DAPI medium (Molecular Probes) at RT.
Online supplemental material
Fig. S1 shows that the right arm of chromosome XII forms a loop restricted to rDNA during metaphase. Fig. S2 shows that rDNA sister chromatid cohesion is maintained in metaphase loops. Fig. S3 shows that cdc14-1 mutants arrest with separated rDNA proximal regions. Fig. S4 shows that nucleolar segregation during late anaphase does not require mitotic spindles. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200408087/DC1.
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Acknowledgments |
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This work was supported by the Medical Research Council (MRC) UK.
Submitted: 13 August 2004
Accepted: 6 December 2004
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References |
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