[Cell Cycle 4:1, 109-112; January 2005]; ©2005 Landes Bioscience
Cdc14 and the Temporal Coordination between Mitotic Exit and Chromosome Segregation Spotlight on the Nucleolus
ABSTRACT Cell division involves the inheritance of a complete set of the genome in the form of chromosomes. One of the strategies employed by eukaryotic cells is to maintain replicated sister chromatids together until the anaphase onset. A protein complex named cohesin holds sisters together following replication until anaphase when cleavage of cohesin by the protease separase initiates segregation. Recent studies in budding yeast have shown that cohesin cleavage alone is not sufficient for the segregation of the entire genome. Instead, repetitive regions, such as the ribosomal DNA (rDNA) array and telomeres, require additional mechanisms during mitotic disjunction. The segregation of such chromosome regions is delayed and needs specific cell cycle regulators such as the FEAR network and the conserved phosphatase Cdc14, all of which orchestrate the timely completion of chromosome segregation before mitotic exit. Future studies will be targeted towards unravelling the nature of the additional segregation requirements for repetitive regions and the specifics of its cell cycle control.
Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=1356
KEY WORDS
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INTRODUCTION
Following DNA replication during S phase, newly replicated sister chromatids are first linked together (cohesion) before being compacted (condensation) and untangled (decatenation) and then moved to opposite poles of the dividing cell (segregation).1 Sister chromatids are actively linked together, or cohesed, through protein complexes named cohesins.2,3 The dissolution of cohesin-mediated linkages at the onset of anaphase1 is brought about by the cleavage of one of its subunits, Mcd1/Scc1, through the Esp1 protease.4 At the metaphase-to-anaphase transition cohesin cleavage is activated through the control of the ubiquitin-protein ligase APC/C (anaphase promoting complex/cyclosome) by its activator Cdc20p.5 The activation of APC/Cdc20 mediates proteolysis of the anaphase inhibitor Pds1p (also known as securin) which liberates the Esp1p protease (also known as separase) to cleave cohesin.1,6 Cohesin cleavage allows the physical separation of sister chromatids to the poles by pulling of the microtubule apparatus. Upon completion of chromosome segregation, mitotic cyclin-dependent kinases (CDKs) are inactivated allowing cells to exit mitosis.7 A key factor in the inactivation of mitotic CDKs is the protein phosphatase Cdc14.8 Cfi1/Net1p is a Cdc14p inhibitor that maintains the phosphatase impounded in the nucleolus during most of the cell cycle.9,10 Cfi1/Net1p phosphorylation is the trigger to release Cdc14.11,12 This release occurs during anaphase in two waves13 spreading Cdc14p throughout the cell to reach its targets. In early anaphase, the FEAR network (Cdc Fourteen Early Anaphase Release), composed of separase (Esp1p), polo-kinase (Cdc5p), Slk19p, Spo12p and Bns1p, stimulates a partial and transient release of Cdc14p.13-15 Recent work shows that Cdc14 release by FEAR is accomplished by mitotic Cdk phosphorylation of Net1p.12 One role of FEAR released Cdc14p is to target the Sli15/Ipl1 complex to the spindle midzone in order to stabilize it.16 Full cdc14p release is achieved in late anaphase and requires the MEN network (Mitotic Exit Network), composed of the GTPase Tem1p, protein kinases Cdc15p, Dbf2p and Cdc5p, the guanine nucleotide exchange factor Lte1p, the GTPase activating proteins Bub2p and Bfa1p and the scaffold protein Nud1p.7 In MEN mutants, although FEAR partially releases Cdc14, the phosphatase is sequestered again. Inactivation of MEN or Cdc14p results in arrest in late anaphase consistent with the key role of the phosphatase in promoting mitotic exit.7 In higher eukaryotes, cohesed chromosomes undergo a dramatic structural reorganization before sister chromatid separation occurs, whereby the chromosome becomes organized
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Cdc14, nucleolus, mitotic exit, chromosome segregtaion, cohesin, condensin
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Received 11/05/04; Accepted 11/10/04
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*Correspondence to: Luis Aragón; Cell Cycle Group; Clinical Sciences Centre, Medical Research Council; Imperial College London; Du Cane Road; London W12 0NN, United Kingdom; Tel.: +44.20.83833708; Fax: +44.20.83833708; Email:
[email protected]
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Cell Cycle Group; Clinical Sciences Centre; Medical Research Council; Imperial College London; London, UK
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Jordi Torres-Rosell Félix Machín Luis Aragón*
www.landesbioscience.com
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into a seemingly packaged state.17 This reorganization occurs during mitosis and is referred to as chromosome condensation. The functional significance of condensation is thought to be two-fold: (1) to mediate the resolution of sister chromatids from each other, and (2) to ensure that the linear length of chromosome arms is sufficiently reduced as to avoid severing of chromosomes during cytokinesis.17 In S.cerevisiae mitotic chromosome condensation is largely dependent on a multi-subunit complex named condensin.18-23 The complex is composed of Smc2p (XCAP-E/hCAP-E/cut14/CeMIX-1), Smc4p (XCAP-C/hCAP-C/cut3/DmSMC4/CeSMC4), Ycs4p (XCAP-D2/ Eg7/hCAP-D2/CNAP-1/cnd3), Ycg1p or Ycs5p (XCAP-G/ hCAP-G/cnd4) and Brn1p (hCAP-H/BRRN1/Spcnd2/DmBarren). Yeast condensin becomes enriched in the nucleolus during mitosis,19,22 suggesting that segregation of this locus requires condensin function.19 In budding yeast, the nucleolus is formed around the highly repetitive rDNA array (1–2 Mbp long) on chromosome XII.24 Chromosome XII is thus the largest chromosome, with 1 Mb plus the rDNA array (RDN1), which can vary in size, 100–200 units of a 9.1 Kb repeat,24 thus reaching a total chromosome size of 2–3 Mb.25 RDN1 is located on the right arm approximately 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,26 but it also holds Cdc14 protein phosphatase.10,27 Interestingly, the terminal phenotype of cdc14 mutants suggests that this phosphatase has a role role in rDNA segregation as cdc14-1 blocked cells segregate nuclear masses but cannot separate nucleoli.28 Furthermore, these cells exhibit abnormally decondensed rDNA29-32 demonstrating that Cdc14 activity is required for rDNA condensation. A set of recent papers have highlighted the importance of Cdc14p in the segregation of rDNA30-31 and telomeres,32 demonstrating that the disjunction of these repetitive regions occurs in mid-anaphase, long after cohesin cleavage, and is regulated by the conserved phosphatase Cdc14. These studies begin dissecting the molecular role of this phosphatase in the segregation of repeats.
FEAR-RELEASED Cdc14 CONTROLS rDNA SEGREGATION IN MID-ANAPHASE
The Cdc14 phosphatase is kept inactive during most of the cell cycle in the nucleolus. At the anaphase onset the FEAR pathway promotes the partial relase of Cdc14 thereby activating the protein. Release of Cdc14 by the FEAR network is transient and is not able to trigger mitotic exit in the absence of MEN activity.13 Once released, Cdc14 itself promotes full Cdc14 release through activation of the MEN network (Mitotic Exit Network).13-15 The fact that Cdc14 mutants arrest with the majority of the nuclear masses segregated except for rDNA28 raised the possibility that the main nuclear mass and the rDNA array require different segregation mechanisms. In recent reports we and others have shown that nucleolar chromatin does not segregate along with the bulk of the DNA, as previously thought, but instead it is spatially separated and temporally delayed from the rest of the genome.30-32,34 We showed that the timing of nucleolar segregation coincides with that of Cdc14 release by the FEAR network in wild type cells. In FEAR mutants nucleolar segregation is further delayed until the time when Cdc14 is released by the MEN network,30 demonstrating that it is the FEAR-controlled release of Cdc14 that is important for the division of the nucleolus and rDNA resolution. Therefore, the first wave of Cdc14 released by the FEAR network ensures proper segregation of the nucleolus after cohesin cleavage. Later on, full and sustained release of Cdc14 by the MEN will allow cells to exit mitosis. 110
Figure 1. Segregation of chromosome XII in budding yeast. At completion of DNA replication, sister chromatids are held together by the cohesin complex. During the metaphase-to-anaphase transition, chromosome segregation is initiated through the cleavage of cohesin. As cells enter mid-anaphase, sister chromatids are fully resolved from each other for most chromosome regions except the rDNA array and telomeres. Activation of Cdc14 by the FEAR network is necessary to mediate the additional requirements for the segregation of these chromosome regions. Cdc14 downstream events include; (1) rDNA resolution through condensin and possibly Topoisomerase II, (2) rDNA condensation through condensin and Aurora B and, (3) telomere disjunction through a yet unknown pathway.
Why is nucleolar segregation delayed with respect to the rest of the genome? Although there is still no answer, it is possible that it has to do with the structure of the chromosome that carries the rDNA locus: the right arm of chromosome XII is by far the longest in budding yeast, and the complete segregation of this long arm in anaphase depends on condensation at the rDNA after dissolution of sister chromatid cohesion and not a late anaphase spindle.35 Functioning between cohesin cleavage and mitotic exit, Cdc14 emerges as an ideal candidate to help activate such mechanisms.
Cdc14 TARGETS RESOLUTION AND CONDENSATION ACTIVITIES TO rDNA
Cohesin cleavage is known to trigger the segregation of sister chromatids by spindle elongation.4 Cdc14 mutants arrest with the majority of the nuclear masses segregated except for rDNA28 suggesting that cells stop after cohesin has been cleaved, however the lack of rDNA segregation raised the possibility that cohesin cleavage within rDNA is impaired in this mutant. Two recent papers show that the lack of rDNA segregation in Cdc14 mutants is totally independent of the cohesin complex.31,32 Instead their work points towards the idea that rDNA segregation involves additional resolution and condensation activities dependent on Cdc14 and condensin. Sullivan et al. make use of an experimental model where an anaphase-like state can be reached by artificially inducing cohesion cleavage between sister chromatids.36 The system utilizes a modified version of the cohesin subunit Scc1 where a TEV protease recognition site has been introduced. The induction of the TEV protease through a controllable promoter leads to Scc1 cleavage, which ends up in cohesion disassembly. Normally, the resolution of cohesed sister chromatids takes place through a similar cleavage mechanism by the Esp1 protease.4 In TEV-induced anaphases Cdc14 is not released from the nucleolus and cells although able to segregate nuclear masses, remarkably fail in separating the nucleolus.37 Now, the same authors show that overexpression of Cdc14 in the TEVanaphase rescues the nucleolar segregation defect in a manner dependent on condensin.31 Both Cdc14 and condensin were previously known to be required for rDNA condensation in mitosis.2,19
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Cdc14 and the Temporal Coordination between Mitotic Exit and Chromosome Segregation
Sullivan et al. revisited this point showing that Cdc14 not only promotes rDNA condensation but it is also required for sister chromatid resolution throughout the locus in a TEV-induced anaphase. Interestingly, these authors find that in TEV-induced anaphases sister chromosome tags inserted in the centromere-proximal and centromere-distal flanks of rDNA do not resolve from each other, however sister tags towards the distal region not only resolve but segregate to opposite poles. These results suggest that in TEV- induced anaphases the rDNA array is fully cohesed throughout. What is more, it implies that in a TEV-induced anaphase there must exist a “centromere independent” mode of telomere segregation. In contrast to this lack of rDNA resolution in TEV anaphases, in a cdc14-1 mutant arrest, sister chromosome tags inserted in the centromereproximal flank of the rDNA array are resolved in most cells35 whereas tags inserted in the centromere-distal flank of rDNA are largely unresolved.35 These results demonstrate that in the cdc14-1 arrest the rDNA is not cohesed throughout its entire length but instead it is largely unzipped from centromere-proximal towards centromeredistal regions. Interestingly, in cdc14-1 mutants the segregation of telomere tags (distal to rDNA) is also severely impaired.35 Differences between the experimental systems might account for this discrepancy. It is worth noting that a striking difference between both systems is the presence of a wild type Cdc14 protein in the nucleolus (in the TEV model) or a released inactive cdc14 protein (in the thermosensitive cdc14 mutant model). The Sullivan et al. study also shows that rDNA resolution and condensation are mediated by different factors: Aurora B kinase is not necessary for rDNA resolution (although it is required to structure the rDNA, as shown recently by Lavoie et al., ref. 23), whereas Topoisomerase II is required for resolution but dispensable for Cdc14-mediated structural changes. Condensin function is essential for both processes. In a parallel paper, D’Amours et al. find that Cdc14 and condensin are not only required for the correct segregation of rDNA but also telomeric regions,32 therefore generalising this function to repetitive regions of the yeast genome. D’Amours et al., used a temperature-sensitive allele of Cdc14, namely cdc14-3, as their model to inactivate Cdc14. cdc14-3 mutants failed to accurately separate sister chromosome tags in the centromere-distal flank of rDNA and the right telomere of chromosome V. Interestingly, removal of the rDNA array from chromosome XII suppressed the resolution of the rDNA tags. D’Amours et al. go one step further and provide an explanation of why rDNA segregation fails in the absence of Cdc14 activity by demonstrating that condensin is targeted to the rDNA in a Cdc14-dependent manner. A novel biochemical mechanism to explain condensin targeting regulation by Cdc14 is also provided by the authors, who show that condensin subunit Ycs4p, which is not targeted to rDNA in cdc14-3 mutants, is sumoylated during anaphase in a Cdc14-dependent manner. D’Amours et al. reached similar conclusion to Sullivan et al. about the necessity of condensin to resolve rDNA through a mechanism different from condensation. However, they show that rDNA resolution does not require topoisomerase II activity, in contrast to Sullivan et al., suggesting that condensin might resolve rDNA through a still unknown function related to neither condensation nor disentanglement. An independent study has also shown that condensin localization to rDNA is impaired in cdc14 mutants. Using chromatin immunoprecipitation Wang et al. showed a reduction of the condensin subunit Brn1 in the rDNA of cdc14-1 arrested cells. Furthermore this report suggests that condensin fails to concentrate on the rDNA www.landesbioscience.com
due to mislocalization of the complex to other genomic sites.33 These authors reached similar conclusions to D’Amours et al. strongly supporting a role for Cdc14 in the regulation of condensin targeting to rDNA.
CONCLUSIONS AND FUTURE DIRECTIONS
Recent studies have established the importance of condensin in the segregation of the rDNA through the FEAR-released Cdc14 regulation. These findings show that condensin and Cdc14 function in the same pathway during rDNA segregation. The key question that now arises is what is the nature of the linkages that delay the segregation of rDNA to late anaphase in wildtype cells and prevents segregation of rDNA and telomeres in the absence of Cdc14 function (Fig. 1). The possible answer to this question is at present very speculative, even after all the recent effort, ranging from the possibility that repetitive regions could be more prone to entanglements and/or mitotic recombination, to a requirement of regions tethered to the nuclear envelope to be released during segregation. Clearly, the actual nature of the chromosomal linkages at the rDNA will receive tremendous attention in the next months. Future findings will strongly contribute to our present understanding of condensin function in vivo as well as how the segregation of the genome is coordinated with the regulatory pathways that promote mitotic exit. References 1. Nasmyth K. Disseminating the genome: Joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu Rev Genet 2001; 35:673-745. 2. Guacci V, Koshland D, Strunnikov A. A direct link between sister chromatid cohesion and chromosome condensation revealed through the analysis of MCD1 in S. cerevisiae. Cell 1997; 91:47-57. 3. Michaelis C, Ciosk R, Nasmyth K. Cohesins: Chromosomal proteins that prevent premature separation of sister chromatids. Cell 1997; 91:35-45. 4. Uhlmann F, Lottspeich F, Nasmyth K. Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 1999; 400:37-42. 5. Zachariae W, Nasmyth K. Whose end is destruction: Cell division and the anaphase-promoting complex. Genes Dev 1999; 13:2039-58. 6. Uhlmann F, Lottspeich F, Nasmyth K. Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 1999; 400:37-42. 7. Bardin AJ, Amon A. Men and sin: What’s the difference? Nat Rev Mol Cell Biol 2001; 2:815-26. 8. Visintin R, Craig K, Hwang ES, Prinz S, Tyers M, Amon A. The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation. Mol Cell 1998; 2:709-18. 9. Shou W, Seol JH, Shevchenko A. Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 1999; 97:233-44. 10. Visintin R, Hwang ES, Amon A. Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus. Nature 1999; 398(6730):818-23. 11. Shou W, Azzam R, Chen SL. Cdc5 influences phosphorylation of Net1 and disassembly of the RENT complex. BMC Mol Biol 2002; 3:3. 12. Azzam R, Chen SL, Shou W. Phosphorylation by cyclin B-Cdk underlies release of mitotic exit activator Cdc14 from the nucleolus. Science 2004; 305:516-9. 13. Stegmeier F, Visintin R, Amon A. Separase, polo kinase, the kinetochore protein Slk19, and Spo12 function in a network that controls Cdc14 localization during early anaphase. Cell 2002; 108:207-20. 14. Yoshida S, Asakawa K, Toh-e A. Mitotic exit network controls the localization of Cdc14 to the spindle pole body in Saccharomyces cerevisiae. Curr Biol 2002; 12:944-50. 15. Pereira G, Manson C, Grindlay J, Schiebel E. Regulation of the Bfa1p-Bub2p complex at spindle pole bodies by the cell cycle phosphatase Cdc14p. J Cell Biol 2002; 157:367-79. 16. Pereira G, Schiebel E. Separase regulates INCENP-aurora B anaphase spindle function through Cdc14. Science 2003; 1-10, (1126/science.1091936). 17. Swedlow JR, Hirano T. The making of the mitotic chromosome: Modern insights into classical questions. Mol Cell 2003; 11:557-69. 18. Strunnikov AV, Hogan E, Koshland D. SMC2, a Saccharomyces cerevisiae gene essential for chromosome segregation and condensation, defines a subgroup within the SMC family. Genes Dev 1995; 9:587-99. 19. Freeman L, Aragon-Alcaide L, Strunnikov A. The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J Cell Biol 2000; 149:811-24. 20. Ouspenski II, Cabello OA, Brinkley BR. Chromosome condensation factor Brn1p is required for chromatid separation in mitosis. Mol Biol Cell 2000; 11:1305-13. 21. Lavoie BD, Tuffo KM, Oh S, Koshland D, Holm C. Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren. Mol Biol Cell 2000; 11:1293-304.
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22. Bhalla N, Biggins S, Murray AW. Mutation of YCS4, a budding yeast condensin subunit, affects mitotic and nonmitotic chromosome behavior. Mol Biol Cell 2002; 13:632-45. 23. Lavoie BD, Hogan E, Koshland D. In vivo requirements for rDNA chromosome condensation reveal two cell-cycle-regulated pathways for mitotic chromosome folding. Genes Dev 2004; 18:76-87. 24. Petes TD. Yeast ribosomal DNA genes are located on chromosome XII. Proc Natl Acad Sci USA 1979; 76:410-4. 25. Mortimer RK, Johnston JR. Genealogy of principal strains of the yeast genetic stock center. Genetics 1986; 113:35-43. 26. Shaw PJ, Jordan EG. The nucleolus. Annu Rev Cell Dev Biol 1995; 11:93-121. 27. Garcia SN, Pillus L. Net results of nucleolar dynamics. Cell 1999; 97:825-8. 28. Granot D, Snyder M. Segregation of the nucleolus during mitosis in budding and fission yeast. Cell Motil Cytoskeleton 1991; 20:47-54. 29. Guacci V, Hogan E, Koshland D. Chromosome condensation and sister chromatid pairing in budding yeast. J Cell Biol 1994; 125:517-30. 30. Torres-Rosell J, Machin F, Jarmuz A, Aragon L. Nucleolar segregation lags behind the rest of the genome and requires Cdc14p activation by the fear network. Cell Cycle 2004; 3:2-14. 31. Sullivan M, Higuchi T, Katis VL, Uhlmann F. Cdc14 phosphatase induces rDNA condensation and resolves cohesin-independent cohesion during budding yeast anaphase. Cell 2004; 117:471-82. 32. D’Amours D, Stegmeier F, Amon A. Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA. Cell 2004; 117:455-69. 33. Wang BD, Yong-Gonzalez V, Strunnikov AV. Cdc14p/fear pathway controls segregation of nucleolus in S. cerevisiae by facilitating condensin targeting to rDNA chromatin in anaphase. Cell Cycle 2004; 3:960-7. 34. Torres-Rosell J, Machin F, Jarmuz A, Aragon L. Nucleolar segregation lags behind the rest of the genome and requires Cdc14p activation by the FEAR network. Cell Cycle 2004; 3:496-502. 35. Machín F, Torres-Rossell J, Jarmuz A, Aragón L. Spindle independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase. J Cell Biol 2005; In press. 36. Uhlmann F, Wernic D, Poupart MA, Koonin EV, Nasmyth K. Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 2000; 103:375-86. 37. Sullivan M, Uhlmann F. A nonproteolytic function of separase links the onset of anaphase to mitotic exit. Nat Cell Biol 2003; 5:249-54.
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