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Chromosome Segregation: Seeing Is Believing

2005; Elsevier BV; Volume: 15; Issue: 13 Linguagem: Inglês

10.1016/j.cub.2005.06.033

1879-0445

Kerry Bloom,

Chromosomal and Genetic Variations

For chromosome segregation in mitosis, each centromere directs assembly of a complex, proteinaceous structure — the kinetochore, which connects the chromosome to microtubules of the mitotic spindle. A recent study has provided important new insights into the mechanism by which kinetochores capture spindle microtubules. For chromosome segregation in mitosis, each centromere directs assembly of a complex, proteinaceous structure — the kinetochore, which connects the chromosome to microtubules of the mitotic spindle. A recent study has provided important new insights into the mechanism by which kinetochores capture spindle microtubules. The mechanisms that contribute to accurate chromosome segregation are manifold and complex. The players have been identified from early genetic mapping studies, cytological observations of autoimmune patients and genetic screens in model organisms. Centromeres are responsible for directing the assembly of a complex proteinaceous structure, the kinetochore. The kinetochore provides the linkage between the chromosome and microtubules of the mitotic spindle. How the kinetochore engages the microtubule, promotes the complex oscillatory dance by which replicated chromosomes attain correct attachment, yet maintain the ability to correct errors has been the subject of intensive study. This is a complex process, but the idea that we might be able to dissect it by genetic and molecular analysis was given a boost when it was realised that a 'point centromere' in budding yeast [1Biggins S. Walczak C.E. Captivating capture: how microtubules attach to kinetochores.Curr. Biol. 2003; 13: R449-R460Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar] — a chromosome with a single microtubule–chromosome attachment — exhibits similar oscillatory behavior as the 20-30 microtubule–chromosome attachments in a mammalian cell [2Pearson C.G. Maddox P.S. Salmon E.D. Bloom K. Budding yeast chromosome structure and dynamics during mitosis.J. Cell Biol. 2001; 152: 1255-1266Crossref PubMed Scopus (178) Google Scholar]. This conjecture has been realized in a tour de force of experimental biology reported recently by Tanaka et al. [3Tanaka K. Mukae N. Dewar H. van Breugel M. James E.K. Prescott A.R. Antony C. Tanaka T.U. Molecular mechanisms of kinetochore capture by spindle microtubules.Nature. 2005; 434: 987-994Crossref PubMed Scopus (214) Google Scholar]. Their success has come from the judicious use of a combination of approaches: high resolution digital microscopy; fusion of multiple copies of the green fluorescent protein (GFP) to key kinetochore components; and a simple genetic switch to regulate centromere activity and therefore the segregation properties of a single chromosome. The yeast kinetochore is composed of more than 70 proteins organized into discrete subcomplexes [4Westermann S. Cheeseman I.M. Anderson S. Yates 3rd, J.R. Drubin D.G. Barnes G. Architecture of the budding yeast kinetochore reveals a conserved molecular core.J. Cell Biol. 2003; 163: 215-222Crossref PubMed Scopus (180) Google Scholar, 5De Wulf P. McAinsh A.D. Sorger P.K. Hierarchical assembly of the budding yeast kinetochore from multiple subcomplexes.Genes Dev. 2003; 17: 2902-2921Crossref PubMed Scopus (226) Google Scholar]. The breakthrough in monitoring chromosome movement came from the use of an indirect labeling strategy employing integrated copies of lac operator and lac-repressor-GFP fusion proteins to visualize the operator at specific chromosomal loci in live cells. In metaphase of mitosis, the centromeres of replicated sister chromatids are bi-oriented on the spindle, and appear as two foci. The kinetochore microtubules are dynamic, resulting in constant oscillatory centromere movements. Mechanisms involving tension-dependent microtubule rescue act to preserve the average separation of 0.6μm (~1/2 total spindle length) between separated centromeres. Centromere oscillations continue, allowing all 16 chromosomes to become bi-oriented — the metaphase configuration — before anaphase ensues. The major unsolved problems concern the mechanisms that lead to the metaphase configuration and how metaphase is maintained. The initial events in centromere attachment have been directly visualized, and the key players in these processes have been identified [3Tanaka K. Mukae N. Dewar H. van Breugel M. James E.K. Prescott A.R. Antony C. Tanaka T.U. Molecular mechanisms of kinetochore capture by spindle microtubules.Nature. 2005; 434: 987-994Crossref PubMed Scopus (214) Google Scholar]. Tanaka et al. [3Tanaka K. Mukae N. Dewar H. van Breugel M. James E.K. Prescott A.R. Antony C. Tanaka T.U. Molecular mechanisms of kinetochore capture by spindle microtubules.Nature. 2005; 434: 987-994Crossref PubMed Scopus (214) Google Scholar] were able to visualize these events because they found conditions where most of the centromeres were already aligned, save for one malpositioned centromere. Yeast centromeres can be inactivated in cis by transcription of a proximal promoter [6Hill A. Bloom K. Genetic manipulation of centromere function.Mol. Cell. Biol. 1987; 7: 2397-2405Crossref PubMed Scopus (149) Google Scholar]. In a still somewhat mysterious process — probably involving the targeted degradation of centromere components — the promoter inactivates the microtubule binding function of the centromere. By arresting cells in mitosis, the bulk of the chromosomes are bi-oriented, while the chromosome with an inactive centromere is detached. The last centromere is activated on switching to conditions that repress its proximal promoter, and the initial events of centromere attachment of this 'lost' chromosome can then be directly visualized. The initial attachments were seen to proceed via interactions between the kinetochore and the side of the microtubule [3Tanaka K. Mukae N. Dewar H. van Breugel M. James E.K. Prescott A.R. Antony C. Tanaka T.U. Molecular mechanisms of kinetochore capture by spindle microtubules.Nature. 2005; 434: 987-994Crossref PubMed Scopus (214) Google Scholar]. This behavior is similar to lateral attachments observed in tissue culture [7Merdes A. De Mey J. The mechanism of kinetochore-spindle attachment and polewards movement analyzed in PtK2 cells at the prophase-prometaphase transition.Eur. J. Cell Biol. 1990; 53: 313-325PubMed Google Scholar, 8Rieder C.L. Alexander S.P. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells.J. Cell Biol. 1990; 110: 81-95Crossref PubMed Scopus (340) Google Scholar] for the initial encounters between mammalian chromosomes and microtubules. Initial kinetochore attachment with the microtubule lattice is facilitated by mechanisms that favor microtubule growth. This growth is stimulated by a class of microtubule plus-end binding proteins, known as +TIPs, and the small GTPase Ran. One of these microtubule binding proteins, Stu2 (XMAP215/ch-TOG) had previously been found to regulate microtubule dynamics [9Kosco K.A. Pearson C.G. Maddox P.S. Wang P.J. Adams I.R. Salmon E.D. Bloom K. Huffaker T.C. Control of microtubule dynamics by Stu2p is essential for spindle orientation and metaphase chromosome alignment in yeast.Mol. Biol. Cell. 2001; 12: 2870-2880Crossref PubMed Scopus (119) Google Scholar, 10Pearson C.G. Maddox P.S. Zarzar T.R. Salmon E.D. Bloom K. Yeast kinetochores do not stabilize Stu2p-dependent spindle microtubule dynamics.Mol. Biol. Cell. 2003; 14: 4181-4195Crossref PubMed Scopus (62) Google Scholar]. In the absence of Stu2, Tanaka et al. [3Tanaka K. Mukae N. Dewar H. van Breugel M. James E.K. Prescott A.R. Antony C. Tanaka T.U. Molecular mechanisms of kinetochore capture by spindle microtubules.Nature. 2005; 434: 987-994Crossref PubMed Scopus (214) Google Scholar] found a reduced density of nuclear microtubules and decreased efficiency of attachment between an unattached chromosome and microtubule. In the absence of core kinetochore components — Ndc10, Ndc80, Mtw1, Ctf19 — there was reduced efficiency of kinetochore–microtubule encounters, but a normal frequency of nuclear microtubule extension. Thus the 'global' regulation of microtubule dynamics by Stu2p, +TIPs and Ran are critical for ensuring the length and number of microtubules sufficient for kinetochore capture. Once a lateral attachment has been formed, the chromosome migrates toward the pole. Interestingly, the rate of chromosome movement (0.5–2.0μm min–1) is slower than that of microtubule depolymerization (2–3μm min–1), so what keeps the microtubule from depolymerizing through the site of chromosome attachment? In an astounding observation, Tanaka et al. [3Tanaka K. Mukae N. Dewar H. van Breugel M. James E.K. Prescott A.R. Antony C. Tanaka T.U. Molecular mechanisms of kinetochore capture by spindle microtubules.Nature. 2005; 434: 987-994Crossref PubMed Scopus (214) Google Scholar] found that Stu2 migrates to the plus-end of an attached microtubule. Stu2 is localized to microtubules and centromeres that have not yet been captured. Upon capture, Stu2 appears at the microtubule plus-end, where it prevents depolymerization. The inference is that Stu2 mediates kinetochore-dependent microtubule rescue. One possibility is that Stu2 on the recently attached kinetochore migrates from the kinetochore to the microtubule plus-end. In this way, the kinetochore regulates the stability of the microtubule to which it is attached. The technique of fluorescence recovery after photobleaching (FRAP) applied to centromere-bound Stu2 should show whether Stu2 released from the attached kinetochore migrates to the plus-end of a microtubule. Kinetochores have been shown to exert local control of the dynamics of attached microtubules [10Pearson C.G. Maddox P.S. Zarzar T.R. Salmon E.D. Bloom K. Yeast kinetochores do not stabilize Stu2p-dependent spindle microtubule dynamics.Mol. Biol. Cell. 2003; 14: 4181-4195Crossref PubMed Scopus (62) Google Scholar]. Perhaps the initial lateral interaction results in a conformational change that signals kinetochore bound +TIPs to disperse. This finding raises an additional consideration for understanding spatial regulatory networks. Various lines of evidence have indicated that Stu2 promotes microtubule growth or shortening [10Pearson C.G. Maddox P.S. Zarzar T.R. Salmon E.D. Bloom K. Yeast kinetochores do not stabilize Stu2p-dependent spindle microtubule dynamics.Mol. Biol. Cell. 2003; 14: 4181-4195Crossref PubMed Scopus (62) Google Scholar, 11Severin F. Habermann B. Huffaker T. Hyman T. Stu2 promotes mitotic spindle elongation in anaphase.J. Cell Biol. 2001; 153: 435-442Crossref PubMed Scopus (97) Google Scholar, 12Shirasu-Hiza M. Coughlin P. Mitchison T. Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification.J. Cell Biol. 2003; 161: 349-358Crossref PubMed Scopus (81) Google Scholar, 13van Breugel M. Drechsel D. Hyman A. Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer.J. Cell Biol. 2003; 161: 359-369Crossref PubMed Scopus (98) Google Scholar]. The finding that Stu2 coming from kinetochores promotes microtubule rescue [3Tanaka K. Mukae N. Dewar H. van Breugel M. James E.K. Prescott A.R. Antony C. Tanaka T.U. Molecular mechanisms of kinetochore capture by spindle microtubules.Nature. 2005; 434: 987-994Crossref PubMed Scopus (214) Google Scholar], while Stu2 coming from elsewhere may promote microtubule shortening (or the release of shortening factors [14Usui T. Maekawa H. Pereira G. Schiebel E. The XMAP215 homologue Stu2 at yeast spindle pole bodies regulates microtubule dynamics and anchorage.EMBO J. 2003; 22: 4779-4793Crossref PubMed Scopus (62) Google Scholar]) indicates that perhaps the history of a protein's location is important:not only is there spatial segregation of function, but spatial 'memory'. The minus-end directed kinesin Kar3 contributes to poleward chromosome movement: mutational disruption of the ATP hydrolysis site in Kar3 leads to a significant increase in 'pauses' along the way. This cannot be the only mechanism of chromosome movement, however, as while kar3 mutant cells are sick, they are still viable, and chromosome translocation can be observed in kar3 mutants. The precise function of Kar3 has been enigmatic, and the Kar3-dependent chromosome translocation observed by Tanaka et al. [3Tanaka K. Mukae N. Dewar H. van Breugel M. James E.K. Prescott A.R. Antony C. Tanaka T.U. Molecular mechanisms of kinetochore capture by spindle microtubules.Nature. 2005; 434: 987-994Crossref PubMed Scopus (214) Google Scholar] is one of the clearest examples of its role in mitosis. Whether Kar3 is at the kinetochores, overlapping anti-parallel microtubules or microtubule plus-ends [15Maddox P.S. Stemple J.K. Satterwhite L. Salmon E.D. Bloom K. The minus end-directed motor Kar3 is required for coupling dynamic microtubule plus ends to the cortical shmoo tip in budding yeast.Curr. Biol. 2003; 13: 1423-1428Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar], or all of the above, is still not clear. Once a kinetochore has migrated to the pole, sister centromeres become randomized and bi-oriented by attachment to a microtubule from the opposite pole (Figure 1). Interestingly, this transition requires an additional set of proteins, the Dam1 complex and Ipl1 [1Biggins S. Walczak C.E. Captivating capture: how microtubules attach to kinetochores.Curr. Biol. 2003; 13: R449-R460Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar]. This is a clear case of different complexes being required for specific processes — lateral interactions versus bi-orientation — and raises the question of how the transition to bi-orientation is established. If the lateral interactions take place early in the formation of the spindle, microtubules from the adjacent spindle pole may interact with the sister kinetochore (see Figure 1 and [3Tanaka K. Mukae N. Dewar H. van Breugel M. James E.K. Prescott A.R. Antony C. Tanaka T.U. Molecular mechanisms of kinetochore capture by spindle microtubules.Nature. 2005; 434: 987-994Crossref PubMed Scopus (214) Google Scholar]). As the poles separate, the lateral interactions may mature into end-on interactions. Alternatively, once Dam1 and Ipl1 are recruited, the biochemistry of these interactions may favor end-on interactions [16Westermann S. Avila-Sakar A. Wang H.W. Niederstrasser H. Wong J. Drubin D.G. Nogales E. Barnes G. Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex.Mol. Cell. 2005; 17: 277-290Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 17Miranda J.J. De Wulf P. Sorger P.K. Harrison S.C. The yeast DASH complex forms closed rings on microtubules.Nat. Struct. Mol. Biol. 2005; 12: 138-143Crossref PubMed Scopus (213) Google Scholar]. Even when bi-oriented, sister kinetochores continue to oscillate, though they maintain an average position midway between the poles. A given kinetochore makes very few excursions to the opposite pole [18Pearson C.G. Yeh E. Gardner M. Odde D. Salmon E.D. Bloom K. Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase.Curr. Biol. 2004; 14: 1962-1967Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar]. What are the mechanisms that give rise to metaphase and do they relate to the mechanisms described for initial encounters? An important consequence of bi-orientation is the tension generated between replicated but unseparated sister chromatids. This tension may promote rescue of microtubule depolymerization. In this view, attached kinetochore microtubules depend on the stretch of chromatin between sister kinetochores for rescue [19Gardner M.K. Pearson C.G. Sprague B.L. Bloom K. Salmon E.D. Odde D.J. Tension-dependent regulation of kinetochore microtubule dynamics can explain metaphase congression in yeast.Mol. Biol. Cell. 2005; : 1Google Scholar], an effect antagonized by increased kinetochore microtubule catastrophe at the spindle equator. Local control of microtubule dynamics by the kinetochore could depend on a combination of a spatial cue, possibly provided by release of Stu2 from the kinetochore, and/or a mechanical tension-sensing cue. This type of regulation could result in highly dynamic kinetochore microtubules while orchestrating the organization of kinetochores into a metaphase configuration with remarkable fidelity to one spindle-half. With the direct visualization of initial encounters and the components in hand, the prospect of understanding the molecular basis for the 'dance of the chromosomes' is on the horizon.