Chromosome Segregation: Correcting Dispatch Improper Attachment

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Tarun M. Kapoor binding site per kinetochore. In syntelic attachments, both sister kinetochores are attached to the same spindle pole [2,6,10]. If any of these on-pathway or off- Accurate chromosome segregation requires that the pathway intermediates in chromosome attachments two sister kinetochores attach to microtubules from remain uncorrected during cell division, chromosomes opposite spindle poles. New work reveals how a will be lost. kinetochore can segregate properly while remaining Distinguishing between monotelic and amphitelic improperly attached to two spindle poles. attachment is conceptually simple. If a kinetochore has a finite number of microtubule binding sites, the occupancy of each binding site can be determined. If The faithful transmission of genomes to daughter any unoccupied binding sites are present, an error can cells requires that each replicated chromosome is be signaled. Components of the spindle assembly correctly transported during cell division. Chromo- checkpoint — a signaling pathway that can delay some movements are achieved through attachments anaphase and contribute to proper chromosome seg- of kinetochores — proteinaceous complexes assem- regation — are targeted to kinetochore sites not occu- bled on centromeric DNA — to microtubules from the pied by microtubules [1]. The displacement of these bipolar, spindle-shaped cell-division apparatus. For proteins from kinetochores by microtubules can accurate segregation, it is generally believed that a satisfy the checkpoint and thereby release the block kinetochore must attach to microtubules from one to anaphase. The anaphase delay allows time for spindle pole, while the sister kinetochore must attach chromosome position to be altered and centrosome- to microtubules from the opposite pole; this is dependent and centrosome-independent pathways, referred to as amphitelic attachment or chromosome with different kinetics, to operate until monotelic bi-orientation [1–3]. attachments are converted to proper amphitelic During mitosis, these kinetochore–microtubule attachments [12,13]. attachments are established in the period between Distinguishing between proper amphitelic nuclear envelope breakdown and anaphase, two attachments and improper merotelic or syntelic irreversible events in vertebrate mitosis [1,4]. Most attachments cannot rely on mechanisms that detect current models suggest that errors in chromosome occupancy of kinetochore–microtubule binding sites attachment are corrected before anaphase by the alone, as it is the orientation, and not number, of the reversal of improper attachments [1,3]. If improper interacting microtubules that is incorrect. Each attachments are not corrected, they may result in kinetochore experiences forces that push it away whole chromosome loss. New work by Cimini et al. [5], from, or pull it towards, a spindle pole through reported in this issue of Current Biology, provides attached microtubules [4]. The kinetochore also direct evidence for how one type of improper experiences forces that are transmitted through the chromosome–microtubule attachment may be chromosome: these include the so-called ‘polar wind’, corrected in mammalian cells. There are number of a force pushing chromosomes away from the pole surprising results in this study, including data showing which is generated by interactions of spindle that a chromosome can segregate correctly while microtubules and chromosome arms, as well as maintaining improper attachments, and that this forces acting on the sister kinetochore to which a mechanism is active after the start of anaphase, a kinetochore is mechanically linked. In amphitelic point-of-no-return during mitosis. attachment, there are forces ‘pulling’ kinetochores in The formation of correct chromosome–microtubule opposite directions which result in increased inter- attachments passes through an ‘on-pathway’ kinetochore distances; these are often about 150% intermediate state of monotelic attachment (Figure 1), greater than when the kinetochores are unattached or chromosome mono-orientation, in which one sister [11]. From the results of a series of seminal micro- kinetochore is attached to microtubules from a spindle manipulation studies, Nicklas and co-workers pole while the other kinetochore is unattached [2,6]. [3,14,15] have suggested that such a mechanical The pathway is not error free, and a number of types tension at kinetochores contributes to stabilizing of ‘off-pathway’ intermediates have also been attachments to microtubule. It has also been pro- characterized (Figure 1). In merotelic attachment, for posed that proper tension across sister kinetochores example, one sister kinetochore has microtubules contributes to the detection and correction of connecting it to both spindle poles; the other sister merotelic and syntelic attachment errors. kinetochore is often attached to only one of the two Cimini, Salmon and co-workers [7–9] have recently spindle poles [7–9]. This error can only occur in examined merotelic attachment in mammalian mitosis organisms that have more than one microtubule and have found this error in ~1% of dividing cells, a frequency which can account for most cases where 1230 York Avenue, New York, New York 10021, USA. chromosomes have been observed to lag behind at E-mail: [email protected] the spindle equator during anaphase in cell culture. Dispatch R1012

observed at later stages of mitosis was significantly A greater than the number of lagging chromosomes Merotelic seen at the completion of cell division. This indicated that at least two mechanisms act during mitosis to correct merotelic attachments: one that operates before anaphase, and surprisingly, one that must operate after the start of anaphase. In their new study, Amphitelic Cimini et al. [5] developed and used live cell imaging methods to track movements of individual merotelic Syntelic Monotelic kinetochores, and observed how merotelic attachment can be corrected after anaphase has begun. B C If a kinetochore has only one microtubule binding site, a correction mechanism acting at the kinetochore would involve release of the microtubule attachment and a completely unattached kinetochore intermediate. But in mammals, the number can be as high as 35 per kinetochore, with an average of ~28 [16]. Cimini et al. [5] found that correction of merotelic attachment during anaphase did not simply involve the release of attachments to the incorrect pole. These kinetochores maintained attachments, but moved towards the correct spindle pole by changing the lengths of microtubules. The microtubule bundle oriented towards the correct spindle pole shortened, while the bundle oriented towards the incorrect pole length- ened, placing the chromosome in the daughter cell opposite to the one with its sister. Measurements Microtubule revealed differences in fluorescence intensities and Chromosome therefore the number of microtubules in the bundles Spindle pole oriented to the two spindle poles from a single merotelic kinetochore, with more microtubules Current Biology extending towards the correct spindle pole. How Figure 1. Chromosome–microtubule attachments during cell these differences in microtubule number are achieved division. before anaphase starts is mysterious, and suggests (A) Four different types of attachment of chromosomes to the cell that a kinetochore itself is ‘smart’ and knows which division apparatus. (B) Cimini et al. [5] report that an improperly the right pole from the wrong one [17]. attached merotelic kinetochore, with microtubules pulling it On the basis of their observations of differences in towards opposite poles, can position itself during anaphase such the number of kinetochore-microtubule attachments, that it reaches the correct daughter cell at the completion of Cimini et al. [5] propose an appealing model for how mitosis. They found that the microtubule bundle connecting a error correction might work. While the forces directing merotelic kinetochore to the spindle pole that would result in proper segregation has more microtubules (thicker line). The a merotelic kinetochore towards the two opposite greater number of microtubules pulling towards a pole results in poles are equal, the tension at each microtubule lower mechanical tension at each microtubule-binding site, binding site is inversely proportional to the number of switching the kinetochore to a state in which it depolymerizes microtubules present. For bundles with more attached microtubules. The thinner bundle, attached to the microtubules, the tension at each binding site is lower. wrong pole, has higher tension per binding site and the kineto- Lower tension could promote microtubule depolymer- chore allows for incorporation of tubulin subunits and increase in the distance of the chromosome from the wrong end of the cell ization and thereby move the kinetochore towards the division apparatus. (C) Syntelic attachments may be corrected by correct pole [18]. Higher tension facilitates lengthen- a similar mechanism. In this case, both kinetochores are likely to ing of the thinner microtubule bundle, facilitating be in a state of low tension and the attachment can be corrected chromosome movement away from that end of the by depolymerizing the microtubule fibers connecting them to spindle (Figure 1B). spindle poles. Complete release of microtubule binding by There is a striking similarity between the correction kinetochores is not observed during the correction of either syntelic or merotelic attachments during vertebrate mitosis. of merotelic orientations during anaphase and a mechanism that corrects syntelic attachments before anaphase. Direct observations [19] revealed that Using high-resolution light microscopy of fixed cells, syntelic kinetochores also did not release all and by carefully analyzing kinetochore and microtubule attachments polewards during early microtubule attachments, they showed that merotelic stages of the correction process, but regulated the attachments can be remarkably common early in lengths of the attached microtubules. Shortening mitosis (~30% at prometaphase). The number of microtubules moved chromosomes towards the merotelic attachments declines over the course of spindle pole. The geometry of kinetochores in syntelic mitosis, and delaying anaphase can help reduce these attachments limits the use of inter-kinetochore errors; however, the number of merotelic attachments distance as a measure of the tension at these sites. It Current Biology R1013

is likely that the tension at each kinetochore binding 18. Maddox, P., Straight, A., Coughlin, P., Mitchison, T.J., and Salmon, site in syntelic chromosomes is low compared to that E.D. (2003). Direct observation of microtubule dynamics at kinetochores in Xenopus extract spindles: implications for spindle at amphitelic attachments, as the only force opposing mechanics. J. Cell Biol. 162, 377–382. the polewards force at a kinetochore are the forces 19. Lampson, M.A., Renduchitala, K., Khodjakov, A., and Kapoor, T.M. acting on chromosome arms (the polar wind). This low (2004). Correcting improper chromosome-spindle attachments during cell division. Nat. Cell Biol. 6, 232–237. tension state may promote microtubule depolymer- 20. Biggins, S. (2004). Correcting SYNful attachments. Nat. Cell Biol. 6, ization at syntelic kinetochores to shorten microtubule 181–183. fibers, initiating the correction of these attachments, similar to what is suggested by Cimini et al. [5] for merotelic attachments (Figure 1C). The tension-sensitive regulators of microtubule polymerization at kinetochores are not known. Favored candidates include Aurora kinases, their activators and substrates [20]. Probes for the functions of these proteins, the amazing microscopy methods developed by Cimini and co-workers [5], combined with micromanipulation techniques described in the classic studies in insect cells [3,15], may provide answers.

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