Centromeres And Kine To Chores Review 2003

Cell, Vol. 112, 407–421, February 21, 2003, Copyright 2003 by Cell Press

Centromeres and Kinetochores: From Epigenetics to Mitotic Checkpoint Signaling Don W. Cleveland,1,* Yinghui Mao,1 and Kevin F. Sullivan2 1 Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine University of California, San Diego 9500 Gilman Drive La Jolla, California 92093 2 The Scripps Research Institute 10555 N. Torrey Pines Road La Jolla, California 92037

The centromere is a chromosomal locus that ensures delivery of one copy of each chromosome to each daughter at cell division. Efforts to understand the nature and specification of the centromere have demonstrated that this central element for ensuring inheritance is itself epigenetically determined. The kinetochore, the protein complex assembled at each centromere, serves as the attachment site for spindle microtubules and the site at which motors generate forces to power chromosome movement. Unattached kinetochores are also the signal generators for the mitotic checkpoint, which arrests mitosis until all kinetochores have correctly attached to spindle microtubules, thereby representing the major cell cycle control mechanism protecting against loss of a chromosome (aneuploidy). Introduction Chromosome movement on the spindle during mitosis and meiosis is powered and regulated by the centromere, a discrete locus on each chromosome. Originally identified as the primary constriction, this region is a complex chromosomal substructure (Figure 1A). While the term centromere is generally taken to refer to the DNA segment that confers centromere function, the cytologically visible centromere is more complex . In mitosis, a proteinaceous structure, the kinetochore, assembles at the surface of the centromere and acts as the site of spindle microtubule binding. By electron microscopy, the kinetochore appears as a narrow band of dense chromatin just at the surface of the primary constriction, the inner kinetochore, and a laminar outer kinetochore domain (frequently misnamed a “plate”) that contains many of the microtubule binding and signal transduction molecules discussed below. The monotonous heterochromatin that links two sister kinetochores has emerged as a distinct functional entity within the centromere-kinetochore complex, important for cohesion and kinetochore regulation. The boundaries of the centromere locus therefore extend beyond the kinetochoreforming region. In this review, we discuss how centromeres and their kinetochores assemble, how they power mitotic chromosome movements, and how, as signaling *Correspondence: [email protected]

Review

elements of the mitotic checkpoint, they control cell cycle advance during cell division. Defining the Locus The centromere challenges the classic view of a genetic locus. Dogma and experience suggest that a chromosomal locus is defined by its DNA sequence, and its function is contained with the information content present in the primary sequence, e.g., recognition sites for DNA binding proteins. This simple situation is correct for the centromere of budding yeast, where point mutations abolish activity. In other organisms, however, including fission yeast, specific centromere-nucleating DNA sequences have not been found, and centromere function exhibits properties of an epigenetic locus, behaving as a self-replicating protein complex that resides on centromere DNA but is not determined by it. Although the details differ markedly, resolution of biochemically and structurally distinct centromere domains in many organisms has revealed common aspects of organization (Figure 1B), providing a sketch of a simple “universal centromere architecture.” Chromatin is the key feature and the centromere domain is built on a distinct type of nucleosome found nowhere else in the genome, in which histone H3 is replaced by a divergent (50% identity) homolog usually referred to as CENP-A (Smith, 2002). This core is flanked by another chromatin domain, a highly phased nucleosome array in S. cerevisiae and heterochromatin in other species that plays a role in cohesion of sister centromeres (Bernard et al., 2001). Both domains are required for complete centromere function. The Single Nucleosomal Centromere of Budding Yeast The budding yeast centromere is by far the simplest and most dissected one (Figure 2). Spanning only ⵑ125 bp of DNA, S. cerevisiae centromeres contain three distinct DNA sequence elements (Figure 2A). Two of these, CDE I (purple, Figure 2A) and CDE III (red, Figure 2A), are conserved at each chromosome and function as sequence-specific elements by the criterion that single point mutations abolish their activity. The other, CDE II (blue in Figure 2A), is not conserved in sequence among the yeast chromosomes, but the approximate length (76–84 bp) and A⫹T rich content (90%) are maintained (reviewed in detail by Cheeseman et al., 2002b). The 125 bp centromere DNA is flanked on either side by a uniquely positioned (i.e., highly phased) nucleosome array rich in cohesins and thought to provide a functional context for centromere activity (Laloraya et al., 2000; Tanaka et al., 1999). Somewhat ironically, CDE II, the unconserved DNA element, interacts with Cse4 (Stoler et al., 1995), the homolog of CENP-A, the most highly conserved protein motif known for the centromere (Figures 2A and 2B). CENP-A/Cse4 nucleosomes are specifically associated with centromere DNA in vivo (Meluh et al., 1998), strongly suggesting their assembly as a nucleosomal complex at

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Figure 1. Organization of Centromeres (A) Overall organization of the centromere. A mitotic chromosome has been sectioned along the plane of the spindle axis, revealing the symmetric bipolar organization of a chromosome fully engaged on the spindle. (Right) Key elements have been pseudo colored. (Violet) The inner centromere, a heterochromatin domain that is a focus for cohesins and regulatory proteins such as Aurora B and Kin I. (Red) The inner kinetochore, a region of distinctive chromatin composition attached to the primary constriction. (Yellow) The outer kinetochore, the site of microtubule binding, is comprised of a diverse group of microtubule motor proteins, regulatory kinases, microtubule binding proteins, and mitotic checkpoint proteins. (B) Schematic illustration of centromere loci. Organization of centromeric DNA sequences from the four example organisms. (Top) Budding yeast with a 125 bp centromere comprised of three sequence domains (pink, red, yellow). Fission yeast centromeres show an organized structure, with a nonconserved central core (red), flanking inner repeats (pink arrows) at which the CENP-A-containing nucleosomes assemble, and conserved outer repeats (stippled purple). The Drosophila centromere spans ⵑ400 kb (red) embedded in constitutive heterochromatin (purple). (Bottom) Human centromeres have sizes approaching 10 Mb and are comprised of ␣-I satellite DNA (red) and a more divergent, less regular ␣-II satellite (pink), flanked by heterochromatin (purple).

the centromere (Figure 2B). A second CDE II interacting protein, Mif2 (Meluh and Koshland, 1995), is the homolog of an essential mammalian kinetochore chromatin binding protein CENP-C (Figure 2B). Centromere function in yeast also depends critically on sequence specific DNA-protein interactions. CDE III binds an essential four subunit protein complex, Cbf3 (Gardner et al., 2001) (Figures 2B and 2C). CDE I binds a nonessential bHLH DNA binding protein, Cbf1, that interacts with Cbf3 components (Hemmerich et al., 2000). The geometry dictates that the Cbf1-Cbf3 interaction must take place across the DNA gyres wound over the Cse4 nucleosome, perhaps stabilizing the higher order complex like a protein clamp (Figure 2B). The Cbf3 complex produces an extended footprint (80 bp) on centromere DNA (Russell et al., 1999; Espelin et al., 1997). The resulting broad interaction surface may be important for stable centromere binding or to provide a large binding surface for more distal kinetochore proteins, or both. The flanking chromatin domain functions in cohesion (Megee et al., 1999; Tanaka et al., 1999). How is the centromeric nucleoprotein complex attached stably to a single, highly dynamic (e.g., He et al., 2000) spindle microtubule? Four key protein complexes comprised of ⬎20 components are central players in outer kinetochore assembly and microtubule binding (Figures 2C and 2D). The Ctf19 complex and the Ndc80 complexes appear to represent “adaptors” that interact

with both core centromere and distal kinetochore or spindle components (Ortiz et al., 1999; Wigge and Kilmartin, 2001; Janke et al., 2001). The Dam1p complex (Cheeseman et al., 2001; Janke et al., 2002) (also known as DASH [Li et al., 2002]) may be the central component of microtubule binding (Figures 2C and 2D), with its activity regulated by the yeast Aurora B kinase (Ipl1) (Tanaka et al., 2002; Biggins et al., 1999). Present in a conserved complex with the yeast homologs of INCENP (Sli15) and survivin (Bir1), respectively (Bolton et al., 2002), Ipl1-dependent phosphorylation of at least three Dam1p components are essential for microtubule capture (Cheeseman et al., 2002a). From the model in Figure 2D, with the components drawn to scale, it is immediately apparent that connecting a large microtubule (25 nm) with a small, 10 nm nucleosomal centromere requires multiple attachments, especially considering that the microtubule-kinetochore linkage is dynamic, with individual connections broken and reformed as the microtubule grows or shrinks. Fission Yeast Centromere: Chromatin Not a Specific DNA Sequence The critical importance of the chromatin environment surrounding the core CENP-A chromatin domain has been most clearly elucidated in S. pombe. Each of the three centromeres, which range in size from 40–100 kb, possess a pair of repeated sequence arrays that are

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Figure 2. Molecular Details of a Simple Kinetochore (A) The centromere DNA of S. cerevisiae is shown at top with the three sequence elements, CDE I (purple), CDE II (blue), and CDE III (red), highlighted. Numbering corresponds to chromosome 11. Proteins that bind to the centromere DNA elements are color coded to match the corresponding DNA binding domain and are drawn to scale assuming a globular structure. Human homologs, where known, are denoted in italics. CBF3 contains two copies of Ctf3 and two dimers of Ndc10, but their arrangement in the complex is not known and is likely to be more asymmetric than shown here. (B) The nucleosomal centromere. Cse4 (blue and purple spacefill) forms a specialized nucleosome at the centromere, modeled here on the structure of the nucleosome-containing histone H3 (Luger et al., 1997). Other histones (gray) are shown only as backbone traces. The position of centromere DNA on the nucleosome is derived from Keith and Fitzgerald-Hayes (2000) by placing CDE I (purple) in contact with one of the two Cse4 molecules. Most of the remaining histone core surface is in contact with CDE II (blue), while CDE III (red, behind) projects into the internucleosomal linker DNA. The divergent N-terminal tail of Cse4 is drawn in purple. (Right) The nucleosome is rotated 180⬚ and is shown with Cbf1 (purple), Mif2 (blue), and Cbf3 (red) placed on DNA elements CDE I, -II and -III, respectively, illustrating the relative sizes and positions of the core centromere components. (C) Kinetochore complexes associate with the centromere nucleoprotein core. Four key kinetochore protein complexes discussed in the text are illustrated. Interactions, genetic or biochemical, are indicated by black arrows, while Ipl1 substrates are shown with red arrows. (D) A model of budding yeast centromere-microtubule interaction. A microtubule (green) is shown binding to the centromere in the context of a 30 nm fiber.

arranged in an inverted repeat around an unconserved central core sequence (Figure 1B) (Clarke, 1998). Both central core and inner repeat sequences are bound by the CENP-A homolog (Cnp1) and two conserved proteins, Mis6 and Mis12, homologs of subunits of the Ctf19 complex from budding yeast (Goshima et al., 1999; Partridge et al., 2000; Takahashi et al., 2000). Unlike budding yeast, no unique sequence within it is sufficient in S. pombe, with flanking sequences critical for the establishment and maintenance of centromere function (Partridge et al., 2000; Ngan and Clarke, 1997; Ekwall et al., 1997). These are modified by methylation of lysine 9 of histone H3, which in turn recruits Swi6, the fission yeast equivalent of vertebrate HP1 (Lachner et al., 2001). These are characteristic biochemical markers of heterochromatin, which likely require elements of the RNAi system in S. pombe by analogy with Cenhomology-dependent heterochromatic silencing at the

mating type locus (Volpe et al., 2002; Partridge et al., 2002). A key function of this heterochromatin domain is the recruitment of the fission yeast cohesin (Rad21) (Bernard et al., 2001; Tomonaga et al., 2000), the central component of the protein glue that holds duplicated chromatids together. Metazoans: The Mega Multisubunit Centromere Metazoan centromere organization is in much more complex than that in the yeasts (Figure 1B). Spanning 0.3–5 Mbp of DNA, human centromeres contain extensive (1,500 to ⬎30,000 copies), tandemly repeated arrays of a 171 bp sequence element termed ␣ satellite (Figure 3A). Centromere function has been mapped to ␣ satellite arrays by centromeric deletions, either naturally occurring on the X chromosome (Schueler et al., 2001) or those induced by telomere insertion into the Y (Brown et al., 1994). Centromeric satellite DNAs are not con-

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Figure 3. Organization of Human Centromeres (A) DNA organization. The hierarchic organization of an ␣ satellite DNA is illustrated, with a 171 bp monomer sequence shown at top. Monomer sequences are iterated with nucleotide sequence variations to form a higher order repeat (colored arrows), which itself is tandemly iterated with high (⬎99%) sequence conservation to form extensive arrays of higher order repeats. At bottom is a diagram of the centromere region of chromosome 10, illustrating the ⵑ2 Mb ␣ satellite array with surrounding pericentric satellite arrays (SAT2 and SAT3). (B) Discontinuous distribution of centromere proteins. Hypotonic stretching of chromosomes followed by immunofluorescence with human anti-centromere antibodies revealed a punctuate distribution of centromere antigens (Blower et al., 2002), suggesting a multiple subunit organization at the centromere/kinetochore complex. (Right) Extended chromatin fibers from human interphase cells demonstrate discontinuous CENP-A domains (red) interspersed with histone H3 domains (green). Images provided by Beth Sullivan (Boston University). (C) Folding centromeric chromatin in mitotic chromosomes. The discontinuous CENP-A chromatin fiber (left) forms a single domain at the surface of the chromosome and therefore must be folded or coiled to achieve the polarized distribution of CENP-A and H3 sites in mature mitotic kinetochores (Sullivan et al., 2001). The coil shown illustrates only one possible folding pathway in the context of an idealized centromere region, showing that CENP-A (red) assembles onto regular ␣-I satellite sequences (green), with ␣-II satellite (teal blue) and pericentric satellite sequences (orange, purple) flanking this site. At right, a sinuous path of chromosomal DNA through the centromere region is proposed on the basis of the differential localization of ␣-I and ␣-II sequences to the outer and inner centromere regions.

served in sequence among metazoans, but share: (1) their presence in very large tandem arrays, and (2) unit repeat lengths that tend toward multiples of the nucleosome repeat length (Henikoff et al., 2001). (An exception is Drosophila, where the functionally mapped centromere consists of simple [5 bp] satellite sequences interspersed with various transposons [Sun et al., 1997]). CENP-A nucleosomes are bound to ␣ satellite DNA in human centromeres (Vafa and Sullivan, 1997), but these are not uniformly distributed. Stretched chromatin fibers reveal interspersed CENP-A- and histone H3-containing nucleosomes (Figures 3B and 3C; Blower et al., 2002). These foci may represent kinetochore “subunits” (for which the budding yeast represents a unitary example) that assemble together (Zinkowski et al., 1991) to form multiple binding sites for the multiple microtubules that attach to metazoan centromeres. Primate centromeres frequently have two major ␣ satellite families adjoining each other (He et al., 1998): a highly regular ␣-I and an ␣-II that varies widely in monomer sequences and repeat structure. CENP-A is bound primarily to ␣-I satellite sequences, at the surface of the chromosome (Ando et al., 2002). In contrast, ␣-II satellite is localized in the interior or central domain of the centromere, where components such as INCENP, Aurora B, and cohesin are concentrated (Figure 3C). This twocomponent organization is evident throughout eukaryotes. A core domain built around CENP-A and centromere-specific chromatin binding proteins establishes the kinetochore-forming component of the centromere, while flanking domains are enriched in proteins involved in chromatid cohesion. The Epigenetic Centromere While in budding yeast centromere DNA alone can nucleate centromere formation de novo, centromeres in

S. pombe depend strongly, and those of metazoan cells primarily, on epigenetic factors rather than DNA sequence for activity. Three lines of evidence support this statement. First, centromere sequences are evolving at an unusually high rate, coevolving with their essential partner CENP-A, and show no obvious sequence conservation that links divergent species or even different chromosomes in the case of Drosophila (Henikoff et al., 2001). Second, centromere DNA sequences by themselves are unable to specify centromere function: stable dicentric chromosomes have been found in which one centromere has been silenced with no obvious rearrangement of centromere DNA (Sullivan and Willard, 1998). Third, acquisition of centromere function has been found on certain rearranged chromosomes lacking an endogenous centromere (Figure 4A). In a well studied case, a stable derivative of human chromosome 10 with a deletion spanning the centromere was found to have a functional centromere at position 10q25. Mitotic stability through development and adult life in this patient demonstrated that this neocentromere functions and binds to ⬎20 kinetochore, centromere, and chromatin proteins (Saffery et al., 2000). This region shows no centromeric DNA or any other distinctive feature (Barry et al., 1999). CENP-A is present over a 460 kb domain (Lo et al., 2001), similar in size to the minimal Drosophila centromere (420 kb; Sun et al., 1997) and the smallest natural human centromeres (500 kb) (found on Y chromosomes, TylerSmith et al., 1993). Thus, noncentromeric DNA segments can acquire full centromere function without DNA sequence changes and be maintained at those sites indefinitely—even through multiple human generations (TylerSmith et al., 1999). Chromatin rather than DNA may be a major determinant of centromere identity (Williams et al., 1998). During

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Figure 4. Epigenetics and DNA Sequences in Centromere Formation (A) A human neocentromere. (Top) Human chromosome 10. (Below) Rare internal recombination has produced the two subfragments in one patient. One (left) is a ring chromosome (r(del)10) that carries the original centromere (red). The other is a linear deletion product, mar(del)10, that has acquired a new centromere at 10q25 (orange). The CENP-A domain of this neocentromere spans about 400 kb. (B) Generation of neocentromeres. A Drosophila chromosome derivative, Dp1187, was mapped for centromere activity using x-irradiation to introduce chromosomal breaks (top). In one product, an inversion placed the normally euchromatic sequences on the left arm (cyan) directly in cis to the functional centromere. Centromeric chromatin is proposed to have spread into the now flanking euchromatic DNA. Subsequent x-irradiation produced deletion that entirely lacks the initial centromere DNA but, nevertheless, retains substantial centromere activity (bottom) by virtue of the centromeric chromatin that has spread into the region. (C) Construction of a human minichromosome. A single copy of the 16mer higher order repeat of chromosome 17 ␣ satellite DNA was amplified to generate arrays of up to 64 repeat units in a bacterial artificial chromosome vector. These were further amplified to generate fragments up to ⵑ1 Mb in length. Synthetic ␣ satellite arrays were combined with a ␤-geo resistance marker gene (green), telomere DNA (yellow), and human genomic DNA (gray) for transfection into HT1080 cells. Linear minichromosomes (6–10 Mb in length, 5%–10% the size of the smallest human chromosomes) containing 17 ␣ satellite (without detectable host chromosomal DNA) were identified at a low frequency (Harrington et al., 1997). (D) Dissecting DNA sequence requirements by minichromosome engineering. (Top) The ␣ satellite domain of human chromosome 21, including a highly regular ␣21-I array with a high frequency of CENP-B boxes (green ovals) and a flanking ␣21-II array that is highly irregular in sequence organization and has a low frequency of CENP-B boxes. (Middle) Construction of large arrays containing (␣21-ICENPB⫹) or lacking (␣21Icenpb-) functional, 11mer CENP-B B boxes were produced in a BAC vector, amplified, and transfected into HT1080 cells. ␣21-ICENPB⫹ yielded minichromosomes in 40%–50% of transformants; ␣21-Icenpb⫺ yielded none. Amplification of a 340 bp non-␣ satellite sequence into which CENP-B boxes were embedded (V340:CENPB⫹) yielded no minichromosomes (Ohzeki et al., 2002).

the mapping of a Drosophila centromere by X-rayinduced chromosome breakage, a chromosome was recovered that placed the centromere directly next to a euchromatic DNA segment (Figure 4B). A subsequent round of x-irradiation produced stable chromosome derivatives that lacked centromere DNA but retained only the euchromatic sequence, indicative of a centromeredetermining chromatin structure that spread into the euchromatin and which retained full centromere function after subsequent X-ray-mediated severing of the natural centromere. The universal association of centromere function with a distinct histone also supports a role for chromatin in centromere determination. CENP-A appears to be at the root of the kinetochore assembly process and is required for assembly of most distal kinetochore components examined (Howman et al., 2000; Oegema et al.,

2001). CENP-A chromatin does not form a specialized domain of DNA replication (Shelby et al., 2000; Sullivan and Karpen, 2001), and its loading is uncoupled from that of the conventional histones (Shelby et al., 1997; Takahashi et al., 2000). CENP-A is distributed conservatively, however, to the two daughter chromatids during S phase (Shelby et al., 2000), providing the potential for carrying a DNA sequence-independent heritable mark through a CENP-A directed, self-replicating chromatin assembly pathway that bears little reference to the actual DNA upon which it sits (for more detailed account, see Sullivan et al., 2001). Determinants of De Novo Centromere Formation It is clear that non-DNA elements play a major role in centromere determination, but do DNA sequences not play a role at all? This has been answered by engineering

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artificial mammalian chromosomes. Minichromosomes were initially formed de novo (Harrington et al., 1997) by transfecting a mixture of a large synthetic chromosome 7 ␣ satellite array, telomeric sequences, a selectable marker, and genomic DNA fragments (for origin and other functions) (Figure 4C). This produced stably transmitted microchromosomes with functional centromeres containing chromosome 7 ␣ satellite sequences and typical kinetochore components, but only in a handful of extremely rare instances. Subsequently, a yeast artificial chromosome (YAC) carrying chromosome 21 ␣ satellite arrays was retrofitted with telomeres by recombination and introduced into human cells, yielding a much enhanced frequency of microchromosomes containing newly assembled functional centromeres, but only when the YAC contained ␣-I type satellite arrays (Ikeno et al., 1998). The homogeneous, highly regular ␣-I DNA has a high frequency of sites for the centromere-associated DNA binding protein CENP-B; ␣-II does not. This suggested a role for CENP-B in centromere formation, an idea previously dismissed by demonstration that CENP-B null mice are viable and fertile with only mild phenotypic effects on gonad size (Hudson et al., 1998). By engineering two ␣-I satellite arrays such that they differed only by the presence of functional CENP-B boxes (Figure 4D), CENP-B was shown to be necessary for de novo centromere formation, but it functions efficiently only in the context of ␣-satellite DNA. Since heterochromatin formation is a highly cooperative process driven by a series of mutually reinforcing reactions (Richards and Elgin, 2002), components such as CENP-B likely function in mediating centromeric chromatin modification, as has been seen for the three CENP-B homologs in S. pombe (Nakagawa et al., 2002). Kinetochores Power Chromosome Movements in Mitosis Since the term “mitosis” was introduced over 100 years ago by Fleming, a challenge has been to understand how cells divide and faithfully transmit chromosomes at each cell division. In a typical somatic cell cycle (Figure 5A), chromosomes in prophase initiate condensation, then, upon disassembly of the nuclear envelope and the interphase microtubule array, the fully compacted chromosomes spill into what was the cytoplasm to produce prometaphase. As a nascent mitotic spindle assembles, a dynamic process of repetitive search by unstable microtubules ensues for capture of chromosomes at their kinetochores (Figure 1A). Initial capture is frequently by binding of one kinetochore of a duplicated chromosome pair along the side of a spindle microtubule (Figure 5B, violet chromatid pair), allowing rapid (up to 1 ␮m/s) poleward translocation along that microtubule powered by a minus-end directed, kinetochore bound microtubule motor, almost certainly cytoplasmic dynein (Rieder and Alexander, 1990). This is followed by attachment of additional microtubules (up to 30 in humans [Rieder, 1981] or seven at mouse kinetochores [Putkey et al., 2002]), motor action at attachment sites, and oscillatory movements linked to continued growth and shrinkage of those kinetochore bound microtubules (Figures 5B and 5C, orange chromatid pair).

Subsequent capture by the unattached kinetochore of a microtubule from the opposite spindle pole produces biorientation and congression to the cell center in a rapid, discontinuous series of movements, again mediated by kinetochore motors (Figure 5B, red chromatids). The presence at kinetochores of active plus and minus end motors has been demonstrated, with net direction of movement switchable in vitro by phosphorylation (Hyman and Mitchison, 1991). In metazoans (Figure 6A), known kinetochore motors include the kinesin family member CENP-E and cytoplasmic dynein. Motors in Congression and Anaphase A long perplexing question has been how congression is achieved. With a common set of motors at both sister kinetochores, how is it that movement of a bioriented pair to the center is dominant, especially for those (like the red pair in Figure 5B) that can have many more microtubules bound to the kinetochore first attached and that are initially much closer to one pole? Laser ablation to disconnect the two chromatids or destroy either kinetochore established that almost all of the force is generated by the leading kinetochore (Khodjakov and Rieder, 1996), i.e., the one whose bound microtubules are shortening and whose motors are moving toward the microtubule minus ends. Two soluble dynein inhibitors (p50 dynamitin and a dynein antibody) disrupt the alignment of kinetochores at metaphase (Sharp et al., 2000). Mutations in the tethers (Rough deal [Rod] and Zeste white 10 [ZW10]) that link dynein to kinetochores attenuate the rate of poleward chromosome movement (Savoian et al., 2000), implicating dynein as a likely primary motor for congression. Other motors, especially the plus end motor CENP-E, stabilize microtubule capture at both sisters (McEwen et al., 2001; Putkey et al., 2002) in part through a contribution of CENP-E in maintaining attachment of kinetochores to the end of a disassembling microtubule (Lombillo et al., 1995). Chromosome alignment is precluded by disruption of CENP-E function in vitro (Wood et al., 1997) or in vivo (Putkey et al., 2002). A final class of kinetochore component, exemplified by the Kin I subgroup of the kinesin family (including XKCM1 in Xenopus [Walczak et al., 1996] and MCAK in mammals [Wordeman and Mitchison, 1995]), is a microtubule disassemblase [Desai et al., 1999]. This novel action acts at and between the two sister kinetochores, using the normal kinesin ATPase cycle to power removal of tubulin subunits. Chromosomes also experience forces exerted along the chromosomes arms as a result of spindle microtubule interaction with plus-end directed microtubule motors bound to chromatin (chromokinesins). The first identified, Drosophila Nod, is required for proper alignment of meiotic chromosomes that have not undergone recombination (Afshar et al., 1995). Immunodepleting another (Kid) prevents normal chromosome alignment (Antonio et al., 2000; Funabiki and Murray, 2000), while antibody-induced inhibition of human Kid blocks chromosome oscillations (like those of the red chromatid pair, Figure 5C), with chromosome arms atypically extending toward spindle poles during congression (Levesque and Compton, 2001).

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Figure 5. Chromosome Movements in Mitosis (A) Summary of the stages of mitosis. (Red) DNA; (green) microtubules. Images provided by Andreas Merdes (University of Edinburgh). (B) Chromosome movements during mitosis: (violet) one kinetochore of a duplicated chromatid pair becomes laterally attached to a single microtubule and rapidly powers the pair poleward. (Orange) The mono-oriented chromosome acquires additional microtubules and then oscillates near the pole. (Red) After capture by the unattached kinetochore of a microtubule from the opposite pole, the now bioriented chromosome congresses to the spindle equator. (Blue) At anaphase, the sister chromatids separate and undergo poleward motion. (C) The speeds of chromosome movements for the chromatids in (B). (Violet) Mono-oriented rapid poleward translocation; (orange) oscillatory movements prior to biorientation; (red) bioriented congression and oscillation; and (blue) poleward motion in anaphase. Adapted from Skibbens et al. (1993). Reproduced from The Journal of Cell Biology, 1993, vol. 122, pp. 859–875 by copyright permission of the Rockefeller University Press.

Modeling Chromosome Congression Among the early models to explain congression were proposals that the force generated was proportional to the length of the attached microtubule (Hays et al., 1982; Ostergren, 1951). The explanation was as perplexing as the initial problem, as no molecular mechanism was apparent for how such a length-dependent force could actually be generated through action at kinetochores. With the demonstration that ZW10 and Rod apparently stream poleward after microtubule capture (Williams et al., 1996), as do many of the kinetochore bound components of the mitotic checkpoint (Howell et al., 2000, 2001), it seems to us that a model compatible with the key evidence is that congression is powered, at least in part, by cytoplasmic dynein asymmetrically bound to the leading kinetochore of a chromatid pair. The 1-50 pN force generated during chromosome movement (Alexander and Rieder, 1991; Nicklas, 1983, 1988) is within the capacity of one or a few dynein molecules (ⵑ2–6 pN per motor molecule [Shingyoji et al.,

1998]). Considering that dynein is tethered to the underlying kinetochore by ZW10/Rod (see model in Figure 6B), congression would be achieved if the affinity of these tethers for kinetochore sites was weakened by increasing microtubule occupancy at that kinetochore. In other words, if distortion of the kinetochore occurs as more microtubules bind and this decreases the strength of ZW10/Rod binding, then dynein’s action would simply pull itself and ZW10/Rod off of the more highly occupied, trailing kinetochore, producing the streaming of ZW10 so prominently seen on both sides of the sister kinetochores after congression (Williams et al., 1996). The continuing oscillations at metaphase (Figures 5B and 5C, red chromatid pair) would simply reflect stoichastic changes in microtubule number on the two sides, thereby switching which kinetochore could more tightly hang onto its dynein. A cautionary note, however, is that there are multiple contributors and multiple solutions to congression so that the model put forward here is likely only be one

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Figure 6. Kinetochore Microtubule Capture and Chromosome Congression (A) Kinetochore microtubules are captured by motor proteins, including CENP-E and cytoplasmic dynein, the latter in associated with dynactin, ZW10, and Rod. Kin I acts as a microtubule disassemblase at and between the two sister kinetochores. Microtubule plus-end tracking proteins (such as EB1, APC) track microtubule “plus” ends and contribute to kinetochore microtubule stability and attachment. Plus-end directed chromokinesins are localized on chromosome arms and are responsible for driving the chromosome arms away from the poles (the “polar ejection” force). (B) A model for chromosome congression in prometaphase driven by dynein-dependent minus-end directed kinetochore motility. Increasing microtubule occupancy at the trailing kinetochore lowers ZW10/Rod affinity for that kinetochore causing dynein to pull itself and ZW10/Rod out of the kinetochore and to stream along the kinetochore microtubule. On the leading kinetochore with fewer microtubules bound, ZW10/ Rod is tethered more tightly, and the dynein power stroke moves the chromosome toward the spindle equator.

aspect. Chromosome fragments produced by laser cutting still congress with only a single kinetochore (Khodjakov et al., 1997). Similarly, in some cells, dynein inhibitors apparently do not affect congression, although they disrupt mitotic checkpoint signaling (Howell et al., 2001). Mitotic Complexities: Nonmotor Attachments In budding yeast where the intranuclear spindle forms prior to centromere duplication during S phase, nonmotor microtubule-associated proteins (MAPs) seem to be primary in microtubule capture, while dynein plays a role in spindle positioning, but not chromosome movement (Yeh et al., 1995). The Dam1p complex (Figures 2C and 2D) interacts physically with central kinetochore proteins of both the Ctf3 and Ndc80 complexes and binds to microtubules directly in vitro (Cheeseman et al., 2001), consistent with a direct role in mediating kinetochoremicrotubule attachments. A group of microtubule plus-end binding proteins (e.g., the EB1 protein family) might also be involved in mediating interactions between microtubules and kinetochores (Figure 6A). The yeast EB1 homolog BIM1 localizes to the plus ends of cytoplasmic microtubules and

increases dynamic instability (Tirnauer et al., 1999). Removal of EB1 has demonstrated that it is important for spindle microtubule stabilization (Rogers et al., 2002). During congression of a chromatid pair, EB1 is found at the ends of kinetochore bound microtubules that are growing, but not at those that are disassembling (Tirnauer et al., 2002). EB1 interacts (Su et al., 1995) with the human adenomatous polyposis coli (APC) tumor suppressor protein (not to be confused with APC/C, the E3 ubiquitin ligase used in controlling anaphase onset— see below). APC also binds to and stabilizes microtubules (Zumbrunn et al., 2001), localizes to the ends of microtubules embedded in kinetochores, forms a complex with mitotic checkpoint proteins Bub1 and Bub3, and is a substrate for both of the Bub1 and BubR1 kinases in vitro (Kaplan et al., 2001). One possible explanation for these observations is that EB1/APC complex may be one of the nonmotor linker(s) that connect microtubule attachment and the spindle checkpoint signaling machinery on the kinetochore. Anaphase Movements Using Motors and Flux Two mechanisms for poleward chromosome movement in anaphase, the primary function of mitosis, are now

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known: kinetochore motors and microtubule flux. That motors are used is hardly surprising, given their contribution to congression. For motors, the most obvious candidate is dynein. Although not required for chromosome segregation in budding yeast (Yeh et al., 1995), disruption of dynein clearly shows an anaphase chromosome segregation defect in Tetrahymena (Lee et al., 1999) and in kinetochore- and chromatid-to-pole movements in flies (Sharp et al., 2000). Flux, however, is much less intuitive. Discovered by photobleaching fluorescent microtubules (Gorbsky et al., 1987) and confirmed using fluorescence photoactivation of tubulin assembled into kinetochore bound microtubules (Mitchison and Salmon, 1992), flux represents continuous addition of tubulin subunits at kinetochores, coupled to disassembly at the poles driven by plus-end directed, pole bound microtubule motors presumably pulling on kinetochore microtubules and sliding them poleward. Subunits are lost from the minus ends of the microtubules, which must be tethered to the spindle pole, but in a way that subunits can be disassembled. The major mechanism for chromosome movement in anaphase in vertebrate somatic cells is motor-powered kinetochore movement coupled to microtubule disassembly at the kinetochore (Mitchison and Salmon, 1992; Walters et al., 1996). Here, poleward flux makes a relatively minor contribution with chromosome-to-pole movement three to eight times faster than flux. Yeast appear to lack microtubule depolymerization at poles and poleward microtubule flux during anaphase (Mallavarapu et al., 1999). In Xenopus egg extracts, however, anaphase A movement occurs at rates similar to poleward spindle microtubule flux (Desai et al., 1998), consistent with flux as the predominant mechanism. Elsewhere, the situation is controversial: fluorescent speckle microscopy has been used to claim a dominant role for flux (Maddox et al., 2002) in syncytial Drosophila embryos, while other efforts have found that dynein inhibitors disrupt chromatid-to-pole movement during anaphase A (Savoian et al., 2000; Sharp et al., 2000). It seems safe to conclude that in general, both mechanisms are used to produce anaphase force at kinetochores. Unattached Kinetochores as Signaling Devices for the Mitotic Checkpoint In order to assure accurate segregation, the mitotic checkpoint (also known as the spindle assembly checkpoint) acts to block entry into anaphase until both kinetochores of every duplicated chromatid pair have attached correctly to spindle microtubules. What has emerged in the decade since its initial description is that unattached kinetochores and/or those not under microtubuleinduced tension are the central signaling elements that produce a “wait anaphase” signal. A trio of cell-biological experiments provided the primary insights that a single, unattached kinetochore is enough to inhibit anaphase onset. By filming mitoses, it was initially found that anaphase ensues about 20 min after the last kinetochore attaches to the spindle (Rieder et al., 1994) and that by repeated detachment of a meiotic chromosome from a spindle by manipulation with a microneedle delayed anaphase indefinitely (Li and Nicklas, 1995). That

it was an unattached kinetochore that was responsible came from the seminal demonstration that laser ablation of the last unattached kinetochore produces anaphase onset within about 15 min (Rieder et al., 1995). A kinetochore-dependent wait anaphase signal is also suggested in budding yeast: blocking centromere assembly (by destruction of the Cbf3 component Ndc10) eliminates mitotic delay in the presence of microtubule assembly inhibitors (Gardner et al., 2001). Generating a Diffusible Inhibitor of Advance to Anaphase Genetics in yeast initially identified seven components of the mitotic checkpoint, Mad1–Mad3 (Mitotic arrest defective) (Li and Murray, 1991), Bub1–Bub3 (Budding uninhibited by benomyl) (Hoyt et al., 1991), and Mps1, a kinase that is also essential for spindle pole body duplication (Weiss and Winey, 1996). There are vertebrate homologs of all of these except Bub2. Initially demonstrated for Mad2 (Chen et al., 1996; Li and Benezra, 1996), all have now been identified to bind to and act at unattached kinetochores including Mad1 (Chen et al., 1998), Bub1 (Taylor and McKeon, 1997), Bub3 (Taylor et al., 1998), BubR1 (the mammalian Mad3) (Taylor et al., 1998), and Mps1 (Abrieu et al., 2001). Additional required contributors without yeast counterparts are known in metazoans. Without the kinetochore-associated microtubule motor protein CENP-E, a binding partner of BubR1, the checkpoint cannot be established or maintained in vitro (Abrieu et al., 2000) or in mice (Putkey et al., 2002). Inhibition by mutation (Basto et al., 2000) or antibody injection (Chan et al., 2000) of ZW10 and Rod have revealed both to also be required for checkpoint signaling. A final component with extensive sequence homology to and overlapping function with Bub3 is Rae1 (Babu et al., 2003), initially identified with an additional role in nuclear transport. Although the nature of the direct molecular interaction(s) between checkpoint proteins and kinetochores and the interdependencies of checkpoint proteins have not been determined (see Musacchio and Hardwick [2002] for details), the basic plan of the signaling cascade is established (Figure 7). Mad2 is recruited to unattached kinetochores in a complex with Mad1 (Chen et al., 1998). BubR1 and Bub1, both kinases, are required for generation and then rapid release from kinetochores of one or more inhibitors of Cdc20 (Fizzy in flies [Dawson et al., 1993], p55 in mammals [Kallio et al., 1998] or Slp1p in fission yeast [Kim et al., 1998]). This sequesters or inactivates Cdc20, which is required for substrate recognition by the anaphase promoting complex/cyclosome (APC/C) (substrates include securin [Zur and Brandeis, 2001] and cyclin B [Raff et al., 2002]), whose ubiquitinmediated destruction by the proteosome is required for anaphase onset (Cohen-Fix et al., 1996; Fang et al., 1998; Zou et al., 1999). The identity of the kinetochore-derived wait anaphase inhibitor (or inhibitors) is not established. An activated form of Mad2 (frequently referred to as Mad2*) has long been thought to be the inhibitory signal released. Microinjection of antibodies to Mad2 first revealed premature anaphase onset and chromosome missegregation (Gorbsky et al., 1998). Mad2 only binds to unattached

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Figure 7. Activating and Silencing the Mitotic Checkpoint Unattached kinetochores bind a collection of mitotic checkpoint components. These include at least four kinases [BubR1 (Chan et al., 1999), Bub1 (Roberts et al., 1994), Mps1 (Abrieu et al., 2001), and Mapk (Takenaka et al., 1998)]. These and the microtubule motor CENP-E are required for rapid recruitment of the Mad1/Mad2 complex. Mad2 cycles rapidly, releasing from kinetochores in a modified form or complex that sequesters Cdc20, thus inhibiting recognition by the ubiquitin ligase APC/C of substrates, including securin, whose destruction is required for advance to anaphase. ZW10/Rod, the kinetochore targeting components for cytoplasmic dynein, are required for generation of the inhibitory signal, possibly because they mitigate the rapid release of the inhibitor from kinetochores. After microtubule attachment, tension is generated between the bioriented kinetochores, checkpoint signaling is silenced, and anaphase ensues after decay of the previously made inhibitor.

kinetochores and the association at those kinetochores is extremely dynamic, with release and rebinding to unattached kinetochores within 25 s (Howell et al., 2000). Since a tetrameric form of recombinant Mad2 forms a ternary complex with Cdc20 and APC/C in vitro, blocks APC/C ubiquitination activity, and arrests Xenopus embryos and egg extracts in mitosis (Fang et al., 1998), it has been proposed as a candidate for the wait anaphase inhibitor. However, no checkpoint-dependent Mad2 oligomer generation has yet been identified in vivo. The recruitment of Mad2 at kinetochores has often been taken as a measure of ongoing checkpoint signal generation (and release). However, inhibition of ZW10/ Rod yields an inactive checkpoint despite prominent Mad2 binding at kinetochores (Chan et al., 2000). Diminution of Hec1, the homolog of yeast Ndc80, on the other hand, yields a chronically activated checkpoint with no Mad2 bound to kinetochores (Martin-Lluesma et al., 2002). Thus, it is now abundantly clear that the steady-state level of Mad2 bound to kinetochores is not a faithful reporter for checkpoint activation or inactivation. Another candidate for the wait anaphase signal is BubR1, which directly binds Cdc20 and APC/C elements Cdc16, Cdc27, and APC7 (Chan et al., 1999; Wu et al., 2000) and by doing so can block Cdc20 activation of APC/C for mitotic substrates. The inhibitory activity has been argued to be BubR1 alone without a contribution of Mad2 (Tang et al., 2001) or in a complex (named MCC) that apparently contains stoichiometric amounts of BubR1, Bub3, Mad2, and Cdc20 (Sudakin et al., 2001). An equivalent quaternary complex containing Mad3 (the yeast BubR1), Bub3, Mad2, and Cdc20 has also been observed in budding yeast (Fraschini et al., 2001; Hardwick et al., 2000). Further, the much higher APC/C inhibitory activity in vitro of MCC (⬎3,000-fold more potent than tetrameric Mad2) (Sudakin et al., 2001) would make this complex an attractive candidate for a diffusible inhibitory signal were it not that it (and its yeast counterpart) is formed in a kinetochore-independent manner

(Sudakin et al., 2001; Fraschini et al., 2001). This has led to the suggestion that the kinetochore contribution may modify APC/C itself to increase its affinity for MCC (Sudakin et al., 2001). While possible, this is unattractive as the only kinetochore-derived inhibitor because in order to keep APC/C inactive, such a model would require a single kinetochore to be capable of rapidly recruiting, and modifying, all cellular APC/C—a daunting task. More plausible are models with built-in signal amplification, for example, where a very abundant component (like Mad2) is activated and released by kinetochores, thereby producing a high concentration of an inhibitor to saturate available Cdc20. Lastly, kinetochores directly attract and may modify the target for inhibition, Cdc20, which cycles at kinetochores (Kallio et al., 2002) with kinetics even faster than reported for Mad2 (complete turnover within about 5 s). How this relates to checkpoint signaling is made less clear by unaltered cycling after checkpoint signal production is silenced by microtubule attachment. The Checkpoint Signal Has Limited Diffusibility While the identity(ies) and mechanism of generation at unattached kinetochores of the Cdc20-APC/C inhibitor(s) remains imperfectly defined, the activated form must be diffusible. The primary evidence for this is simple logic: in order to delay anaphase so as to prevent loss of a single chromosome, one unattached kinetochore (for example, on a chromatid pair trapped by chance behind one of the spindle poles) must be able to inhibit APC/C-dependent destruction of securin. This requires that the lone unattached kinetochore release a sufficiently strong (diffusible) signal to inhibit all Cdc20APC/C. But, there are strict limits to such diffusion. By analysis of cells containing two distinct spindles (after cell fusion), one or a few unattached kinetochores have been found unable to prevent anaphase in an adjacent spindle in which all kinetochores are attached (Rieder et al., 1997). The limited range of the APC/C inhibition almost

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certainly reflects the limited signal strength produced by a single unattached kinetochore, coupled to the short natural half-life of the inhibitor(s), whose destruction is complete within about 15 min after silencing production (Rieder et al., 1995). In addition, after release and local diffusion the inhibitor may be concentrated by trafficking to spindle poles by cytoplasmic dynein (see below), where APC/C is enriched (Tugendreich et al., 1995). This is compatible with the finding that many checkpoint components are rapidly trafficked poleward by dynein after release (Howell et al., 2001). Silencing the Checkpoint Signal: Attachment and Tension There is a continuing controversy as to whether the mitotic checkpoint is silenced by microtubule attachment (Rieder et al., 1995) or by the tension exerted between bioriented kinetochore pairs after attachment (Li and Nicklas, 1995) and whether activities of subsets of the known components are selectively silenced by one or the other (Skoufias et al., 2001; Zhou et al., 2002). McIntosh formally proposed the tension model under which the mechanical tension generated by polewarddirected forces acting on both kinetochores of a bioriented chromatid pair turns off checkpoint signaling (McIntosh, 1991). Compelling evidence for a tension requirement initially emerged using praying mantid spermatocytes in meiosis I, which have a Y chromosome and two genetically different X chromosomes. Although the Y is supposed to pair with both X chromosomes, occasionally it pairs only with one, thus yielding a monooriented X. When this occurs, anaphase onset is delayed by up to 9 hr but can be triggered to initiate almost immediately by application of mechanical tension applied across the mono-oriented kinetochore by use of a force-calibrated microneedle (Li and Nicklas, 1995). This is a general finding in meiosis: eliminating tension between homologous chromosomes by preventing recombination delayed anaphase onset in yeast, presumably from checkpoint activation. This delay was eliminated by genetically allowing sister kinetochores to inappropriately separate during meiosis I, thereby allowing each homolog to biorient in the absence of recombination and restore tension across the sisters (Shonn et al., 2000). Similarly, in cells that enter mitosis without a prior round of DNA replication, the unreplicated chromatids attach to spindle microtubules, but no tension can be developed and the mitotic checkpoint is chronically activated (Stern and Murray, 2001). This is not the whole story, however. In the initial demonstration that kinetochores are the signaling elements for the checkpoint, destruction of the last unattached kinetochore eliminated checkpoint signaling despite lack of tension on the kinetochore of the sister chromatid (Rieder et al., 1995). Similarly, in maize, satisfying the checkpoint requires tension in meiosis, but in mitosis, attachment is sufficient (Yu et al., 1999). Furthermore, in PtK1 cells, loss of Mad2 recruitment to kinetochores depends on microtubule attachment, not tension (Waters et al., 1998). Similarly, very low doses of the microtubule inhibitor vinblastine produce loss of tension and kinetochore bound Mad2 and a checkpoint arrest that, unlike the case after inhibition of spindle assembly

(Gorbsky et al., 1998), is insensitive to inactivation with Mad2 antibody injection (Skoufias et al., 2001). This has lead to the proposal that there are two branches of checkpoint signaling and silencing. One, in which the signal released is an activated, inhibitory form of Mad2, is silenced by microtubule attachment, while the other, presumably involving conversion of BubR1 into a Cdc20 inhibitor, is silenced by tension. This is not unappealing, but is at best a very murky argument, since attachment and tension are intimately interrelated. It has been long known that tension stabilizes attachment (Ault and Nicklas, 1989). CENP-E elimination, for example, yields loss of kinetochore tension and fewer (McEwen et al., 2001; Putkey et al., 2002), less stably bound microtubules. Moreover, BubR1 (Mad3), Bub1, or Mad2 cannot suppress the Cdc20-APC/C activity independently of the others. Thus, it seems to us that rather than separate parts of checkpoint silencing, attachment and tension really represent two halves of the same coin, integrally interrelated. Mitotic Checkpoint Defects Promote Aneuploidy and Tumorigenesis The mitotic checkpoint in budding yeast, where the intranuclear spindle forms quickly after centromeres are duplicated in S, is a real checkpoint activated only to arrest mitosis in the relatively rare instances when attachment is delayed. In most other organisms, it is not a checkpoint at all. Rather, it is an essential cell cycle control pathway activated at every mitosis/meiosis immediately upon nuclear envelope disassembly. Loss of Mad2, Bub3, or Rae1 in mice is lethal early, with cells accumulating mitotic errors and undergoing apoptosis by embryonic day 5 or 6 (Dobles et al., 2000; Kalitsis et al., 2000; Babu et al., 2003). Similarly, in Drosophila, loss of Bub1 causes chromosome missegregation and lethality (Basu et al., 1999). Microinjection of antibodies to Mad2 yields premature anaphase onset and chromosome missegregation (Gorbsky et al., 1998). Haploinsufficiency in Mad2 provokes late onset, self-limiting lung tumors (Michel et al., 2001). Reduction in Bub3 or Rae1 generates aneuploidy in vitro and a sharply increased susceptibility to chemically induced tumorigenesis (Babu et al., 2003). Thus, the primary mission of the checkpoint is to prevent such errors in chromosome segregation, a hallmark of human tumor progression (Hartwell and Kastan, 1994). Conclusions It is now clear that the centromere and its associated kinetochore are much more than simple attachment sites for spindle microtubules. Mammalian centromeres are much more complex than initially imagined, representing repeated assemblies of the simple, one nucleosome centromeres of budding yeast. Central to genetic inheritance, in almost all examples known they are themselves determined not by DNA sequence, but by one or more epigenetic elements. They are active components in microtubule capture, stabilization, and in powering chromosome movements essential to proper segregation. More than that, they are the signaling elements for controlling cell cycle advance through mitosis.

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