Towards a unifying model for the metaphase ...

Mntageuesis vol.13 DO.4 pp.321-335, 1998

REVIEW

Towards a unifying model for the metaphase/anaphase transition

M.Kirsch-Volders1, E.Cundari and B.Verdoodt Laboratory for Anthropogenetics, Free University of Brussels, Pleinlaan 2, 1050 Brussels, Belgium

Introduction Mitosis is a general name referring to division of a cell which has replicated its DNA into two daughter cells. In fact, it covers many different events at the level of the cell membrane, the cytoplasm, the nuclear membrane and the chromosomes and is functionally divided into six steps: prophase, prometaphase, metaphase, anaphase A and B and telophase, followed by cytokinesis (for a review see Alberts et ai, 1994). Recently attention has been focused more and more on the checkpoints which control the transition from one stage of the cell cycle to the next (for reviews see Gorbsky, 1995, 1997; Elledge, 1996; Wells, 1996). In this context a cell cycle transition is a unidirectional change of state by which a given cell that was performing one set of processes shifts its activity to perform another set of processes. The term 'checkpoint'

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The term mitosis actually covers a complex sequence of events at the level of the cell membrane, the cytoplasm, the nuclear membrane and the chromosomes; recently attention has been focused more and more on the checkpoints that control their orderly progression. The term 'checkpoint' refers here to the inhibitory pathways that coordinate coupling between the sequence of events, ensuring dependence of the initiation of each upon successful completion of others. This paper will mainly focus upon the possible checkpoint which controls a brief but essential step, dissociation of the sister chromatids into two identical chromosomes. This step will be called the metaphase/ anaphase transition. First, the molecular components that are important in metaphase/anaphase transition will be reviewed: accurate segregation of sister chromatids between the daughter cells is dependent on coordinated interaction of centrosomes, centromeres, kinetochores, spindle fibres, topoisomerases, proteolytic processes and motor proteins. Deficiencies in or impairment of any of these structures or in their control systems may lead to a more or less important genomic imbalance. A model combining the ultrastructural components, the molecular components and the controlling molecules will be proposed. The unifying concept emerging from this synthesis indicates that sister chromatids separate independently of the tubulin fibres, as a result of proteolytic processes controlled by the anaphase promoting complex. The spindle fibres are thus necessary to move the separated chromatids to the spindle poles but probably not to initiate separation. A number of remaining questions are also highlighted.

refers to the inhibitory pathways that coordinate coupling between the sequences of events, ensuring dependence of initiation of each upon successful completion of others (Elledge, 1996). These pathways can be detected by observation of a 'relief of dependence' in mutant or chemically treated cells, i.e. that later events should not take place as long as an earlier, prerequisite event has not been carried out successfully (HartweU and Weinert, 1989). As far as mitosis is concerned, the terminology 'mitotic checkpoint' has been proposed; this, however, often refers to the whole sequence which spans DNA replication in one cell cycle to Gl of the subsequent cell cycle. It is our aim to concentrate on the possible checkpoints which control a brief but essential step, dissociation of two sister chromatids into two identical chromosomes. This step is usually called the metaphase/anaphase transition. The transition from metaphase to anaphase is a key cell cycle event that commits the cell to distribute the genetic information equally between both daughter cells, exit mitosis and enter a new interphase. Accurate segregation of sister chromatids between the daughter cells is dependent on the coordinated interaction of centrosomes, centromeres, kinetochores, spindle fibres, topoisomerases, proteolytic processes and motor proteins. Deficiencies in or impairment of these structures or of their control systems may lead to a more or less important genomic imbalance. Different mechanisms leading to segregation errors have been described: (i) The absence of sister chromatid separation is called nondisjunction. Chromatids that normally separate during cell division stick together and are transported during anaphase to one pole. This may occur in a mitotic division, but is observed more frequently during meiosis. The resulting daughter cells of a mitotic division with non-disjunction contain respectively In + x and 2n - x chromosomes at the next mitosis. (ii) Loss of single chromosomes may be due either to nonattachment of the chromosome kinetochore to the spindle microtubules or to delayed migration of the chromosome (anaphase lagging). Such chromosomes become isolated from the main nucleus and form a micronucleus. Chromosome loss leads to one euploid and one monosomic daughter cell. A micronucleus might eventually re-associate randomly with one of the daughter nuclei: the chromosomal content of the daughter cells will depend on which nucleus incorporated the micronucleus. These two first mechanisms involve a single (or more) chromosome with its two sister chromatids and lead to aneuploidy; they are functionally distinguishable from errors which concern all chromosomes and are described in (iii). (iii) A third mechanism is polyploidization. Here all chromosomes are present more than twice in every cell. Polyploidy

M.Kirsch-Volders, E.Cundari and B.Verdoodt

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of the nuclear membrane assembly, chromosome anatomy, centromere/kinetochore structure, tubulin polymerization/ depolymerization, DNA decatenation by topoisomerase II and chromosome transport is progressing very quickly. As yet no unifying model which links the ultrastructural and the molecular components of the mitotic machinery with the molecules in charge of the specific metaphase/anaphase transition checkpoint has been proposed. It is our aim to summarize the most recent findings concerning the complex network of structural and regulatory cellular factors involved in the metaphase/anaphase transition. The model focuses on controlled separation of sister chromatids and centromeres. This basic knowledge is essential to fully understand the observations describing chromosome loss, chromosome non-disjunction and polyploidization after in vitro or in vivo exposure to aneuploidy-inducing chemicals. This class of mutagens is characterized by a broad range of intracellular targets, the respective role of which will be better understood when molecular and structural components of the mitotic machinery are considered together. The same considerations are applicable to several anticancer drugs, e.g. taxol, the chemotherapeutic efficiency of which will depend on the genetic background of the tumour cells. Ultrastructural components of the cell division machinery The chromosome The centromere. Centromeres are repetitive DNA sequences, keeping both sister chromatids of one chromosome together; they are adjacent to the kinetochores which attach the chromosome to the mitotic spindle fibres. As such they are essential for proper segregation of meiotic and mitotic chromosomes. In mammalian cells they are defined cytologically as the primary constrictions. Yeast centromeres. Two species have been extensively studied, Saccharomvces cerevisiae (budding yeast) and Schizosaccharomvces pombe (fission yeast); although these species are rather closely related, their centromeres are very different in structure. In S.cerevisiae the centromeres are sequences of only 125 bp (point centromeres); in S.pombe they are SXIO^IO 5 bp in size (Bloom, 1993). The organization of the centromeric DNA of budding yeast has been well described. Three domains are needed for its function. CDEI, a palindromic sequence, is only required for centromeric function in certain genetic backgrounds. CDEII is a sequence of -80 bp that consists of 90% AT and which varies between chromosomes. It is necessary for centromere function, as its deletion inactivates the centromere, but its precise sequence does not seem to be very important. Instead, it might serve a structural function in combination with histonelike proteins. This domain seems to play an important role in cohesion between sister chromatids. The third domain, CDEIII, is a highly conserved palindromic sequence of 25 bp that binds a protein complex, Cbf3, on which the kinetochore proteins assemble. Point mutations in this part abolish its function, (reviewed in Hyman and Sorger, 1995; Pluta et al. 1995). Vertebrate centromeres. The human centromere consists mainly of tandem repeats of a 171 bp sequence; the total sequence varies in length, following the chromosome from 0.5 to 10 Mb long. Very few other sequences than the a-satellites are found in the centromere; between the repeat units sequence differences of up to 35% exist, both within and between


can be achieved in normal tissues in plants, insects and also in mammalian liver (Gerlyng et al., 1993). It may also result from different mitotic abnormalities. The mechanisms that lead to polyploidization are outlined below (for a review see Parry et al., 1993). In the normal rat liver polyploidization occurs via an intermediate stage of binucleate cells. Just after birth the liver parenchyma consists almost exclusively of diploid mononucleate cells (Gerlyng et al., 1993). In a next stage their nuclei divide, but cytokinesis does not take place and binucleate cells are formed. If such cells divide further a common spindle is formed for both nuclei, division proceeds otherwise normally, but both daughter nuclei are tetraploid (Kirsch-Volders et al., 1988). Endopolyploidy (for a review see Therman et al, 1983) is usually the result of endocycles which include processes in which the chromosomes replicate but a spindle is absent (Nagl, 1978). The most common of these is endoreduplication, in which at mitosis the chromosomes consist of 2n chromatids instead of the normal two, as more than one round of DNA synthesis has taken place without intervening cell division. Treatment with mitotic inhibitors may interfere with chromatid separation and may induce diplochromosomes, i.e. chromosomes with four parallel chromatids instead of the normal two. It is thought that in plants endoreduplication is the main mechanism of polyploidization (for a review see Parry et al, 1993). If the chromatids are extended and more or less paired, polytene chromosomes are formed. These range from cablelike structures consisting of several extended chromatid strands to the typical banded chromosomes of diptera, which combine a high degree of multiplicity with a tendency to somatic pairing which aligns the homologous chromomeres into bands. A less frequent type of an endocycle is so-called endomitosis, which implies that the chromosomes undergo a condensation and division cycle as in mitosis, however, these processes take place inside the nuclear membrane without spindle formation or anaphase and telophase movements. This was originally observed to occur spontaneously in mouse tumours (for a review see Parry et al, 1993). Of more sporadic occurrence is restitution, which implies that chromosomes do not segregate as in mitosis but are included in one nucleus, most often in anaphase. C-Mitosis, a metaphase figure showing randomly distributed condensed chromosomes and no spindle, was originally observed after treatment with sufficiently high concentrations of colchicine. This type of mitotic abnormality very rarely occurs in non-malignant cells, but also sometimes leads to formation of a polyploid nucleus, although the result is more often formation of several micronuclei. Because aneuploidy was shown to play an important role in carcinogenesis, especially for deletion of tumour suppressor and mutator genes (for a review see Levine, 1995), and since polyploidization in tumours is often followed by aneuploidy, which in turn is associated with higher grade invasive tumours and poorer prognosis (Sandberg, 1977; Segers et al, 1994; Verdoodt et al, 1994), the mechanisms underlying the mitotic checkpoints have become of increasing interest (Li,X. and Nicklas, 1995). On the one hand, several attempts have been made to identify the genes involved in control of the checkpoints, in particular p53. which has been implicated in the spindle assembly checkpoint (Cross et al, 1995) and in the surveillance of genome stability (for a review see Shimamura and Fisher, 1996). On the other hand, the understanding

A unifying model for the metaphase/anaphase transition

yeast all forms of a gene are written in lower case; the wildtype indicated with a +.) The presence of CBF3 seems to be necessary, but not sufficient, for microtubule assembly at the kinetochores. A number of as yet unidentified proteins also appear to be required; CBF3 may also provide a structure onto which proteins that bind the CDEII sequences can assemble. All components of CBF3 are needed for vegetative growth. Mutations in them increase the frequency of chromosome loss (Hyman and Sorger, 1995). Mutants of CBF3A show asymmetrical chromosome segregation, while ctfl3 temperature-sensitive mutants, which are deficient in CbOC, delay the cell cycle in G2/M at the restrictive temperature (Wang,Y. and Burke, 1995). Kinetochore structure in vertebrates. Electron microscopy studies revealed that vertebrate kinetochores consist of a three layered structure. The inner layer follows the surface of the chromosome; it consists of chromatin and proteins and is only detectable when the chromosomes are not too condensed. The middle zone appears empty in electron microscopy; it is mainly composed of centromeric heterochromatin. The outer plate appears fibrous in structure, is ~0.5 |im in diameter and 4060 run in thickness and contains DNA as well as proteins. It is to this part that the microtubules attach; some tubulin binding proteins, known as microtubule-associated proteins or MAPs, have been identified. Moreover, motor proteins like kinesin and cytoplasmic dynein are known to be present in the outer kinetochore plate (Tomkiel and Earnshaw, 1993; Sullivan et al, 1996). The characteristic kinetochore proteins can already be detected in cells during interphase, but they are not yet organized into kinetochore structures. Assembly of kinetochores was seen to start in early prophase in HeLa cells, when the nuclear membrane was still intact. At this stage they were present in a still immature form; as the chromosomes condensed further the kinetochores developed a more mature structure. However, this did not occur in a very synchronized manner over the different chromosomes of a single cell (Schroeter et al, 1993). Of the proteins at the centromere, the function of some is at least in part known. These are known as the centromere proteins (CENPs). Six different types are known, designated A-F. A-D are structural components of the kinetochore, whereas CENP-E and -F are motor proteins (reviewed in Sullivan et al, 1996). CENP-A is structurally related to histone H3. It appears to be involved in conformational changes in the chromatin, at the level of the nucleosomes, in formation of centromeres. CENP-B associates with human a-satellite sequences; it recognizes a specific sequence of 17 bp, known as the CENPB box. It is probably also involved in structural organization of the centromeres, being placed in between the nucleosomes (Sullivan et al, 1996). This protein may play a role, possibly together with CENP-A, in linking the chromatids together at the level of the centromeres (Tomkiel and Earnshaw, 1993). CENP-C is a component of the inner plate of the kinetochore and seems to be present in similar amounts at all centromeres (Sullivan et al, 1996). When anti-CENP-C antibodies were microinjected during interphase in HeLa cells chromosomes appeared to migrate normally to the metaphase plate but the cells remained blocked in metaphase. These cells eventually attempted to complete division, but various abnormalities occurred. Also, in cells injected early in interphase no CENP323


chromosomes. These 171 bp sequences are organized in higher order repeats, which are mostly chromosome specific and are >95% similar within the same locus (Arn et al, 1989; Warburton and Willard, 1996). The direction of the a-satellite sequences does not seem to be important for their function: isochromosomes act as normal. Other structural sequences do not seem to be necessary either, as various rearrangements also behave normally during division. If those exist, it is most likely that multiple copies are distributed over the length of the centromere (Willard et al, 1989). The overall organization of centromeric DNA in 170 bp asatellite repeat units is known to be conserved in many primates. Sequences from chimpanzee and gorilla show in some cases >90% homology with certain human centromeric DNA sequences, but the most similar sequences do not always occur on homologous chromosomes between species. A cause for this may be different rates of interchromosomal exchange between non-homologous chromosomes (for a review see Warburton and Willard, 1996). Experimental constructs of a-DNA inserted into chromosomes of various organisms often formed functional centromeres; centromere proteins (CENPs) associated with these sites. This would indicate that a-satellite DNA alone is sufficient to form a functional centromere. However, there are indications that more complexity may exist, as stable marker chromosomes are known that contain no a-satellite DNA detectable by in situ hybridization (Sullivan et al, 1996). In the mouse the organization of centromeric DNA is different. The centromeric regions there consist of two types of sequences: major and minor satellites. The major satellite consists of repeats of a 234 bp sequence and is present in ~106 copies over the genome, whereas the minor satellite DNA is made up of repeats of a 120 bp sequence that occurs -50 000 times throughout the genome (Vig, 1993). The minor satellite DNA seems to be located in the centre of the centromeres, whereas the major satellite spans a wider region. However, minor satellite sequences do not seem to be required for formation of the primary constriction nor do they determine the time of separation of the chromatids (Vig, 1993). Another peculiarity of the mouse is that although it does not seem to produce CENP-B protein, its minor satellite DNA does contain the CENP-B box. Less is known about the sequence of rat centromeric DNA; recent data indicate that they are highly variable between chromosomes (Hoebee and de Stoppelaar, 1996). The kinetochores. The kinetochores are structures consisting of protein and DNA that attach the chromosomes to the microtubules of the spindle and move them along it. Kinetochore structure in yeasts. In yeasts the structure of the centromere cannot be distinguished by electron microscopy, as is possible in mammalian metaphases, but a number of its proteins have been identified using biochemical methods (Table I). Budding yeast Cbfl protein binds the CDEI sequence; if the CBF1 gene is deleted the rate of chromosome loss is greatly increased. The CBF3 protein complex binds to CDEHI and consists of four components: CBF3A, CBF3B, CBF3C and CBF3D. CbOd corresponds to Skpl protein, which is involved in cyclin A degradation in mitosis, both in yeast and in humans (Bai et al, 1996). (The general convention for S.cerevisiae is that the wild-type alleles are indicated in capital letters, mutant forms in lower case and proteins with the first letter in upper case and all following in lower case. In fission


Table I. The main components of the kinetochores in budding yeast (S.cerevisiae) Important characteristics DNA sequences CDEI CDEn CDEin Proteins Cbfl Cbf3 Cbf3A Cbf3B Cbf3C CbOD

8 bp imperfect palindrome, if deleted mitotic chromosome loss increases 10- to 30-fold, it is not essential for centromore function 78—86 bp, 90% AT; essential for centromere function, but point mutations do not inactivate the centromere 25 bp imperfect palindrome; point mutations in this part inactivate the centromere Binds the CDEI sequence This complex, consisting of Cbf3A, Cbf3B, CbO and CbOD, binds the CDEIII sequence Required for symmetrical chromosome segregation Probably a zinc finger protein; mutants delay in G2/M phase Mutants delay in G2/M phase (other name Ctfl3) Involved in degradation of cyclin A (other name Skpl)

The spindle Structure. Three categories of spindle fibres can be distinguished: interpolar, kinetochore and astral fibres. The interpolar fibres are those that start from the centrosomes at the pole, extend to the cellular equator and contact each other; the kinetochore fibres form the connection between the centromeres of the chromosomes and the spindle poles. The first two categories form the spindle tubules; the last type, the astral fibers, serves to bring the centrosomes and the remainder of the spindle into a correct position for cytokinesis. The number of kinetochore fibres per chromosome depends on the organism: human chromosomes have -30, but budding yeast cells typically have only one (for reviews see Alberts et al, 1994; Inoue, 1997). Tubulin and the spindle fibres. The spindle fibres consist of mainly tubulin, together with a number of associated proteins. The spindle fibre tubulin molecules are in dynamic equilibrium with a pool of free tubulin in the cell (for reviews see Alberts et al., 1994; Kirsch-Volders and Parry. 1996). Microtubules can move objects, either by polymerization/ 324

depolymerization (Lombillo et al, 1995) or with the help of motor proteins. The addition of tubulin dimers to microtubules is thought to move the chromosomes towards the metaphase plate by pushing on the arms, a phenomenon known as 'polar ejection force' or 'polar wind'. At the same time the microtubules attached to the kinetochores grow and shorten in alternation, causing oscillatory movements of the chromosomes around the centre of the spindle. During anaphase A the chromosomes are mainly moved poleward by shortening microtubules, which lose tubulin molecules at the level of the kinetochores. Some disassembly also occurs at the spindle poles (this is valid for vertebrate cells, in other organisms other mechanisms exist) (reviewed in Inoue and Salmon, 1995). A recent study has followed the behaviour of the microtubules over the cell cycle in the rat kangaroo PtKl epithelial cell line. In interphase cells contain long microtubules that extend to the cell membrane, only part of which are attached to the centrosome. In early prophase, although the microtubule network retains the same general appearance as in interphase, more tubules originate from the centrosomes, which now start to separate. As the nuclear envelope begins to be broken down, the microtubule structure changes abruptly, disappearing in the cytoplasm and with only short microtubules originating from the centrosomes. At prometaphase the spindle organization is initiated and more tubulin is again found in microtubules (Zhai et al, 1996). Kinetochore fibre (K fibre) microtubules are known to be more stable than odiers, even when they are not directly attached to a kinetochore. The kinetochore causes organization of these microtubules into bundles, with a more or less constant wall to wall spacing; associated proteins may bind adjacent microtubules together. The association with any type of chromatin seems to stabilize microtubules as well (Heald et al.. 1996). Research has been done into what causes organization of the microtubules around the chromosomes. In several studies it was found that the kinetochore stabilizes the microtubules. However, in meiosis in grasshopper sperm cells the size of the chromosome seemed to have more effect than the number of kinetochores present on stabilizing the microtubules. A similar effect had been observed before in Xenopus eggs. However, results concerning the relative roles of kinetochores. chromatin and centrosomes in organizing the spindle vary greatly between experimental systems, so at present no generally applicable conclusions can be drawn (Zhang and Nicklas, 1995a). In this same grasshopper system formation of the spindle


C protein could be detected at the centromeres in the metaphase that followed (Tomkiel et al, 1994). CENP-C thus appears to be required during interphase for assembly of functional kinetochores and when they are already present in prometaphase or later stages of the cell cycle microinjection of antibodies against CENP-C cannot make them disassemble again. CENP-D protein is less well characterized and appears to correspond to RCC1 protein, which is a negative regulator of chromosome condensation. However, as antibodies produced against one form do not recognize the other the protein must exist in different conformations or undergo post-translational modifications (Eamshaw and Tomkiel, 1992). Concerning its function in the kinetochore little is known (Sullivan et al, 1996). RCC1 functions by preventing premature entry into mitosis. In temperature-sensitive mutants mitosis is started before completion of DNA replication at the non-permissive temperature (Ponstingl and Bischoff, 1993). Dynein and kinesin-related molecules have also been located at the centromere. Kinesin itself seems mainly to be involved in transport of membrane vesicles (Moore and Endow, 1996). CENP-E, a kinesin-like motor protein, and CENP-F appear to be involved in movements of the chromosomes during anaphase (Tomkiel and Earnshaw, 1993). CENP-E apparently is only present at the kinetochores in prometaphase and metaphase. In anaphase it appears to be associated with microtubules in the centre of the spindle (Tomkiel and Earnshaw, 1993).


The centrosome serves as a centre of microtubule organization and consists of a pair of centrioles and some pericentriolar material, also known as the centrosome matrix. The centrioles are structures of ~0.2 |im width; they are usually found at right angles to each other. They appear to have the same structures as the basal bodies at the starting point of many cilia. When the centrioles are replicated first the two members of a pair separate and the new centrioles are formed at right angles to the old ones; they are very rarely synthesized de novo, the old centrioles probably functioning as a template for the new ones. Replication in fibroblasts starts at about the start of S phase. Cells blocked in Gl tend to accumulate more than the normal number of centrosomes, but not as many as would be expected given the duration of the cell cycle block. Replication of the centrosomes seems connected to progression of the cell cycle. Some brake on multiple replications per cell cycle appears to exist (Balczon et al, 1995).

Whereas the microtubules from cilia start directly at the centriole, in the more complexly organized cytoskeleton they seem to be connected to the pericentriolar material. Also, in mitosis this part seems to be most important in organizing the spindle: in three polar spindles one of the poles does not contain a centriole, yet functions well (for a review see Kellogg et al, 1994). In cases where mitosis is so disordered that multipolar spindles form usually, but not always, a pair of centrioles is present at each pole. Some disorganized centrosomal material can then be found at those poles. However, the presence of more than two pairs of centrioles in a cell does not automatically give rise to a multipolar spindle (Paweletz et al, 1989). Moreover, one should note that plants have no centrioles and yet mitosis works well. Also, in situations where a normal bipolar spindle should be formed the centrosomes are not absolutely required for this in all systems. Such cases are rare, however. An example is meiosis in Drosophila, in which system it is thought that kinesin-like proteins and the chromatin itself help to organize the spindle. A similar mechanism exists in the first divisions of the mouse embryo, which is also one of the rare cases of de novo formation of centrioles (reviewed in Kellogg etal, 1994). Incomplete chromosomes may function in spindle organization. In an experimental system derived from Xenopus eggs it was possible to obtain assembly of a spindle around DNAcovered magnetic beads in the presence of nuclear extract but without centrosomes. Cytoplasmic dynein seemed to be required for organization of the microtubules into a spindle in this system (Heald et al, 1996). In grasshopper spermatocytes at least one chromosome had to be present for spindle formation, but in this system the length of the chromosome arms exerted a stronger influence than the number of kinetochores present (Zhang and Nicklas, 1995a,b). Both studies are consistent with the conclusion that organization into a bipolar structure is an intrinsic property of the combination of chromatin and microtubules. In principle duplication of the centrosomes should be coupled to other cell cycle events. Nevertheless, through application of certain treatments these events can be uncoupled. For example, cells that are blocked in G1 by hydroxyurea treatment tend to accumulate more than the normal number of centrosomes, but not as many as would be expected given the extended duration of the cell cycle block (Balczon et al, 1995). The p53 gene appears to be required for correct centrosome replication. pSi^' mouse embryonic fibroblasts accumulated more than the normal number of centrosomes in the second passage in vitro without any special treatment. A high fraction of abnormal divisions also resulted (Fukasawa et al, 1996). This indicates that p53, directly or indirectly, must be involved in regulation of replication of the centrosomes. Another indication for a role of p53 in function of the centrosome is that in a few transformed cell lines part of the p53 protein appeared to be localized at the centrosomes (Brown et al, 1994). A recent study showed that Cdc2 remains associated with the centrosomes throughout the cell cycle in a number of human cell lines. Two of these were derived from normal lung cells. The protein also appeared to be associated with the intermediate filaments of the nuclear matrix. However, when a different antibody specific for the PSTAIRE domain was used to detect Cdc2 only cells in early Gl were labelled. This 325


can be prematurely initiated by disrupting the nuclear membrane. The resulting metaphase appears normal on first sight, but the cell cannot divide properly, as the chromosomes form a tangled mass. For spindle formation the chromosomes have to be present in the cell. They are not needed, however, to maintain at least a partial spindle once it has been formed (Zhang and Nicklas, 1995b). Microtubule-associated proteins (MAP) and passenger proteins. These proteins serve to stabilize the microtubules and to mediate their interaction with other components of the cell, in the interphase cytoskeleton as well as in the mitotic spindle. They all interact with the C-terminal domain of tubulin, although the protein structure differs among MAPs. Also, some types of MAP are specific for certain tissues, whereas others have a more widespread distribution. Neurones especially are a rich source (reviewed in Maccioni and Cambiazo, 1995). The effect of different types of mutations on the function of a MAP could be studied in the budding yeast protein Mhpl. This protein seems essentially to stabilize microtubules throughout the cell cycle; its overexpression leads to abnormally long cytoplasmic microtubules. Overexpression of the microtubule binding region of the protein alone leads to a delay in division with impairment of spindle elongation (Irminger-Finger et al, 1996). A functionally related class of proteins are the so-called passenger proteins. These proteins have as a common characteristic that they associate with different cellular structures over the cell cycle. During prometaphase and metaphase they are found at the centromeric regions of the chromosomes. In anaphase they dissociate from the chromosomes, to move to the metaphase plate region; they remain there, associated with the overlapping microtubules, as the sister chromatids move to the spindle poles. This group includes, in addition to CENPE and the inner centromere proteins (INCENPs), which are discussed separately, MAb 6C6, a pericentriolar protein, the telophase disk protein TD-60 and the nuclear matrix protein &>?>&. Most of these proteins, except CENP-E, can be detected in interphase nuclei (reviewed in Maccioni and Cambiazo, 1995). The motor protein CENP-E contains two microtubule binding domains and may thus serve to cross-link microtubules in the anaphase spindle. This cross-linking activity is suppressed through phosphorylation (Liao et al, 1994). CENP-E is degraded at the end of mitosis (Brown et al, 1994). The centrosomes

M.Klrsch-VoWers, E.Cundari and B.Verdoodt

could indicate a change in conformation of Cdc2 at the centrosome during the cell cycle (Pockwinse et al., 1997).

Topoisomerase II The function of topoisomerase II (topoll) is to disentangle DNA double helices, by cutting both strands, allowing them to unwind and re-annealing the break afterwards. In vitro the enzymatic activity is rather non-specific and can lead both to disentangling or to more entangled DNA afterwards, depending on the experimental conditions. This makes it probable that its activity is regulated in vivo; its gene expression seems to be down-regulated by p53 (Wang,O. et al., 1997). Aberrant expression of topoll may occur in malignant cells. In breast cancer it was seen to be associated with low hormone receptor

Table D. Overview of the motor proteins involved in mitotic processes Phase of mitosis

Protein

Direction*

Species

Prophase

bimC KAR3 Ned Eg5 Cytoplasmic dynein MCAK XkJpl NOD MKLP1 bimC KAR3 XkJpl CENP-E KAR3 MKLPI bimC Cytoplasmic dynein

+

Aspergillus S.cerevisiae Drosophila Xenopus Rat + other species Chinese hamster Xenopus Drosophila Human Aspergillus S.cerevisiae Xenopus Human S.cerevisiae Human Aspergillus Rat + other species

Prometaphase: capture of chromosomes by the spindle Prometaphase congression of chromosomes Metaphase Anaphase A Anaphase B

+ NDb ND ND + + _ ND + + -

T h e plus sign indicates movement towards the plus end of the microtubules. i.e towards the kinetochores: a minus sign, towards the spindle poles •"ND. not determined.

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Molecular components Motor proteins Motor proteins bind to and move on microtubules; they obtain the energy they need to produce mechanical force from ATP. Two well-known types of motor proteins are kinesin and dynein: kinesin and most of the kinesin-related proteins move towards the plus ends of the microtubules, whereas dynein moves towards their minus ends (microtubules attach with their plus ends to the kinetochores) (Sawin and Endow, 1993). Kinesin and related proteins share a similar overall structure, consisting of a motor domain of -350 amino acids, an ahelical stalk region and a C-terminal domain. The globular motor domain has ATPase activity and also binds to the microtubules, whereas the a-helical part is responsible for formation of dimers (Moore and Endow, 1996). Dynein is an unrelated protein of ~4500 amino acids, with four ATPase domains (Gibbons, 1996). Although much remains to be further elucidated, motor proteins appear to be involved in all stages of mitosis, starting from separation of the centrosomes in prophase. Table II gives an overview of the most important motor proteins involved in mitosis. In prometaphase they are involved in movement of the chromosomes towards the metaphase plate, at metaphase they help to stabilize the spindle and in anaphase they function both to move the chromosomes to the spindle poles and to move the spindle poles apart (Barton and Goldstein, 1996).

counts, high histological grade tumours, a high proliferation rate measured by the S phase fraction and aneuploidy (Jarvinen etal, 1996). Correct functioning of this enzyme appears to be necessary for correct separation of the sister chromatids in metaphase. However, inhibitors of topoll also cause extensive DNA breakage, making the influence of this enzyme on chromosome structure difficult to determine (Sumner et al., 1993). The cellular distribution of topoll over the cell cycle has been studied in Chinese hamster ovary (CHO), mouse and human cells. In prophase the enzyme was found throughout the chromosomes, whereas in metaphase it was restricted to the centromeres, to be lost from them in anaphase. This would be consistent with its presumed role in sister chromatid separation, as in metaphase the chromatids are normally only connected at the centromere (Sumner, 1995, 1996). In Drosophila melanogaster the function of topoll on the metaphase chromosomes was shown to be inhibited via a regulatory protein, Barren. When this regulator was active sister chromatids could not separate correctly at anaphase. However, the centromeres often did separate in these cells (Bhat et al., 1996). Similar effects were observed in a topolldeficient mutant of S.pombe (Funabiki et al., 1993) and in Chinese hamster C1 -1 cells treated with etoposide (Parry et al., 1996). In these cases it would seem that the centromeres are not held together by intertwined DNA strands, but maybe by some proteins. This is in contrast to the standard model, where the centromeres are the last regions of the replicated DNA which is disentangled. In the Drosophila Barren mutant metaphases with a normal appearance were formed (Bhat et al., 1996). The defect probably only concerned that fraction of topoll that functions in sister chromatid separation at anaphase, not its activity in chromosome condensation during prophase. If chemical inhibitors (among others, ethidium bromide, amsacrine and etoposide) are used the resulting metaphase chromosomes were often also abnormal, not being able to condense correctly or containing DNA breaks (Sumner et al., 1993; Andreassen et al., 1997). Many of these products are known to also have effects on other cellular components, which may help to explain the variability in their effects (Sumner et al., 1993).

A unifying model for the metaphase/anaphast transition

Chemical treatment of vertebrate cells with various inhibitors of the topoll enzyme, like Hoechst 33342, amsacrine, etoposide and mitoxantrone, caused a slower passage through metaphase and production of tetraploid cells in the next cycle. These cells were not able to separate the sister chromatids and eventually re-entered interphase. At the next division diplochromosomes, i.e. chromosomes with four parallel chromatids instead of two, could be seen (Sumner, 1996).

The mitotic cyclins and their regulation The basic units involved in regulation of the cell cycle are a group of protein kinase complexes, consisting of a cyclin and a cyclin-dependent protein kinase (Cdk). The Cdks need to bind a cyclin to be active (for a review see Sherr, 1996). The cyclins form the regulatory subunits; their levels fluctuate greatly, depending on the position of the cell in the cycle (Hall and Peters, 1996). As far as the mitotic checkpoint is concerned, two cyclins are considered to play important roles. Cyclin A expression is necessary for cells to progress through S phase (Guadagno and Newport, 1996; Sherr, 1996). At the Gl/S transition and during S phase cyclin A associates

Proteolytic processes in mitosis Many events of the cell cycle are regulated through ubiquitinmediated proteolysis, at the Gl-S transition as well as at exit from mitosis. Ubiquitin is a small (8.5 kDa) protein that is present in all eukaryotic cells and serves to label other proteins for destruction (Stryer, 1988). Although different enzymes are active at different points of the cell cycle, a general pattern emerges: a chain of ubiquitin molecules is coupled to the substrate; this serves as a marker for degradation by proteases. For this, ubiquitin is first activated by the ubiquitin activating enzyme El and then transesterified to ubiquitin conjugating enzyme E2, with the assistance of a ubiquitin-protein ligase, called E3. E3 function at anaphase is carried out by a large enzyme complex, the cyclosome or anaphase promoting complex (APC) (reviewed in King et al, 1996). Inhibition of APC activity, whether it is through substrate competition in Xenopus egg extracts (Holloway et al, 1993), microinjection of antibodies against APC proteins in human HeLa cells (Tugendreich et al, 1995) or through mutation in budding yeast (Irniger et al, 1995), prevents chromosome segregation. These findings create a paradox: although the initiation of anaphase does not require degradation of mitotic cyclins, it still remains dependent upon D box-mediated proteolysis catalysed by the APC. The simplest resolution of this dilemma is to postulate the existence of non-cyclin substrates that inhibit anaphase until they are degraded via APC-mediated proteolysis. Pdsl and Cut2 might play such a role in budding yeast. In fact, the role of PDS1 and CUT2 in inhibiting anaphase is not currently understood. One hypothesis is that such proteins might function as a chromosomal 'glue' that holds chromosomes together until the glue is dissolved at anaphase, releasing the chromatids and initiating other anaphase movements (Holloway etal, 1993). Surprisingly, meiotic spindles that lack chromosomes still undergo anaphase spindle movements on schedule (Zhang and Nicklas, 1996), indicating that chromosome separation itself cannot be the sole trigger of other anaphase events. Tugendreich et al. (1995) propose that normal anaphase spindle movements are triggered by APC-dependent degradation of at least two different classes of proteins: one class that is involved in holding sister 327


INCENPs and CLIPs During metaphase the chromatids seem to be held together at the centromere by proteins that complex with the centromeric DNA. Several proteins specific for the pairing domain have been described. They are called inner centromere proteins (INCENP) and chromatid linking proteins (CLIP) (Rattner et al, 1988). These proteins are different from the CENP proteins, as both their molecular masses and their precise localizations differ (Sullivan et al, 1996). In some cases the human X chromosome separates prematurely; this may be due to a lack of these proteins. This has also been observed for other human chromosomes, 18 and 21 (Fitzgerald, 1993). Although these proteins are located at the expected location to serve to hold the chromatids together until anaphase, no direct proof exists as yet that they actually carry out this function (Bickel and Orr-Weaver, 1996). The INCENP proteins are found between the chromatids at the centromere of colcemid-arrested cells. Little is as yet known about their function. Their pattern of distribution in the cell changes over the cell cycle: while they are found at the centromeres and telomeres during early metaphase, they are released from the chromosomes at late metaphase. During anaphase they were detected at the central spindle and near the cell cortex at the future location of the cleavage furrow (Earnshaw and Cooke, 1991; Mackay et al, 1993). They may therefore participate in cleavage furrow formation and cytokinesis. This has been confirmed by a recent study, where the INCENP proteins were prevented from moving to the cellular cortex by linking them covalently to CENP-B. In those cells cytokinesis could not be completed: a midbody-like structure that did not contain INCENP proteins was formed, but cells did not proceed further than this stage (Eckley et al, 1997). CLIPs remain located at the kinetochores throughout the cell cycle, but are lost from the chromosome arms once they separate. In colcemid-treated cells CLIPs can only be detected at the centromeres (Rattner et al, 1988; Miyazaki and OrrWeaver, 1994). As few anti-CLIP autoimmune sera are available, only a limited amount is known about these proteins, apart from their molecular masses, i.e. 50 and 63 kDa (Rattner et al, 1988; Sullivan et al, 1996).

with Cdk2. Cyclin A also forms a complex with Cdc2 and in this form is involved in entry into mitosis (Nigg, 1995). Cyclin B, the synthesis of which starts at the beginning of S phase, complexes with Cdkl (Cdc2); this complex is known as maturation promoting factor (MPF) and is necessary for entry into mitosis. To exit the telophase of mitosis and reenter Gl degradation of cyclin B is needed (King et al, 1996). The events of anaphase occur in a strict order. The first mitotic cyclin to be degraded is cyclin A. Next, the MPF is deactivated by degradation of cyclin B, the centromeres split and sister chromatids separate and movement of the chromosomes towards the spindle poles is initiated. Initiation of anaphase was initially seen as resulting from inactivation of the MPF and the cell returns to interphase, the stable state at low MPF activity (Murray and Kirschner, 1989). However, non-degradable forms of mitotic cyclin arrest the cell cycle in telophase rather than metaphase (as would be predicted by the model of Murray and Kirschner) in both Xenopus egg extracts (Holloway et al, 1993) and budding yeast (Surana et al, 1993). Therefore, inactivation of the MPF cannot serve as the trigger for sister chromatid segregation: this still proceeds in the abscence of cyclin B degradation.


chromatids together (such as CUT2 and PDS1) and a second class that directly influences the behaviour of the mitotic spindle, where a portion of the APC appears to be located. However, budding yeast mutants with unreplicated DNA still exhibit some types of anaphase movement when the APC is also mutated (Irniger et al., 1995), indicating that certain aspects of anaphase may be controlled by APC-independent mechanisms. In most cells the presence of unattached chromosomes or defects in spindle assembly activates an internal cellular signalling pathway, known as the spindle assembly checkpoint, which blocks the onset of anaphase and stabilizes APC substrates (Gorbsky, 1995; Rieder et al., 1995).

Table III. Overview: comparison between the different metaphase checkpoint genes BUB1 BUB2 BUB3 MAD1 MAD2 MAD3 MPS1 MPS2 RAD9

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Hypersensitivity to benzimidazoles. no arrest on treatment with these agents, abnormal regulation of HI kinase activity, phosphorylation of Madlp (Hoyt et al.. 1991: Roberts et al. 1994; Hardwick and Murray. 1995) Hypersensitivity to benzimidazoles, no arrest on treatment with these agents (less sensitive than the other bub mutants), abnormal regulation of HI kinase activity (Hoyt et al. 1991. Wang and Burke. 1995) Hypersensitivity to benzimidazoles, no arrest on treatment with these agents, phosphorylation of Madlp (Hoyt et al.. 1991; Hardwick and Murray. 1995) Hypersensitivity to benzimidazoles. no arrest on treatment with these agents, cell cycle delay in the presence of minichromosomes (Li.R. and Murray, 1991; Hardwick and Murray. 1995; Wells and Murray. 1996) Hypersensitivity to benzimidazoles. no arrest on treatment with these agents, abnormal regulation of HI kinase activity, phosphorylation of Madlp, cell cycle delay in the presence of minichromosomes (Li,R. and Murray. 1991. Hardwick and Murray, 1995) Hypersensitivity to benzimidazoles. no arrest on treatment with these agents, cell cycle delay in the presence of minichromosomes (Li.R. and Murray. 1991) Duplication of the spindle pole body, cell cycle arrest, phosphorylation of Madlp (Hardwick et al.. 1996: Weiss and Winey. 19%) Duplication of the spindle pole body (Winey et al. 1991) Arrest of the cell cycle after DNA damage at any stage of the cell cycle Gl. S. G2 and mitosis, anaphase arrest in case of breaks in dicentnc chromosome (Neff and Burke. 1992)


The mitotic checkpoint In a variety of organisms checkpoints have been characterized that prevent cells which have failed to separate their sister chromatids in mitosis (due to, for example, the absence of a spindle) from re-replicating in the next S phase. This checkpoint is thus essential to prevent irreversible polyploidization. The genes possibly involved are documented below. Yeast (S.cerevisiae and S.pombe) More is known about the genetic basis of this checkpoint in yeasts than in mammalian cells; several mutants that are defective in this process have been isolated. In budding yeast {S.cerevisiae) it has been found that mutations in the three MAD (MAD1, MAD2 and MAD3), the three BUB (BUB1, BUB2 and BUB3) and the MPS gene inactivate the spindle assembly checkpoint, allowing cells with a defective spindle to proceed through division (Hoyt et at., 1991; Hardwick et ai, 1996). Comparison of the different metaphase checkpoint genes is summarized in Table III. Three different complementation groups, corresponding to three different genes, were isolated for the BUB (budding uninhibited by benzimidazole) genes. These are required for cell cycle arrest in the case of spindle dysfunction caused by treatment with benzimidazoles, such as benomyl or nocodazole. If these genes are mutated the cells will start to replicate their DNA and form a new bud in the presence of inhibitors of microtubule formation, without being able to complete nuclear division. These cells are therefore also abnormally sensitive to these agents (Hoyt et al, 1991). The three MAD (mitotic arrest deficient) genes seem to have similar functions: their mutation leaves cells sensitive to the spindle inhibitors benomyl and nocodazole, as nuclear division

is then initiated with a non-functional spindle. It has been shown that the Mad gene product is necessary to maintain high histone 1 kinase activity during the prolonged stay in mitosis in the presence of spindle inhibitors. In mad2 mutants this activity decreases inappropriately if cells are treated with benomyl and no functional spindle could be formed. This is also the way in which these genes were first isolated, i.e. in cells unable to stop in mitosis under these circumstances until the spindle is completed (Li,R. and Murray, 1991). Other important genes in this respect are MPS1 (monogolar spindle) and MPS2, also implicated in replication of the spindle pole body, and RAD9. Mammalian cells (and other multicellular eukaryotes) In the Mammalia, including humans, far less is known about functioning of the mitotic checkpoint. In the following paragraphs four mechanisms specifically controlling the metaphase/ anaphase transition will be considered: (i) attachment of the chromosomes to the spindle and spindle integrity; (ii) control of sister chromatid separation; (iii) the Mad/Bub pathway; (iv) p53 and the relation of the checkpoint function to apoptosis. The spindle assembly checkpoint: control of the correct attachment of chromosomes to the spindle in mammalian cells The mechanism to detect correct connection of all chromosomes to the spindle appears to depend on the tension on the spindle fibres, the absence of tension signalling loose chromosomes. This model was mainly derived from experiments on mantid spermatogenesis, which has a special arrangement of the sex chromosomes in the first meiotic metaphase. In many species the male has three sex chromosomes, two different X and a Y. In spermatogenesis they arrange as a trivalent, of which one daughter cell should receive both X chromosomes and the other the single Y. Errors in which one X chromosome pairs with the Y are quite common. Cells with unpaired sex chromosomes do not further divide and eventually degenerate; this can, however, be prevented if the experimenter pulls with a micromanipulation needle on the loose chromosome, thus providing tension (Li,X. and Nicklas, 1997). This poses the question of the biochemical mechanisms that signal detection of badly aligned chromosomes. From experiments in various cell types it could be deduced that phosphorylation of certain kinetochore proteins plays an important role; these can be detected by antibody 3F3/2. This antibody detects a phosphorylated protein at the kinetochores; if cells are treated with phosphatase PP1 the 3F3/2 epitope is no longer detectable


eres and chromosome arms (see sections Topoisomerase II and INCENPs and CLIPs). In Drosophila meiosis, in both males and females, mei-S332 protein is required to keep the centromeres together until onset of anaphase II. Mutants of this gene separate their sister chromatids in anaphase I, leading to non-disjunction and chromosome loss in the second meiotic division. The protein, however, appears to have no function at all in mitosis (Kerrebrock et al., 1995; reviewed in Sekelsky and Hawleyn, 1995). Centromeric cohesion might be necessary for correct alignment to the metaphase plate; if the centromeric connection is disrupted both kinetochores moved independently, although the chromosome arms were still attached to each other (Skibbens etal, 1993, 1995). The mad/bub pathway in vertebrates. Recently homologues of MAD2 and BUB1 have been identified in vertebrates. MAD2 homologues could be cloned in Xenopus (XMAD2) and human (HsMAD2); both were involved in metaphase arrest after nocodazole treatment (Chen et al., 1996; Li,Y. and Benezra, 1996). Antibodies against XMAD2 prevented metaphase arrest in Xenopus egg extracts; added sperm chromosomes decondensed and formed interphase nuclei (Chen et al., 1996). Human HeLa cells treated with such antibodies failed to arrest after nocodazole treatment. Moreover, T47D, a human breast cancer cell line that is sensitive to nocodazole and taxol, showed reduced expression of this gene (Li,Y. and Benezra, 1996). By die use of these antibodies it was also possible to study the cellular distribution of these MAD homologues. Human MAD2 in HeLa cells was localized to the kinetochores in prometaphase, but was not detectable in metaphase or anaphase cells (Chen et al, 1996; Li,Y. and Benezra, 1996). It appeared that anti-XMAD2 antibodies only labelled chromosomes that were not yet properly attached to the metaphase spindle; XMAD2 does not seem to correspond to the 3F3/2 epitope (Chen et al, 1996). A mouse homologue of BUB1, mBubl, has also been isolated; it is also involved in the metaphase checkpoint activated by nocodazole treatment and shows a similar localization during the cell cycle to XMAD2 and HsMAD2. Blocking the innate human BUB function in a cell line derived from HeLa cells with a dominant negative form of mBubl caused cells to progress through the cell cycle after nocodazole treatment. Untreated HeLa cells eventually exit mitosis in the absence of a functional spindle, but then die by apoptosis in the next cycle; with non-functional mBub expression they were able to exit mitosis faster and to replicate their DNA, eventually even entering the next mitosis (Taylor and McKeon, 1997). The role of p53 in control of the mitotic checkpoint. The p53 gene has previously been found to be involved in Gl and G2 arrest in various cell types. Usually its expression is induced by DNA damage, like that caused by ionizing radiation (Kastan et al, 1991; Yin et al., 1992; Aloni-Grinstein et al, 1995; Stewart et al, 1995). However, overexpression of the gene through an inducible promotor alone will also cause the cells to arrest (Agarwal et al, 1995). Recently indications have been found that the p53 gene is probably also involved in the mechanism of metaphase arrest and thus in prevention of polyploidy and aneuploidy (Cross et al, 1995; Minn et al, 1996). Introduction of a p53 mutant allele into a diploid p53 wildtype human colon cancer cell line caused it to develop 329


(Gorbsky and Ricketts, 1993). The relevant protein appears to be located in the central zone of the kinetochore (Campbell and Gorbsky, 1995), but it has not yet been isolated. The antibody can be used in a wide range of cell types. In a rat kangaroo kidney cell line, Ptkl, the kinetochores in an undisturbed cell cycle become phosphorylated early in mitosis and are then dephosphorylated when the chromosomes become attached to the spindle. Kinetochores that did not attach properly, remained phosphorylated (Gorbsky and Ricketts, 1993). When living Ptkl cells were microinjected with antibody 3F3/2 they continued to express the phosphoepitope long after the chromosomes reached the metaphase plate and cells did not enter anaphase. Eventually the immunofluorescence disappeared and only then did the cells enter anaphase. This effect was even observed after injection of antibodies in metaphase. Treatment with this antibody did not inhibit chromosome movements towards the metaphase plate (Campbell and Gorbsky, 1995). In mantid spermatocytes it was shown that kinetochores of unattached chromosomes were more strongly phosphorylated that those of properly attached chromosomes (Li,X. and Nicklas, 1997). As the chromosomes can be experimentally manipulated in this cell type, phosphorylation of the kinetochores was seen to depend on the tension on the chromosome (Nicklas et al, 1995; Li,X. and Nicklas, 1997). Also important is that phosphorylation can be made to reappear by relieving the tension by pushing on a chromosome which remains correctly attached. This indicates that the relevant proteins are not lost from the chromosomes after correct alignment on the spindle (Li,X. and Nicklas, 1997). In mantid and grasshopper spermatogenesis tension on the chromosomes was required to alleviate the metaphase block caused by chromosomes that were poorly attached to the spindle. In the Ptkl system other factors seem to play a role, as in these cells it is possible to destroy the unattached side of the kinetochore of a mono-attached chromosome by means of a laser. This destruction allows anaphase to start, although that chromosome was never under tension (Rieder et al, 1995). As in Ptkl mitotic cells phosphorylation of the same 3F3/2 epitope does seem to signal to the cell not to proceed to anaphase, another explanation might exist, taking into account the mantid meiosis model. It may be that tension on the kinetochore is not the factor regulating the checkpoint, but, for example, attachment of microtubules. In meiosis a frequent error is that both kinetochores attach to the same spindle pole, which would mean that they are both correctly attached to microtubules yet they are not under tension. In mitotic cells this error does not occur as easily as in meiosis, rendering control for this type of error less important. There are some indications that difficulties with attachment of the microtubules are also a signal for the metaphase checkpoint. In cells treated with anti-CENP-E antibodies kinetochores became unstable and many chromosomes appeared to no longer bind microtubules, and these cells also arrest in metaphase (Tomkiel et al., 1994). Control of sister chromatid separation. Until now it has not been clear what holds the sister chromatids together until anaphase. In part this is performed by intertwined DNA, as occurs when two replication forks meet. Proteins that complex with the DNA must also be involved and it seems that there are differences between the centromeres and the chromosome arms in this respect, as certain chemical treatments or certain mutations can cause asynchronous separation of the centrom-


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of taxol are also strongly dependent on the treatment protocol, such as the duration of treatment. Indications exist that mitotic checkpoint control is more stringent in humans than in rodents and that it does not depend entirely on p53 alone in this species. In contrast to the original cells, SV40-transformed human cells retained their resistance to polyploidization after 72 h colcemid treatment, (Kung et ai, 1990). Human HeLa S3 cells were also more resistant than CHO cells (Schimke etai, 1991) and p53~'~ engineered human embryonic fibroblasts were seen to accumulate less aneuploid cells than equivalent mouse p53~*~ cells after the same treatment with nocodazole (Di Leonardo, 1997). For these reasons studies were undertaken in our laboratory with the aim of characterizing the role of p53 in the mitotic checkpoint in human cells and in the absence of forced gene expression (Cundari et ai, 1998). Several hours after induction of the mitotic block the cell exited mitosis, duplicated centromeres and separated sister chromatids to enter a Gl-like tetraploid state. Only p53-deficient cells then resumed DNA replication and progressed further to a polyploid cell cycle, while p53expressing cell lines underwent a durable arrest in the tetraploid condition. Confirmation of these data was recently obtained in primary human lymphocytes, where nocodazole treatment was also shown to cause mitotic slippage and tetraploidization (Elhajouji et ai, 1997). A unifying model of the metaphase/anaphase transition Combining the information described in detail above leads us to the development of a model (Figure 1) in which the interactions between the ultras true rural components, the molecular components and the controlling molecules at the metaphase/anaphase transition are combined. The figure gives an overview of the most important phenomena in the different stages of mitosis, from prometaphase until early anaphase. The critical features of the proposed model are detailed below. Prometaphase. The mitotic spindle is being formed, the chromosomes are not yet all attached to the spindle microtubules and the 3F3/2 phophoepitope is detectable at the kinetochores. TopoII is probably still active and able to disentangle the DNA between the chromosome arms. As in cells blocked in prometaphase by colcemid, the chromosome arms only separate after a certain time span (Rieder and Palazzo, 1992). The sister chromatids remain connected at the centromere as long as the cells remain arrested. The connection between the sister chromatids at the centromere might be formed by proteins and to remove them the APC is probably required (reviewed in King et ai, 1996). CLIP proteins remain detectable between the chromosome arms until metaphase (Rattner et ai, 1988). The INCENPs are at this time mainly found at the level of the centromeres and the telomeres and, in lesser amounts, on the euchromatin (Earnshaw and Cooke, 1991). The motor protein CENP-E appears to be a substrate for phosphorylation by cyclin B/Cdc2 and in its phosphorylated form it does not cross-link the microtubules. In prometaphase and metaphase (Tomkiel and Earnshaw, 1993) this protein is found at the kinetochores, where it might serve to couple the chromosomes to the spindle microtubules, presumably in its phosphorylated form (Blangy et ai. 1995). Metaphase. When all chromosomes are connected to both spindle poles 3F3/2 is "switched off. A signal to start anaphase


hyperdiploid (47-53 chromosomes) and tetraploid cells over 20 passages in vitro (Agapova et ai, 1996). In an earlier study fibroblasts from Li-Fraumeni patients developed aneuploidy and tetraploidy in vitro after 5-15 population doublings, whereas control cells contained few tetraploid cells in the same circumstances. The Li-Fraumeni cells also spontaneously became transformed in culture, whereas controls senesced after 20-30 population doublings (Bischoff et ai, 1990). These findings are not unique for humans, as homozygous p53~ mouse embryonic fibroblasts spontaneously developed aneuploidy at early passage (passage 9) in culture. This also occurred in heterozygous cells, but to a lesser degree (Livingstone et ai, 1992). p53-negative cells not only show spontaneous aneuploidy more frequently than p53-positive cells, they have also been observed to be more sensitive to chemical disturbance of the spindle. For instance, Cross et al. (1995) found that p53^l+ mouse embryonic fibroblasts accumulated with a 4C DNA content after treatment with nocodazole or colcemid, indicating arrest in G2 or during mitosis. However, p53~*~fibroblastsreentered S phase without having been able to complete mitosis. The data recently obtained by Di Leonardo et al. (1997) on the same cell type confirm these earlier results. Similar results were obtained with a p53-negative derivative of the murine prolymphocytic FL5.12 cell line, which also continued cycling after nocodazole treatment. However, in the original p53+l+ cell line the protein was hardly expressed when mitotic proteins were present and arrest in mitosis was transient. Cyclin B1 levels, the degradation of which is required for exit from mitosis, had already declined after 24 h and p53 levels only rose later on, together with those of cyclin E, indicating that the cells had entered the next Gl phase. The cells could not keep up a high level of p53 in the continuing presence of nocodazole and it started to decrease after 48 h. Arrest in mitosis was independent of p53 expression in these cells (Minn et ai, 1996). This indicates that cells which fail to arrest in metaphase when they are not able to properly complete mitosis can still be detected during the next cycle and are eventually removed through apoptosis. A comparison between the human erythroleukemia cell line K562, which does not express p53, and its subclone KS, which does express this gene, in their response to nocodazole treatment gave interesting results. Neither cell line arrested to any significant degree in mitosis, but whereas K562 accumulated polyploid cells, KS only doubled its DNA content. KS probably arrested in the following Gl phase, whereas K562 continued cycling (Cundari et ai, 1998). This confirms in human cells the results of Minn et al. (1996), providing another case where the p53-dependent Gl checkpoint protects the cell against polyploidization as well as against DNA damage. In parallel with the p53-dependent checkpoint pathway, p53independent mechanisms for G2/M phase arrest also appear to exist. Human and mouse p53-negative primary fibroblasts were shown to be more sensitive to taxol treatment than equivalent cells that were p53-positive. These cells appeared to arrest in G2/M, but eventually escaped arrest, to form micronucleated cells in the next cycle; apoptosis also occurred. In contrast, p53-positive cells were able to divide further, after transient arrests, both at mitosis and in the next Gl phase (Wahl et ai, 1996). Sensitivity to taxol seems to be higher in cells that are nearer to mitosis; the difference in toxicity may also be due to differences in induction of apoptosis. The effects


Changes In Activity of the anaphase chromosome structure promoting complex Prometaphase

INCENP (partial)

Metaphase


Metaphaseanaphase transition

Legend: • • Chromosome scaffold = ^

INCENPs/CLIPs/DNA 3F3/2 phosphorylated epltope

0 Klnetochore proteins | | [ Cdc2 >. Topolsomerase II ESS Mlcrotubules CU Chromatln (&& Anaphase Promoting Complex

Fig. 1. Proposal for a unifying model of the metaphase/anaphase transition.

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M.Klrsch-VoWers, E.Cundari and B.Verdoodt

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could conclude from this that cyclin A degradation is not very important in exit from metaphase. However, in Drosophila embryos that express a non-degradable form of cyclin A cells had difficulty in leaving metaphase and abnormal anaphases were seen (Sigrist et al, 1995). The precise regulation thus appears to depend on the cell type. Protein phosphatase 2A (PP2A) function is required for cyclin B destruction in budding yeast. In the presence of a defective spindle mutants of cdc55, the regulatory subunit of PP2A, fail to arrest the cell cycle, although cyclin B is stable. This is due to a failure to remove inhibitory phosphates on Cdc28 (Minshull et al, 1996). Also, in clams PP2A is thought to be able to inactivate the APC (Lahav-Baratz et al, 1995). Anaphase. The chromatids start to move to their respective spindle poles and the mitotic cyclins are degraded. When sister chromatid separation starts the INCENPs remain behind in the position of the metaphase plate; later they move to the cell cortex, where the cleavage furrow will be formed (Earnshaw and Cooke, 1991). CENP-E moves in late anaphase A from the kinetochores to the microtubules of the spindle midbody, presumably in its non-phosphorylated form. During early anaphase A it is probably involved in moving the chromatids via stimulating depolymerization of the microtubules at the kinetochores (Lombillo et al, 1995; Brown et al, 1996). Degradation of cyclin B has been observed to be required for exit from anaphase (Holloway et al, 1993). The model schematizes separation of the chromatids in subsequent steps related to cyclins A and B respectively. It essentially leads to abandoning the cytogenetic paradigm which described chromatid separation at the centromeres as resulting from the pulling forces exerted by the spindle fibres on the chromosomes. Indeed, the unifying concept emerging from this synthesis indicates that sister chromatids separate independently of the tubulin pulling forces, rather as a result of proteolytic processes controlled by the APC. The spindle fibres are thus necessary to move the separated chromatids to the spindle poles but probably not to initiate separation. Integrity of the spindle, phosphorylation of the kinetochores and alignment of the chromosomes (not their number) are the main metaphase/anaphase checkpoints. In some cases abnormalities at the level of these checkpoints may lead to p53-independent apoptosis, eventually regulated by a Raf-1/Bcl2 phosphorylation pathway. p53 itself should interfere only in the subsequent Gl/S transition to eliminate those cells which divided in the absence of a spindle, escaped the metaphase/anaphase checkpoint, underwent sister chromatid separation and became tetraploid. In addition to providing an overview of the current information on separation of chromatids, our model also indicates some of the missing links. These are summarized below. • Is chromatin condensation induced by phosphorylation of hi stone 1? « Are the centromeres of the sister chromatids duplicated before prophase or at metaphase? • Does the scaffold exist at the level of the centromere? If not, what happens during chromatid separation? e Are the chromatid loops circling around each chromatid scaffold or are they attached only at one side, such that the scaffolds can directly face each other? o What is the nature of and what are the binding sites (chromatin or the scaffold) of the glue proteins? o Which event or checkpoint decides about progressive release of INCENP and CLIP at the centromeres?


is given shortly after. The nature of this signal is not yet known, but it must be distinct from the mechanisms that prevent premature anaphase (Rieder et al, 1997). INCENPs in mid-metaphase are mainly found at the centromeres; they seem to dissociate from the chromosomes in late metaphase and may become associated with the spindle microtubules (Earnshaw and Cooke, 1991). Metaphase/anaphase transition. Anaphase is typically initiated in mammalian cells ~2O-30 min after the last chromosome has attached to the metaphase plate and is connected to microtubules of both spindle poles (Rieder et al, 1994, 1997). The APC is activated; sister chromatids separate fully in untreated normal cells. The CLIP proteins are lost from the chromosome arms at this stage, although they remain detectable at the level of the kinetochores until telophase (Rattner etai, 1988). Several proteins are involved in activating the APC: apart from the MPF (Lahav-Baratz et al., 1995), cAMP-dependent protein kinase (PKA; Grieco et al., 1996) and the Drosophila fizzy gene product (Dawson et al., 1995) are also involved. It appears that activation by PKA depends indirectly on the MPF; PKA activity starts later than Cdc2 activity and the MPF is required for PKA activation. The cyclin A-Cdc2 complex was unable to activate PKA (Grieco et al., 1996). However, dephosphorylation is also required for APC activation, as mutations in protein phosphatase 1 (PP1) block the cells in metaphase in fission yeast (Ishii et al., 1996) and in other organisms (King et al., 1996). The mechanism by which the fizzy gene product stimulates the APC is as yet unknown; it is a homolog of budding yeast CDC20 (Dawson et al., 1995). The proteins holding the chromosomes together, the socalled 'glue proteins', which must be removed for sister chromatid separation to take place, are not yet well characterized. Proteins that have to be degraded by the APC for initiation of anaphase have been described, but they are more likely to be regulatory proteins. In budding yeast there is PDS1, which appears also to be involved in the spindle checkpoint (Yamamoto et al., 1996); in fission yeast cutl + and cut2 + (Funabiki et al., 1996). The pimples gene product of Drosophila has similar effects: cyclin degradation occurs as normal in mutant cells, but sister chromatids cannot separate (Stratmann and Lehner, 1996). Given the behaviour of the INCENPs in metaphase, it appears unlikely that these are the 'glue proteins' that hold the sister chromatids together until anaphase. The CLIPs remain associated with the kinetochores during anaphase, but these proteins disappear from the inner surface of the chromosome arms (Rattner et al, 1988), which makes them better candidates for being the 'glue proteins'. When considering deactivation of the cyclins some differences appear to exist between cell types or animal species. For example, it has been found that degradation of cyclins A and B1 occurred later in normal human and rat cells than in transformed cell lines derived from these tissues. In normal cultured cells cyclin A remained present at nearly metaphase concentrations until early anaphase, to be lost from the cell abruptly in late anaphase. Cyclin Bl disappeared completely at telophase in these cells. In various transformed cell lines cyclin A was often no longer present in metaphase cells; in human HeLa cells cyclin Bl was already degraded at the metaphase/anaphase transition (Girard et al, 1995). However, a human lymphoblastoid cell line derived from normal lymphocytes behaved like normal cells (Widrow et al, 1997). One


• Which event or checkpoint decides about APC-dependent proteolysis, simultaneously or sequentially of glue proteins cyclin A and cyclin B? • How is topoisomerase II activity regulated? • Is p53-independent apoptosis related to control of chromosome number or to correct alignment? • Do diplochromosomes observed in the prophase {An, 4C cells) which follows mitotic slippage posses one or two centromeres? • If homologous chromosomes recognize each other through sequence homology of centromeric DNA and minisatellites, why does this mechanism then not function in mitosis?

Acknowledgements The authors wish to thank Prof. J.M.Parry of the University of Wales, Swansea, for his critical reading of the manuscript and for his helpful comments. This work was supported by the EU-ENY4-CT97-O471 research programme.

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It is evident that more questions need to be answered about the regulation and checkpoint controls of this key mechanism in cell biology before definitive conclusions can be drawn about the sequence of events deciding on the metaphase/ anaphase transition. Besides the importance of gaining a better basic knowledge of the control of chromatid separation in metaphase/anaphase, our model also provides indications for the nature of potential cellular targets for the development of chemicals capable of modifying progression of cells through the cell cycle. Such modifications of the separation of chromosomes during the metaphase/anaphase transition may also be critical to production of aneuploid and polyploid cells. Application of this understanding could be very helpful in risk assessment of mutagens/carcinogens and in the development of new chemotherapeutic protocols.

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