Download 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
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.
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'
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
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© UK Environmental Mutagen Society/Oxford University Press 1998
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M.Kirsch-Volders, E.Cundari and B.Verdoodt
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
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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
A unifying model for the metaphase/anaphase transition
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
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
M.Kirsch-Volders, E.Cundari and B.Verdoodt
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)
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 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
A unifying model for the metaphase/anaphase transition
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
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
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).
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).
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
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).
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.
326
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).
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).
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
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.
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
M.Kirsch-Volders, E.Cundari and B.Verdoodt
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).
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
Table III. Overview: comparison between the different metaphase checkpoint genes
BUB1
BUB2
BUB3
MAD1
MAD2
MAD3
MPS1
MPS2
RAD9
328
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)
A unifying model for the metaphase/anaphase transition
(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-
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
M.Kirsch-Volders, E.Cundari and B.Verdoodt
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
330
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
A unifying model for the metaphase/anaphase transition
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.
351
M.Klrsch-VoWers, E.Cundari and B.Verdoodt
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
332
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?
A unifying model for the metaphase/anaphase transition
• 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?
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.
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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Received on November 5, 1997; accepted on January 7, 1998
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