Chromosoma (2005) 114: 252–262
DOI 10.1007/s00412-005-0010-z
MINI-REVIEW
Jeffrey M. Craig . K. H. Andy Choo
Kiss and break up—a safe passage to anaphase in mitosis
and meiosis
Received: 23 March 2005 / Revised: 20 May 2005 / Accepted: 24 May 2005 / Published online: 13 July 2005
# Springer-Verlag 2005
Abstract Eukaryotic chromosomes have many challenges
to overcome between DNA replication and sister chromatid
segregation. If these challenges are not met, cell death or
unregulated cell division (cancer) may result. During prophase, chromosomes condense, the nuclear membrane
breaks down and cohesins are removed from chromosome
arms. In prometaphase, initial spindle attachments are made
by sister kinetochores followed by correction of erroneous
attachments, centromere oscillation between spindle poles
and congression towards the cell’s equator. In metaphase,
all chromosomes attain stable bipolar spindle attachments
and align at the metaphase plate, ready for the metaphase–
anaphase transition when all ties between sister chromatids
are broken. This review concentrates on recent developments that have revealed the intricacies of these processes.
We now know more about how the mechanisms of cohesin
removal differ between prophase and the metaphase–anaphase transition, the processes for detection and correction
of improper spindle-kinetochore attachments and the concept that tension between sister kinetochores is the driving
factor for satisfying the spindle checkpoint. We are also
beginning to gain some understanding of the mechanisms
behind the co-segregation of sister chromatids at the first
meiotic division.
Introduction
After DNA replication in eukaryotes, pairs of sister chromatids are linked along their lengths by catenated (topologCommunicated by D. Griffin
Review related to the 15th International Chromosome Conference
(ICC XV), held in September 2004, Brunel University, London, UK
J. M. Craig (*) . K. H. A. Choo
Chromosome Research Group, Department of Paediatrics,
Murdoch Childrens Research Institute, Royal Children’s
Hospital, University of Melbourne,
Flemington Road,
Parkville, 3052, Australia
e-mail: [email protected]
ically entangled) double-stranded DNA fibres—inevitable
products of the replication process—and by cohesin complexes that most likely form a ring around the sisters (see
below). Before cell division and chromosome segregation,
links between sister chromatids have to be removed to enable chromosomes to segregate to opposite poles. Most of
the DNA catenations on chromosome arms are lost between
replication and mitosis, whereas most cohesins are released
from chromosome arms during prophase. Centromeric catenations and cohesin complexes remain until the metaphase–anaphase transition.
In single-celled eukaryotes such as the budding yeast
Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe, sister chromatids are separated along
their lengths at metaphase, but in multicellular eukaryotes
such as vertebrates, release of arm cohesion precedes loss
of centromere cohesion. In meiosis, these processes differ.
During the first meiotic division (meiosis I), homologous
pairs of chromosomes are segregated rather than sister
chromatids, which are not segregated until the second
round of meiotic division (meiosis II). The sequence of
events that accompany loss of cohesion at mitosis and
meiosis has long puzzled cell biologists, but the past year
has seen an expansion in our knowledge of these processes.
This review discusses this knowledge and focuses on the
events that occur between the onset of mitosis and the onset
of anaphase. Events occurring immediately before and after
this period will be described briefly and are reviewed
elsewhere (Ohi and Gould 1999; Stegmeier and Amon
2004). Where relevant, we will compare and contrast the
pathways underpinning mitosis and meiosis. The understanding of the fidelity of these pathways is paramount
because defects at any stage can lead to aneuploidy or
apoptosis that both contribute to cancer and other genetic
diseases.
We will begin with an outline of the key structural and
protein components that are pertinent to the present discussion. Proteins will be listed by their human and budding
yeast nomenclature where different, e.g. Rad21/Scc1. The
names and functions of the proteins referred to in the text
and figures of this review are also summarised in Table 1.
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Table 1 Proteins involved in
the passage from prophase to the
onset of anaphase
The table lists human and budding yeast homologues and
briefly summarises their functions. Only the proteins listed in
the accompanying figures are
listed due to space constraints
Nh no homologue identified
a
Budding yeast Kip2 and 3 are
both similar to MCAK
Human
Budding
yeast
Functions
APC/C
APC/C
Aurora B
Ipl1
Cdc20
Cdc20
CENP-A
Hec1
MCAK
Cse4
Ndc80
Kip2/3a
Nh
Dam1
Plk1
Cdc5
Rad21
Rec8
SA (1, 2)
Securin
Separase
Sgo1
Scc1
Rec8
Scc3
Pds1
Esp1
Sgo1
SMC1
SMC3
Smc1
Smc3
Anaphase promoting complex/cyclosome; targets Securin, cyclin B and
possibly Sgo1 for degradation via polyubiquitination
Core passenger protein; protein kinase influencing many mitotic
processes including generation of tension at kinetochores
Spindle checkpoint protein; activates APC/C when not inhibited by
soluble checkpoint proteins
Centromere-specific histone H3 variant
Ndc80 complex subunit; regulates microtubule attachment
Kinesin and microtubule depolymeriser; contributes towards correction
of aberrant kinetochore–microtubule attachments and inter-kinetochore
tension
Complex that directly links microtubules to kinetochores
in budding yeast
Kinase; regulates prophase removal of chromosome arm cohesins and
kinetochore tension
Core cohesin subunit; cleaved by separase
Meiosis-specific Rad21 paralogue
Core cohesin subunit
Inhibits separase
Cleaves Rad21 when not bound to Securin
Shugoshin; microtubule-binding protein; prevents mitotic prophase
removal of cohesins; protects Rec8 from cleavage by Separase/Esp1
during meiotic anaphase I; involved in sensing kinetochore tension at
mitosis
Core cohesin subunit
Core cohesin subunit
The kinetochore
Pericentric heterochromatin
The kinetochore is a multiprotein complex that is the
structural and functional hub of the centromere (Cleveland
et al. 2003; Maiato et al. 2004). It contains constitutive
proteins that provide a foundation structure throughout the
cell cycle (Amor et al. 2004) and transient proteins that
contribute to the different stages of cell division (Vagnarelli
and Earnshaw 2004). Basic kinetochore structure consists
of three zones that roughly correspond to the three layers
seen with electron microscopy: the inner, middle and outer
layers. The inner layer comprises constitutive, structural
proteins such as the centromere-specific histone H3 homologue CENP-A/Cse4. The outer layer consists of proteins
involved in microtubule contact, dynamics and spindle
checkpoint signalling. The middle layer contains multiprotein complexes, some relatively stable and some more dynamic, that connect the inner and outer layers in a structural
and functional manner (Maiato et al. 2004; McAinsh et al.
2003). Many proteins in the inner layer are necessary for
binding of proteins in the middle and outer layers, but the
hierarchical order of kinetochore protein deposition both
within and between the layers remains to be fully elucidated.
Heterochromatin is a multi-subunit complex that causes
chromatin to remain condensed and transcriptionally inactive throughout the cell cycle (Craig 2005; Fukagawa et al.
2004). As a rule, multicellular eukaryotes have their kinetochores embedded in tandemly repetitive DNA and/or
regions highly enriched in retrotransposons. These sequences can be transcribed and have the potential to disrupt
centromere structure and function if they are not silenced
by heterochromatin (Craig 2005; Fukagawa et al. 2004;
Wong and Choo 2004). Although the simple “point” centromeres of budding yeast have no heterochromatin, domains of heterochromatin are seen in the centromeres of
the fission yeast. Wherever it occurs, pericentric heterochromatin has been found to be essential for mitosis. It is
responsible for establishing sister chromatid cohesion via
the cohesin complex and DNA topoisomerase II (Topo II)
(Craig 2005). Furthermore, the heterochromatin cross-linking protein HP1 (known in fission yeast as Swi6), in addition to binding the fission yeast cohesin subunit SA/Scc3,
has also been found to bind other kinetochore proteins
(see below).
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The cohesin complex
Mitotic cohesin has four subunits: SMC1, SMC3, Rad21/
Scc1 and SA/Scc3 (Haering and Nasmyth 2003). Humans
have two mitotic paralogues of SA/Scc3 (SA1 and SA2),
and meiotic paralogues have been identified for all cohesins except SMC3. SMC1 and SMC3 form a ring structure that is sealed by Rad21/Scc1 and SA/Scc3, and it has
been proposed that each cohesin complex encircles one
DNA strand from each sister chromatid (Haering and
Nasmyth 2003). The mechanism of cohesin removal at the
metaphase–anaphase transition involves cleavage of the
Rad21/Scc1 cohesin subunit by the protease Separase/
Esp1. The mechanism of cohesion loss on chromosome
arms is discussed below.
The passenger protein kinase complex
Passenger proteins were originally defined as being nuclear
in G2, associating with condensing chromosomes during
prophase, concentrating at the centromere in metaphase
and transferring to the spindle at the onset of anaphase
(Earnshaw and Cooke 1991). Of the known passenger
proteins, four form a stable complex with a protein kinase
function: Aurora B/Ipl1 kinase, INCENP/Sli15, Survivin/
Bir1 and Borealin. This complex influences a number of
mitotic events including the removal of chromosome condensation, arm cohesion, correction of aberrant kinetochore–microtubule attachments, bipolar spindle assembly,
targeting of checkpoint proteins to kinetochores and cytokinesis (Vagnarelli and Earnshaw 2004).
Topoisomerase II
Topo II catalyses the passing of one double-strand of DNA
through another by breaking and rejoining one of the strands,
and can thereby catenate or decatenate two strands of DNA.
Topo II localises to chromosomes during interphase and
through anaphase. Centromeric Topo II increases between
prophase and metaphase and decreases during anaphase
(reviewed by Porter and Farr 2004). Identifying specific
functions for Topo II has proven to be challenging. From
studies using Topo II inhibitors or RNAi-mediated knockdown in different metazoan species, it was found that Topo
II contributes to chromatin compaction prior to metaphase (Porter and Farr 2004). The gene knockdown of the
two isoforms of human Topo II or the single isoform of
Drosophila Topo II resulted in extended mitotic chromosomes and chromosome mis-segregation (Chang et al.
2003; Sakaguchi and Kikuchi 2004). However, the precise
contribution of Topo II to the separation of sister chromatids remains to be fully elucidated. As Topo II can catenate and decatenate DNA, it is unclear whether it remains
at centromeres until signalling from the spindle checkpoint
to decatenate and release sister centromeres, or whether it
actively catenates sister centromeres until it is removed in
response to checkpoint cues. Current evidence appears to
favour the latter possibility because during the metaphase–
anaphase transition, Topo II is modified by the addition of
the ubiquitin-like molecule SUMO, causing its removal
from chromatin without affecting its activity (Azuma et al.
2003; Bachant et al. 2002). Although the mechanisms for
the subsequent loss of sister centromere catenation are unknown, this finding implicates the presence of a Topo IIindependent pathway that decatenates DNA in its absence.
In addition, the temporal and spatial relationships between
Topo II and cohesins remain to be investigated.
The stages leading to anaphase
During G2 phase of the eukaryotic cell cycle, checkpoint
mechanisms ensure that a cell has accurately replicated its
DNA and repaired any DNA damage. Also during G2, the
level of mitotic cyclins begins to rise and culminates in the
assembly of M-phase-promoting factor (MPF), a complex
of mitotic cyclins and cyclin-dependent kinases. MPF initiates assembly of the mitotic spindle, breakdown of the
nuclear envelope and chromosome condensation (reviewed
in Ohi and Gould 1999).
After the onset of mitosis, a number of stages have to be
traversed before anaphase chromosome segregation is possible. These stages have traditionally been classified as
prophase, prometaphase and metaphase. In prophase, the
mitotic spindle starts to form as asters in the cytoplasm and
spindle fibres start to radiate from these asters. In prometaphase, kinetochores capture microtubules first from
one pole then from the other, correct aberrant microtubule
capture and oscillate until they line up at the metaphase
plate. To aid the discussion of the underlying mechanisms
leading to the separation of sister chromatids during these
early mitotic phases, we have defined the following six
functional stages (Fig. 1) and discussed each below:
1. Condensation and removal of links between chromosome arms
2. Microtubule capture by one of a pair of kinetochores
3. Correction of erroneous microtubule attachments
4. Bi-orientation, stabilisation and generation of tension.
5. Passing the spindle checkpoint
6. Separation of sister chromatids by removal of centromeric cohesin and DNA decatenation
Stage 1. Condensation and removal of links between
chromosome arms
Chromosome condensation occurs during prophase and
prometaphase and utilises condensin protein complexes
(Ono et al. 2004). Condensin distribution changes throughout chromosomes in early prophase to an axial distribution
in early prometaphase (Kireeva et al. 2004). Condensinmediated chromosome condensation is essential for resolution of sister chromatids and for the correct apposition of
sister kinetochores towards opposite poles (Moore et al.
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Fig. 1 Mitotic events leading to the safe passage of prophase
through anaphase. The stages (1–6) of mitosis, from the chromosomal point of view, from prophase through the onset of anaphase
are numbered as in the text. Before stage 1, Topo II catenation (blue
ovals) has been removed from most of the chromosome excluding
the centromere and some sites on chromosome arms. During stage 1,
chromosomes condense and cohesins (red ovals) are removed from
most sites along chromosome arms and remain concentrated at
centromeres (yellow circles). At stage 2, microtubules (purple rods)
are captured by one kinetochore. At stage 3, incorrect spindle attachments, syntelic (top) or merotelic (bottom), are corrected, re-
sulting in stable bi-orientation and generation of tension by spindle
dynamics that include a “Pac-man” depolymerisation of kinetochore
microtubules (stage 4). The dotted arrow between syntelic and
merotelic configurations indicates the possibility that one may
change into the other with the addition or removal of a kinetochore–
microtubule interaction. At metaphase (stage 5), all chromosomes
bi-orient and align on the metaphase plate. At the onset of anaphase
(stage 6), cohesins and Topo II are removed from centromeric and
residual sites along chromosome arms and the chromosome starts to
migrate towards the spindle poles
2005; Ono et al. 2004). Chromosomes lacking condensin
can delay condensation, fail to resolve sister chromatids,
fail to associate with nonhistone centromere proteins and/
or exhibit aberrant chromosome alignment and segregation
(Hudson et al. 2003; Moore et al. 2005; Ono et al. 2004).
Furthermore, at least in Caenorhabditis elegans, Aurora B/
Ipl1 kinase is likely responsible for recruiting centromeric
condensins (Moore et al. 2005).
Topo II-mediated DNA catenation of chromosome arms
is almost complete by the onset of mitosis. The situation is
different with cohesin-mediated sister chromatid cohesion,
which is not lost from arms until prophase. Removal of
cohesin in prophase involves the phosphorylation of the
cohesin subunit SA/Scc3, most likely by Polo-like kinase 1
(Plk1/Cdc5) and/or Aurora B/Ipl1 (Hauf et al. 2005; Kitajima
et al. 2005; McGuinness et al. 2005; Sumara et al. 2002)
(Fig. 2). The reason that cohesins remain at centromeres
during prophase is that they are protected by the recently
identified protein Shugoshin (Sgo1, Japanese for “guardian
spirit”) (Kitajima et al. 2005; McGuinness et al. 2005; Tang
et al. 2004). Sgo1 protects centromeric cohesion until the
metaphase–anaphase transition (see below). It is targeted to
the kinetochore by the spindle checkpoint protein Bub1
and is degraded at the metaphase–anaphase transition
(Kitajima et al. 2005; Salic et al. 2004; Tang et al. 2004). It
is also possible that Sgo1 protects a small subset of chromosome arm cohesin complex in prophase and may also be
a structural link between the inner kinetochore and microtubules (see below and Figs. 2 and 4). Some cohesins have
been observed at chromosome arms at metaphase, and
chromosome arms not treated with spindle poisons usually
stay together at metaphase and are resistant to physical
separation (Gimenez-Abian et al. 2004; Paliulis and Nicklas
2004).
Stage 2. Microtubule capture by one of a pair
of kinetochores
Even though more than 60 different centromere proteins
have now been described, it has not been until very recently
that the search for the protein that tethers microtubules to
kinetochores has yielded results. Study of the budding
yeast has shown that microtubules are attached to kinetochores via the Dam1 complex (Westermann et al. 2005).
This ten-subunit complex oligomerises to form rings and/or
spirals around microtubule plus ends thereby stabilising
them (Westermann et al. 2005) (Fig. 2). These rings can
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slide along microtubules and thus tether microtubules to
kinetochores even when they are in flux. However, Dam1
homologues have yet to be found in fission yeast or complex eukaryotes. Furthermore, even though a number of
microtubule-associated proteins are known in complex eukaryotes, whether any of them are involved in a Dam-like
function remains to be seen.
What are the proteins that have been shown to link the
Dam complex to centromeres? Dam1 interacts with proteins from the Ctf19 and Ndc80 kinetochore protein complexes (Shang et al. 2003; Westermann et al. 2005) (Fig. 2).
The Ctf19 complex interacts with all three budding yeast
Fig. 2 Interactions of some of
the proteins involved in the
steps between prophase cohesin
removal and stable bi-orientation and generation of tension
between kinetochores. In stage
1, Sgo1, deposited on chromosomes by Bub1, protects cohesin subunit SA/Scc3 against
Aurora/Ipl1 and PLK1/Cdc5mediated phosphorylation and
loss from DNA. In stage 2,
microtubules are captured by
kinetochores via the Dam1
complex (Dam1c) in budding
yeast and unknown proteins in
other organisms. Microtubules
are further anchored to centromere (cen) DNA via the Ndc80,
Ctf19 and KNL complexes and
by DNA-binding proteins such
as CENP-A/Cse4 and CENP-C/
Mif2. In stage 3, syntelic kinetochore–microtubule interactions
are corrected by Aurora B/Ipl1mediated phosphorylation of
target proteins such as Ndc80
and MCAK and by Sgo1 by an
unknown mechanism. In stage
4, stable bi-orientation is accompanied by inter-kinetochore
tension induced by a number of
different proteins including mitotic kinases, cohesins and Topo
II. Tension-induced separation
of Aurora B and MCAK has
been proposed to make MCAK
available for dephosphorylation
and subsequent reinforcement of
microtubule depolymerisationinduced inter-kinetochore tension, making ready for the onset
of anaphase
centromere DNA-binding complexes but has so far not
been identified in other eukaryotes (Ortiz et al. 1999). The
Ndc80 complex has homologues in all eukaryotes so far
studied and is named after one of its four subunits, known
in humans as Hec1 (Kline-Smith et al. 2005). Studies in C.
elegans have found that the Ndc80 complex binds to kinetochores via the kinetochore null (KNL) protein complex
that is localised to kinetochores via the DNA-binding inner
kinetochore proteins CENP-A and CENP-C (Cheeseman et
al. 2004) (Fig. 2) and to the cohesin complex via an interaction between Hec1 and SMC1 (Zheng et al. 1999). It has
also been shown from work with C. elegans that the con-
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served nine-subunit Mis12/Mtw1 protein complex assists
microtubule attachments most likely by regulating the rate
at which they are formed (Cheeseman et al. 2004). Interestingly, the Mis12/Mtw1 complex interacts directly with
mammalian HP1 paralogues HP1α and HP1γ, linking heterochromatin formation with microtubule capture (Obuse
et al. 2004).
Stage 3. Correction of erroneous kinetochore
attachments
Immediately after microtubule capture of one of a pair of
sister kinetochores, chromosomes move towards the pole
from which the microtubule arose. This movement is caused
by depolymerisation of the polar (minus) end of the microtubule and by kinetochore-mediated depolymerisation of
the plus end. The latter, so-called “Pac-man” action, happens because kinetochore proteins such as CENP-E and
cytoplasmic dynein and its binding partner dynactin hold
on to microtubules during the process of depolymerisation
(Cleveland et al. 2003; Heald 2000). However, when these
processes are occurring, many other microtubules are also
radiating from the same pole. It is therefore understandable
that both sister kinetochores may be captured by microtubules from the same pole (so-called syntelic attachment,
Fig. 1). The spindle checkpoint monitors bipolar kinetochore
attachment by not allowing cells to progress to anaphase if
any kinetochores have not achieved bi-orientation (see
Fig. 3 Models for generation of tension at kinetochores with Aurora
B, MCAK and Shugoshin. The centromeric region of a pair of
mitotic sister chromatids is illustrated in a and b, with spindles
(purple rods) attaching to centromeres via outer kinetochore proteins
(yellow ovals). Prior to stable bi-orientation, microtubules form unstable attachments to kinetochores (double-headed arrows). a Aurora
B/Ipl1 (blue) and MCAK (red) co-localise to a large extent with the
former localising more to the inner centromere region. (Andrews et
al. 2004; Tanaka et al. 2002). Aurora B/Ipl1 can phosphorylate
MCAK and weaken its microtubule depolymerisation activity. After
the generation of tension through bipolar spindle attachment at
metaphase, MCAK is pulled away from Aurora B/Ipl1 at the inner
centromere region, freeing MCAK to be dephosphorylated and activating its microtubule depolymerisation function, therefore enabling
below). However, if cells relied purely on this checkpoint,
anaphase would never be allowed to proceed if even one
kinetochore had syntelic microtubule attachments. Recently, light has been shed on this conundrum with the
discovery that Aurora B/Ipl1 kinase can correct syntelic
microtubule attachments. Aurora B/Ipl1 differentiates between bi-oriented and syntelic microtubule attachments
because the latter lacks tension (Dewar et al. 2004). Sensing
this lack of tension at a syntelic kinetochore, Aurora B/Ipl1
disassembles one of the two kinetochore–microtubule attachments probably by phosphorylating one or more of its
many substrates. Aurora B/Ipl1 phosphorylation of microtubule centromere-associated kinesin (MCAK) localises it
to kinetochores (Fig. 2), and MCAK phosphorylation decreases its microtubule-depolymerising activities. However, it seems that residual MCAK depolymerisation activity
is enough to destabilise microtubules at one of the syntelic
sister kinetochores, resulting in attachment of only one sister to one spindle pole (Andrews et al. 2004; Kline-Smith
et al. 2005; Lampson et al. 2004). MCAK is probably
aided in this task by Aurora B/Ipl1-mediated phosphorylation of Ndc80 and Dam1 thereby disrupting their interaction (Shang et al. 2003) (Fig. 2). Aurora B/Ipl1 may
also be involved in this process through phosphorylation of
the phosphatase Cdc14/Clp1 (Trautmann et al. 2004).
Merotelic kinetochore attachments, where one of a pair
of bi-oriented sisters is attached to two spindle poles
(Fig. 1), are more troublesome to deal with than syntelic
attachments. This is because kinetochores are still under
it to pull the kinetochore and chromosome towards the spindle pole.
At the onset of anaphase, sister chromatids separate and MCAK
remains at kinetochores whereas Aurora B transfers to the spindle
midzone. b Shugoshin (green), in addition to protecting centromeric
cohesins cleavage during prophase, has been shown to interact with
microtubules (Indjeian et al. 2005). When kinetochores are not under
tension, Shugoshin is able to interact with microtubules and sends a
signal to destabilise them. As centromeres achieve stable bi-orientation, microtubules are pulled away from Shugoshin, which is now
unable to destabilise them. Shugoshin is degraded at the onset of
anaphase. These two models are not mutually exclusive and were
adapted from Andrews et al. (2004), Indjeian et al. (2005), Tanaka et
al. (2002)
258
tension from bi-orientation and therefore unable to trigger
the anaphase checkpoint. For this reason, merotely is a
major mechanism for generating aneuploidy in mammalian
cells (Cimini et al. 2001), and therefore, correction of
merotely is paramount to prevent genetic instability and
carcinogenesis. Merotely is more common in species such
as C. elegans with holocentric chromosomes (centromeres
span whole chromosomes). Studies of C. elegans have
implicated two mechanisms in prevention and correction of
merotelic attachments. Firstly, correct chromosome condensation is a prerequisite for correct apposition of sister
kinetochores. Secondly, the KLP-19 protein ensures that
bi-oriented chromosomes are under constant tension, maintaining their apposition to opposite poles (Powers et al.
2004). Other mammalian proteins shown to be important
for correction of merotelic attachments include the passenger protein Borealin (Vagnarelli and Earnshaw 2004) and
MCAK (Kline-Smith et al. 2005).
Stage 4. Bi-orientation, stabilisation and generation
of tension
After avoiding or correcting abnormal spindle attachments,
a centromere bi-orientates and its position oscillates until it
Fig. 4 Interactions of some of
the proteins involved in release
of the mitotic checkpoint and
subsequent release of centromeric cohesion. Unattached kinetochores release a soluble
complex of checkpoint proteins
that inhibit Cdc20. When every
kinetochore has been captured,
Cdc20 is free to activate the
APC/cyclosome, a ubiquitin ligase that targets mitotic cyclin B
and Securin for degradation.
Separase, previously bound and
inactivated by Securin, is then
free to cleave the cohesin subunit Rad21/Scc1, resulting in
the breakdown of centromeric
cohesin complexes. At the same
time, Topo II is sumoylated and
released from sister chromatids.
At this stage, sister chromatids
can be segregated to opposite
poles (red arrows) through kinetochore (k)–microtubule (m)
interactions
congresses at the metaphase plate. Microtubule depolymerisation is minimised giving each kinetochore pair some
stability (Fig. 3). Proteins involved in this stabilisation
include the EB1 protein and its binding partner adenomatous polyposis coli (APC), the Ran GTPase activating
protein RanGAP1 and its binding partner RanBP2, and the
Rho GTPase CDC42 and its effector mDia3 (Maiato et al.
2004; Yasuda et al. 2004). Further illustrating the interactivity of the vast network of mitotic and kinetochore proteins, mDia3 binds to CENP-A and HP1 in human cells
(Yasuda et al. 2004). Microtubule stabilisation helps promote tension between bi-oriented kinetochores. Tension
between physically connected kinetochores is more important than cohesion per se to satisfy the spindle checkpoint
because two kinetochores on the same circular minichromosome generated tension in the absence of functional
cohesins and Topo II (Dewar et al. 2004). Dewar et al.
(2004) also found that Aurora B/Ipl1 kinase is essential for
the generation of tension between kinetochores. It has been
proposed that tension between kinetochores pulls sister
kinetochores apart enough to separate the Aurora B/Ipl1 at
the inner centromere region from its substrate MCAK at the
kinetochore (Andrews et al. 2004; Tanaka et al. 2002)
(Fig. 3). It was proposed that MCAK would then be free to
be dephosphorylated, possibly by the protein phosphatase 1
259
(PP1) protein, thus, activating its microtubule depolymerisation function (Fig. 2). This depolymerisation would presumably activate Pac-man-like movement of the kinetochore
towards the pole. However, due to sister kinetochore links
made by cohesins and Topo II, each sister kinetochore
would not be able to travel far but would be primed for
quick segregation at the onset of anaphase.
In addition to Aurora B/Ipl1 kinase, a role in the creation
of tension at kinetochores has been implicated for Plk1/
Cdc5 kinase (Gruneberg et al. 2004; Sumara et al. 2004)
(Fig. 2). However, it is not known which targets of Plk1/
Cdc5 are phosphorylated or whether Plk1/Cdc5 acts at
kinetochores or spindle poles. Sgo1 has also been recently
implicated in tension sensing (Indjeian et al. 2005) (Fig. 2).
Budding yeast cells with mutation in the SGO1 gene were
unable to arrest in response to a lack of tension between
sister kinetochores. As Sgo1 has been found to be directly
involved in interactions between kinetochores and microtubules (Salic et al. 2004), it has been suggested that it acts
as a tension sensor (Indjeian et al. 2005) (Fig 2). In addition, KNL proteins have been shown to be essential for
kinetochores to sustain tension (Cheeseman et al. 2004)
(Fig. 2). Current evidence has therefore unveiled a network
of proteins that perform the tension generating and sensing
function.
Stage 6. Separation of sister chromatids by removal
of centromeric cohesin and DNA decatenation
DNA in sister kinetochores and at residual sites on chromosome arms is freed from being bound by the cohesin
complex after cleavage of its Rad21/Scc1 subunit (reviewed in Haering and Nasmyth 2003), and this cleavage is
enhanced by prior phosphorylation of Rad21 (Hauf et al.
2005) (Fig. 4). During the period leading up to the successful passage through the spindle checkpoint, Topo II has
been actively catenating DNA at sister centromeres and
possibly at a small number of regions along chromosome
arms. At this point, Topo II is modified by the addition of
the ubiquitin-like molecule SUMO, which causes its removal from chromatin but does not affect its activity (reviewed in Porter and Farr 2004) (Fig. 4). This loss of Topo
II is accompanied by a loss of catenation through a largely
unknown mechanism.
The transition from metaphase to anaphase is aided by
the APC/C-dependent, ubiquitin-mediated destruction of
cyclin B (Fig. 4). Free Securin/Pds1 induces Cdc14-mediated inactivation of the cyclin-dependent kinase Cdk1 via
the FEAR and MEN networks of proteins (reviewed in
Stegmeier and Amon 2004). In budding yeast, Cdc14 also
controls the segregation of repetitive DNA and telomeres in
a cohesin-independent but Aurora B- and condensin-dependent manner (D’Amours et al. 2004; Sullivan et al. 2004).
Stage 5. Passing the spindle checkpoint
The spindle checkpoint does not allow decatenation and
loss of cohesion of any sister kinetochores until all kinetochores have achieved bi-orientation at the metaphase plate
with kinetochores under tension (reviewed in Taylor et al.
2004). In brief, during prophase and prometaphase, a complex of at least nine proteins assembles at the kinetochore
aided at least in part by Aurora B/Ipl1 kinase (Lampson et
al. 2004; Taylor et al. 2004). These proteins include Mad2,
CDC20, Bub3 and BubR1 that together form the soluble
mitotic checkpoint complex (MCC) that most likely directly inhibits the onset of anaphase (Fig. 4). Kinetochore
checkpoint proteins also include CENP-E, which binds
microtubules, and other spindle attachment sensors such as
Mad2 and Bub1. The anaphase inhibition signal is downregulated in a stepwise fashion as mono- and bi-oriented
microtubule attachments are made and tension is generated.
Only at this final stage is the soluble MCC no longer produced and the kinetochores are free to segregate (Fig. 4).
At the same time as the anaphase inhibition signal is
down-regulated, Cdc20 becomes liberated from the kinetochore checkpoint complex and is free to activate the anaphase promoting complex/cyclosome (APC/C). The APC/
C activates Separase/Esp1 by ubiquitinating and targeting
the Separase/Esp1 inhibitor Securin/Pds1 for degradation
(Fig. 4). Separase/Esp1 is then free to cleave the cohesin
complex. The APC/C also induces the degradation of mitotic cyclin B (see below) and Sgo1 (Salic et al. 2004), the
loss of the latter removing the protection from centromeric
cohesins.
Mitosis vs meiosis
In the light of the preceding discussion, it would be of
interest to compare and contrast the relevant features of
mitosis and meiosis. In meiosis I, chromosome homologues are segregated to opposite poles whereas sister chromatids remain joined until meiosis II where sister chromatids
segregate to opposite poles (reviewed in Allshire 2004;
Marston and Amon 2004). Current data indicate that whereas some meiosis-specific mechanisms exist, numerous functional components have a common counterpart in mitosis
and meiosis, suggesting that functional adaptation of these
components has occurred during the evolution of these
processes. Firstly, maternal and paternal chromosomes pair
via chiasmata produced during recombination, with this
pairing being aided by chromosome condensation (Chan
et al. 2004) and, at least in the budding yeast, by centromere pairing (Kemp et al. 2004). Secondly, some cohesin subunits have meiotic counterparts: Rec8 substituting
for Rad21/Scc1, STAG3 for SA/Scc3 and SMC1β for
SMC1 (Marston and Amon 2004; Revenkova et al. 2004).
Interestingly, Rad21/Scc1 is also present at centromeres
during mouse meiosis from prophase I to the start of
anaphase II, prompting the suggestion that meiosis II is
more similar to mitosis than first thought (Xu et al. 2004).
Thirdly, at least in fission yeast, the Spo13 protein and the
monopolin protein complex, both present only during
meiosis I, ensure that sister chromatids are orientated towards the same pole and help in the protection of cen-
260
as a major controller of mitotic events. It can influence loss
of arm cohesion during prophase, correction of inappropriate kinetochore–microtubule interactions, generation of
inter-kinetochore tension, Rad21/Scc1 cleavage, and removal of meiosis-specific protection proteins. The concept
of disruption of protein interaction by spatial separation has
also emerged as a mechanism of control of protein activity.
At each stage of mitosis or meiosis, correct associations
need to be made between different proteins and protein
complexes if genomic stability is to be maintained. Most
importantly, the events that occur at each stage have to be
choreographed precisely if cancer or genetic disease is to
be avoided.
tromeric cohesins, possibly by maintaining high levels of
Securin/Pds1 (Katis et al. 2004; Lee et al. 2004). Fourthly,
Sgo1 protects Rec8 from cleavage by Separase/Esp1 during anaphase I but not anaphase II, where at least in Drosophila, the Mei-S332 protein, an earlier identified weakly
conserved homologue of Sgo1, is delocalised from centromeres by phosphorylation by the kinase Plk1/Cdc5 (Clarke
et al. 2005). These results, however, disagree with the implication of Plk1/Cdc5 in the removal of arm cohesion in
mitotic prophase (see above). Possible reasons could be
that either Sgo1 and Mei-S332 have overlapping but
non-identical functions, and/or that phosphoregulation of
STAG3 differs from that of SA/Scc3. Finally, it has recently been shown that in fission yeast, the Mes1 protein
is expressed in mitosis and meiosis but alternatively spliced
and highly expressed between late MI and late MII (Izawa
et al. 2005). During this time, its role is to inhibit APC/Cmediated degradation of MPF during anaphase of meiosis I
but not during anaphase of meiosis II.
Acknowledgements J.M.C. is an NH&MRC RD Wright Postdoctoral Fellow and an Honorary Fellow of the University of
Melbourne Department of Paediatrics. K.H.A.C. is an NH&MRC
Senior Principal Research Fellow. Thanks to Paul Canham, Paul
Kalitsis and Richard Saffery for helpful comments on the manuscript.
Cancer
References
It is not surprising that problems with proteins involved in
cell division can lead to oncogenesis. In a microarray metaanalysis of expression of genes in more than 3,700 separate
cancer samples, Rhodes et al. (2004) identified a small
subset of genes that are universally activated in most cancer
types or activated in undifferentiated cancer (Rhodes et al.
2004). Present in this list were Topo II, Plk1, Rad21,
Cdc20, Survivin and CENP-A. Overexpression of Aurora
B (Meraldi et al. 2004), INCENP and Securin (Zou et al.
1999) has also been linked to oncogenesis, and chromosomal instability (CIN) has been attributed to mutations in
mitotic checkpoint proteins (Rajagopalan and Lengauer
2004).
Conclusions
This review has focussed on recent findings related to the
events that occur between the onset of mitosis and the onset
of anaphase. These events include the removal of connections between chromosome arms, chromosome condensation, microtubule capture at kinetochores, correction
of aberrant microtubule attachments, bi-orientation, stabilisation and generation of tension at kinetochores, overcoming the spindle checkpoint and separation of sister
chromatids at the metaphase–anaphase transition. The reasons behind the differences between mitosis and meiosis I
have also been discussed, revealing the interactions between meiosis-specific factors and proteins involved in
both processes. We have discussed recent important discoveries including that of Shugoshin and its homologues
that protect mitotic centromeres from cohesin cleavage, the
mechanisms involved in the generation and sensing of tension between kinetochores and the mechanisms involved in
protection and orientation of sister kinetochores during the
first meiotic division. Protein phosphorylation has emerged
Allshire R (2004) Cell division: guardian spirit blesses meiosis.
Nature 427:495–497
Amor DJ, Kalitsis P, Sumer H, Choo KH (2004) Building the centromere: from foundation proteins to 3D organization. Trends
Cell Biol 14:359–368
Andrews PD, Ovechkina Y, Morrice N, Wagenbach M, Duncan K,
Wordeman L, Swedlow JR (2004) Aurora B regulates MCAK
at the mitotic centromere. Dev Cell 6:253–268
Azuma Y, Arnaoutov A, Dasso M (2003) SUMO-2/3 regulates topoisomerase II in mitosis. J Cell Biol 163:477–487
Bachant J, Alcasabas A, Blat Y, Kleckner N, Elledge SJ (2002) The
SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion
through SUMO-1 modification of DNA topoisomerase II. Mol
Cell 9:1169–1182
Chan RC, Severson AF, Meyer BJ (2004) Condensin restructures
chromosomes in preparation for meiotic divisions. J Cell Biol
167:613–625
Chang CJ, Goulding S, Earnshaw WC, Carmena M (2003) RNAi
analysis reveals an unexpected role for topoisomerase II in
chromosome arm congression to a metaphase plate. J Cell Sci
116:4715–4726
Cheeseman IM, Niessen S, Anderson S, Hyndman F, Yates JR III,
Oegema K, Desai A (2004) A conserved protein network controls assembly of the outer kinetochore and its ability to sustain
tension. Genes Dev 18:2255–2268
Cimini D, Howell B, Maddox P, Khodjakov A, Degrassi F, Salmon
ED (2001) Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells. J Cell
Biol 153:517–527
Clarke AS, Tang TT, Ooi DL, Orr-Weaver TL (2005) POLO kinase
regulates the Drosophila centromere cohesion protein MEIS332. Dev Cell 8:53–64
Cleveland DW, Mao Y, Sullivan KF (2003) Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell
112:407–421
Craig JM (2005) Heterochromatin—many flavours, common themes.
BioEssays 27:17–28
D’Amours D, Stegmeier F, Amon A (2004) Cdc14 and condensin
control the dissolution of cohesin-independent chromosome
linkages at repeated DNA. Cell 117:455–469
Dewar H, Tanaka K, Nasmyth K, Tanaka TU (2004) Tension between two kinetochores suffices for their bi-orientation on the
mitotic spindle. Nature 428:93–97
261
Earnshaw WC, Cooke CA (1991) Analysis of the distribution of the
INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation. J Cell Sci
98(Pt 4):443–461
Fukagawa T, Nogami M, Yoshikawa M, Ikeno M, Okazaki T,
Takami Y, Nakayama T, Oshimura M (2004) Dicer is essential
for formation of the heterochromatin structure in vertebrate
cells. Nat Cell Biol 6:784–791
Gimenez-Abian JF, Sumara I, Hirota T, Hauf S, Gerlich D, de la
Torre C, Ellenberg J, Peters JM (2004) Regulation of sister
chromatid cohesion between chromosome arms. Curr Biol 14:
1187–1193
Gruneberg U, Neef R, Honda R, Nigg EA, Barr FA (2004) Relocation of Aurora B from centromeres to the central spindle at
the metaphase to anaphase transition requires MKlp2. J Cell
Biol 166:167–172
Haering CH, Nasmyth K (2003) Building and breaking bridges
between sister chromatids. BioEssays 25:1178–1191
Hauf S, Roitinger E, Koch B, Dittrich CM, Mechtler K, Peters JM
(2005) Dissociation of cohesin from chromosome arms and loss
of arm cohesion during early mitosis depends on phosphorylation of SA2. PLoS Biol 3:e69
Heald R (2000) Motor function in the mitotic spindle. Cell 102:399–
402
Hudson DF, Vagnarelli P, Gassmann R, Earnshaw WC (2003)
Condensin is required for nonhistone protein assembly and
structural integrity of vertebrate mitotic chromosomes. Dev
Cell 5:323–336
Indjeian VB, Stern BM, Murray AW (2005) The centromeric protein
Sgo1 is required to sense lack of tension on mitotic chromosomes. Science 307:130–133
Izawa D, Goto M, Yamashita A, Yamano H, Yamamoto M (2005)
Fission yeast Mes1p ensures the onset of meiosis II by blocking
degradation of cyclin Cdc13p. Nature 434:529–533
Katis VL, Matos J, Mori S, Shirahige K, Zachariae W, Nasmyth K
(2004) Spo13 facilitates monopolin recruitment to kinetochores
and regulates maintenance of centromeric cohesion during
yeast meiosis. Curr Biol 14:2183–2196
Kemp B, Boumil RM, Stewart MN, Dawson DS (2004) A role for
centromere pairing in meiotic chromosome segregation. Genes
Dev 18:1946–1951
Kireeva N, Lakonishok M, Kireev I, Hirano T, Belmont AS (2004)
Visualization of early chromosome condensation: a hierarchical
folding, axial glue model of chromosome structure. J Cell Biol
166:775–785
Kitajima TS, Hauf S, Ohsugi M, Yamamoto T, Watanabe Y (2005)
Human Bub1 defines the persistent cohesion site along the
mitotic chromosome by affecting Shugoshin localization. Curr
Biol 15:353–359
Kline-Smith SL, Sandall S, Desai A (2005) Kinetochore-spindle
microtubule interactions during mitosis. Curr Opin Cell Biol
17:35–46
Lampson MA, Renduchitala K, Khodjakov A, Kapoor TM (2004)
Correcting improper chromosome-spindle attachments during
cell division. Nat Cell Biol 6:232–237
Lee BH, Kiburz BM, Amon A (2004) Spo13 maintains centromeric
cohesion and kinetochore coorientation during meiosis I. Curr
Biol 14:2168–2182
Maiato H, DeLuca J, Salmon ED, Earnshaw WC (2004) The dynamic kinetochore–microtubule interface. J Cell Sci 117:5461–
5477
Marston AL, Amon A (2004) Meiosis: cell-cycle controls shuffle
and deal. Nat Rev Mol Cell Biol 5:983–997
McAinsh AD, Tytell JD, Sorger PK (2003) Structure, function, and
regulation of budding yeast kinetochores. Annu Rev Cell Dev
Biol 19:519–539
McGuinness BE, Hirota T, Kudo NR, Peters JM, Nasmyth K (2005)
Shugoshin prevents dissociation of cohesin from centromeres
during mitosis in vertebrate cells. PLoS Biol 3:e86
Meraldi P, Honda R, Nigg EA (2004) Aurora kinases link chromosome segregation and cell division to cancer susceptibility.
Curr Opin Genet Dev 14:29–36
Moore LL, Stanvitch G, Roth MB, Rosen D (2005) HCP-4/CENP-C
promotes the prophase timing of centromere resolution by
enabling the centromere association of HCP-6 in Caenorhabditis elegans. Mol Cell Biol 25:2583–2592
Obuse C, Iwasaki O, Kiyomitsu T, Goshima G, Toyoda Y, Yanagida
M (2004) A conserved Mis12 centromere complex is linked to
heterochromatic HP1 and outer kinetochore protein Zwint-1.
Nat Cell Biol 6:1135–1141
Ohi R, Gould KL (1999) Regulating the onset of mitosis. Curr Opin
Cell Biol 11:267–273
Ono T, Fang Y, Spector DL, Hirano T (2004) Spatial and temporal
regulation of condensins I and II in mitotic chromosome assembly in human cells. Mol Biol Cell 15:3296–3308
Ortiz J, Stemmann O, Rank S, Lechner J (1999) A putative protein
complex consisting of Ctf19, Mcm21, and Okp1 represents a
missing link in the budding yeast kinetochore. Genes Dev 13:
1140–1155
Paliulis LV, Nicklas RB (2004) Micromanipulation of chromosomes
reveals that cohesion release during cell division is gradual and
does not require tension. Curr Biol 14:2124–2129
Porter AC, Farr CJ (2004) Topoisomerase II: untangling its contribution at the centromere. Chromosome Res 12:569–583
Powers J, Rose DJ, Saunders A, Dunkelbarger S, Strome S, Saxton
WM (2004) Loss of KLP-19 polar ejection force causes misorientation and missegregation of holocentric chromosomes.
J Cell Biol 166:991–1001
Rajagopalan H, Lengauer C (2004) Aneuploidy and cancer. Nature
432:338–341
Revenkova E, Eijpe M, Heyting C, Hodges CA, Hunt PA, Liebe B,
Scherthan H, Jessberger R (2004) Cohesin SMC1 beta is
required for meiotic chromosome dynamics, sister chromatid
cohesion and DNA recombination. Nat Cell Biol 6:555–562
Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh
D, Barrette T, Pandey A, Chinnaiyan AM (2004) Large-scale
meta-analysis of cancer microarray data identifies common
transcriptional profiles of neoplastic transformation and progression. Proc Natl Acad Sci U S A 101:9309–9314
Sakaguchi A, Kikuchi A (2004) Functional compatibility between
isoform alpha and beta of type II DNA topoisomerase. J Cell
Sci 117:1047–1054
Salic A, Waters JC, Mitchison TJ (2004) Vertebrate shugoshin links
sister centromere cohesion and kinetochore microtubule stability in mitosis. Cell 118:567–578
Shang C, Hazbun TR, Cheeseman IM, Aranda J, Fields S, Drubin
DG, Barnes G (2003) Kinetochore protein interactions and their
regulation by the Aurora kinase Ipl1p. Mol Biol Cell 14:3342–
3355
Stegmeier F, Amon A (2004) Closing mitosis: the functions of the
Cdc14 phosphatase and its regulation. Annu Rev Genet 38:
203–232
Sullivan M, Higuchi T, Katis VL, Uhlmann F (2004) Cdc14
phosphatase induces rDNA condensation and resolves cohesinindependent cohesion during budding yeast anaphase. Cell 117:
471–482
Sumara I, Vorlaufer E, Stukenberg PT, Kelm O, Redemann N, Nigg
EA, Peters JM (2002) The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase. Mol
Cell 9:515–525
Sumara I, Gimenez-Abian JF, Gerlich D, Hirota T, Kraft C, de la
Torre C, Ellenberg J, Peters JM (2004) Roles of polo-like kinase 1 in the assembly of functional mitotic spindles. Curr Biol
14:1712–1722
Tanaka TU, Rachidi N, Janke C, Pereira G, Galova M, Schiebel E,
Stark MJ, Nasmyth K (2002) Evidence that the Ipl1–Sli15
(Aurora kinase-INCENP) complex promotes chromosome biorientation by altering kinetochore-spindle pole connections.
Cell 108:317–329
262
Tang Z, Sun Y, Harley SE, Zou H, Yu H (2004) Human Bub1
protects centromeric sister-chromatid cohesion through Shugoshin during mitosis. Proc Natl Acad Sci U S A 101:18012–
18017
Taylor SS, Scott MI, Holland AJ (2004) The spindle checkpoint: a
quality control mechanism which ensures accurate chromosome
segregation. Chromosome Res 12:599–616
Trautmann S, Rajagopalan S, McCollum D (2004) The S. pombe
Cdc14-like phosphatase Clp1p regulates chromosome biorientation and interacts with Aurora kinase. Dev Cell 7:755–762
Vagnarelli P, Earnshaw WC (2004) Chromosomal passengers: the
four-dimensional regulation of mitotic events. Chromosoma
113:211–222
Westermann S, Avila-Sakar A, Wang HW, Niederstrasser H, Wong
J, Drubin DG, Nogales E, Barnes G (2005) Formation of a
dynamic kinetochore–microtubule interface through assembly
of the Dam1 ring complex. Mol Cell 17:277–290
Wong LH, Choo KH (2004) Evolutionary dynamics of transposable
elements at the centromere. Trends Genet 20:611–616
Xu H, Beasley M, Verschoor S, Inselman A, Handel MA, McKay
MJ (2004) A new role for the mitotic RAD21/SCC1 cohesin in
meiotic chromosome cohesion and segregation in the mouse.
EMBO Rep 5:378–384
Yasuda S, Oceguera-Yanez F, Kato T, Okamoto M, Yonemura S,
Terada Y, Ishizaki T, Narumiya S (2004) Cdc42 and mDia3
regulate microtubule attachment to kinetochores. Nature 428:
767–771
Zheng L, Chen Y, Lee WH (1999) Hec1p, an evolutionarily conserved coiled-coil protein, modulates chromosome segregation
through interaction with SMC proteins. Mol Cell Biol 19:5417–
5428
Zou H, McGarry TJ, Bernal T, Kirschner MW (1999) Identification
of a vertebrate sister-chromatid separation inhibitor involved in
transformation and tumorigenesis. Science 285:418–422