708
Progression into and out of mitosis
Wolfgang Zachariae
Progression through mitosis is controlled by cyclin-dependent
kinases, which drive cells into metaphase, and by the anaphasepromoting complex/cyclosome, a ubiquitin ligase that triggers
sister chromatid separation and exit from mitosis. Recent work
has shown how the mutual regulation between cyclin-dependent
kinases and the anaphase-promoting complex/cyclosome
ensures that cell-cycle events occur in the right order. The
analysis of complexes required for sister chromatid cohesion and
chromosome condensation has revealed how cyclin-dependent
kinases and the anaphase-promoting complex/cyclosome
control the behaviour of chromosomes.
Addresses
Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden,
c/o EMBL, Meyerhofstrasse. 1, D-69117 Heidelberg, Germany;
e-mail: [email protected]
Current Opinion in Cell Biology 1999, 11:708–716
0955-0674/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
APC/C anaphase-promoting complex/cyclosome
CDK
cyclin-dependent kinase
M-CDK mitotic CDK
RENT
regulator of nucleolar silencing and telophase
S-CDK S-phase-promoting CDK
SCF
Skp1–cullin–F-box complex
SMC
structural maintenance of chromosomes
Introduction
During mitosis, sister chromatids, generated by chromosome duplication during S phase, segregate towards
opposite poles of the cell, which then divides in a process
known as cytokinesis. The motility of chromatids is mediated by microtubules that emanate from opposite poles of
the mitotic spindle and attach to a structure on each sister
chromatid called the kinetochore [1]. Accurate segregation
of sister chromatids during anaphase depends on cohesion
between sister chromatids. Cohesion is established during
S phase and persists until metaphase. During early mitosis,
this cohesion counteracts the pulling force exerted by
microtubules and thereby creates the tension that allows
congression of chromosomes to the spindle equator. The
sudden dissolution of cohesion, rather than an increase in
microtubule forces, is thought to trigger separation of sister
chromatids at the metaphase-to-anaphase transition [2].
Accurate segregation of sisters requires a second rearrangement of chromosome architecture: chromosome
condensation. Interphase chromosomes are dispersed
throughout the nucleus and their DNA is much longer
than the mitotic spindle. During early mitosis, condensation compacts the DNA of each chromosome into a small
package which follows the kinetochore on its way to the
spindle pole.
In recent years, there has been considerable progress in
understanding the molecular mechanisms that govern cell
cycle progression. Two types of enzymes have key roles in
controlling chromosome behaviour and its co-ordination
with cell division: cyclin-dependent kinases (CDKs) and a
ubiquitin protein ligase called the anaphase-promoting
complex or cyclosome (APC/C). The production of sister
chromatids and their preparation for segregation during
anaphase requires a period of high CDK activity which lasts
from late G1 until anaphase [3]. S-phase-promoting CDKs
(S-CDKs) trigger chromosome duplication, which produces
sister chromatids held together by cohesion. In yeast, initiation of DNA replication requires association of the
catalytic subunit Cdk1 with the B-type cyclin Clb5, whereas in animal cells S phase is promoted by Cdk2 binding to
cyclin E and also to cyclin A. By switching off proteolysis
mediated by the APC/C, S-CDKs prepare the stage for activation of mitotic CDKs (M-CDKs) in late G2. Activation of
M-CDKs requires accumulation of mitotic cyclins which
associate with an appropriately phosphorylated Cdk1.
Mitotic cyclins include cyclin A and B in animal cells and a
set of four B-type cyclins (Clb1,2,3,4) in budding yeast. MCDKs trigger breakdown of the nuclear envelope (in
animal cells), condensation of chromosomes, and the formation of a mitotic spindle that can capture microtubules.
Finally, by promoting activation of the APC/C shortly
before anaphase, M-CDKs initiate their own destruction.
Cyclin degradation by the APC/C causes inactivation of
Cdk1, which triggers cytokinesis and is a prerequisite for a
new round of chromosome duplication. Formation of replication-competent complexes at origins only occurs in a
state of low CDK activity. Concurrently with cyclin degradation, the APC/C triggers separation of sister chromatids
by mediating the degradation of an inhibitory protein called
securin. Proteolysis mediated by the APC/C therefore coordinates cytokinesis and chromosome re-duplication with
the separation of sister chromatids produced in the previous
S phase. These studies left open the question of how CDKs
and the APC/C change the architecture of chromosomes,
however. Insight into this question was provided recently
by the discovery of molecular machines that mediates cohesion of sister chromatids and their condensation.
This review focuses on recent work that helps to explain
how cells prepare their chromosomes for segregation during anaphase and how APC/C-dependent proteolysis
serves to co-ordinate sister chromatid separation with
cytokinesis and chromosome re-duplication. It is largely
based on data from yeast but the evolutionary conservation
of key molecules suggests that ‘what is true for yeast is also
true for the animal’. Several recent reviews give a more
general introduction to sister chromatid cohesion [4], chromosome condensation [5], and cell cycle regulation by the
APC/C [6,7].
Progression into and out of mitosis Zachariae
Preparing sisters for separation
What are the molecular mechanisms responsible for
cohesion between sister chromatids?
Proteins essential for sister chromatid cohesion were identified in yeast by screening for mutants that rapidly lose
chromosomes and prematurely separate sister sequences
[8,9,10••,11••]. Four proteins — Smc1, Smc3, Scc1 (also
called Mcd1) and Scc3 — are subunits of a cohesin complex that is essential for both the establishment of
cohesion during S phase and its maintenance until
metaphase [11••]. Cohesin was found to associate with
chromosomes at centromeric regions and at many distinct
sites along the arms [12•,13•]. The DNA-binding activity
of cohesin is thought to be provided by Smc1 and Smc3,
which are members of the SMC (structural maintenance of
chromosomes) family of proteins [14]. These evolutionary
conserved proteins comprise an N-terminal NTP-binding
domain, a coiled-coil region with a central hinge, and a
DNA-binding domain at the C-terminus [15•,16,17].
Bacterial SMCs form antiparallel dimers [18•], suggesting
that Smc1 and Smc3 might form a similar V-shaped complex with two globular DNA-binding domains at the end
of each ‘leg’. Cohesin is a conserved constituent of eukaryotic cells. In Xenopus extracts, a similar complex was
identified whose immunodepletion from interphase
extracts abrogates sister chromatid cohesion in the mitosis
that follows [19••].
How is cohesion established between sister chromatids?
The establishment of cohesion during S phase, but not its
maintenance until metaphase, requires several proteins
that are not associated with the cohesin complex. Scc2
forms a complex with a novel protein called Scc4. In scc2 or
scc4 mutants, cohesin is assembled normally but fails to
associate with chromatin suggesting that the Scc2/4 complex functions as a cohesin loading factor or CLF (R Ciosk,
K Nasmyth, personal communication). Mis4, fission yeast’s
Scc2 homologue, was also found to function during S phase
[20••]. In yeast, cohesin can bind to chromosomes from late
G1 until metaphase but cohesion between sisters is only
established when cohesin is loaded during S phase [21•]. A
link between cohesion and DNA replication might be provided by the Eco1/Ctf7 protein [10••,11••]. In eco1/ctf7
mutants, cohesin binds to chromosomes but then fails to
hold sisters together. Genetic interactions between mutations in ECO1/CTF7 and in DNA replication genes suggest
that Eco1/Ctf7 functions at replication forks. Eco1/Ctf7
might crosslink cohesin complexes bound to sister
sequences to form the bridges that actually hold sister
chromatids together. In summary, S-phase not only generates sister chromatids, it also introduces a structural cue
that is essential for segregation of sisters during anaphase.
What causes chromosomes to condense when cells
enter mitosis?
Complexes required for chromosome condensation, called
condensins, were first purified from Xenopus extracts and
more recently from fission yeast [22,23••,24••]. These
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complexes contain an SMC heterodimer comprising Smc2
(or Cut14/XCAP-E) and Smc4 (or Cut3/XCAP-C) and
three non-SMC subunits, Cnd1 (XCAP-D2), Cnd2
(XCAP-H) and Cnd3 (XCAP-G). Cnd1 and Cnd3 show
weak homology to the β-subunit of the adapter protein
complex involved in the assembly of clathrin [24••]. Like
SMCs, clathrin contains two coiled-coil regions separated
by a hinge. The flexible Smc2–Smc4 dimer might adopt its
functional conformation by associating with Cnd1 and
Cnd3. Xenopus and fission yeast condensin are both regulated by the Cdk1–cyclin B kinase; however, the
mechanisms are different. In animal cells, where the
nuclear envelope disassembles in early mitosis,
Cdk1–cyclin B regulates condensin’s activity. Xenopus condensin introduces positive supercoils into DNA in an
ATP-dependent reaction in vitro [25]. This activity
requires phosphorylation of the non-SMC subunits by
Cdk1–cyclin B [23••]. In fission yeast, where the nuclear
membrane stays intact during mitosis, Cdk1–cyclin B regulates access of condensin to the nucleus [24••]. During
interphase, condensin resides in the cytoplasm.
Phosphorylation of Smc4/Cut3 by Cdk1–cyclin B promotes accumulation of condensin in the nucleus. These
mechanisms explain the dependence of chromosome condensation on Cdk1–cyclin B activation and
decondensation on cyclin B degradation observed in vivo.
Activation of the anaphase-promoting
complex/cyclosome
By promoting the formation of mitotic chromosomes that
attach to a bipolar spindle, the state of high Cdk activity drives cells into metaphase. Further cell-cycle progression into
anaphase depends on activation of the APC/C [26]. By mediating degradation of securin the APC/C triggers sister
chromatid separation, and by degrading cyclins it triggers
cytokinesis and entry into the next cell cycle. The APC/C is
a ubiquitin protein ligase of surprising complexity which
contains at least ten (if not more) conserved subunits [7].
Little is known about the functions of individual components, however. The cullin-related subunit Apc2 [27–29] and
the RING-finger protein Apc11 [29] might function as
APC/C’s catalytic centre. These proteins associate which
each other [30] and show homology to components of the
SCF complex (Skp1–cullin–F-box), another ubiquitin ligase
with essential roles in cell-cycle regulation. The
cullin–RING-finger complex within the SCF (Cdc53 and
Hrt1/Rbx1/Roc1) was recently found to constitute a minimal
ubiquitin ligase [31•]. These data suggest that APC/C and
SCF use similar mechanisms to ubiquitinate their substrates.
A key question concerns the regulation of the APC/C during the cell cycle. APC/C-dependent proteolysis requires
conserved Trp–Asp repeat (WD) proteins, Cdc20 (or
Fizzy/Slp1/p55 Cdc) and Cdh1 (or Hct1/Fizzyrelated/Srw1/Ste9), which function as rate-limiting and
cell-cycle stage-specific activators of the APC/C. Cdc20
initiates APC/C-dependent degradation at the metaphaseto-anaphase transition [32–35,36 ••]. By mediating
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degradation of securin and mitotic cyclins, Cdc20 has a
central role in controlling entry into and exit from
anaphase. Once cyclins are destroyed, Cdc 20’s activation
of the APC/C declines and is replaced by Cdh1 until cells
enter S phase [34,37,38]. In yeast and human cells, Cdc20
and Cdh1 both bind to the APC/C but their association is
regulated differently. Cdc20 levels fluctuate during the
cell cycle so that it only binds to the APC/C during mitosis
[39•,40••,41•]. Cdh1 levels are constant but it only binds to
the APC/C during late anaphase and G1. When cells enter
S phase, binding of Cdh1 is blocked owing to its phosphorylation by S-CDKs such as Cdk1–Clb5 in yeast [42•] and
Cdk2-cyclin A in human cells [43•]. Expression of cyclin A
is controlled by the transcription factor E2F and the
retinoblastoma tumor suppressor protein which are known
to govern progression from G1 into S phase. By inactivating
APC/C-dependent proteolysis, S-CDKs establish conditions for the accumulation of mitotic cyclins. Switching
from Cdc20 to Cdh1 is important for somatic cells to link
preparations for mitosis to the accumulation of S-phase
cyclins whose expression is controlled by extracellular signals such as nutrients or growth factors. It was recently
observed that the WD proteins associate with APC/C substrates, suggesting that they activate ubiquitination of their
targets by recruiting them to the APC/C core particle
(M Schwab, W Seufert, personal communication).
Sister separation: a Gordian cut
How does APC/CCdc20 promote the sudden loss of cohesion
in all pairs of sister chromatids? In budding yeast, sister separation is triggered by the disappearance from
chromosomes of the cohesin subunit Scc1 [9]. Although
this event depends on APC/CCdc20, it is not triggered by
degradation of Scc1 by the APC/C itself. Instead, removal
of Scc1 is caused by its proteolytic cleavage at two related
sites [52••]. A noncleavable Scc1 variant is able to establish
cohesion but then persists on chromosomes which prevents
sister chromatid separation. Scc1’s cleavage is mediated by
an essential and evolutionary conserved ‘separin’ protein
called Esp1. Whether Esp1 itself is an unconventional protease or whether it activates an unknown protease remains
to be determined. From late G1 until metaphase, Esp1’s
activity is blocked by its association with the securin Pds1,
an anaphase inhibitor whose degradation by APC/CCdc20 is
essential for sister separation [53,54]. In fact, degradation of
Pds1 appears to be the sole essential role of APC/CCdc20
with regard to sister separation [35,54,55].
What triggers the sudden activation of APC/CCdc20 at the
metaphase-to-anaphase transition? Recent experiments
suggest that activation of APC/CCdc20 depends on the prior
phosphorylation of APC/C ‘core’ subunits by Cdk1–cyclin
B. Cdc20 can only promote the activity of the phosphorylated, mitotic form of the APC/C [44•] (E Kramer, J-M Peters,
personal communication). Activation of APC/CCdc20
requires Cks1 (also known as p9 or Suc1), a small conserved
subunit of Cdk–cyclin complexes that seems to target the
Cdk1–cyclin B kinase to the APC/C [45•–47•].
In summary, the APC/C promotes sister separation only
indirectly by destroying the securin Pds1, thereby liberating the separin Esp1, which then literally cuts the sisters
apart. Interestingly, Pds1 is only essential to prevent premature sister separation in the presence of unattached
kinetochores or damaged DNA [55,56•]. In an unperturbed
cell cycle, Pds1 degradation appears to be an essential precondition rather than the direct trigger for sister separation.
Therefore, sister separation must be regulated by additional mechanisms that might control, for example, the activity
of Esp1 or the susceptibility of Scc1 to Esp1-dependent
cleavage. Cleavage of Scc1 might be a conserved mechanism for inactivating cohesin. Rad21, fission yeast’s Scc1
homologue, and Rec8, a cohesin subunit that replaces
Scc1/Rad21 during meiosis, contain sequences related to
the cleavage sites at similar positions [52••].
Phosphorylation of Cdc20 during mitosis has been
observed in animal systems but its role remains controversial [40••,48,49]. It was reported that activation of a
purified mammalian APC/C requires phosphorylation of
Cdc20 by Cdk1–cyclin B [47•], whereas another study
shows efficient activation of mitotic APC/C by a nonphosphorylatable version of Cdc20 (E Kramer,
J-M Peters, personal communication). Phosphorylation
might augment Cdc20’s activity but is probably not
essential for the activity of APC/CCdc20 in vitro.
Activation of APC/CCdc20 by Cdk1–cyclin B renders
anaphase onset dependent on the previous entry into
mitosis; however, cyclin degradation must be delayed
until after Cdk1 kinases have triggered events such as
spindle assembly and chromosome condensation. Such a
delay could result from additional mechanisms required
for the activation of APC/CCdc20. There is evidence that
initiation of cyclin proteolysis has to await the activation
of polo-like kinase and a decline in the activity of protein
kinase A [47•,50,51].
Complexes with properties similar to Esp1–Pds1 have also
been found to control anaphase onset in fission yeast and
vertebrate cells, suggesting that sister chromatid separation is regulated by separin–securin complexes in all
eukaryotes. In fission yeast, segregation of sisters depends
on Cut1, an Esp1 homologue, and on degradation by the
APC/C of an associated inhibitor called Cut2 [57,58].
Whereas Esp1/Cut1 homologues share a conserved C-terminal region, securin proteins are unrelated apart from
some similarity in the distribution of charged amino acids.
A vertebrate securin was recently discovered in a screen for
potential APC/C substrates [59 ••]. It binds to an
Esp1/Cut1 homologue and a nondegradable variant blocks
segregation of sisters. Human securin is encoded by the
pituitary tumor-transforming gene PTTG, which is overexpressed in some tumors and shows transforming activity in
cultured cells. This finding adds to the growing suspicion
that malfunctions of genes controlling sister chromatid
separation are a major cause for the genetic instability of
tumor cells [60].
Progression into and out of mitosis Zachariae
It is currently unclear whether sister separation is the only
aspect of anaphase controlled by separin–securin complexes. In fission yeast, Cut2 is required for Cut1’s localization
to the mitotic spindle [61]. It has been proposed that Cut1,
upon degradation of Cut2, might coordinate sister separation with changes in spindle dynamics.
A unifying picture of sister separation in yeast and animal
cells has been threatened by the finding that in animal
cells most of cohesin disappears from chromosomes at
prophase, considerably earlier than sisters actually separate
[19••]. This might be related to the fact that animal chromosomes condense much more than those of budding
yeast. During extensive condensation in early mitosis,
most sister sequences become separated from each other;
this requires dissolution of a large fraction of the cohesion
established during S phase. A small fraction of these cohesive bonds (or indeed a different type of bond) is essential
to maintain cohesion of condensed chromatids until its
final destruction at the metaphase-to-anaphase transition.
In animal cells, as in yeast, sister separation, but not condensation, requires degradation of securin by APC/CCdc20
[59••]. Thus, separin might only mediate the final separation of condensed sister chromatids but is probably not
required for the partial separation of sister sequences during condensation. Although this process coincides with the
activation of condensin by the Cdk1–cyclin B kinase, it
also occurs in Xenopus extracts depleted of condensin
[19••]. What then triggers the removal of cohesin at
prophase? Chromosome condensation is tightly correlated
with phosphorylation of histone H3 (but not histone H1)
by an as yet unknown kinase [62,63]. It was shown recently that mitotic phosphorylation of histone H3 is also
essential for proper chromosome segregation [64••]. The
mitotic histone H3 kinase is therefore a promising candidate for controlling cohesin’s removal at prophase and its
identification is eagerly awaited.
Exit from mitosis: what goes up must
come down
Exit from mitosis collectively refers to several events that
occur when cells transit from late anaphase into G1 (or the
next S phase in embryonic cells). These events, which
include disassembly of the mitotic spindle, chromosome
decondensation, cytokinesis and re-formation of replication-competent complexes at origins all depend on the
inactivation of mitotic Cdk1 kinases. Co-ordinating cyclin
degradation, which causes Cdk1 inactivation, with securin
degradation, which triggers anaphase, is therefore crucial
to ensure that sister separation, cell division and chromosome re-duplication occur in the right order. In animal
cells, securin and the mitotic cyclins A and B are presumably all degraded by APC/CCdc20 shortly before anaphase
onset. The other APC/C activator Cdh1 is only required to
maintain APC/C activity once cells have entered G1.
Budding yeast, however, contains an unusual mitotic
cyclin called Clb2 whose degradation late in anaphase is
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mediated by APC/CCdh1 [34,37]. The participation of
Cdh1 in mitotic cyclin degradation explains what has long
been a puzzling difference between yeast and animal cells.
Endogenous cyclin B molecules are unstable in Xenopus
and clam extracts arrested in mitosis due to addition of a
nondegradable cyclin B variant. In this case, cyclin degradation persists because Cdk1 activates APC/CCdc20. In a
comparable experiment in yeast, Clb2 remains stable [65]
because Cdh1 cannot bind to the APC/C until Cdk1 has
been inactivated.
In budding yeast, Cdk1 inactivation during mitosis
involves three different mechanisms: proteolysis mediated
by APC/CCdc20 which destroys Clb3 and Clb5; proteolysis
by APC/CCdh1, which destroys Clb3 and Clb2; and accumulation of the CDK inhibitor Sic1, which inactivates any
surviving Cdk1–B-type-cyclin complexes. Nevertheless,
as in animal cells, it is Cdc20 and not Cdh1 that is essential for Cdk1 inactivation during mitosis. Deletion of PDS1
allows cdc20 mutants to undergo anaphase but not to inactivate Cdk1 [35,66]. In contrast, cdh1 mutants that fail to
degrade Clb2 exit from mitosis because they can inactivate
Cdk1 with the help of Sic1 [37]. Surprisingly, the S-phase
cyclin Clb5 appears to be the sole cyclin whose degradation by APC/CCdc20 is essential for exit from mitosis [36••].
By degrading Clb5, Cdc20 permits the Cdc14 phosphatase
to dephosphorylate Cdh1 and Sic1, which causes both formation of active APC/CCdh1 complexes and stabilization of
Sic1 [67••,68•]. From S phase until late anaphase, Sic1 is
targeted for degradation by SCF through its phosphorylation by CDKs. Cdc14 also provokes a burst of Sic1
synthesis by removing phosphates that block nuclear accumulation of Swi5, an activator of SIC1 transcription [67••].
Clb5 differs from other cyclins whose degradation is apparently less important in that it is especially potent in
counteracting Cdc14’s phosphatase activity. Therefore, it
has to be degraded by a Cdc14-independent pathway. In
summary, the conserved property of Cdc20 to mediate
both degradation of securin and inactivation of Cdk1
kinases helps to ensure that cells cannot attempt cell division or chromosome re-duplication until they have already
separated sister chromatids during anaphase.
The Cdc14 phosphatase
Activation of the Cdc14 phosphatase is central to Cdk1
inactivation in budding yeast [67••]. During most of the
cell cycle, Cdc14’s catalytic activity is inhibited by the
Net1/Cfi1 protein, which sequesters Cdc14 to the nucleolus [69••,70••]. Net1/Cfi1 is part of a multifunctional
complex called ‘RENT’ (regulator of nucleolar silencing
and telophase), which also contains a chromatin silencing
factor [70••]. During anaphase, Cdc14 is suddenly released
from its ‘prison’ and spreads throughout the cell to dephosphorylate Swi5, Sic1 and Cdh1. The finding that
Net1/Cfi1 is a phospho-protein that can be dephosphorylated by Cdc14 led to the proposal of a negative feedback
loop [70••]: phosphorylation of Net1/Cfi1 by an unknown
mitotic kinase might trigger Cdc14’s release during
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anaphase. Once cells are in G1, this kinase activity ceases,
Cdc14 then dephosphorylates Net1/Cfi1, which in turn
recaptures the phosphatase. The molecular trigger for
Cdc14’s activation remains to be identified. Activation of
Cdc14 occurs in esp1 mutants indicating that sister chromatid separation per se is not required [69••]. Instead, both
processes are independent consequences of APC/CCdc20
activity. Release of Cdc14 requires proteolysis of Pds1,
whereas dephosphorylation of Cdc14 substrates depends
on proteolysis of Clb5 [36••]. The dependence of Cdc14
on APC/CCdc20 ensures that all pathways for Cdk1 inactivation, whether or not they depend on cyclin proteolysis,
remain linked to sister chromatid separation. Homologues
of Cdc14 do exist in animal cells but whether they have a
role in Cdk1 inactivation remains to be investigated [71].
This is indeed an appealing hypothesis because dephosphorylation is required for both binding to the APC/C of
human Cdh1 [43•] and stabilization of CDK inhibitors
such as p27Kip1 [72].
In addition to proteolysis mediated by APC/C Cdc20,
Cdc14’s release requires a group of proteins tentatively
called the ‘late mitotic network’. This group includes proteins that resemble components of a signaling cascade such
as several protein kinases (Cdc15, Dbf2, Dbf20 and the
polo-like kinase Cdc5) and the Ras-like GTPase Tem1,
which might be regulated by a GDP/GTP exchange factor
called Lte1(see [6] and references therein). However, the
nature of the signal remains mysterious. The kinase activities of Dbf2 and Cdc5 are cell-cycle-regulated and reach a
maximum during anaphase [73,74,75•]. It is therefore possible that they trigger Cdc14’s release by phosphorylating
Cdc14 or Net1/Cfi1. In fission yeast, homologues of Tem1
(Spg1), Cdc15 (Cdc7), Dbf2 (Sid2) and Cdc5 (Plo1) are
required for cytokinesis but not for a new round of DNA
replication [76,77]. Whether they control cytokinesis by
activating a Cdc14 homologue is not known. Fission yeast
and budding yeast might use the same conserved pathway
to control different aspects of exit from mitosis.
Keeping destruction in check
The APC/C has emerged as the key target of a surveillance
mechanism known as the ‘mitotic checkpoint’. This system
transiently blocks cell-cycle progression beyond metaphase
until sister kinetochores of all duplicated chromosomes
have been attached to microtubules from opposite spindle
poles. The mitotic checkpoint is dispensable during an
unperturbed cell cycle in yeast but appears to be essential
for preventing premature entry into anaphase in mammalian cells. The same mechanism arrests cells in a
metaphase-like state upon artificial destruction of the spindle by microtubule depolymerizing drugs. Components of
the mitotic checkpoint in yeast were identified by looking
for mutants that rapidly die when exposed to microtubule
drugs. These proteins include Mad1, Mad2, Mad3, Bub2,
Bub3, the protein kinases Bub1 and Mps1, and the securin
Pds1 (see [78] and references therein). The mitotic checkpoint is highly conserved: in vertebrates, Mad1, Bub1,
BubR1 (a Bub1-related kinase) and Bub3 are required for
cell-cycle arrest upon spindle damage [79,80,81•,82•] and
for restraining anaphase entry in a normal cell cycle
[80,82•,83]. In both yeast and animal cells, Mad2 associates
with Cdc20 and the APC/C [41•,84,85••,86–88]. Moreover,
Mad1, Mad3 [86] and BubR1 [82•] were found to interact
with Cdc20 and the APC/C, but whether all these proteins
bind to APC/CCdc20 as a large ‘checkpoint complex’ is
unclear. There is compelling evidence that the
Mad2–Cdc20 interaction is required for blocking
APC/CCdc20’s ubiquitin ligase activity, which then prevents
proteolysis of securin and cyclin B. Variants of Cdc20 that
fail to associate with Mad2 cause cells to enter anaphase in
the presence of spindle damage [56•,86,88]. How Mad2
blocks the activity of APC/CCdc20 is unknown. Mad2 was
found in a ternary complex with APC/C and Cdc20, suggesting that it does not inhibit binding of the activator to
the APC/C core. The finding that Mad2 tetramers are more
potent than monomers in blocking cyclin degradation in
Xenopus extracts suggests that inhibition of APC/C activity
might involve a structural rearrangement of the
Mad2–Cdc20 complex [85••].
One of the puzzling aspects of checkpoint control is that
checkpoint proteins are involved in both blocking cell
cycle progressing and sensing microtubule attachment at
kinetochores. In vertebrate cells, Mad1, Mad 2, Bub1,
BubR1 and Bub3 selectively accumulate at unattached
kinetochores and disappear from them upon microtubule
attachment [79,80,81•,89,90•,91]. There has been some
debate as to whether the checkpoint proteins are regulated by tension or by the occupancy of kinetochores by
microtubules. Recent studies indicate that, at least in animals, both mechanisms have a role. Binding of Mad2 is
stimulated by phosphorylation that occurs on kinetochores
in prophase when they are not under tension [92•]. Mad2’s
disappearance seems to be promoted by the attachment of
microtubules even under circumstances that presumably
prevent the generation of tension [91]. It has been proposed that unattached kinetochores assemble and then
release a Mad2-containing complex which inhibits
APC/CCdc20 throughout the cell [81•,83]. Such complexes
must be unstable and are regenerated as long as unattached kinetochores are present. When all kinetochores
have acquired microtubules, the inhibitory signal ceases
and APC/CCdc20 becomes active.
How does the checkpoint pathway block both sister chromatid separation and Cdk1 inactivation, which, at least in
budding yeast, involves not only APC/CCdc20 but also
APC/CCdh1 and the CDK inhibitor Sic1? Recent work has
shown that cell-cycle arrest upon microtubule depolymeriztion is mediated by two distinct pathways
[56•,75•,93•,94]. Together, Mad1, Mad2, Mad3, Bub1,
and most likely also Bub3 are required to block
APC/CCdc20, which prevents degradation of Pds1. As a
result, Esp1 remains inactive and sister chromatids cannot separate. The persistence of the Cdc20 substrates
Progression into and out of mitosis Zachariae
Clb5 and Pds1 also blocks Cdk1 inactivation by inhibiting the activation of Cdc14 [36••]. However, mad2 or pds1
mutants still halt cytokinesis and chromosome re-duplication, suggesting that there is an additional pathway
blocking inactivation of Cdk1. This pathway depends on
Bub2 and Byr4 and is thought to block Cdc14’s release
from the nucleolus. The fission yeast homologues of
Bub2 (Cdc16) and Byr4 form a two-component GTPaseactivating protein for the Tem1-related GTPase Spg1
[95]. Thus, Bub2–Byr4 might inactivate Tem1 by converting it from the GTP-bound to the presumably
inactive GDP-bound form. Bub2 is required for inhibition of Dbf2, suggesting that this kinase is located
downstream of Tem1 in the pathway for Cdc14 activation
[75•]. Although the Mad and the Bub2 pathway are both
activated by (artificial) microtubule depolymerization,
they might normally respond to different signals. In contrast to Mad proteins, Bub2 and Byr4 are associated with
spindle poles where they might co-ordinate cytokinesis
with the arrival of chromosomes at the poles [93•,94].
Acknowledgements
I thank Jiri Bartek, Rafel Ciosk, Edgar Kramer, Kim Nasmyth, Jan-Michael
Peters, Michael Schwab, Wolfgang Seufert and Masaki Shirayama for
unpublished data, Kazuhisa Kinoshita for fruitful discussions, and
Simonetta Piatti and Tony Hyman for critical comments on this manuscript.
References and recommended reading
Papers of particular interest, published within the annual period of review,
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