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R613
Cell cycle: Checkpoint proteins and kinetochores
Aaron F. Straight
Vertebrate homologs of yeast spindle assembly
checkpoint proteins are localized to kinetochores and
may act as a sensor for proper chromosome attachment
to the mitotic spindle.
Address: Department of Physiology, Box 0444, School of Medicine,
University of California, San Francisco, California 94143-0444, USA.
E-mail: [email protected]
Current Biology 1997, 7:R613–R616
http://biomednet.com/elecref/09609822007R0613
© Current Biology Ltd ISSN 0960-9822
Cells carefully monitor the alignment of chromosomes on
the mitotic spindle to ensure that their chromosomes are
properly segregated at anaphase. Detachment of kinetochores from microtubules or depolymerization of the
mitotic spindle activates a checkpoint mechanism that
arrests the cell cycle prior to the initiation of anaphase. This
‘spindle assembly checkpoint’ allows the cell time to
correct the offending lesion so that it can proceed through
mitosis without chromosome missegregation [1,2]. Proteins
involved in the spindle assembly checkpoint have been isolated in budding yeast and in vertebrates [3–7]. Cells
lacking components of the spindle assembly checkpoint can
proceed through mitosis in the absence of a spindle or in
the presence of unattached chromosomes [3,5,8].
Recent work has shown that two of the spindle assembly
checkpoint proteins, Mad2 and Bub1, localize to the kinetochores of vertebrate cells for part of mitosis. Prophase
and prometaphase chromosomes show kinetochore localization of Mad2 and Bub1, but as chromosomes align on
the metaphase plate the kinetochore staining disappears
and does not reappear until the next cell cycle (Figure 1a).
Misaligned chromosomes show Mad2 and Bub1 localization at the kinetochore even when all other chromosomes
reside on the metaphase plate (Figure 1b,c) [4,6,7]. These
results suggest that the kinetochore both monitors the
attachment of the chromosome to the spindle and signals
the cell to arrest until chromosomes are properly aligned.
The spindle assembly checkpoint
Early experiments in the 1930s showed that, when vertebrate cells are treated with drugs that depolymerize the
mitotic spindle, they arrest in mitosis [9,10]. The mitotic
arrest in these cells was attributed to the removal of the
mechanical apparatus that separates the chromosomes —
the mitotic spindle. Recent work has shown that the
absence of the spindle itself is not the cause of the mitotic
arrest, but that the arrest is mediated through the activation of a checkpoint that monitors the integrity of the
spindle and the alignment of the chromosomes. Cells with
an intact mitotic spindle will still arrest in mitosis if a
single chromosome is not properly attached to the spindle
(Figure 1b) [11]. This arrest is caused by an inhibitory
signal emanating from kinetochores that are not attached
to the spindle apparatus [12].
Genetic studies in yeast have identified some of the
molecules involved in the spindle assembly checkpoint.
Like vertebrate cells, budding yeast cells arrest in mitosis
in response to spindle depolymerization [13]. Yeast
mutants were isolated that died rapidly when treated with
the spindle-depolymerizing drug benomyl. These
mutants — mad1, mad2 and mad3, and bub1, bub2 and bub3
— all fail to activate the spindle assembly checkpoint and
continue through mitosis without a spindle. Although the
mutants die rapidly in the presence of benomyl, the mad
and bub gene products do not appear to be directly
involved in spindle integrity, as the mutant strains have
normal spindles [3,5]. Instead, mad and bub mutants are
thought to be defective in the signalling mechanism that
arrests the cell cycle in response to spindle depolymerization. Wells and Murray [8] showed that excess kinetochores in budding yeast cells cause pronounced cell cycle
delays in mitosis. These cell cycle delays are absent in the
mad and bub mutants, suggesting that the Mad and Bub
proteins are involved in monitoring chromosome attachment to kinetochores as well as spindle integrity.
The kinetochore as the mechanism and the sensor for
mitotic spindle attachment
Kinetochores mediate attachment of the chromosomes to
the mitotic spindle. When a microtubule contacts a kinetochore, the kinetochore captures the microtubule [14,15].
These kinetochore–microtubule attachments rearrange
until sister kinetochores are attached to opposite poles of
the spindle. In insect cells, tension on the kinetochores
further stabilizes kinetochore–microtubule interactions,
thus promoting bipolar attachment of the chromosomes to
the spindle [16,17]. Once kinetochores are attached to the
spindle microtubules, the kinetochores mediate congression of the chromosomes to the metaphase plate [18] and
cells do not initiate anaphase until all chromosomes are
properly aligned.
Kinetochores can sense proper attachment to the mitotic
spindle. Proteins at the kinetochore are specifically phosphorylated dependent upon their proper attachment to
the mitotic spindle. In insect cells, kinetochores that are
not attached to the mitotic spindle, and kinetochores
that have only attached to one pole of the spindle, stain
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Current Biology, Vol 7 No 10
Figure 1
(a)
Interphase
Prophase
Mad
Bub
Metaphase
Anaphase
Misaligned chromosome
Error correction
Mad
Bub
M
Buad
b
M
Buad
b
(b)
Mad
Bub
Mad
Bub
M
Buad
b
Mad
Bub
M
Buad
b
(c)
Mitotic arrest
Spindle depolymerization
Mad
Bub
M
Buad
b
M
Buad
b
Mad
Bub
Mad
Bub
M
Buad
b
Adaptation
Mad
Bub
M
Buad
b
Mitotic arrest
© 1997 Current Biology
Mad2 and Bub1 proteins reside at kinetochores and mediate checkpoint
arrest of the cell cycle. (a) During a normal mitosis, Mad2 and Bub1
localize to kinetochores in prophase and prometaphase. Once all
chromosomes are attached to the spindle, cells proceed through
anaphase. (b) Misaligned chromosomes signal spindle assembly
checkpoint arrest. Chromosomes that are not properly attached to the
spindle maintain Mad2 at the kinetochore and the cell arrests in mitosis.
(c) Spindle depolymerization causes mitotic arrest. In the absence of a
spindle, Mad2 and Bub1 remain at the kinetochores of chromosomes
and the cell arrests in mitosis. The checkpoint arrest can be overcome by
two mechanisms: by error correction, the cell can correct the offending
lesion and process through a normal mitosis; by adaptation, the cell can
override the checkpoint arrest, in the presence of the defect, resulting in
an abnormal mitosis with the potential for chromosome loss.
brightly with the 3F3/2 antibody, which recognizes a
phospho-epitope at the kinetochores of mammalian and
insect cells. When chromosomes achieve a bipolar attachment to the spindle and the sister kinetochores come
under tension, the staining of the 3F3/2 epitope is diminished. This loss of 3F3/2 staining can be mimicked by
pulling on a mono-attached chromosome so as artificially
to apply tension to the kinetochore. Cells that are
microinjected with the 3F3 antibody delay dephosphorylation of the kinetochore and pause in mitosis. Thus, the
kinetochore responds to proper attachment to the
spindle by modifying the resident kinetochore proteins
(reviewed in [19]).
The spindle assembly checkpoint meets the kinetochore
Three recent papers report that spindle assembly checkpoint proteins reside at the kinetochore and regulate
passage through mitosis. Chen et al. [6] and Li and
Benezra [4] isolated, respectively, the Xenopus (XMAD2)
and human (hsMAD2) homologs of the yeast Mad2, and
Taylor and McKeon [7] isolated the mouse homolog
(mBub1) of yeast Bub1. All of these proteins localize to
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the kinetochores of chromosomes in cultured cells of the
organisms from which they were isolated. Interestingly, in
all three organisms, the checkpoint proteins only localize
to the kinetochores of cells in prophase and prometaphase,
prior to proper chromosome alignment on the metaphase
plate, or of cells that have been treated with nocodazole to
depolymerize the mitotic spindle (Figure 1a,c).
To investigate further the relationship between chromosome attachment to the spindle and XMAD2 staining,
Chen et al. [6] took advantage of cells that had attached all
but one or two chromosomes to both spindle poles. Chromosomes that had only attached a single kinetochore to
the spindle showed no XMAD2 staining on the attached
kinetochore, but bright XMAD2 staining on the unattached sister kinetochore (Figure 1b). Thus, the checkpoint proteins at the kinetochore respond to microtubule
attachment to the kinetochore.
Mad2 and Bub1 are required for the spindle assembly
checkpoint in vertebrate cells. Chen et al. [6] and Li and
Benezra [4] showed that antibodies against Mad2 blocked
the spindle assembly checkpoint. The spindle assembly
checkpoint can be reconstituted in vitro using extracts of
Xenopus laevis eggs [20]. Using X. laevis egg extracts, Chen et
al. [6] showed that the addition of anti-XMAD2 antibodies
to a checkpoint-arrested extract inactivated the checkpoint
and allowed progression through mitosis. Additionally, cells
that were treated with anti-XMAD2 prior to checkpoint
activation were unable to establish a checkpoint arrest. Li
and Benezra [4] took a related approach to test the function
of hsMAD2 in tissue culture cells. Antibodies to hsMAD2
were electroporated into tissue culture cells, then the cells
were treated with nocodazole to depolymerize the spindle.
Cells that received the hsMAD2 antibodies failed to arrest
in the presence of nocodazole.
The kinetochore-localization domain of mBub1 resides in
its amino terminus. Taylor and McKeon [7] overexpressed
the this amino-terminal mBub1 domain in tissue culture
cells. The overexpressing cells arrested poorly compared
to wild-type cells in the presence of nocodazole. Overexpression of full-length Bub1 in yeast confers a similar
phenotype, causing cells to become hypersensitive to
spindle depolymerization [21]. It is likely that Bub1 overexpression disrupts the ability of the kinetochore to signal
checkpoint arrest in the absence of a spindle. Checkpointarrested tissue culture cells eventually recover from the
arrest and die, exhibiting characteristics of apoptosis
(Figure 1, adaptation). In contrast, cells that express the
amino-terminal mBub1 fragment lack the checkpoint,
continue into the next cell cycle, and become polyploid
without signs of apoptotic elimination. The spindle
assembly checkpoint may serve a dual role in mediating
arrest so that the cell can correct errors, and in the elimination of damaged cells.
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Does the lack of a spindle assembly checkpoint affect the
timing of a normal cell cycle? Taylor and McKeon [7]
investigated this question by examining the effect of
overexpressing the mBub1 amino-terminal domain on the
timing of the cell cycle. They found that the overexpression had no effect on the timing of G1, S or G2 phases of
the cell cycle, but delayed the exit from mitosis by
approximately 25 minutes. Yeast cells that lack the spindle
assembly checkpoint protein Mad1 are also accelerated in
their exit from mitosis (P. Dann and A. Rudner, personal
communication). Thus, the spindle checkpoint may have
a role during the normal cell cycle in restraining the
passage through mitosis.
Other proteins involved in the spindle assembly checkpoint have been found to be resident components of the
kinetochore. Studies with budding yeast have shown that
the Mad2 protein interacts tightly with another spindle
assembly checkpoint protein, Mad1, and studies with
Xenopus extracts have shown that XMAD2 interacts with a
Mad1-like protein (R. Chen and K. Hardwick, personal
communication). The Mad1 protein has also been shown
to be phosphorylated by the protein kinase Mps1, and
overexpression of Mps1 causes constitutive activation of
the spindle assembly checkpoint [22]. The Bub1 protein
kinase also interacts with another checkpoint protein,
Bub3, forming an active kinase complex [21]. Thus, the
kinetochore may contain as many as five proteins directly
involved in the spindle assembly checkpoint, two of which
are protein kinases.
Integrating information at the kinetochore with the cell
cycle engine
Exit from mitosis is normally mediated by ubiquitin-dependent proteolysis of B-type cyclins, resulting in a drop in
cyclin-dependent kinase levels [23]. Activation of the
spindle assembly checkpoint blocks exit from mitosis,
arresting cells with high B-type cyclin levels and elevated
cyclin-dependent kinase activity [20,24]. The question
remains as to how the kinetochore generates the signal to
arrest the cell cycle, and how that signal is transduced to the
cell cycle machinery. Studies with X. laevis extracts have
shown that a well known component of kinase cascades, the
mitogen-activated protein (MAP) kinase ERK-2, is
required for both the establishment and maintenance of the
spindle assembly checkpoint [20]. In addition, studies with
yeast have shown that mutations in a B-type subunit of a
type-II protein phosphatase cause a spindle assembly
checkpoint phenotype [24,25]. Recent experiments suggest
that the inhibitory signal for checkpoint arrest may not be
mediated by a soluble factor, because the signal is not transferred between two spindles in the same cytoplasm [26].
One of the most attractive targets of the checkpoint signal
is the cyclososme/anaphase-promoting complex (APC),
the enzyme that ubiquitinates cyclins, targeting them for
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destruction [23]. Cyclin ubiquitination activity is inhibited
in extracts from checkpoint-arrested yeast cells, but it is
not known whether this is a direct or indirect effect of the
checkpoint on the cyclosome/APC [27]. Other targets,
such as the proteasome itself or the hydrolases that
remove ubiquitin from proteins, are still potential candidates for regulation. Although the signalling pathway from
the kinetochore to the cell cycle engine is unknown,
current studies suggest that checkpoint proteins on kinetochores are able to sense the attachment of the chromosomes to the spindle microtubules and, in response to
improper attachment, delay the cell cycle in order to
promote faithful chromosome segregation.
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