(The FASEB Journal. 1999;13:S231-S234.)
© 1999 FASEB
Protein dynamics at the kinetochore: cell cycle regulation of the metaphase to anaphase transition
GARY J. GORBSKY1,
MARKO KALLIO,
JOHN R. DAUM and
LEANA M. TOPPER
Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA
1Correspondence: Box 439 UVA Health Sciences Center, Charlottesville, VA 22908. E-mail: GJG5y{at}virginia.edu
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ABSTRACT
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The spindle checkpoint blocks the initiation of anaphase in mitosis and
meiosis if chromosomes are not aligned at the metaphase plate. The
checkpoint functions by preventing a ubiquitin ligase called the
anaphase-promoting complex/cyclosome (APC/C) from ubiquitinylating
proteins whose destruction is required for anaphase onset. The spindle
checkpoint signal originates at the kinetochores of unaligned
chromosomes and is broadcast to the rest of the cell. Although the
spindle checkpoint is not understood in detail, several components of
the checkpoint-signaling pathway have been identified. Many of these
components associate transiently with the kinetochores of unaligned
chromosomes. We propose a model in which kinetochores that lack stable
attachments to the spindle microtubules serve as catalytic staging
areas for the assembly of inhibitor complexes. These inhibitor
complexes then leave the kinetochores and block activity of the APC/C
throughout the cell. We suggest that microtubule occupancy at
kinetochores or physical tension induced by microtubule capture turns
off the capability of the kinetochore to produce the APC/C inhibitor.
Subsequently, the inhibitor concentration in the cell wanes and
anaphase initiates.—Gorbsky, G. J., Kallio, M., Daum, J. R., Topper,
L. M. Protein dynamics at the kinetochore: cell cycle regulation of the
metaphase to anaphase transition.
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THE SPINDLE CHECKPOINT AND THE REGULATION OF M PHASE
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THE UNBALANCED SEGREGATION of chromosomes during cell
division plays important roles in human disease. In cancer, the
tendency of certain tumor cells to develop abnormal nondiploid
karyotypes is a source of biochemical and behavioral variation among
cells. Some of these variants have more aggressive growth
characteristics, and they can become resistant to body defenses and
applied therapies. Outgrowth of these variants leads to increasing
tumor malignancy. In the development of germ cells, the mis-segregation
of chromosomes in meiosis generates aneuploid gametes. Embryos produced
from these gametes cause miscarriage and the birth of infants with
genetic abnormalities.
The segregation of the chromosomes is not without safeguards. In normal
cells a monitoring system, termed the spindle checkpoint, inhibits the
progression of M phase if the mitotic or meiotic machinery is not
properly assembled. The spindle checkpoint is most clearly manifest
when cells are treated with anti-microtubule drugs such as vinblastine,
colchicine, or nocodazole that cause microtubule disassembly or with
drugs such as Taxol that hyperstabilize microtubules, preventing their
normal dynamic assembly and disassembly. Under these circumstances, the
spindle checkpoint is activated and cell cycle progression is arrested
at M phase at a point before the separation of the chromatids at
anaphase.
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THE 3F3/2 KINETOCHORE PHOSPHOEPITOPE
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Genetic studies in yeast (1)
and laser ablation
experiments in mammalian cells (2)
indicate that
kinetochores are the source of the checkpoint signal that arrests M
phase when cells are treated with microtubule drugs. The checkpoint
signaling capability is a property of kinetochores that lack normal
microtubule attachments to the spindle. While all the steps of the
signaling pathways involved are not known, several important proteins
and markers have been identified. Several years ago we found that a
phosphoepitope recognized by the 3F3/2 monoclonal antibody, originally
produced by Cyert et al. (3)
, was expressed at the
kinetochores of prometaphase chromosomes until the chromosomes moved to
the metaphase plate (4)
. We then found that microinjecting
the 3F3/2 antibody into living mitotic cells would protect the
kinetochore phosphoepitope causing it to persist even after chromosomes
had assembled at the metaphase plate (5)
. As long as the
antibody protected the kinetochore phosphoepitope from intracellular
phosphatases, cells remained arrested at metaphase. Eventually the
cells entered anaphase, coincident with the disappearance of the 3F3/2
phosphoepitope from kinetochores. Intriguingly, Nicklas et al.
(6
, 7)
found in cultured grasshopper and praying mantid
spermatocytes, where micromanipulation of the chromosomes is possible,
tension artificially applied to kinetochores of unaligned chromosomes
could both down-regulate the expression of the 3F3/2 phosphoepitope and
abrogate the spindle checkpoint, allowing progression to anaphase.
In collaboration with Stuart Tugendreich and Phil Hieter, we have found
that the 3F3/2 antibody can quantitatively immunoprecipitate a complex
of proteins, termed the anaphase-promoting complex or cyclosome
(APC/C), from mitotic but not interphase cell extracts. The ability to
bind this complex is lost if the extracts are treated with phosphatase.
The APC/C is an E3 or ubiquitin ligase component of a ubiquitinylation
cascade that targets mitotic substrates such as cyclins and anaphase
inhibitor proteins for cell cycle-regulated degradation. Evidence
suggests that the spindle checkpoint functions by keeping the APC/C
inactive (reviewed in 8
, 9
). As a result, anaphase
inhibitor proteins such as the budding yeast Pds1 and the fission yeast
Cut2 are maintained, and chromatid separation is blocked. We
hypothesize that the 3F3/2 antibody recognizes a phosphorylation site
that is inhibitory to the ubiquitin ligase activity of the APC/C.
Presumably, this phosphorylation is maintained as long as the spindle
checkpoint is on. Under normal conditions chromosome attachment to the
spindle leads to the removal of this inhibitory phosphorylation,
allowing the APC/C to become active. We envision that microinjection of
the 3F3/2 antibody into living mitotic cells artificially maintains the
inhibitory phosphorylation, presumably protecting it from phosphatases,
and thus delaying activation of the APC/C and degradation of the
anaphase inhibitor proteins.
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PROTEINS OF THE SPINDLE CHECKPOINT PATHWAY
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Another element involved in inhibiting the activity of the APC/C
is the protein Mad2. Mad2 is one of a series of proteins first
identified through screening in budding yeast for mutants that fail to
arrest the cell cycle when treated with microtubule inhibitors. Three
MAD (mitotic arrest deficient) and, independently, three
BUB (budding uninhibited by benzimidazole) genes were
identified (10
, 11)
. Homologues of several of these genes
have been found in fission yeast, amphibians, and mammals where the
proteins appear to play similar roles in regulating the spindle
checkpoint (12
13
14
15)
. In addition, several of these
proteins are associated, at least in part, with mitotic kinetochores.
The Mad2 protein appears to bind and inhibit the ubiquitin ligase
activity of the APC/C (16
, 17)
. Interestingly, in both
budding yeast and fission yeast, the Mad2 protein is not essential for
normal growth in culture. Thus, in yeast it appears that the spindle
checkpoint is reserved for cell cycle ‘emergencies,’ (e.g., when
environmental conditions cause spindle damage).
Immunolocalization has shown that the Mad2 protein is found in several
regions in mitotic cells, in the cytoplasm, at spindle poles, and at
kinetochores before their attachment to spindle microtubules (12
, 13
, 18)
. To determine if the Mad2 protein plays a role in normal
mitosis in mammalian cells, we microinjected function-blocking antibody
to Mad2 into cultured cells (18)
. The antibody did not
affect prophase and early prometaphase events. The condensation of the
chromosomes, separation of the spindle poles, breakdown of the nuclear
envelope, and attachment of the chromosomes to the mitotic spindle
occurred normally. However, in cells injected with anti-Mad2 antibody,
a premature initiation of anaphase was induced during prometaphase well
before all the chromosomes had assembled at the metaphase plate. Some
chromosomes that had achieved bipolar attachment to the spindle poles
underwent relatively normal segregation, but those that had stable
attachments to only one pole failed to segregate their chromatids. All
events downstream of anaphase onset were initiated, including chromatid
movement to the poles (anaphase A), separation of the poles (anaphase
B), cytokinesis, reconstitution of the interphase nuclei, and exit to
G1. The premature initiation anaphase induced by the anti-Mad2 antibody
caused massive nondisjunction of the chromosomes, and the resulting
progeny cells were aneuploid.
We interpret these results to indicate that during normal mitosis in
mammalian cells the Mad2 protein functions to restrain anaphase onset
until chromosomes are properly aligned at the metaphase plate. Thus,
unlike yeast, mammalian cells appear to require Mad2 protein, and thus
the spindle checkpoint, at every mitosis. This essential role for Mad2
in mitosis in cultured mammalian cells does not appear to result from
selection for the spindle checkpoint in permanent cell lines because
primary human keratinocytes exhibited identical responses when injected
with the anti-Mad2 antibody (18)
. Perhaps the lesser
reliance of budding yeast and fission yeast on the spindle checkpoint
for normal mitosis is a consequence of the fact that microtubule
attachment to the kinetochores occurs within the intact nuclear
envelope, which does not break down in mitosis as it does in higher
eukaryotes. Thus, perhaps in the yeasts the likelihood of chromosomes
failing to attain initial bipolar attachment is low, and there is
little need for the spindle checkpoint except when environmental
conditions cause spindle damage.
One interesting aspect of the experiments in mammalian cells is that
microinjection of the anti-Mad2 antibody never induced anaphase onset
in prophase or early prometaphase. Instead, anaphase initiated only
after several minutes in prometaphase even when the anti-Mad2 antibody
was injected during prophase. Although we can not eliminate trivial
explanations for observation of this delay in the effect of the
antibody (e.g., the injected antibody may only partially inactivate
Mad2 function), a more interesting interpretation is that cells require
some period in prometaphase before they develop competency to enter
anaphase. We hypothesize that anaphase onset may be regulated by two
sequential mechanisms. We suggest that during prophase and early
prometaphase, cells are incapable of entering anaphase onset because
some essential machinery necessary for performing anaphase onset is not
assembled until midprometaphase. By then, under normal circumstances,
the spindle checkpoint would have been active and would prevent
premature anaphase onset before chromosome alignment at the metaphase
plate. However, when the checkpoint system is preempted by injection of
anti-Mad2 antibody, then anaphase onset ensues as soon as anaphase
competency is acquired.
The precise mechanism by which the Mad2 protein regulates the APC/C is
not yet fully known. However, our work and that of other laboratories
have shown that the Cdc20 protein (called p55CDC in mammalian systems)
serves to mediate the binding of Mad2 protein to the APC/C
(19
20
21)
. Immunolocalization and the expression of Green
Fluorescent Protein-Cdc20 chimeras reveal that the protein is found
throughout the cytoplasm but concentrated at mitotic kinetochores, at
spindle poles, and along spindle fibers. When antibodies to Cdc20 were
microinjected into living mitotic cells, several aspects of mitotic
progression were impacted. Cells were arrested or delayed at metaphase,
anaphase was prolonged, and cells were slow to exit to G1
(19)
. These results are consistent with the role proposed
for yeast Cdc20 in targeting the APC/C to its substrates for
ubiquitinylation and degradation (22
23
24
25)
.
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CHECKPOINT PROTEIN DYNAMICS AT THE KINETOCHORE
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Mitotic progression from metaphase onward is dependent on
regulated proteolysis. In the presence of unaligned chromosomes, the
spindle checkpoint blocks proteolytic events required for the onset of
anaphase. Signaling of the spindle checkpoint appears to originate from
kinetochores that lack proper attachment to the mitotic spindle. We
hypothesize that such kinetochores are catalytic assembly/activation
sites for an inhibitor of the APC/C that is then released into the
cytoplasm to inhibit progression to anaphase throughout the cell
(Fig. 1
). We hypothesize that production of this inhibitor involves the
transient association of the kinetochore with spindle checkpoint
proteins such as Mad2 and Cdc20. We further suggest that the inhibitor
is at some rate spontaneously inactivated in the cytoplasm. However, as
long as unattached kinetochores persist, the APC/C inhibitor continues
to be generated. Proper attachment of all the kinetochores turns off
production of the inhibitor. After the existing inhibitor drops below a
threshold, the APC/C becomes functional and anaphase initiates. Many of
the component proteins of this complex mechanical and chemical
signaling pathway have been identified. How microtubule attachment and
kinetochore tension regulate their interactions and activities remains
to be deciphered.
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Figure 1. A proposed model for the role of kinetochore protein dynamics in the
spindle checkpoint. A) The unattached kinetochore
(black) catalyzes the assembly and/or activation of a complex
containing Mad2, Cdc20, and likely other components not illustrated.
The active inhibitor complex (indicated by black shading) is released
from the kinetochore and blocks the ability of the anaphase-promoting
complex/cyclosome (APC/C) to ubiquitinylate target anaphase inhibitor
proteins such as Pds1 in budding yeast and Cut2 in fission yeast. In
the cytoplasm the Mad2/Cdc20 inhibitor complex is spontaneously
inactivated at some rate (indicated by color change from black to
gray), perhaps by release of the Mad2 component. However, as long as
unattached kinetochores persist, active inhibitor complex is
continuously regenerated, perhaps by recycling inactivated subunits.
B) Attachment of the final kinetochore halts
assembly/activation of the inhibitor complex (indicated by stretched,
gray color of kinetochores attached to microtubule bundles). Residual
inhibitor complex is slowly inactivated, thus allowing time for the
final attaching chromosome to move to the metaphase plate. The APC/C,
released from inhibition by loss of the Mad2 protein but requiring the
Cdc20 protein for targeting, ubiquitinylates the Pds1/Cut2-type
anaphase inhibitor proteins. C) These proteins are then
degraded by the proteasome and anaphase is initiated. [Modified from
Gorbsky et al. (18)
]
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ACKNOWLEDGMENTS
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We thank Drs. Bruce Nicklas, Jasminder Weinstein, Dan Burke,
Rey-Huei Chen, and Andrew Murray for providing reagents, expertise and
advice. This work has been supported by a grant from the National
Institute of General Medical Sciences.
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ABSTRACT
THE SPINDLE CHECKPOINT AND...
