In Drosophila cells cyclin B is normally degraded in two phases: (1) destruction of the spindle-associated cyclin B initiates at centrosomes and spreads to the spindle equator, and (2) any remaining cytoplasmic cyclin B is degraded slightly later in mitosis. The APC/C regulators Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1 bind to microtubules in vitro and associate with spindles in vivo. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A destruction box-mutated form of cyclin B (cyclin B triple-point mutant [CBTPM]-GFP) that cannot be targeted for destruction by Fzy/Cdc20, is no longer degraded on spindles in syncytial embryos. However, CBTPM-GFP can be targeted for destruction by Fzr/Cdh1. In cellularized embryos, which normally express Fzr/Cdh1, CBTPM-GFP is degraded throughout the cell but with slowed kinetics. These findings suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell. These observations have important implications for the mechanisms of the spindle checkpoint (Raff, 2002).
This study follows the subcellular localization of Fzy/Cdc20 and Fzr/Cdh1 throughout the cell cycle in living Drosophila embryos. GFP-Fzy is concentrated on kinetochores, centrosomes, and spindles early in mitosis, and starts to disappear from these structures once the chromosomes align at the metaphase plate. This localization is similar to that reported for p55cdc20 in fixed human cells, and it fits in well with the proposed role of Fzy/Cdc20 in linking the spindle assembly checkpoint to the APC/C. In higher eukaryotes, the spindle checkpoint system consists of several proteins, including the Mad and Bub proteins as well as CenpE, Mps1, Rod, and ZW10. As cells enter mitosis, most of these proteins accumulate on unattached kinetochores, and are then lost from the kinetochores once the chromosomes align at the metaphase plate. Several of these checkpoint proteins can bind to Fzy/Cdc20, and this appears to inhibit the ability of Fzy/Cdc20 to activate the APC/C. Therefore, an unattached kinetochore is thought to continuously generate inhibitory checkpoint protein/Fzy (Cdc20) complexes, thus ensuring that the APC/C is not activated until all of the chromosomes have aligned properly at the metaphase plate (Raff, 2002).
The checkpoint proteins Mad2, BubR1, CENP-E, Rod, and ZW10 have all been shown to bind to kinetochores and then move along microtubules to the centrosomes in a dynein-dependent manner. During mitosis, the localization of GFP-Fzy to kinetochores is microtubule independent, whereas its localization at centrosomes is microtubule dependent. This is consistent with the possibility that Fzy/Cdc20 may also load onto kinetochores and then move along microtubules to the centrosomes (Raff, 2002).
In contrast to GFP-Fzy, GFP-Fzr is strongly concentrated at centrosomes throughout the cell cycle, apparently in a microtubule-independent fashion. The concentration of Fzr/Cdh1 at centrosomes was unexpected, since it had been previously proposed that Fzr/Cdh1 catalyzes the second phase of cyclin B destruction that occurs in the cytoplasm. However, the Fluorescence Redistribution After Photobleaching (FRAP) analysis suggested that Fzr/Cdh1 is rapidly turned over at centrosomes. Although the significance of this turnover is unclear, it is possible that Fzr (Cdh1)-APC/C complexes activated at centrosomes could diffuse throughout the cell to catalyze the destruction of cyclin B (Raff, 2002).
Fzy/Cdc20 protein is abundant in syncytial embryos, whereas Fzr/Cdh1 protein is virtually undetectable. Moreover, a D-box-mutated form of cyclin B (CBTPM-GFP), which cannot be targeted for destruction by Fzy/Cdc20, is not degraded on spindles in syncytial embryos. CBTPM-GFP can be targeted for destruction by Fzr/Cdh1, and, in cellularized embryos, where Fzr/Cdh1 is normally present, CBTPM-GFP is destroyed throughout the cell but with slowed kinetics. Taken together, these findings indicate that Fzy/Cdc20 alone is responsible for catalyzing the destruction of cyclin B on the spindle in syncytial embryos, whereas Fzr/Cdh1 can catalyze the destruction of cyclin B throughout the cell in cellularized embryos (Raff, 2002).
These results suggest a model of how the destruction of Drosophila cyclin B is regulated in space and time. Early in mitosis, inhibitory checkpoint protein/Fzy (Cdc20) complexes form at unattached kinetochores. It is proposed that these complexes are restricted to the spindle microtubules, and spread from the kinetochore to the centrosome, and then throughout the spindle. As the kinetochores align at the metaphase plate, inhibitory complexes no longer form, and this leads to the activation of Fzy (Cdc20)-APC/C complexes. Exactly where and how this activation occurs is unclear, but it is proposed that only the specific pool of Fzy/Cdc20 that has passed through the kinetochore (and so is restricted to the spindle) is activated to degrade cyclin B. The destruction of cyclin B on the spindle then initiates the second phase of cyclin B destruction by activating Fzr/Cdh1-APC/C complexes. Unlike the Fzy/Cdc20 complexes, activated Fzr/Cdh1 complexes are not restricted to spindle microtubules, and can target cyclin B for destruction throughout the cell (Raff, 2002).
Since the destruction of cyclin B appears to initiate at centrosomes, it is suspected that the Fzy (Cdc20)-APC/C complexes initially become activated to degrade cyclin B at centrosomes. Presumably, the activated complexes then spread along the microtubules toward the spindle equator. This would explain why, in syncytial Drosophila embryos where only Fzy/Cdc20 is present, the attachment between centrosomes and spindles appears to be essential for the destruction of the spindle-associated cyclin B. Why Fzy/Cdc20 might initially be activated at centrosomes is unclear. Perhaps the disassembly of the inhibitory checkpoint protein/Fzy (Cdc20) oligomers that form at the unattached kinetochores requires some activity that is concentrated at centrosomes (Raff, 2002).
It is stressed that this model applies only to the destruction of cyclin B. For example, cyclin A is also targeted for destruction by Fzy (Cdc20)-APC/C complexes, but it is not concentrated on spindles. It seems unlikely that Fzy/Cdc20 also catalyzes the destruction of cyclin A only on the spindle. Therefore, it is speculated that there must be separate pools of Fzy/Cdc20 that are responsible for degrading cyclin A and B. An attractive aspect of this model is that it explains how these different pools are generated. Only the pool of Fzy/Cdc20 that passes through the kinetochore is inhibited from activating the APC/C by the spindle checkpoint system, and only this pool of Fzy/Cdc20 is competent to catalyze the destruction of cyclin B. In this way, the destruction of cyclin B is inhibited by the spindle checkpoint system, whereas the destruction of cyclin A is not (Raff, 2002).
Could this mechanism for regulating cyclin B destruction in Drosophila embryos apply to other systems? If two vertebrate mitotic cells are fused to form a single cell, the presence of an unattached kinetochore on one spindle (spindle A) does not block the exit from mitosis on the other spindle (spindle B) once the chromosomes on spindle B have aligned. Moreover, once spindle B exits mitosis, spindle A exits mitosis soon afterwards, even if some of its kinetochores remain unattached. These observations are consistent with the model. It would be predicted that the Fzy (Cdc20)/checkpoint-protein complexes generated at the unattached kinetochores of spindle A are restricted to microtubules and so cannot inhibit the exit from mitosis on the neighboring spindle B. Moreover, the activation of Fzy/Cdc20 on spindle B would eventually activate Fzr (Cdh1)-APC/C complexes on spindle B. These complexes can then spread throughout the cell, ultimately degrading cyclin B on spindle A and forcing it to exit mitosis. The degradation of clb2 in S. cerevisiae also occurs in two phases that appear to be catalyzed sequentially by Fzy/Cdc20 and Fzr/Cdh1, although the spatial organization of this destruction has not been investigated (Raff, 2002).
However, the model cannot explain how cyclin B is degraded in early Xenopus embryo extracts. Like early Drosophila embryos, these extracts contain Fzy/Cdc20, but lack Fzr/Cdh1. Nonetheless, cyclin B is completely degraded at the end of mitosis in these extracts, even if no nuclei or spindles are present. Thus, Fzy/Cdc20 can catalyze the destruction of cyclin B that is not spindle associated in Xenopus extracts. The reason for this apparent difference is unclear. However, it is noted that early Xenopus extracts do not have a functional spindle checkpoint. The mechanisms that link the destruction of cyclin B to the spindle checkpoint may also be required to restrict Fzy/Cdc20 complexes to the mitotic spindle (Raff, 2002).
The finding that Fzy/Cdc20 and Fzr/Cdh1 are concentrated at centrosomes highlights the potential importance of this organelle in regulating the exit from mitosis. It is speculated that the concentration of these proteins at centrosomes might serve two purposes. (1) It might enhance the fidelity of their sequential activation. The inactivation of cyclin B/cdc2 triggered by Fzy/Cdc20 seems to start at centrosomes, and cyclin B levels might only have to fall below a certain threshold level at the centrosome (rather than throughout the whole cell) to trigger the activation of the centrosomal Fzr/Cdh1. (2) In budding yeast there is a second, Bub2-dependent checkpoint that monitors the positioning of the spindle between the mother and daughter cell. Bub2 is concentrated at the spindle pole body where it is thought to suppress the activation of the mitotic exit network, and so block the activation of Fzr/Cdh1 and the exit from mitosis. It is not clear if mammalian cells also have a spindle orientation checkpoint, but if they do, the concentration of Fzr/Cdh1 at centrosomes may be important for the function of this checkpoint (Raff, 2002).
Sister chromatid separation during exit from mitosis requires separase. Securin inhibits separase during the cell cycle until metaphase when it is degraded by the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, sister chromatid separation proceeds even in the presence of stabilized securin with mutations in its D-box, a motif known to mediate recruitment to the APC/C. Alternative pathways might therefore regulate separase and sister chromatid separation apart from proteolysis of the Drosophila securin PIM. Consistent with this proposal and with results from yeast and vertebrates, it is shown in this study that the effects of stabilized securin with mutations in the D-box are enhanced in vivo by reduced Polo kinase function or by mitotically stabilized Cyclin A. However, PIM is shown to contain a KEN-box, which is required for mitotic degradation in addition to the D-box; sister chromatid separation is completely inhibited by PIM with mutations in both degradation signals (Leismann, 2003).
Embryos homozygous for pim null mutations but equipped with a
maternal pim+ contribution from pim heterozygous
mothers progress normally through the initial embryonic cycles. Entry into
mitosis 15 and progression to metaphase are still normal. Moreover, the
transition from metaphase to anaphase is triggered as well, as evidenced by
the degradation of the mitotic Cyclins A, B and B3. However, sister chromatid
separation during mitosis 15 is completely inhibited.
This block of sister chromatid separation after the exhaustion of the maternal
pim+ contribution is almost completely prevented when
pim embryos inherit a gpimdba-myc transgene.
gpimdba-myc drives expression of Pim with C-terminal myc
epitopes and a mutant D-box (AKPAGNLDA instead of
KKPLGNLDN). Pimdba-myc is stable during
mitosis according to confocal immunofluorescence microscopy, in contrast to
Pim-myc with the wild-type D-box. gpimdba-myc expression
is controlled by the normal pim regulatory region, and the resulting
level of Pimdba-myc before mitosis 15 was found to be comparable to
wild-type Pim levels. Because stabilized Pimdba-myc protein
promotes sister chromatid separation in pim mutants, it appears that
sister chromatid separation is not dependent on degradation of the
Drosophila securin Pim. Analogous experiments with a
gpimdba transgene driving expression of a D-box mutant Pim
version without myc epitopes also revealed rescue of mitosis 15 in
pim mutants, excluding the possibility that sister
chromatid separation in the presence of stabilized Pimdba-myc
occurs simply because C-terminal myc epitopes specifically abolish the
inhibitory Pim function (Leismann, 2003).
Instead of being required during each mitosis, Pim degradation might be
important to keep protein levels below a critical threshold. Moderate overexpression of wild-type pim (about fivefold) is sufficient to block sister chromatid separation. Moreover, although gpimdba rescues sister chromatid separation during mitosis 15 and 16 in pim mutants, it does not allow later divisions, perhaps because the levels of
stabilized Pimdba have built up beyond the critical threshold (Leismann, 2003).
If degradation of the securin Pim was not an obligatory process required
during each mitosis, separase bound to securin would be expected to have
sufficient basal activity to allow sister chromatid separation. In this case,
premature sister chromatid separation during interphase and early mitosis
would have to be prevented by securin-independent regulation. Since
securin-independent regulation at the level of Scc1 phosphorylation by
Cdc5/Polo kinase has been described in yeast, whether a reduction
in polo function enhances the effects of stabilized
Pimdba was investigated. Within the CNS of polo-mutant embryos,
many abnormal cells were observed with very large polyploid nuclei, when these embryos also carried gpimdba. Similar abnormal cells are almost never observed in either polo+ sibling embryos with gpimdba or in
polo- sibling embryos without gpimdba. In the presence of stabilized Pimdba, therefore, the remaining level of maternal polo+ contribution is no longer sufficient to mask
phenotypic abnormalities in polo-mutant embryos. Moreover, reduced
polo+ function enhances the effects of stabilized
Pimdba (Leismann, 2003).
In addition to Scc1 regulation by Cdc5/Polo kinase, vertebrate Cdk1 has
been shown to regulate separase independently of securin. The
effects of stabilized Cyclin A in Drosophila embryos are
consistent with the finding that vertebrate Cdk1 phosphorylates and thereby
inhibits separase. Mutant Cyclin A versions that cannot be degraded during
mitosis delay progression through the embryonic cell divisions during
metaphase before sister chromatid separation. Therefore, Drosophila
Cyclin A-Cdk1 complexes might inhibit separase activity. Accordingly, the
effects of stabilized Cyclin ADelta1-53 are expected to be enhanced by
expression of stabilized Pimdba. Labeling with antibodies against
tubulin and a DNA stain clearly reveal an increased number
of metaphase figures in epidermal regions of embryos expressing both Cyclin
ADelta1-53 and Pimdba, compared with embryos expressing only Cyclin ADelta1-53. The stabilized
Cyclin ADelta1-53 therefore results in a more pronounced metaphase delay in
the presence of the stabilized Pimdba (Leismann, 2003).
In principle, stabilized Cyclin A might delay cells in metaphase because it
results in an inhibition of Pim degradation during mitosis. However, cells
delayed in metaphase by stabilized Cyclin ADelta1-170 no longer contain
Pim-myc according to immunolabeling experiments, whereas metaphase cells that
do not express Cyclin ADelta1-170 are always positive for Pim-myc. It is concluded, therefore, that the metaphase delay induced by stabilized Cyclin A does not result from delayed Pim degradation (Leismann, 2003).
The phenotypic interactions between stabilized Pimdba and Polo
or Cyclin A are consistent with the notion that separase complexed with
non-degradable securin might have sufficient activity to allow sister
chromatid separation and that the timing of this process is controlled by
pathways other than securin degradation. However, the sister
chromatid separation in Pimdba-expressing cells might also be
supported by residual mitotic Pimdba degradation. A KEN motif,
which is found close to the N-terminus in all of the securins, might allow some
limited mitotic Pimdba degradation, escaping detection by confocal
microscopy as applied in the previous experiments (Leismann, 2003).
To determine whether the KEN motif of Pim functions as a degradation
signal, the mitotic stability of a myc-tagged Pim version with a
mutant KEN-box (Pimkena-myc with AAA instead of KEN) was analyzed.
Pimkena-myc, and Pim-myc for control, were expressed in the
anterior region of embryos during cycle 14. Immunolabeling at the stage of mitosis 14 indicate that Pimkena-myc is largely stable throughout mitosis, in contrast to Pim-myc, which is detected before but not after the metaphase-to-anaphase transition.
Progression beyond the metaphase-to-anaphase transition was monitored by the
labeling of DNA and Cyclin B, which is rapidly degraded when cells enter
anaphase. These results show that the KEN-box is required and that the variant
D-box (KKPLGNLDN), which is still present in Pimkena-myc, is not
sufficient for normal mitotic Pim degradation (Leismann, 2003).
Overexpression of Pimkena-myc results in mitotic defects.
Normal anaphase and telophase figures are not observed in
Pimkena-myc-positive cells that have progressed beyond the
metaphase-to-anaphase transition according to the absence of anti-Cyclin-B
labeling. Instead of pairs of well-separated telophase daughter nuclei, which
are readily observed in Cyclin-B-negative regions in the Pim-myc control
experiments, Cyclin-B-negative regions of Pimkena-myc-expressing
embryos display decondensing metaphase plates or chromatin bridges between
partially separated nuclei. These abnormalities caused by Pimkena-myc
are indistinguishable from those previously observed with
Pimdba-myc, which has been shown to inhibit sister chromatid
separation (Leismann, 2003).
Sister chromatid separation is also inhibited by strong overexpression of
wild-type Pim-myc. By contrast, at low physiological expression levels,
Pim-myc and, remarkably, also the stabilized versions Pimdba-myc
and Pimkena-myc, can promote sister chromatid separation in pim mutants (Leismann, 2003).
To analyze the function of Pim with mutations in both D- and KEN-box, additional transgenes (g>stop>pimkenadba
and g>stop>pimkenadba-myc) were constructed, allowing the expression
of Pimkenadba or Pimkenadba-myc under the control of the
normal pim regulatory region. To establish chromosomal insertions of
these potentially detrimental transgenes, a stop cassette flanked
by FLP recombinase target sites (>stop>) was inserted into the 5'
untranslated region. This stop cassette was eventually excised by transmitting
the established insertions via males expressing FLP recombinase specifically
in spermatocytes. Expression of the paternally recombined transgenes
(g>pimkenadba and g>pimkenadba-myc) started at the onset of zygotic expression during cycle 14 of embryogenesis. Expression of g>pimkenadba and
g>pimkenadba-myc in pim-mutant embryos did not
allow sister chromatid separation during mitosis 15.
Instead of normal mitotic figures, which were readily apparent in
pim+ sibling embryos, only
decondensing metaphase plates were observed during exit from mitosis. Thus,
pim-mutant embryos expressing g>pimkenadba and
g>pimkenadba-myc display the same phenotype as
pim mutants without transgene or with the non-recombined
g>stop>pimkenadba transgene (Leismann, 2003).
Control experiments with g>stop>pim transgenes encoding
wild-type Pim show that expression after stop-cassette removal is
sufficient to promote normal sister chromatid separation in pim
mutants. Moreover,
additional control experiments show that the recombined
g>pimkenadba-myc transgene is expressed as expected.
Anti-myc immunoblotting clearly show expression, and
co-immunoprecipitation experiments indicate that the Pimkenadba-myc protein
associates efficiently with Separase (SSE) and Three rows (THR), a
Drosophila protein known to form trimeric complexes with SSE and Pim. In
addition, although g>pimkenadba-myc expression in
pim+ sibling embryos has little effect during the initial
embryonic cell divisions (mitosis 14-16), it results in a severe mutant
phenotype in the CNS where additional cell divisions occur. Wild-type Pim
therefore appears to protect cells from the effects of
Pimkenadba-myc but only as long as the latter has not yet
accumulated to high levels (Leismann, 2003).
In summary, the experiments with g>pimkenadba and
g>pimkenadba-myc in pim mutants show that
sister chromatid separation does not occur in the presence of physiological
levels of the double mutants Pimkenadba and
Pimkenadba-myc, in contrast to the findings with the single mutants
Pimdba, Pimdba-myc and Pimkena-myc (Leismann, 2003).
It is concluded that mutations in either the D- or the KEN-box result in significant stabilization of Pim protein during mitosis. Neither the D- nor the KEN-box, therefore, are sufficient for normal degradation during the embryonic cell divisions in Drosophila. Similar observations have been described for
human securin. However, in contrast to Drosophila, mitotic degradation of human securin still occurs quite effectively when either only the D- or the KEN-box is intact. The D- and KEN-boxes of Drosophila
Pim, therefore, might function less independently than the corresponding
motifs in human securin. Eventually, the understanding of D- and KEN-box
function will require structural analyses of their interactions with
Fizzy/Cdc20 and Fizzy-related/Cdh1, which recruit proteins with these
degradation signals to the APC/C. Fizzy and Fizzy-related are clearly both involved in Pim degradation, at least indirectly, since Pim is stabilized in both
fizzy and fizzy-related mutants (Leismann, 2003).
Under the assumption that Pimkenadba and
Pimkenadba-myc are still capable of providing the positive Pim
function, these results with these stabilized mutants suggest that Pim must be
degraded during each and every mitosis to allow sister chromatid separation.
Although not detectable by confocal microscopy, the single mutants
Pimdba and Pimkena might not be completely stable in
mitosis. After low-level expression in pim-mutant embryos, residual
mitotic degradation of single-mutant proteins might free some separase
activity sufficient for sister chromatid separation. Similar results have been
observed with the fission yeast securin Cut2, which is completely stabilized
in a Xenopus extract destruction assay by mutations in either of the
two D-boxes, and yet, low-level expression of single-but not double-mutant
proteins is able to complement growth of cut2-ts strains at the
restrictive temperature. It is emphasized that even in wild-type cells, mitotic Pim
degradation appears to be far from complete, and it can be speculated that it
is the Pim protein of a special pool of separase complexes that is more
efficiently degraded, perhaps on kinetochores or during transport on spindles
towards kinetochores. At high expression levels of Pim with or without single
mutations, free excess of this securin might rapidly re-associate and inhibit
the activated separase, resulting in the observed block of sister chromatid
separation (Leismann, 2003).
These results also point to alternative pathways that might regulate separase
activity and sister chromatid separation independently of Pim degradation. As
in yeast, the success of mitosis in cells with reduced separase function is
dependent on Polo kinase in Drosophila embryos. Moreover, since
expression of mitotically stabilized Cyclin A versions result in a metaphase
delay without inhibiting Pim degradation, Cyclin A appears to contribute
independently of Pim to the inhibition of premature sister chromatid
separation. Even though it remains to be analyzed whether Polo kinase and
Cyclin A-Cdk1 act during Drosophila divisions as proposed for Polo
homologs and vertebrate Cyclin B-Cdk1, these results indicate that separase and sister chromatid separation are unlikely to be regulated exclusively by securin degradation (Leismann, 2003).
The spindle assembly checkpoint is essential to maintain genomic stability during cell division. The role of the putative Drosophila Mad2 homologue was examined in the spindle assembly checkpoint and mitotic progression. Depletion of Mad2 by RNAi from S2 cells shows that it is essential to prevent mitotic exit after spindle damage, demonstrating its conserved role. Mad2 has been shown to block mitotic exit by sequestering Cdc20 (Fizzy in Drosophila). Mad2-depleted cells also show accelerated transit through prometaphase and premature sister chromatid separation, fail to form metaphases, and exit mitosis soon after nuclear envelope breakdown with extensive chromatin bridges that result in severe aneuploidy. Interestingly, preventing Mad2-depleted cells from exiting mitosis by a checkpoint-independent arrest allows congression of normally condensed chromosomes. More importantly, a transient mitotic arrest is sufficient for Mad2-depleted cells to exit mitosis with normal patterns of chromosome segregation, suggesting that all the associated phenotypes result from a highly accelerated exit from mitosis. Surprisingly, if Mad2-depleted cells are blocked transiently in mitosis and then released into a media containing a microtubule poison, they arrest with high levels of kinetochore-associated BubR1, properly localized cohesin complex and fail to exit mitosis revealing normal spindle assembly checkpoint activity. This behavior is specific for Mad2 because BubR1-depleted cells fail to arrest in mitosis under these experimental conditions. Taken together these results strongly suggest that Mad2 is exclusively required to delay progression through early stages of prometaphase so that cells have time to fully engage the spindle assembly checkpoint, allowing a controlled metaphase-anaphase transition and normal patterns of chromosome segregation (Orr, 2007).
The spindle assembly checkpoint (SAC) is a carefully orchestrated quality control mechanism required to ensure accurate chromosome segregation during cell division. The SAC is responsible for preventing anaphase onset in cells whose chromosomes have not yet reached a stable bipolar attachment. SAC activation/maintenance is thought to be mediated by a signal continuously generated at unattached or improperly attached kinetochores during prometaphase. Studies in primary spermatocytes demonstrated that not only microtubule occupancy but also tension across kinetochore pairs is required in order to satisfy the SAC. The delayed metaphase-anaphase transition imposed by the SAC is ultimately controlled by the anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase that targets several mitotic substrates (including mitotic cyclins and securin) for destruction by the 26S proteasome to allow sister chromatid separation and mitotic exit (Orr, 2007).
The main components of the SAC molecular machinery were originally identified by genetic screens in budding yeast. MAD1-3 and BUB1 and BUB3 were shown to be required for a mitotic arrest in the presence of spindle damage. These genes have been found to be conserved from yeast to man with the exception of Mad3, which in higher eukaryotes is called Bub1-related kinase (BubR1; see Drosophila Bub1) because it is highly similar to Bub1 and unlike Mad3 contains a protein kinase domain within the C-terminal half (Orr, 2007).
Significant progress has been made in unraveling the molecular mechanism by which SAC proteins like Mad2 impose the mitotic arrest in response to inappropriately attached kinetochores. Mad2 is required for the establishment of a checkpoint-mediated arrest in response to spindle damage in Xenopus egg extracts and in mammalian cells in culture. Studies in Xenopus and human cells have also shown that Mad2 blocks mitotic exit by sequestering Cdc20, an APC/C activator. Mad2 localizes to kinetochores early in mitosis after binding Mad1 where it undergoes rapid turnover. This rapid turn over at kinetochores is thought to underlay the formation of Mad2-Cdc20 inhibitory complexes that signal abnormal microtubule-kinetochore attachment. Extensive work using fixed cells in a variety of organisms has supported this model by showing that indeed Mad2 accumulates strongly at kinetochores in the absence of microtubules. However, recent studies in Drosophila using a green fluorescent protein (GFP)-Mad2 transgene and time-lapse microscopy have suggested an alternative view. The data indicates that even after microtubule kinetochore attachment takes place, a low level of Mad2 continues to enter the kinetochore and is removed mostly along spindle microtubules in a poleward direction. These results suggest that perhaps the inhibitory signal provided by unattached kinetochores results not from the absence of kinetochore microtubule attachment per se but from the inability of Mad2 (and maybe other checkpoint proteins) to exit the kinetochore through microtubules, causing the accumulation of Mad2 at the kinetochore and the consequent formation of complexes that can now freely diffuse throughout the cytoplasm and inhibit the APC/C (Orr, 2007 and references therein).
Although the role of kinetochores in the generation of a soluble inhibitory signal that delays metaphase-anaphase transition is consistent with most published data, recent experiments have suggested that cytoplasmic Mad2 is also required for the proper timing of early prometaphase independently of kinetochores (Meraldi, 2004). Studies in human tissue culture cells show that when Mad2 is depleted in cells with disrupted kinetochores, sister chromatid separation follows very shortly after nuclear envelope breakdown (NEBD). However, if kinetochore-deficient cells now contain cytosolic Mad2, prometaphase is extended significantly, even though these cells still show a defective SAC response (Meraldi, 2004). These results suggest that Mad2 has a kinetochore-associated function in maintaining SAC activity and a kinetochore-independent function in timing mitotic progression (for discussion see Kops, 2005). Mad2 might therefore perform additional, SAC-unrelated functions during progression through mitosis. Interestingly, recent studies have also shown that besides their role in maintaining SAC activity, other checkpoint proteins also perform additional roles during mitosis progression. Bub3 has been shown to be required for the accumulation of cyclins during G2 and early mitosis. Furthermore, BubR1 and Bub1 were shown to be required for maintaining proper microtubule kinetochore interaction and chromosome congression (Orr, 2007).
Therefore, to gain insight into the primary role of Mad2 during mitosis, Drosophila S2 tissue culture cells were treated with double-stranded RNA against Mad2, and mitotic progression was analyzed in detail. Consistent with previous studies in other organisms, it was found that depletion of Mad2 causes loss of the SAC response in Drosophila S2 cells. Moreover, Mad2-depleted cells fail to reach metaphase, exit mitosis very soon after NEBD, and show highly abnormal chromosome segregation that is characterized by the formation of extensive chromatin bridges and severe aneuploidy. However, the results indicate that Mad2 is unlikely to have any specific role in either chromosome condensation or microtubule kinetochore interaction because a checkpoint-independent arrest in mitosis allows normal chromosome condensation and congression. Also, release from the mitotic arrest allows cells to exit mitosis without chromatin bridges and with chromatid segregation profiles that are indistinguishable from controls. More significantly, if Mad2-depleted cells are released from the mitotic arrest into media containing the microtubule-depolymerizing agent colchicine, cells arrest in mitosis with intact sister chromatid cohesion and strong kinetochore accumulation of SAC proteins, suggesting an active SAC response. Taken together these results suggest that Mad2 has a major role in delaying mitotic progression during early stages of prometaphase so that the SAC can be activated and chromosome segregation can be properly conducted (Orr, 2007).
This study found that all Mad2-associated phenotypes can be reverted and the checkpoint is effectively activated if cells are subjected to a transient mitotic arrest. Thus, contrary to current models which view Mad2 at the center of the inhibition of the APC/C by the SAC, it is hypothesize that Mad2 is required for proper timing of mitotic progression only during prometaphase, allowing cells to fully engage the SAC through kinetochore accumulation of other checkpoint proteins so that complete chromosome condensation and congression can be achieved before the controlled metaphase-to-anaphase transition takes place (Orr, 2007).
Checkpoint proteins have been shown to be essential for the fidelity of mitosis because they are responsible for sensing errors in microtubule kinetochore interaction. Loss of the Mad2 homologue causes inactivation of the SAC in Drosophila S2 cells. To find out whether Mad2 is required for the kinetochore localization of other checkpoint components, immunolocalization studies of other checkpoint proteins was performed. All three proteins tested (Bub1, Bub3, and BubR1) show strong accumulation at kinetochores, demonstrating that they do not require Mad2 for their localization and also that proper kinetochore localization of these checkpoint components does not per se prevent premature mitotic exit. Previous studies in Xenopus and HeLa cells were performed in the presence of microtubule poisons, and therefore it was unclear whether the absence of protein localization reflected a hierarchical relationship or the inability to analyze a large number of mitotic cells because of fast mitotic exit. Because individual depletion of Bub3 in Drosophila and analysis of the hypomorphic allele of BubR1 resulted in a nonfunctional SAC response, it is very likely that these proteins work through parallel signaling pathways that are mutually required at some stage to sustain checkpoint activity. Consistent with previous work, it seems probable that removing Mad2 may abrogate the spindle checkpoint not only because a sensor is being removed, but because the MCC, a multisubunit complex containing BubR1-Mad2-Bub3-Cdc20 (a far more potent APC/C inhibitor; Sudakin, 2001) cannot form in its absence (Orr, 2007).
Previous results have shown that inactivation of Mad2 by antibody microinjection during either prophase or prometaphase induced abnormal sister chromatid segregation in PtK1 cells (Gorbsky, 1998). This phenotypic analysis revealed that Drosophila S2 cells lacking Mad2 also display severe abnormalities during mitotic progression. Mad2-depleted cells fail to reach metaphase and exit mitosis with extensive chromatin bridges. The anaphase bridges formed by Mad2-depleted cells appear to be exclusively due to a premature exit from mitosis because extending the time spent in mitosis is enough to revert this phenotype. This is fully consistent with recent data suggesting that proper chromosome condensation and sister chromatid resolution is only fully completed during early prometaphase. Furthermore, the results suggest that in S2 cells, full chromosome condensation is achieved only late in prometaphase, after the minimal time cells spend in prometaphase when the SAC is inactivated. This is in full agreement with a previous analysis of mitotic progression in Bub3-depleted cells, where it was shown that in the absence of Bub3 the SAC is inactive but that cells do not exit mitosis with inappropriately condensed chromosome because of an extended period in prophase (Orr, 2007).
Several studies have shown that loss of SAC proteins causes premature sister chromatid separation (PSCS) and significant aneuploidy. This study found that loss of Mad2 causes premature degradation of cohesins during prometaphase resulting in high levels of PSCS. In addition, quantification of kinetochore segregation at anaphase shows that loss of Mad2 results in a high frequency of cells showing unequal kinetochore segregation. Furthermore, FACS analysis shows that the DNA content of the mitotic population changes significantly over time in the absence of Mad2. These results suggest that unlike in yeast where MAD2 is not essential for chromosome segregation, Drosophila Mad2 is required to maintain the long viability of cells (Orr, 2007).
Early studies on the role of Mad2 in the SAC response using cultured animal cells revealed premature anaphase onset (Gorbsky, 1998). More recently, it was found that the mitotic clock of unsynchronized rat basophilic leukemia cells has a marked precision in which ~80% of cells complete mitosis in 32 ± 6 min and that Mad2 inactivation in these cells consistently shortened mitosis. Furthermore, depletion of Mad2 by RNAi showed that HeLa cells exit mitosis prematurely (Meraldi, 2004). The current results are fully consistent with this data because depletion of Mad2 in S2 cells causes a threefold reduction in the time from NEBD to anaphase onset. Interestingly, these same studies proposed a role of Mad2 in timing mitotic progression that is more complicated that previously expected. It was shown in HeLa cells that inactivation of kinetochore-bound Mad2 disrupts the SAC without significantly affecting the timing of mitotic progression. However, when the cytosolic pool of Mad2 present in these cells is depleted, then both the SAC response is abnormal and the timing of NEBD to anaphase onset is severely reduced, suggesting that Mad2 is also required to time mitotic progression in a kinetochore-independent manner. This contrasts with current models which propose that Mad2 plays an essential role in SAC activation and maintenance by providing a kinetochore-based signal that inhibits the APC/C. The observations with Drosophila suggest a much more subtle role for Mad2 in ensuring a SAC response. Surprisingly, it was found that after a transient mitotic arrest, Mad2-depleted cells were able to respond to spindle damage and arrest in mitosis with cohesin still located at the centromere of chromosomes and high kinetochore levels of BubR1, suggesting that the SAC is fully functional. Thus, providing time in a checkpoint-independent and transient manner appears to be sufficient for Mad2-depleted cells to either reactivate or maintain SAC activity and respond correctly to microtubule depolymerization. Given that this sustained SAC activity cannot be observed after depletion of other SAC proteins such as BubR1, it is hypothesized that the APC/C inhibitory signal provided by Mad2 is specifically required during early stages of prometaphase to ensure maintenance of SAC activity. Subsequently, SAC activity could be ensured by other checkpoint proteins such as BubR1 that could then accumulate strongly at kinetochores. These observations are in full accordance with previous biochemical studies that identified at the G2-M transition the MCC, a multisubunit complex containing BubR1-Mad2-Bub3-Cdc20. The MCC was shown to be the most powerful APC/C inhibitor). Interestingly, formation of the MCC does not require unattached kinetochores given that it is present well before the NEBD. Taken together, these observations have suggested a 'two-step' model for the activation and maintenance of SAC activity. This model proposes a first step involving the formation of the MCC as cells reach the G2/M transition, allowing cyclin accumulation and mitotic entry. Subsequently, in a second step after NEBD, SAC proteins can bind unattached kinetochores and produce additional inhibitory complexes that sustain SAC activity until all kinetochore pairs are properly attached and congression is achieved. Subsequent studies both in yeast and Drosophila provide strong support for this model. The results reported in this study, provide a further refinement of this model in that the second step can be separated into two events: one at NEBD when cytoplasmic Mad2 is required to extend prometaphase and provide enough time so that in a second event, SAC proteins such as BubR1 and Bub3 can fully engage checkpoint activity. Further studies on the role of Mad2 and other SAC proteins in the inhibitory activity of the MCC before and during early stages of mitosis will be required to unravel how the different levels of regulation are organized. Nevertheless, the current observations provide new insights into how the signals provided by different SAC proteins might contribute to a fully integrated checkpoint response (Orr, 2007).
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