Article |
Address correspondence to Jordan W. Raff, Department of Genetics, Wellcome/Cancer Research UK Institute, Cambridge CB2 1QR, UK. Tel.: 44-122-333-4114. Fax: 44-122-333-4089. E-mail: j.raff{at}welc.cam.ac.uk
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Abstract |
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Key Words: Cdc20; Cdh1; anaphase-promoting complex; cyclin B; mitosis
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Introduction |
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As well as being temporally regulated, the destruction of cyclin B also appears to be spatially regulated. For example, in fused vertebrate cells that contain two spindles, the spindles can exit mitosis independently of each other, suggesting that cyclin B is not being degraded everywhere in the cell at the same time (Rieder et al., 1997). In syncytial Drosophila embryos, the destruction of cyclin B is essential for the exit from mitosis (Su et al., 1998), but Western blotting experiments revealed that cyclin B is only partially degraded at the end of mitosis (Edgar et al., 1994). This suggests that only a specific sub-population of cyclin B is degraded at the end of mitosis in these embryos. More recently, the destruction of cyclin Bgreen fluorescent protein (GFP) fusion proteins has directly been observed to be spatially regulated in Drosophila (Huang and Raff, 1999), human (Clute and Pines, 1999), and yeast (Yanagida et al., 1999; Decottignies et al., 2001) cells.
In Drosophila-cellularized embryos, the destruction of cyclin BGFP appears to initiate at centrosomes and spreads to the equator of the spindle. The degradation of the cytoplasmic cyclin B is then initiated slightly later in mitosis, and continues into the next cell cycle. Unfortunately, the question of how cyclin B is only partially degraded in syncytial embryos could not be directly addressed, as the destruction of cyclin BGFP could not be followed in syncytial embryos. This is probably because GFP has to undergo an intramolecular rearrangement before it becomes fluorescent, and, in flies, this takes 1 h (Hazelrigg et al., 1998). Thus, in syncytial embryos (where cyclin B is continually synthesized and then partially degraded approximately every 10 min [Edgar et al., 1994]), many cyclin BGFP molecules may not survive long enough to become fluorescent, so the signal is very weak. Nevertheless, when the behavior of the endogenous cyclin B was analyzed in fixed embryos, cyclin B was clearly degraded on the spindle, but it did not appear to be degraded in the cytoplasm (Huang and Raff, 1999). This suggests that in Drosophila there are two phases of cyclin B destruction that are temporally and spatially separable: the first phase destroys the spindle-associated cyclin B, whereas the second phase destroys the cytoplasmic cyclin B. In syncytial embryos, only the first phase of destruction seems to be initiated.
An attractive explanation for this spatially regulated destruction of cyclin B is that the APC/C is globally activated to degrade cyclin B, but is itself spatially restricted. Thus, the APC/C might initially be concentrated at centrosomes, move into the spindle, and finally be released into the cytoplasm. In support of this possibility, two core APC/C components, Cdc16 and Cdc27, have previously been shown to be concentrated on centrosomes and spindles in mammalian cells (Tugendreich et al., 1995). However, we have shown that only a small fraction of the APC/C associates with spindles in Drosophila embryos (Huang and Raff, 1999, 2002), suggesting that the APC/C cannot be globally activated to degrade cyclin B.
Two proteins, Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1, bind to the APC/C and are thought to target the APC/C to its various substrates (Pfleger et al., 2001). Experiments in yeasts and flies have led to the suggestion that Cdc20APC/C complexes target proteins for destruction early in the exit from mitosis, whereas Cdh1APC/C complexes target proteins for destruction later in the exit from mitosis and into G1 (Sigrist and Lehner, 1997; Visintin et al., 1997; Kramer et al., 1998, 2000). Therefore, we proposed that the subpopulation of the APC/C that associates with Fzy/Cdc20 might be responsible for the first phase of cyclin B destruction (that is restricted to the spindle), whereas a different subpopulation of the APC/C that associates with Fzr/Cdh1 might be responsible for the second phase of cyclin B destruction (that occurs in the cytoplasm) (Huang and Raff, 1999, 2002). In this paper we set out to test the respective roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time.
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Results |
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Fzy/Cdc20 and Fzr/Cdh1 interact with microtubules, but only Fzy/Cdc20 requires microtubules for its centrosomal localization during mitosis
As both GFP-Fzy and GFP-Fzr associate with centrosomes and spindles in mitosis, we tested whether the endogenous proteins physically interacted with microtubules. We added taxol to 024-h embryo extracts to polymerize the microtubules, and then pelleted the microtubules (together with any associated proteins) through a sucrose cushion. In control extracts, where no taxol was added, both Fzy/Cdc20 and Fzr/Cdh1 remained in the soluble fraction (Fig. 1 B, lane 1). In the presence of taxol, a significant fraction of both proteins (5070%) was detectable in the microtubule pellet (Fig. 1 B, lane 4). Thus, both proteins can interact with microtubules, although it is not clear whether this interaction is direct.
As both proteins interacted with microtubules, we tested whether either protein required microtubules for their localization at kinetochores and/or centrosomes. We injected living GFP-Fzy or GFP-Fzr embryos with colcemid during interphase of nuclear cycle 1012, and observed the embryos on the confocal microscope. GFP-Fzy remained concentrated at interphase centrosomes after colcemid injection, although the localization was more diffuse than normal (Fig. 3 A, 0:0). As the embryos entered mitosis, GFP-Fzy levels decreased at centrosomes and increased at kinetochores (Fig. 3 A, 1:002:00). Injected embryos then appeared to terminally arrest in this mitotic state, with elevated levels of GFP-Fzy on the kinetochores (Fig. 3 A, 6:40). In some of these embryos we inactivated the colcemid with a short pulse of UV light (at 7:00 min for the embryo shown in Fig. 3 A). GFP-Fzy levels increased on the centrosomes and microtubules, and decreased on the kinetochores as the chromosomes became aligned on the reforming spindles (Fig. 3 A, 8:4012:00). Thus, the localization of Fzy/Cdc20 to centrosomes during mitosis appears to be microtubule dependent, whereas its localization at kinetochores appears to be microtubule independent.
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Fzy/Cdc20 catalyzes the destruction of the spindle-associated cyclin B in syncytial embryos
In fixed syncytial embryos, cyclin B appears to be degraded only on the spindle (Huang and Raff, 1999). In this previous study, the destruction of cyclin BGFP in living syncytial embryos could not be followed directly (Introduction). However, by expressing four copies of the cyclin BGFP transgene in cyclin Bnull mutant embryos, we have now followed the behavior of cyclin BGFP in living syncytial embryos (Fig. 4). In these embryos, the spindle-associated cyclin B is degraded at the end of mitosis, but the levels of cytoplasmic cyclin B remain unchanged, directly confirming that only the spindle-associated cyclin B is degraded in syncytial embryos.
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To confirm that cyclin B in syncytial embryos was only being targeted for destruction by Fzy/Cdc20 (and that there was no contribution from a small pool of Fzr/Cdh1 that was undetectable by Western blotting), we tested whether a form of cyclin B that can only be degraded by Fzr/Cdh1 could be degraded in syncytial embryos. We noticed that Drosophila cyclin B has a KEN box as well as a destruction box (D-box). It has previously shown that Fzy/Cdc20 requires that its targets contain a D-box, whereas Fzr/Cdh1 can also recognize other, less well-defined sequences, such as a KEN box (Pfleger and Kirschner, 2000). Therefore, we reasoned that cyclin B molecules containing a mutated D-box would no longer be targeted for degradation by Fzy/Cdc20, but would be targeted for degradation by Fzr/Cdh1 (see below). We previously constructed transgenic flies containing an inducible form of cyclin BGFP that is mutated at all three of the most conserved D-box residues (called cyclin B triple-point mutant [CBTPM]GFP) (Wakefield et al., 2000). When CBTPMGFP was expressed in syncytial embryos, the vast majority of embryos failed to develop significantly (Fig. 5 A) and arrested in mitosis during the early syncytial divisions with their spindles in an anaphase-like state (as shown previously [Wakefield et al., 2000]). A small number of CBTPMGFP-expressing embryos did develop (presumably because CBTPMGFP was only expressed at relatively low levels in these embryos; Fig. 5 B). When we observed these developing embryos by TLCM, we found that CBTPMGFP was not detectably degraded, and that it remained concentrated on centrosomes and spindles throughout the exit from mitosis (Fig. 6 A, compare with wild-type [WT] cyclin BGFP in Fig. 4). Thus, in syncytial embryos, CBTPMGFP cannot be degraded, strongly suggesting that Fzy/Cdc20 alone is normally responsible for degrading cyclin B in these embryos.
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Our finding that CBTPMGFP can be degraded by Fzr/Cdh1, but not by Fzy/Cdc20, allowed us to test how cyclin B might normally be degraded by Fzr/Cdh1 alone. We expressed CBTPMGFP in otherwise wild-type cellularized embryos (that contain both Fzy/Cdc20 and Fzr/Cdh1). In cellularized embryos the nuclei no longer enter mitosis in synchrony (as occurs during the syncytial divisions). Instead, small domains of cells enter and exit mitosis at approximately the same time (Foe, 1989). We found that expressing CBTPMGFP in cellularized embryos did not arrest cells in mitosis, and the protein was completely degraded at the end of mitosis (Fig. 6 B, arrows highlight the two domains of cells that have exited mitosis and have low levels of CBTPMGFP fluorescence). However, on closer inspection it was clear the CBTPMGFP was not degraded with normal kinetics. In cellularized embryos expressing WT cyclin BGFP, the fusion protein initially started to disappear from the spindle, whereas protein levels in the cytoplasm remained relatively constant (Fig. 7 A). As the level of spindle fluorescence approached that of the cytoplasm, the cell entered anaphase and cyclin BGFP started to disappear throughout the cell. In cellularized embryos expressing CBTPMGFP (Fig. 7 B), the kinetics of destruction were more variable. In most cells, the rate of disappearance of CBTPMGFP from the spindle was much slower than normal. As a result, when cells entered anaphase, CBTPMGFP was almost always still detectable on the spindle, and the spindle remnants (the midbody, or central spindle) always contained high levels of CBTPMGFP long after mitosis had finished (Fig. 6 B, arrowheads). Thus, Fzr/Cdh1 appears capable of catalyzing the destruction of CBTPMGFP throughout the cell, although with slowed kinetics.
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Discussion |
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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 (Basto et al., 2000; Chan et al., 2000; Howell et al., 2001; Scaerou et al., 2001; Wojcik et al., 2001). We show that 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.
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, as we had previously proposed that Fzr/Cdh1 catalyzed the second phase of cyclin B destruction that occurs in the cytoplasm (Huang and Raff, 1999). However, our FRAP analysis suggests 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.
The respective roles of Fzy/Cdc20 and Fzr/Cdh1 in degrading cyclin B
We find that Fzy/Cdc20 protein is abundant in syncytial embryos, whereas Fzr/Cdh1 protein is virtually undetectable. Moreover, a D-boxmutated form of cyclin B (CBTPMGFP), which cannot be targeted for destruction by Fzy/Cdc20, is not degraded on spindles in syncytial embryos. CBTPMGFP can be targeted for destruction by Fzr/Cdh1, and, in cellularized embryos, where Fzr/Cdh1 is normally present, CBTPMGFP 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.
These results suggest a model of how the destruction of Drosophila cyclin B is regulated in space and time (Fig. 9). Early in mitosis (Fig. 9 A), inhibitory checkpoint protein/Fzy (Cdc20) complexes form at unattached kinetochores. We propose 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 (Fig. 9 B), 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 we propose 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/Cdh1APC/C complexes (Kramer et al., 2000). 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 (Fig. 9 C).
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We stress 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 (Dawson et al., 1995), but it is not concentrated on spindles (Pines and Hunter, 1991). It seems unlikely that Fzy/Cdc20 also catalyzes the destruction of cyclin A only on the spindle. Therefore, we speculate that there must be separate pools of Fzy/Cdc20 that are responsible for degrading cyclin A and B. An attractive aspect of our 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 (Whitfield et al., 1990; den Elzen and Pines, 2001; Geley et al., 2001).
A general mechanism for regulating the destruction of cyclin B?
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 (Rieder et al., 1997). 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 our model. We would predict 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 (Yeong et al., 2000), although the spatial organization of this destruction has not been investigated.
However, our 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 (Kramer et al., 2000). 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, we note that early Xenopus extracts do not have a functional spindle checkpoint (Minshull et al., 1994). As discussed above, 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.
The role of the centrosome in regulating the exit from mitosis
Our finding that Fzy/Cdc20 and Fzr/Cdh1 are concentrated at centrosomes highlights the potential importance of this organelle in regulating the exit from mitosis (Rieder et al., 2001). We speculate that the concentration of these proteins at centrosomes might serve two purposes. First, 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. Second, in budding yeast there is a second, Bub2-dependent checkpoint that monitors the positioning of the spindle between the mother and daughter cell (Bardin et al., 2000; Daum et al., 2000; Pereira et al., 2000). 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 (Gardner and Burke, 2000). 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.
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Materials and methods |
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Construction of GFP-Fzy and GFP-Fzrexpressing lines
Full-length cDNAs for Fzy and Fzr were obtained from Research Genetics and from Christian Lehner (University of Bayreuth, Bayreuth, Germany), respectively. The coding sequences were modified by PCR so that mGFP6 (Schuldt et al., 1998) could be cloned, in frame, onto the NH2 terminus of both proteins. The GFP fusion proteins were then subcloned into the pWRpUBq Drosophila transformation vector, putting their expression under the control of the polyubiquitin promoter that is expressed at relatively high levels throughout Drosophila development (Lee et al., 1988). Full details of these cloning procedures are available upon request. These plasmids were then used to generate stable fly lines using standard P-elementmediated transformation (Roberts, 1986).
Transgenic lines expressing CBTPMGFP
The transgenic lines expressing CBTPMGFP under the control of the UASp promoter (Rorth, 1998) have been described previously (the conserved D-box sequence RXXLXXXXN has been mutated to GXXAXXXXA, Wakefield et al., 2000). The expression of this protein in cellularized embryos was achieved by crossing males carrying the UAS-CBTPMGFP transgene to females carrying a transgenic Gal4/VP16 fusion protein whose expression was under the control of the 67C maternal -tubulin promoter (that drives high levels of expression during oogenesis, Micklem et al., 1997). In this way, the early embryo has high levels of the Gal4/VP16 fusion protein, but it only drives significant transcription of the CBTPMGFP fusion protein (that comes into the embryo on the male chromosomes) at cellularization, when bulk transcription is initiated. To observe the effect of expressing CBTPMGFP in syncytial embryos, embryos were collected from the females produced in this first cross. These embryos are derived from females carrying both the UAS-CBTPMGFP and Gal4/VP16 transgenes, so expression of CBTPMGFP is driven throughout oogenesis and early embryos express significant amounts of this protein.
Microtubule spindown experiments
Microtubule spindown experiments were performed with extracts made from 024-h embryos as described previously (Raff et al., 1993).
Observation of living embryos by TLCM
Living embryos expressing GFP-Fzy, GFP-Fzr, cyclin BGFP, or CBTPMGFP were observed using a Bio-Rad Radiance confocal system mounted on a Nikon microscope as described previously (Huang and Raff, 1999). Embryos that were to be injected with colcemid were observed using a Bio-Rad 1024 confocal system mounted on a Nikon inverted microscope. The embryos were observed on the confocal system until they entered interphase of nuclear cycle 1012. They were then injected with colcemid (100 mM, dissolved in water), using our own manual injection system mounted on the inverted microscope. The embryos were then followed again on the confocal system. In some embryos, the colcemid was subsequently inactivated by opening the shutter on the microscopes UV lamp and exposing the embryo to either a single pulse of UV light (for 30 s) or to several pulses of UV light (20 s pulses at 2-min intervals). To make movies of these embryos, the image stacks were imported into Adobe® Photoshop® and adjusted to use the full range of pixel intensities. Images were made into movies using Adobe® Premier® .
If the images were to be used to quantitate fluorescence levels, only the raw images were used. Sequential images of embryos were imported into NIH Image, and the average pixel intensity in a manually defined area either on the spindle or in the cytoplasm was calculated. In addition, images were taken of several non-GFPexpressing embryos using the same settings on the confocal microscope. The average pixel intensity from these embryos was used to calculate a zero pixel intensity. For example, in Fig. 4, the pixel intensity on the spindle falls during mitosis until it reaches the same pixel intensity of the cytoplasm. However, the pixel intensity in the cytoplasm is not zero, indicating that this cytoplasmic fluorescence is likely due to cyclin BGFP and not to a background fluorescence in the embryo.
FRAP analysis
Living embryos expressing either GFP-Fzy, GFP-Fzr, GFP--tubulin, or D-TACC-GFP were observed on the Bio-Rad Radiance confocal system. Using our own Macro (written by Alex Sossick) an embryo was imaged, and a small area of the embryo was then selected manually on the computer screen. This area was then photobleached by exposing only this area of the embryo to several passes of the scanning laser on 100% power. Photobleaching was monitored visually, and, when complete, the whole embryo was then imaged again on normal laser power (usually 15% of full power). The images acquired during the recovery period were imported into NIH Image. To calculate the half life of the fusion proteins at the centrosome, a line was defined manually that passed through the area of interest (i.e., through the middle of two centrosomes, one in the nonbleached area and one in the bleached area) and the pixel intensity along this line was calculated. The time at which the pixel intensity on the bleached centrosome reached half of the pixel intensity on the nonbleached centrosome could then be estimated.
Western blotting
SDS-PAGE and Western blotting were performed as described previously (Towbin et al., 1979). Blots were probed with affinity-purified antibodies at 12 µg/ml, or with DM1 anti-tubulin mouse ascites fluid (Sigma-Aldrich) or with JLA20 mouse monoclonal anti-actin antibody (both at 1/1,000 dilution; Developmental Studies Hybridoma Bank, Iowa City, IA), and then with the appropriate peroxidase-conjugated secondary antibody at 1/2,000 dilution (Amersham Pharmacia Biotech). Blots were developed using the SuperSignal enhanced chemiluminescence kit (Pierce Chemical Co.) according to the manufacturer's instructions.
Supplemental Material
Movies of the embryos shown in Figs. 2, 3, 4, and 6 are available online at http://www.jcb.org/cgi/content/full/jcb.200203035/DC1. Video 1 shows GFP-Fzy in a WT embryo (Fig. 2 A). Video 2 shows GFP-Fzr in a WT embryo (Fig. 2 B). Video 3 shows GFP-Fzy in a colcemid-injected embryo (Fig. 3 A). Video 4 shows GFP-Fzr in a colcemid-injected embryo (Fig. 3 B). Video 5 shows cyclin BGFP in a WT syncytial embryo (Fig. 4). Video 6 shows CBTPM-GFP in a WT syncytial embryo (Fig. 6 A). Video 7 shows CBTPM-GFP in a WT cellularized embryo. Note that due to memory limitations, only parts of some of the movies are included in these files.
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Footnotes |
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Kim Jeffers's present address is Virology Research and Development, Commonwealth Serum Laboratories, 45 Poplar Rd., Parkville 3052, Australia.
* Abbreviations used in this paper: aa, amino acid(s); APC/C, anaphase-promoting complex/cyclosome; CBTPM, cyclin B triple-point mutant; cdk, cyclin-dependent protein kinase; D-box, destruction box; Fzy, Fizzy; Fzr, Fizzy-related; GFP, green fluorescent protein; SPB, spindle pole body; TLCM, time-lapse confocal microscopy; WT, wild-type.
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Acknowledgments |
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This work was supported by a Wellcome Trust Senior Research Fellowship in Basic Biomedical Sciences (J.W. Raff).
Submitted: 7 March 2002
Revised: 17 May 2002
Accepted: 17 May 2002
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References |
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