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Address correspondence to Marko J. Kallio, University of Oklahoma Health Sciences Center, 975 N.E. 10th St., Biomedical Research Center, Rm. 266, Oklahoma City, OK 73104. Tel.: (405) 271-3404. Fax: (405) 271-7158. E-mail: marko-kallio{at}ouhsc.edu
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Abstract |
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Key Words: ubiquitin; cell cycle; mitosis; spindle checkpoint; FRAP
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Introduction |
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Many of the mitotic checkpoint proteins and regulators of the APC/C, including Cdc20, Bub1, Mad3/BubR1, Bub3, Mad1, and Mad2, associate with kinetochores and centrosomes, often at specific times in the cell cycle (Chen et al., 1996; Li et al., 1997; Kallio et al., 1998; Taylor et al., 1998). These data have led to increasingly detailed models that unattached kinetochores foster the assembly of inhibitory proteins onto the APC/C and/or Cdc20, thus restraining APC/CCdc20 from targeting securins (Kallio et al., 1998; Chen et al., 1998; Sudakin et al., 2001, Sironi et al., 2002). Howell et al. (2000) used FRAP measurements to show that Mad2 is a transient component of the spindle poles and kinetochores with a turnover halftime of 2428 s. Furthermore, the authors observed that Mad2 is transported between the kinetochores and spindle poles, and this transfer depends on intact spindle microtubules. Here, we quantitatively examine the subcellular localization and dynamics of full length and mutant Cdc20 in living cells using high-resolution fluorescence microscopy and FRAP.
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Results and discussion |
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In interphase cells, Cdc20GFP was concentrated in the nucleus but excluded from the nucleolus. Substantial amounts were also cytoplasmic with a strong concentration at the centrosome. Association with the centrosome persisted throughout the entire cell cycle (Fig. 2 A). In early mitosis, Cdc20GFP accumulated at the kinetochores, at the spindle poles, and along kinetochore microtubules (Fig. 2 A). During prometaphase, kinetochores near the spindle poles were more intense compared with kinetochores near the spindle equator (Fig. 2 A). We did not detect differences in the fluorescence intensities of the leading and trailing kinetochores of prometaphase chromosomes during their migration to the metaphase plate (Fig. S2 and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200201135/DC1). The Cdc20GFP colocalized with the Crest kinetochore marker in M phase (Fig. 2 B). When late prometaphase or metaphase cells were treated with the spindle-disrupting agent nocodazole, the intensity of all kinetochore signals increased to the maximal level (Fig. 2 C), suggesting that the amount of Cdc20 associated with kinetochores is partially and inversely dependent on microtubule occupancy. In nondrug-treated cells, the peak intensity of fluorescence at kinetochores was reached 13 min after nuclear envelope breakdown (NEB; Fig. 2 D), and Cdc20GFP remained associated with the kinetochores from late prophase to late telophase (Fig. 2, B and D). After onset of anaphase, the kinetochore Cdc20GFP signal diminished rapidly and became indistinguishable from the background as the cells exited mitosis (Fig. 2, B and D). The intensity of diffuse cytoplasmic Cdc20GFP also decreased substantially during anaphase and telophase, consistent with the findings that Cdc20 is proteolyzed as cells progress to G1 (Weinstein, 1997). In contrast to the loss of Cdc20 at kinetochores and in the cytoplasm, the intensity of Cdc20GFP at the spindle poles remained high throughout mitosis and after the cells had exited mitosis (Fig. 2 B).
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Distinct domains of Cdc20 mediate localization to kinetochores and centrosomes
In cell extracts, the Cdc20 protein is found in a variety of complexes with spindle checkpoint proteins and with the APC/C. It is unclear if these different populations localize to different subcellular sites. The Cdc20 protein consists of an NH2-terminal region (amino acids 1167) that contains putative destruction boxes and domains involved in binding to the APC/C and to the checkpoint protein Mad2 (Zhang and Lees, 2001). After that are seven WD-40 repeats that form a ß-propeller structure and may be involved in binding to substrates (Hilioti et al., 2001). We found that the NH2-terminal region containing the Mad2-binding domain is required for localization to centrosomes, whereas the WD-40 repeats are necessary for localization to kinetochores and spindle microtubules (Fig. 3). A short deletion of the NH2 terminus (1110), which retains the Mad2-binding domain, had no obvious effect on localization in interphase and mitosis. In contrast, a longer NH2-terminal deletion (
1167), which removes the Mad2/APC/C-binding domain eliminated binding to interphase and mitotic centrosomes but preserved localization at mitotic kinetochores (Fig. 3). A construct containing only the first 167 amino acids and lacking all WD-40 repeats (
168499) showed centrosome and spindle pole localization but did not bind kinetochores. Deletion of the entire NH2 terminus, including the first WD-40 repeat, eliminated binding to both kinetochores and centrosomes. These findings indicate that association of Cdc20 with kinetochores requires the WD-40 repeats. In contrast, Cdc20 localization to centrosomes requires the Mad2/APC/C-interacting domain and thus may be due to interaction with Mad2 or APC/C concentrated there.
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First we determined that photobleaching of Cdc20GFP did not induce defects in chromosome movements or cell cycle progression (unpublished data). The recovery of Cdc20GFP was very rapid at kinetochores and centrosomes with average half-times of 5.1 ± 3.6 s (n = 11) and 4.7 ± 3.6 s (n = 7), respectively (Fig. 4 A; Table I; Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200201135/DC1). At kinetochores, recovery rates were similar from prometaphase to metaphase. In anaphase cells (n = 5), recovery of kinetochores was somewhat faster (t1/2 = 3.3 ± 1.4 s). Recovery of Cdc20GFP at interphase centrosomes was also rapid (t1/2 = 6.9 ± 3.3 s, n = 7; Fig. 4 B; Table I; Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200201135/DC1). The average turnover of Cdc20GFP in the cytoplasm was significantly faster (P < 0.05, 2.7 ± 1.0 s, n = 8; Table I) than that of kinetochores and centrosomes. The total extent of recovery was from 80 to 94% at centrosomes and kinetochores (Table I), suggesting that most Cdc20 associated with these structures exchanges rapidly. The treatment of cells with microtubule drugs, nocodazole or taxol, did not significantly affect recovery at kinetochores or centrosomes (Table I; Fig. 4 C; Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200201135/DC1).
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The role of Cdc20 protein dynamics in the spindle checkpoint
Like Mad2, Cdc20 associates with mitotic kinetochores and spindle poles transiently. However, Cdc20 exchange occurs approximately four times as fast as the published rates for Mad2. Kinetochore-associated Cdc20 exchanges rapidly with the cytoplasmic pool, independent of spindle microtubules. Thus, the trafficking of Cdc20 is distinct from that of Mad2 and may relate to how the checkpoint signal is broadcast throughout the cell from unattached kinetochores.
Some years ago, we proposed a model whereby transient association of Cdc20 with unattached kinetochores served to maintain inhibition of the APC/C toward targets whose ubiquitylation and degradation are required for anaphase onset (Kallio et al., 1998). The work reported here and studies from other laboratories support this basic conceptual framework. We now propose refinements to this model (Fig. 5). Kinetochores lacking microtubule attachment or tension contain high concentrations of spindle checkpoint proteins (e.g., Mad1, Mad2, Bub1, BubR1, and Bub3). We suggest that Cdc20 circulates rapidly at all kinetochores, binding via its WD-40 repeats, possibly through interaction with other WD-40 proteins, such as Bub3 (Fraschini et al., 2001). Binding to the kinetochore by the WD-40 domain leaves free the Cdc20 NH2-terminal domain that contains binding sites for inhibitory checkpoint proteins Mad2 and BubR1. While at the kinetochore, Cdc20 accepts the inhibitory checkpoint proteins, and binding of anaphase inhibitors (securins) to the APC/C is blocked. In contrast, the targeting of early M phase substrates of APC/CCdc20, such as cyclin A, is not inhibited, possibly because the early substrates interact with COOH-terminal regions of Cdc20. Other kinetochore-associated checkpoint proteins (e.g., Mad1, Bub1, Mps, and CenpE) facilitate transfer of the checkpoint inhibitor proteins to Cdc20 (for Mad1 see Sironi et al., 2002). Kinetochore-bound kinases, phosphatases, or other enzymes might also modify Cdc20 or its associated proteins to inhibit APC/CCdc20 targeting of securins. Microtubule attachment or tension at kinetochores induces the dynein-mediated transport of certain checkpoint proteins from the kinetochore toward the poles along spindle fiber microtubules (Howell et al., 2001). The very high exchange rates for Cdc20 (more rapid than those of the inhibitor Mad2) may allow Cdc20 to continuously sample all kinetochores within the cell and thus maintain cell-wide inhibition of APC/CCdc20, even in cells with only one or a few unattached kinetochores. Finally, during metaphase and anaphase, the continued concentration of Cdc20 at kinetochores, centrosomes, and spindle microtubules may additionally serve to maintain high levels of the active APC/CCdc20 near substrates concentrated within the spindle.
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Materials and methods |
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Transfection and expression of Cdc20GFP chimeras
Cell culture was performed as previously described (Kallio et al., 1998). LLC-PK and HeLa cells were transiently transfected with plasmids encoding full-length or mutated Cdc20GFP by LipofectAMINE PLUS reagent (Life Technologies) or Fugene 6 reagent (Roche Diagnostics Corp.) according to the manufacturer's recommendations. 2448 h after transfection, the cells were fixed and processed for microscopic analysis or subjected to biochemical studies or live cell video analysis.
Immunoprecipitation and immunoblotting
HeLa cells expressing full-length or mutated Cdc20GFP chimeras were treated with nocodazole or taxol for 812 h. Mitotic cells were collected. Cell extracts and supernatants were prepared and used for immunoprecipitation using anti-GFP antibodies (2.5 µg; Molecular Probes and Abcam Ltd.) or for Western blotting experiments as described previously (Kallio et al., 1998).
Fixation and immunofluorescence
LLC-PK cells expressing Cdc20GFP were fixed with 3% formaldehyde in PHEM (60 mM Pipes, 25 mM Hepes, pH 6.9, 10 mM EDTA, 4 mM MgCl2) for 15 min. After washes with MBST (10 mM MOPS, 150 mM NaCl, pH 7.3, 0.05% Tween 20), DNA was stained with DAPI and the cells on coverslips were mounted with Vectashield (Vector Laboratories). For immunofluorescence, LLC-PK and HeLa cells on coverslips were simultaneously fixed and extracted for 15 min in 0.25% CHAPS in PHEM containing 2% formaldehyde and 100 nM microcystin LR. Coverslips were labeled with antibodies as previously described (Kallio et al., 1998) and analyzed with a ZEISS Axioplan IIi microscope equipped with a Hamamatsu Orca II camera and Metamorph Imaging system (Universal Imaging Corp.).
Analysis of living cells
For live cell observation, a planapochromat 60x (N.A. 1.4) objective (Nikon) was used with a SenSys CCD camera (Photometrics Ltd.) connected to a Nikon Diaphot microscope and imaged with Metamorph software. The average signal intensity of kinetochore-bound Cdc20GFP at different mitotic phases was analyzed from 10 live cell sequences by measuring the integrated fluorescence intensity minus the background from the 10 brightest kinetochores per each cell and time point. For detailed methods on live cell analysis and FRAP see the supplemental Materials and methods (available at http://www.jcb.org/cgi/content/full/jcb.200201135/DC1).
Online supplemental material
Supplemental materials and methods, supplemental figures (Figs. S1 and S2), and Quicktime movies of the time-lapse fluorescence and phase contrast images accompanying Fig. S2 (Video 1) and Fig. 4 (Videos 24) are available at http://www.jcb.org/cgi/content/full/jcb.200201135/DC1.
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Footnotes |
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Victoria A. Beardmore's present address is Medical Research Council Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland.
* Abbreviations used in this paper: APC/C, anaphase-promoting complex/cyclosome; NEB, nuclear envelope breakdown.
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Acknowledgments |
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This study was supported by grants from the Helsingin Sanomat Foundation (M.J. Kallio) and from the National Institute of General Medical Sciences (G.J. Gorbsky).
Submitted: 30 January 2002
Revised: 10 July 2002
Accepted: 16 July 2002
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