Article |
Address correspondence to Jonathon Pines, Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Rd., Cambridge CB2 1QR, England, UK. Tel.: (44) 1223-334-093. Fax: (44) 1223-334-089. email: j.pines{at}welc.cam.ac.uk or Catherine Lindon, Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Rd., Cambridge CB2 1QR, England, UK. Tel.: (44) 1223-334-093. Fax: (44) 1223-334-089. c.lindon{at}welc.cam.ac.uk
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Abstract |
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Key Words: Aurora; cytokinesis; mitosis; APC/C; Cdh1
Abbreviations used in this paper: APC/C, anaphase-promoting complex/cyclosome; D-box, destruction box; DIC, differential interference contrast; FP, fluorescent protein; Plk1, polo-like kinase 1.
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Introduction |
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Here, we have begun to examine the role and regulation of proteolysis during mitotic exit in mammalian cells, through studying fluorescent protein (FP)tagged substrates in living cells. We find that different mitotic regulators are degraded at different times, indicating that APC/CCdh1 activity may be modulated to coordinate mitotic exit and cytokinesis.
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Results |
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Nondegradable Plk1 frequently interfered with exit from mitosis. Furthermore, cells expressing high levels of YFP-Plk1 frequently were unable to degrade the protein properly, and in these cells mitotic exit was perturbed (Fig. 5). In these cells, anaphase and cytokinesis were slowed down; spindle elongation was impaired and cleavage furrow ingression was delayed, with many cells exhibiting extensive blebbing of the plasma membrane during this delay (Fig. 5 A). Although spindle elongation was frequently slow, maximum elongation (measured as the extent of sister chromatid separation observed by differential interference contrast [DIC] microscopy) was usually unaffected. There was frequently a delay after maximum spindle elongation before the cleavage furrow began to ingress, whereas these events occurred concurrently in uninjected cells. We did not observe chromosome decondensation during this delay; therefore, both mitotic exit and cytokinesis appeared to be delayed. The delay in mitotic exit in cells unable to degrade Plk1 was frequently accompanied by abnormal movement of the anaphase spindle during prolonged cleavage furrow ingression (Videos 5 and 6 and supplemental data, available at http://www.jcb.org/cgi/content/full/jcb.200309035/DC1), indicating that the coordination between microtubule and actin cytoskeletons might be compromised in these cells. Overexpression of Plk1 or YFP-Plk1 also delayed mitosis before anaphase, and this delay was aggravated by a constitutively active form of the kinase, but not by nondegradable forms. However, the delay in mitotic exit was independent of the total time spent in mitosis (unpublished data).
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In budding yeast, redundant pathways have been demonstrated to inactivate mitotic cyclin-Cdks after mitosis: APCCdh1-mediated degradation is not required for cell viability as long as the Cdk inhibitor Sic1 is present (Schwab et al., 1997; Visintin et al., 1997). Thus, we considered the possibility that there might be parallel pathways to inactivate APCCdh1 targets during exit from mammalian mitosis. Because Plks are activated in mitosis by phosphorylation at a threonine residue in the activation loop (Kelm et al., 2002), we inserted the activating T210D mutation (Qian et al., 1999) at this site to generate a version of Plk1 that could not be inactivated by dephosphorylation. We found that, like nondegradable db-Plk1, the constitutively active T210D-Plk1 delayed mitotic exit compared with wild-type Plk1. Furthermore, a double mutant of Plk1 that was constitutively active and nondegradable, dbT210D-Plk1, significantly increased the frequency with which we observed this phenotype (Fig. 5 B, see P values).
We thought that the large variation in mitotic exit times (Fig. 5 B) might arise from variability between cells in the level of Plk1, which we were unable to measure because the proteins were not fluorescently tagged. Therefore, we examined mitotic exit times in cells expressing YFP-tagged Plk1, where we could select cells expressing comparable levels of the different constructs. In these cells, we observed similar delays in mitotic exit, but also saw a similar variation in mitotic exit times (Fig. 5 C). Additionally, we found an increased delay in cells expressing wild-type YFP-Plk1 compared with untagged Plk1. This indicated that the YFP tag could reduce the rate of degradation of the protein. Perhaps because of this, mutating the D-box alone in YFP-Plk1 had a less significant effect on mitotic exit times (Fig. 5 C). To confirm the link between Plk1 degradation and mitotic exit times in cells expressing YFP-tagged Plk1, we examined the rates of YFP-Plk1 and YFP-T210D-Plk1 degradation and correlated this with the time taken to exit mitosis. We found that degradation was systematically impaired in cells displaying very prolonged mitotic exit times (Fig. 5 D). This occurred more frequently in YFP-T210D-Plk1 cells than in YFP-Plk1 cells, indicating that inactivation of Plk1 in anaphase may be required for its proper degradation.
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Discussion |
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Why should there be a requirement for Plk1 inactivation during anaphase? This appears to contradict the well-documented requirement for Plk activity in cytokinesis and mitotic exit (for review see Bahler et al., 1998; Carmena et al., 1998; Heitz et al., 2001; Lee et al., 2001; Song and Lee, 2001; Mulvihill and Hyams, 2002; Seong et al., 2002). We propose that in addition to being required for assembly of the contractile ring, Plk1 may inhibit cleavage furrow ingression in mammalian cells, perhaps to ensure that ingression cannot occur before anaphase (Shuster and Burgess, 2002). According to the model most recently proposed (Dechant and Glotzer, 2003), this inhibition could be achieved by regulating microtubule bundling in the central spindle. Work published by Neef et al. (2003) during the preparation of this manuscript allows us to identify the kinesin-like protein MKLP-2 as a candidate to mediate the inhibitory effect of Plk1 on cell cleavage. MKLP-2 localizes to the central spindle during anaphase in a complex with Plk1, and has microtubule bundling activity in vitro that can be negatively regulated by Plk1.
Although previous work by others has described the appearance of multinucleated cells after transfection of Plk1 (Mundt et al., 1997), a failure of cytokinesis is a rare event under our experimental conditions, perhaps reflecting the lower levels of expression achieved with microinjection. We find that almost all cells attempt cytokinesis within 50 min of anaphase. This 50-min interval corresponds to the previously described period of "cortical contractility," or C-phase (Martineau et al., 1995; Canman et al., 2000). C-phase can be extended by inhibiting proteolysis with MG132 in the presence of blebbistatin, an inhibitor of myosin II (Straight et al., 2003), indicating that the length of C-phase is regulated by proteolysis. Our results show that the relevant substrate does not appear to be Plk1 because nondegradable Plk1 delays cytokinesis without altering the length of C-phase (Fig. 5, B and C). Moreover, cells starting with excess Plk1 almost always eventually perform cytokinesis. Thus, we suggest that C-phase is not just the window of opportunity during which cytokinesis can occur, but also represents the maximum period for which cytokinesis can be delayed in response to inappropriate conditions.
Our preliminary analyses indicate that the delay in cytokinesis correlates with delayed recruitment of cleavage furrow components (unpublished data). An understanding of the pathways that coordinate cytokinesis with mitotic exit remains an important challenge, and our finding that there is ordered degradation of mitotic regulators as cells exit from mitosis may provide important clues to these pathways.
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Materials and methods |
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Immunoblotting
Extracts were made from cell populations at the indicated time points after nocodazole release. Cells were washed with PBS. Extracts prepared by addition of boiling SDS sample buffer directly to culture dishes (for reattached cells in G1), and to cells pelleted from the culture medium (for unattached mitotic cells), were pooled. Samples were heated at >95°C for 3 min and then sheared through 21G needles. Approximately 5 µg of each sample was blotted by the standard semi-dry transfer technique onto ImmobilonTM-P (Millipore). Filters were processed for immunoblotting using standard techniques. Rabbit polyclonal antisera used were raised against (1) human Plk1 amino-terminal peptide (Upstate Biotechnology); (2) cyclin B1 (Hagting et al., 1998); (3) human AIK1 (Aurora A) and mouse AIK2 amino terminus (Aurora B; both gifts of Peter Donovan, Thomas Jefferson University, Philadelphia, PA); (4) BubR1 (a gift of Gordon Chan, Fox Chase Cancer Center, Philadelphia, PA); (5) CENP-E (a gift of Tim Yen, Fox Chase Cancer Center, Philadelphia, PA); and (6) PRC1 (a gift of Tony Hunter, Salk Institute, La Jolla, CA). Goat polyclonal antihuman p55Cdc20 was obtained from Santa Cruz Biotechnology, Inc.
Construction of cDNA plasmids
Plk1 cDNA was cloned from pCMX-GFP10c-Plk1 (Arnaud et al., 1998) into pEYFP-C3 (CLONTECH Laboratories, Inc.) for expression as a fusion protein with YFP at the amino terminus, and into pcDNA3 for expression as an untagged protein.
New point mutations were constructed by whole-plasmid PCR using Pfu DNA polymerase (Stratagene) and confirmed by automated sequencing. Constitutively active versions of Plk1 were constructed by swapping in sequences from pRcCMV-Plk1-T210D (a gift of Erich Nigg, Max Planck Institute, Martinsried, Germany).
Human Aurora A sequence was generated by PCR from pCRUZ-myc-Aurora A (a gift of Claude Prigent, University of Rennes, Rennes, France) and was cloned into pEYFP-N1, and is expressed as a fusion protein linked at its carboxy terminus to YFP. pECFP-N1-cyclin B1 and pECFP-N1-securin61-68 have been described previously (Hagting et al., 2002). Histone 2B-YFP was the gift of Claire Acquaviva (Wellcome Trust/Cancer Research UK Institute, Cambridge, UK). Further details of all constructs used are available upon request.
Microinjection and time-lapse imaging and analysis
Cells were injected and analyzed using time-lapse DIC fluorescence microscopy with different filter cubes to distinguish YFP- and CFP-associated fluorescence as described previously (Clute and Pines, 1999; Hagting et al., 1999, 2002), but with the addition of a programmable XY stage (Prior Scientific Instruments Ltd.) to allow concurrent filming of several fields of cells. Images were collected every 2 or 3 min and saved in IP Lab Spectrum (Scanalytics) format as 16-bit data using a reference look-up table with a preset linear pixel intensity scale. ImageJ software (National Institutes of Health; modified by Jean-Yves Thuret) was used for quantifying CFP and YFP fluorescence. Fluorescence levels in whole cells were measured as pixel values within a region of interest (ROI) drawn around each cell and applied to all images in a series. The ROI drawn in each case was large enough to allow for changing cell shape during mitotic exit. Because we subtracted background pixel values from our measured values, this method gave accurate measurements of total cell fluorescence. DIC images were used to determine the onset of anaphase. Images were then converted to PICT format and exported to Adobe Photoshop®, or processed via ImageJ to make QuickTime® movies.
Online supplemental material
A sequence alignment of human Plk family members, showing the position of the nonconserved D-box motif, is shown in Fig. S1. Fig. S2 shows anti-Plk1 staining of G1 cells injected with untagged versions of Plk1, and confirms that untagged db-Plk1 is not degraded in mitosis and/or G1 phase. Videos available online show examples of cells degrading Plk1 (Video 1), Aurora A (Video 2), or both (Video 3), cells expressing nondegradable Plk1 (Video 4), and cells exhibiting delayed mitotic exit in response to nondegraded Plk1 (Videos 5 and 6). All supplemental videos, supplemental figures, and an associated Materials and methods section are available online at http://www.jcb.org/cgi/content/full/jcb.200309035/DC1.
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Acknowledgments |
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This work was made possible through a Wellcome Trust Advanced Training Fellowship to C. Lindon, by an FP5 RTN from the European Union (contract number QLG1-CT-2001-02026), and by Programme Grant C29/A1782 to J. Pines from Cancer Research UK.
Submitted: 5 September 2003
Accepted: 10 December 2003
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