Report |
Address correspondence to Simonetta Piatti, Dipartimento di Biotecnologie e Bioscienze, Piazza della Scienza 2, 20126 Milano, Italy. Tel.: 39-02-6448-3547. Fax: 39-02-6448-3565. E-mail: simonetta.piatti{at}unimib.it
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Abstract |
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Key Words: anaphase; anaphase-promoting complex; microtubules; securin; spindle
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Introduction |
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Why do metaphase spindles elongate to a certain length and wait before elongating during anaphase B? One possibility is that the mechanical link established by interactions of sister chromatids with microtubules emanating from opposite spindle poles prevents spindle elongation. Mechanistically, this hypothesis suggests that the forces acting on spindles do not change between metaphase and anaphase; rather it is the loss of sister chromatid cohesion at anaphase onset that removes the barrier to spindle elongation. Another possibility is that cell cycle events at the metaphase to anaphase transition are also required for successful spindle elongation. The metaphase to anaphase transition is controlled by the APC, a multisubunit complex with ubiquitin-ligase (E3) activity, which triggers entry into anaphase, exit from mitosis, and cytokinesis (for reviews see Yanagida et al., 1999; Zachariae and Nasmyth, 1999). APC activity can be inhibited by the spindle checkpoint, which monitors correct bipolar attachment of chromosomes to the spindle, preventing entry into anaphase (for review see Wassmann and Benezra, 2001). The APC triggers degradation of securin (Yanagida et al., 1999; Zachariae and Nasmyth, 1999), resulting in activation of the separase (Amon, 2001), which cleaves the cohesin thereby promoting sister chromatid separation (Uhlmann et al., 2000; Waizenegger et al., 2000). Several different proteins, besides securin, are known to be APC substrates (Peters, 1999), but the physiological relevance of their proteolysis has not been established for all of them.
Relatively little is known about the differential role of cell cycleregulated events and chromosome-based pole-to-pole links in spindle mechanics at anaphase. Experiments in Xenopus (Shamu and Murray, 1992) and yeast (Holm et al., 1985) with inhibition of topoisomerase II have shown that if the link between sister chromatids is not broken at the metaphase-anaphase transition, spindles do not elongate, supporting the mechanical link hypothesis. In contrast, insect spermatocytes from which all chromosomes have been removed maintain metaphase spindles and undergo anaphase spindle elongation with kinetics similar to normal spindles (Zhang and Nicklas, 1996). Furthermore, spindles formed in Xenopus egg extracts by plasmid DNA incompetent to assemble kinetochores are the same length as spindles formed by sperm nuclei that assemble kinetochores (Heald et al., 1996). In Saccharomyces cerevisiae, spindle elongation and integrity is affected in cohesion-defective mutants, despite premature separation of sister chromatids (Guacci et al., 1997; Michaelis et al., 1997; Skibbens et al., 1999). Furthermore, the separase Esp1 appears to be required for spindle elongation, besides for separation of sister chromatids (Uhlmann et al., 2000; Jensen et al., 2001). However, in Schizosaccharomyces pombe and S. cerevisiae mutations affecting the pole-to-pole links result in an increased spindle length at metaphase (Goshima et al., 1999; Skibbens et al., 1999).
One way to address the role of bipolar attachment of chromosomes in spindle elongation would be to prevent establishment of sister chromatid cohesion during S phase and assay the effect on spindle length and structure. In this paper, we disrupt bipolar attachment using mutants in S. cerevisiae affecting sister chromatid cohesion (Tanaka et al., 2000) or DNA replication (Piatti et al., 1995) and show that though the spindles elongate eventually they are unable to stabilize their midzones. Our data suggest that in addition to sister chromatid separation, successful anaphase B requires an APC-dependent event that stabilizes the microtubules of the elongating spindle. Stabilization requires destruction of the securin Pds1 but not activity of the separase Esp1, suggesting that Pds1 proteolysis is necessary for stabilization of the central spindle at mitosis independently of Esp1.
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Results and discussion |
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In principle, the rescue of the spindle defects in cells with monopolarly attached chromosomes by a MAD2 deletion could be due to a direct effect of Mad2 on spindle stability rather than to the restoration of the normal timing of APC activation with respect to chromosome segregation. To check whether the rescue of the spindle stability defect by deletion of MAD2 was mediated by the APC, we introduced in the scc1-73 mad2 double mutant a loss-of-function mutation affecting the APC activator Cdc20. We then analyzed spindle structure in a synchronous culture of a cdc20-3 scc1-73 mad2
triple mutant obtained by elutriation and released at 37°C. The spindles in the triple mutant were as unstable as in scc1-73 single mutant cells (Fig. 2 A), suggesting that rescue of the spindle instability in scc1-73 cells by MAD2 deletion is mediated by the APC. This result was confirmed by measuring the intensity of spindle staining along its length in cdc20-3 scc1-73 mad2
versus scc1-73 mad2
cells (Fig. 2 B). The figure shows that unlike wild-type and scc1-73 mad2
, cdc20-3 scc1-73 mad2
central spindles, like those of scc1-73, lack the increase in spindle staining due to overlapping microtubules (Fig. 2 B).
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Taken together, the analysis of cdc6 and scc1 mutants shows that the simple mechanical link hypothesis is not sufficient to explain how correct spindle elongation is triggered. Our experiments suggest rather that spindle elongation must be coupled to APC activation for correct formation of the spindle midzone. The most likely role for APC activation in spindle stabilization is via destruction of the securin PDS1, which is both degraded at the metaphase/anaphase transition in a Cdc20/APC-dependent manner (Yanagida et al., 1999; Zachariae and Nasmyth, 1999) and localized on spindles in S. cerevisiae (Jensen et al., 2001) and S. pombe (Funabiki et al., 1996). Therefore, we deleted PDS1 in scc1-73 cells to check whether removal of Pds1 would bypass the requirement of APC activation in spindle stability. We found that PDS1 deletion was sufficient to stabilize spindles in scc1-73 cells (Fig. 4). Furthermore, scc1-73 pds1 cells underwent cytokinesis and spindle elongation with wild-type kinetics (Fig. 4). This result suggests that the spindle checkpoint-dependent accumulation of Pds1 in scc1 cells is responsible for destabilizing anaphase spindles and for delaying mitotic exit and cytokinesis.
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Materials and methods |
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Western blot analysis
Western blot analysis was performed as described (Piatti et al., 1996). 50 µg of protein were transferred to nitrocellulose membranes and detected using chemiluminescence detection (ECL; Amersham Pharmacia Biotech). Myc-tagged Pds1 was detected with 9E10 Mab; polyclonal antibodies were used to detect Clb2 (Amon et al., 1994), Sic1 (Skowyra et al., 1997), and Swi6 (Moll et al., 1992).
Other techniques
Centrifugal elutriations were performed as described (Piatti et al., 1995). Flow cytometric DNA quantitation was determined according to Epstein and Cross (1992). Visualization of Tet operators integrated at the URA3 locus of chromosome V (35 Kb from the centromere) using the GFP-tetR fusion was performed as described (Michaelis et al., 1997). Immunofluorescence was performed according to Nasmyth et al. (1990). Images were captured with either a Coolsnap CCD camera (Photometrics) mounted on an Eclipse E600 microscope (Nikon).
Online supplemental material
In Fig. S1, the esp1-1 mutation does not affect spindle stability of scc1-73 mad2 cells. In Fig. S2, a double scc1-73 esp1-N5 mutant elongates spindles with similar kinetics to scc1-73 single mutant cells.
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Footnotes |
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* Abbreviations used in this paper: APC, anaphase-promoting complex.
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Acknowledgments |
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Submitted: 23 April 2001
Revised: 10 October 2001
Accepted: 10 October 2001
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References |
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