The proteolysis-dependent metaphase to anaphase transition - NCBI

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Jul 8, 1994 - Nathalie Morin, Ariane Abrieu,. Thierry Lorca, Fran9ois Martin and. Marcel Doree. CNRS UPR 9008/INSERM U 249, Route de Mende, BP 5051 ...
The EMBO Journal vol.13 no. 18 pp.4343-4352, 1994

The proteolysis-dependent metaphase to anaphase transition: calcium/calmodulin-dependent protein kinase 11 mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs Nathalie Morin, Ariane Abrieu, Thierry Lorca, Fran9ois Martin and Marcel Doree CNRS UPR 9008/INSERM U 249, Route de Mende, BP 5051, Montpellier Cedex, France Communicated by M.Dor6e

It has been shown, using spindles assembled in vitro in extracts containing CSF (the cytostatic factor responsible for arresting unfertilized vertebrate eggs at metaphase), that onset of anaphase requires Ca2+dependent activation of the ubiquitin-dependent proteolytic pathway that destroys both mitotic cyclins and an unknown protein responsible for metaphase arrest (Holloway et al., 1993, Cell, 73, 1382-1402). We showed recently that Ca2+/calmodulin-dependent protein kinase II (CaM KII) activates the ubiquitin-dependent cyclin degradation pathway in CSF extracts (Lorca et al., 1993, Nature, 366, 270-273), but did not investigate its possible effect on sister chromatid segregation. In this work we identify CaM KII as the only target of Ca2+ in inducing anaphase in CSF extracts, and further show that transition to anaphase does not require the direct phosphorylation of metaphase spindle components by CaM KII. A possible interpretation of the above results could have been that the ubiquitin-dependent degradation pathway is required for onset of anaphase only when spindles are clamped at metaphase due to CSF activity, and not in the regular cell cycle that occurs in the absence of CSF activity. We ruled out this possibility by showing that competitive inhibition of the ubiquitin-dependent degradation pathway still prevents the onset of anaphase in cycling extracts that lack CSF and do not require Ca2+ for sister chromatid separation. Key words: anaphase/Ca2+/calmodulin-dependent proteinkinase/cell cycle/cyclin proteolysisiXenopus egg

Introduction A major difficulty in elucidating the mechanisms responsible for spatio-temporal coordination of mitotic events is that cells generally do not arrest at a specified stage of mitosis before progressing to the next one; thus, major events involved in different regulatory processes may overlap. Even in individual cells at metaphase, when chromosomes achieve metaphase alignment, kinetochores still move themselves and their attached chromosomes on the ends of relatively stationary but shortening/elongating microtubules, and alignment on the so-called 'metaphase plate' is not maintained for more than a few minutes,

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making it, for example, difficult to separate the events involved in chromosome congression and onset of anaphase. An important exception is metaphase of the second meiotic cell cycle in females of vertebrates, when chromosomes are maintained for hours on the metaphase plate until fertilization. Unfertilized frog eggs have been useful to identify two important factors involved in mitosis control (Masui and Markert, 1971). The first is maturationpromoting factor (MPF), a heterodimeric protein kinase comprising p34cdc2 as the catalytic subunit and a B-type cyclin as a regulatory subunit (reviewed in Doree, 1990; Nurse, 1990). MPF universally controls the G2 to M phase transition in eukaryotic cells. The second factor responsible for metaphase arrest in unfertilized vertebrate eggs is called cytostatic factor (CSF). It has been identified as the product of the c-mos proto-oncogene that is expressed during oocyte maturation and blocks cyclin degradation (reviewed by Masui, 1992). Spindles assembled in vitro in frog egg extracts from permeabilized sperm nuclei also arrest at metaphase if extracts contain active CSF. Recently, such extracts were used to investigate the control mechanisms involved in the onset of anaphase. It was shown that the addition of CaCl2 to CSF extracts containing in vitro-assembled metaphase spindles triggers sister chromatid segregation. Ca2+ acts, at least in part, by activating ubiquitin-mediated proteolysis of an unidentified target (Holloway et al., 1993). Sister chromatid separation could be competed by adding an N-terminal fragment of cyclin B, which acts as a specific competitor for cyclin degradation. Thus the ubiquitin pathway responsible for anaphase may be identical to that responsible for MPF inactivation (reviewed by Hershko et al., 1994; Lorca et al., 1994), even though MPF inactivation itself is not required for anaphase. Although the above results demonstrated convincingly that a ubiquitin-mediated proteolytic process is required for anaphase to be triggered, at least in CSF extracts, they did not address the question as to whether activation of this process is the only event required for Ca2+ to initiate anaphase. Besides activating ubiquitin-mediated proteolysis, Ca2+ could also activate a distinct process also required for sister chromatid segregation. An important progression towards the elucidation of the mechanisms involved in the onset of anaphase would be the identification of the Ca2+ target. Recently, we showed that Ca2+/calmodulin-dependent protein kinase II (CaM KII) activates the ubiquitin-dependent cyclin degradation pathway in CSF extracts and mediates the effects of Ca2+ at fertilization in inactivating MPF and CSF activities (Lorca et al., 1993). However, we did not investigate if CaM KII can also trigger sister chromatid segregation. In this work we demonstrate that the specific inhibition

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Fig. 1. Constitutively active CaM KII triggers anaphase in CSF extracts even in the absence of elevated Ca2+. Metaphase spindles were assembled using the two-step procedure described in the text (A and B) and induced to enter anaphase (C and D) by adding the CaM KII mutant. (A and C) Chromosomes are visualized by the fluorescence of the DNA binding dye Hoechst 33 342. (B and D) Microtubules are visualized with rhodaminelabeled tubulin. The images in (C) and (D) were taken 20 min after CaM KII addition. The white bar in (A) corresponds to 5 gm (same magnification in B-D).

of CaM KII prevents Ca2' from triggering sister chromatid segregation in CSF extracts containing in vitro-assembled metaphase spindles, while the addition of a constitutively active CaM KII mutant readily triggers anaphase movements and subsequent mitotic events, even in the absence of Ca2+. Moreover, metaphase spindles readily undergo anaphase, even in the absence of CaM KII activity, when transferred in extracts where the ubiquitin-dependent degradation pathway has been turned on. Thus activation of CaM KII, that itself acts through activation of the ubiquitin-dependent pathway, is the only Ca2+-dependent event required for sister chromatid segregation to be triggered in CSF extracts. Finally, we extend to metaphase spindles formed in the absence of CSF the finding that the onset of anaphase requires activation of the ubiquitindependent proteolysis pathway.

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Results Constitutively active CaM K!l triggers anaphase in CSF extracts even in the absence of elevated Ca2+ To investigate the possible involvement of CaM KII in triggering the metaphase to anaphase transition, mitotic spindles arrested at metaphase were first assembled according to the two-step procedure described by Shamu and Murray (1992). In the first step, permeabilized Xenopus sperm nuclei were added to a CSF-arrested extract, and subsequently calcium was added to inactivate MPF and CSF and to induce the formation of interphase nuclei and DNA replication. In the second step (after the extract had sequestered added calcium), 0.5 vol of untreated CSFarrested extract was added to trigger nuclear envelope breakdown and the assembly of metaphase spindles (Figure IA and B). Then 0.1 vol of a constitutively active mutant

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min Fig. 2. Histogram showing cumulated frequencies of anaphase figures as a function of time elapsed from the addition of either CaC12 or constitutively active CaM KII to a CSF extract. Samples were taken at the indicated times and percentages of anaphase estimated in each sample from 25-35 spindles taken at random (only metaphase and anaphase figures were observed during the first 25 min).

of CaM KII (see Materials and methods) were added in the absence of Ca2 . The enzyme, produced in reticulocyte lysate from in vitro-transcribed mRNA, had a specific activity of 3-5 pmol of phosphate transferred/min/,l, using syntide II as a specific substrate. Thus it caused only a modest increase of CaM KII activity in CSF extracts, as compared with the much higher (10- to 20fold) but transient increase of endogenous CaM KII activity observed after fertilization (Lorca et al., 1993). Nonetheless, this was sufficient to cause sister chromatid separation (Figure IC), whilst microtubules increased in size around the growing asters and depolymerized between migrating sister chromatids (Figure 1D). In contrast, spindles remained arrested with the metaphase conformation and sister chromatid did not segregate when the same volume of unprogrammed reticulocyte lysate was added to the incubation mixture; the same negative result was obtained in the absence of added Ca2+ when the reticulocyte lysate was programmed with wild-type CaM KII (data not shown). As shown in Figure 2, the timing of the metaphase to anaphase transition caused by the constitutively active CaM KII compared well with that due to Ca2+ addition: it took -18 min (temperature 23°C) for 50% of the in vitro-assembled spindles to enter anaphase after CaM KII addition, as compared with 15 min after CaCl2 addition. After a longer incubation (30-35 min) following addition of either constitutively active CaM KII or CaCl2, chromosomes decondensed and formed caryomers near asters (Figure 3A) that fused together to reform nuclei (Figure 3C). Meanwhile, an abundant network of cytoplasmic microtubules appeared. Microtubule density and length were much higher around nuclei (Figure 3B and D), but they were also clearly detected more distantly from nuclei, contrasting with their absence outside metaphase spindles in untreated extracts (data not shown). Nuclei assembled upon the addition of constitutive CaM KII replicated DNA as efficiently as those assembled upon Ca2+ addition (Figure 3E, lanes 2 and 4). As expected, extracts incubated either with unprogrammed reticulocyte lysate, or in the absence of added CaCl2 with wild-type CaM KII, did not replicate DNA (Figure 3E, lanes 1 and 3).

Inhibiting CaM Kll activity prevents Ca2+ from triggering anaphase in CSF extracts The above results showed that constitutively active CaM KII is able to trigger anaphase and subsequent events in CSF-arrested extracts. They did not prove, however, that activation of endogenous CaM KII upon CaCl2 addition (or fertilization) is responsible for anaphase. To suppress CaM KII activation in extracts following CaCl2 addition we used CaM KII (281-302), a conserved peptide corresponding to the auto-inhibitory domain of Xenopus CaM KII that specifically inhibits this enzyme. Like most peptides, CaM KII (281-302) is not very stable in egg extracts, and these extracts are highly variable in their ability to rapidly sequester added Ca2+. This is probably the reason why in preliminary experiments we observed a 10-15 min delay for sister chromatid segregation rather than its suppression. To circumvent this difficulty, we took advantage of the fact that an elevated level of Ca2+ is likely to be required for only a short period of time for anaphase to be triggered, as it is for the cyclin degradation machinery to be turned on in CSF extracts (Lorca et al., 1991). Thus, in the next experiment 2 mM EGTA were added 2 min after 0.4 mM CaCl2 to terminate the Ca2+ transient and inactivate Ca2+-dependent enzymes. Even in such conditions, anaphase occurred in CSF extracts lacking CaM KII (281-302) earlier than 25 min after CaCl2 addition (Figure 4A and B). In contrast, spindles still remained arrested with the metaphase configuration as late as 60 min after CaCl2 addition in extracts containing the inhibitory peptide (Figure 4C and D), and no anaphase movements were detected later on (not shown). Taken together, the above experiments show that CaM KII activation is both necessary and sufficient for anaphase to be triggered in CSF-arrested extracts. This indicates that the unique function of Ca2+ in inducing the metaphase to anaphase transition in this assay is to activate CaM KII. Transition to anaphase does not require direct phosphorylation of metaphase spindle components by CaM K!l It has been shown that Ca2+-induced segregation of sister chromatids in CSF extracts requires activity of the ubiquitin-dependent proteolytic pathway (Holloway et al., 1993). If the only function of CaM KII in triggering anaphase was to activate the ubiquitin-dependent cyclin degradation pathway, its activity could be predicted to be no longer required for anaphase once the proteolytic pathway had been turned on, even if metaphase spindles were never brought into direct contact with the active kinase. To evaluate this prediction, metaphase spindles were formed using the two-step procedure in an aliquot of CSF-arrested extract. CaCl2 was added to another aliquot of the same extract (in which no sperm chromatin had been added) to turn on the ubiquitin-dependent pathway. Then EGTA was added in turn to terminate the Ca21 transient; as expected, this was found to inactivate endogenous CaM KII (data not shown). Finally, metaphase spindles (Figure 5A and B) formed in the first aliquot (4 ,ul) were transferred in the second aliquot (16 ,l). As shown in Figure SC and D, anaphase readily occurred a few minutes later. Similar results were obtained when constitutive CaM KII, used to turn on the ubiquitindependent proteolytic pathway, was inactivated with the 4345

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Fig. 3. Return to interphase following induction of anaphase in CSF extract. (A and C) Chromosomes stained by Hoechst 33 342. (B and D) Microtubules visualized with rhodamine-labeled tubulin. The (A), (B) and (C), (D) pairs of pictures were taken 30 and 35 min after the onset of gnaphase, respectively (white bars = 5 gim). The arrowhead in (A) points to a typical caryomere formed from an individual decondensing chromosome. Micrograph (C) shows two interphase nuclei newly formed from fused caryomeres. (E) Autoradiogram showing the incorporation of la-32P]dCTP in DNA of newly formed interhase nuclei. Samples were taken from non-treated CSF extract (lane 1), CaCl2-treated CSF extract (lane 2), CSF extract treated in the absence of Ca + with the wild-type CaM KII (lane 3), CSF extract treated in the absence of Ca> with the constitutively active CaM KII mutant (lane 4), and incubated for I h with [a-32P]dCTP (see Materials and methods).

CaM KII (281-302) inhibitory peptide prior to metaphase spindle addition (data not shown). In contrast, spindles remained arrested at metaphase when transferred in egg

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extracts in which neither CaC12 nor CaM KII had been added. These experiments show that there is no requirement for the direct action of CaM KII (or for any other

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Fig. 4. Inhibiting CaM KII activity prevents Ca2+ from triggering anaphase in CSF extracts. Metaphase spindles were assembled as described in the legend to Figure 1. Anaphase was induced in >95% of the spindles (25 spindles analyzed) by adding first 0.4 mM CaC12, then 2 mM EGTA 2 min later to terminate the Ca2+ transient (A and B; micrographs taken 25 min after CaCI2 addition). Anaphase did not occur and all spindles remained arrested at metaphase (30 spindles analyzed) when the same experiment was made in the presence of the CaM KII (281-302) inhibitory peptide (C and D; micrographs taken 60 min after CaC12 addition). (A and C) Chromosomes are stained with Hoechst dye. (B and D) Microtubules are visualized with rhodamine-labeled tubulin.

Ca2l-dependent enzyme) on metaphase spindles for sister chromatid separation to be triggered in egg extracts, and strongly suggest that the only function of CaM KII in triggering anaphase is indeed to activate the ubiquitindependent pathway. Ubiquitin-dependent proteolysis is also required for anaphase to occur in cycling extracts that lack CSF activity In the above experiments [as in those reported previously by Holloway et al. (1993); see Discussion], spindles were assembled and maintained arrested at metaphase in extracts containing the c-mos protein kinase acting as the effective cytostatic factor. The proto-oncogene has been shown to undergo degradation after fertilization, and this degradation is now believed to be mediated by the ubiquitindependent pathway (Nishizawa et al., 1993). Thus, avail-

able evidence did not rule out the possibility that activity of the ubiquitin-dependent proteolytic pathway could be required to inactivate CSF, and not for the onset of anaphase in the regular cell cycle that proceeds in the absence of CSF activity. To address this question, we designed the experiment depicted in Figure 6 using 'cycling extracts' prepared from parthenogenetically activated frog eggs that underwent cmos degradation in ovo (Figure 7) and therefore lacked CSF activity. When added to such extracts (Figure 6), sperm chromatin decondenses and forms nuclei within 30 min that undergo at least two rounds of DNA replication separated by a typical mitosis (micrographs in Figure 8A-C). To monitor ubiquitin-dependent proteolysis, we used a small amount of highly [35S]methionine-labeled B1 cyclin translated from the corresponding mRNA in reticulocyte lysate. As expected, cyclin degradation 4347

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Fig. 5. Transition to anaphase does not require direct phosphorylation of metaphase spindle components by CaM KII. Metaphase spindles formed using the two-step procedure described in the text (A and B) were transferred in a CSF extract that had been treated first with CaC12 to turn on the ubiquitin-dependent pathway, then with EGTA to inactivate endogenous CaM KII. Pictures of anaphase spindles (C and D) were taken 15 min after transfer. At that time, sister chromatid segregation had already occurred in 10 out of 18 spindles analyzed. (A and C) Chromosomes are stained with Hoechst dye. (B and D) Microtubules are visualized with rhodamine-labeled tubulin.

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Fig. 7. The c-mos proto-oncogene responsible for CSF activity is degraded in ovo following parthenogenetic activation of Xenopus eggs, but not in vitro following CaCl2 addition. Lanes 1-3: a CSF extract prepared from inactivated eggs was treated (lane 2) or not (lanes 1 and 3) for 60 min with 0.4 mM CaCl2, and analyzed by SDS-PAGE and Western blotting for c-mos content. Lane 4: extract was prepared 25 min after electric stimulation of unfertilized eggs. The same eggs were used (without electric stimulation) to prepare the CSF extract analyzed in lane 3.

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Fig. 8. Exit from metaphase and ubiquitin-dependent proteolysis in 'cycling' extracts. Samples were taken at the indicated times (see Figure 6) from a control 'cycling' extract and analyzed (A-C) by fluorescence microscopy for progression of mitosis (upper panels, microtubules visualized with rhodamine-labeled tubulin; lower panels, DNA stained with Hoechst dye). (D) The extent of [35S]methionine-labeled B1 cyclin degradation (CycB*, added at time 50 min) was monitored by fluorography in aliquots taken at the indicated times. At time 100 min, 16 spindles were analysed: 14 had completed congression of chromosomes and 12 were typical metaphase (A). At time 110 min, 12 spindles were analyzed: 10 were typical anaphase (B). At time 140 min, all spindles had disappeared and interphase nuclei reformed (30 nuclei scored).

occurred readily at anaphase (Figure 8D). In contrast, spindles remained arrested at metaphase for at least 1.5 h (Figure 9B and C) and labeled cyclin B underwent

only slow and limited degradation (Figure 9A) if high concentrations (40 jig/ml) of cyclin B -GST were added at the time of nuclear envelope breakdown. In this experiment 4349

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cyclin B -GST, which is poorly degraded in egg extracts, acts by competing efficiently for degradation of mitotic cyclins, as well as other putative substrates of the ubiquitindependent proteolytic pathway. Therefore, these results strongly suggest that degradation of one of these substrates is required for the onset of anaphase, even in the absence of CSF. In agreement with this view, methylated ubiquitin, which prevents formation of polyubiquitin chains required for protein degradation (reviewed by Ciechanover and Schwartz, 1994), was also found to inhibit sister chromatid separation in cycling extracts (data not shown). Wild-type ubiquitin had no such effect.

Discussion

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Fig. 9. Inhibiting ubiquitin-dependent proteolysis arrests cell cycle at metaphase in 'cycling' extracts. The cyclin B-GST fusion protein was added (40 ,ug/ml) at time 90 min (see Figure 6) to compete for degradation with substrates of the ubiquitin-dependent proteolytic pathway. (A) The extent of [35S]methionine-labeled B1 cyclin degradation (CycB*, added at time 50 min) was monitored by fluorography in aliquots taken at the indicated times. Progression of the cell cycle was still arrested at metaphase (25 spindles analyzed: 100% metaphase) as late as 150 min after sperm addition (B, microtubules; C, chromosomes), when the control cycling extract had already completed mitosis and reformed interphase nuclei (see Figure 8C).

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Although evidence for transient changes in free Ca2+ during mitosis is complex and conflicting (reviewed by Hepler, 1989), a role for Ca2> at the onset of anaphase has been proposed (reviewed in McIntosh and Hering, 1991). This led us to examine how Ca2+ drives into anaphase spindles in vitro-assembled in Xenopus egg extracts containing CSF. We first showed that Ca2+/calmodulin-dependent protein kinase II mediates the effect of Ca2> in this process. Indeed, the addition of a truncated and constitutively active CaM KII was shown to be sufficient to release egg extracts from metaphase arrest and to allow subsequent cell cycle events to occur, including anaphase, telophase and reconstitution of interphase nuclei able to replicate DNA. Conversely, specific inhibition of endogenous CaM KII by a specific peptide inhibitor corresponding to its auto-inhibitory domain suppressed induction of the metaphase to anaphase transition by Ca2+ in egg extracts. Next, we investigated if transition to anaphase requires the direct phosphorylation of metaphase spindle components by CaM KII. In vitro-assembled spindles were found to readily undergo anaphase, even in the absence of a Ca2+ transient, when transferred in extracts where CaM KII had already returned to its basal activity following its transient activation by Ca2+. Moreover, cycling extracts prepared from parthenogenetically activated eggs that do not contain anymore CSF activity readily underwent anaphase in the absence of Ca2+ transient. Finally, several rounds of cleavage with normal anaphase occur in the early starfish embryo injected with EGTA (A.Picard, unpublished result). These results suggest that Ca2+_ dependent modification of spindle components, including phosphorylation by CaM KII, may not be essential at the onset of anaphase for sister chromatid segregation. At first sight they contrast with a previous report that microinjection of antibodies to a 62 kDa spindle-associated substrate of a Ca2+/calmodulin-dependent protein kinase blocks the metaphase to anaphase transition in sea urchin embryos (Dinsmore and Sloboda, 1989). This protein may, however, either play a role in the onset of anaphase, independentof its Ca2+/calmodulin-dependent phosphorylation, or if required this phosphorylation may occur before metaphase. Thus, the only function of CaM KII in driving into anaphase spindles otherwise arrested at metaphase may be to activate the ubiquitin-dependent cyclin degradation pathway in cells or cell extracts containing CSF activity (Lorca et al., 1993). In the absence of CSF activity, MPF kinase has been shown to induce cyclin degradation even

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in extracts containing high levels of EGTA (Felix et al., 1990). Thus, CaM KII activity is not required in this case to turn on the ubiquitin-dependent degradation pathway. Whilst activation of MPF has been unambiguously shown to be the key event responsible for promoting the G2 to M transition of the eukaryotic cell cycle, the exact role its inactivation plays in exit from mitosis is still unclear (reviewed by Lorca et al., 1994). Cyclin B degradation was found to occur within a narrow time window coincident with the metaphase to anaphase transition in the developing surf clam embryo (Hunt et al., 1992), and the HI kinase activity of MPF was observed to drop at the same time in early starfish and sea urchin embryos (Picard et al., 1987; Meijer and Pondaven, 1988). Moreover, an undegradable cyclin lacking the destruction box was found to prevent cleavage in Xenopus embryos and to arrest cell cycle progression when cells still contained condensed chromosomes, at a stage that was first interpreted as a metaphase arrest (Murray et al., 1989). More recently, however, it was shown using spindles assembled in vitro in Xenopus egg extracts containing CSF, that MPF inactivation is not required for anaphase to occur (Holloway et al., 1993). Thus MPF inactivation would be required only later in the cell cycle for chromosomes to decondense and cytokinesis to occur (Gallant and Nigg, 1992; Satterwhite et al., 1992; Galas et al., 1993). A similar conclusion was drawn by other investigators in budding yeast where, of all known B-type cyclins, CLB2 plays the most important role in mitosis (Surana et al., 1993). They first showed that anaphase occurs before any drop of HI kinase activity of CLB2-CDC28 can be detected in conditional mutants that arrest at telophase, and second that overproduction of CLB2 causes arrest not in metaphase but in telophase. Although both reports reached the same conclusion that MPF inactivation is not required for the onset of anaphase, they basically differ because activation of the cyclin B degradation pathway is apparently not required for anaphase to occur in budding yeast. In contrast, sister chromatid separation was shown to be competed for in frog egg extracts by adding an N-terminal fragment of cyclin B, which acts as a specific competitor for cyclin degradation, and delayed by methylubiquitin, that prevents formation of the polyubiquitin chain required for protein

degradation. A possible interpretation of the above result could have been that the ubiquitin-dependent cyclin degradation pathway is required for the onset of anaphase in frog egg extracts only when spindles are clamped at metaphase due to CSF activity, and not in the regular cell cycle that occurs in the absence of CSF activity. Holloway et al. (1993) could not satisfactorily address this question, even when they used Ca2+-treated extracts arrested at interphase by cycloheximide and then driven to a 'metaphase-like' state by the addition of cyclin BA90 (chromosomes failed to congress properly to a metaphase plate in such conditions). Indeed, such extracts still contained some CSF activity that had first to be inactivated by Ca2+ addition (or prolonged incubation at room temperature that slowly

releases Ca2+ from altered reticulum vesicles, and therefore progressively increases basal level of Ca2+) for sister chromatid segregation to occur. In agreement with their results, we showed previously, and have confirmed in this

report, that raising free Ca2> to the micromolar range readily triggers cyclin degradation in CSF extracts, but fails to induce c-mos proteolysis (Lorca et al., 1991). In this work, however, we have been able to show that competitive inhibition of the ubiquitin-dependent degradation pathway also prevents the onset of anaphase in cycling extracts that lack CSF and do not require Ca2+ for sister chromatid separation. Even in yeast, mutants of the 26S proteasome defective in chromosome segregation were isolated recently (Ghislain et al., 1993; Gordon et al., 1993). Although some proteins are degraded by the 26S proteasome without ubiquitination (Murakami et al., 1992), the above results strongly suggest that ubiquitindependent proteolysis may be universally required in eukaryotes for anaphase to occur. The target for ubiquitindependent degradation has yet to be identified. When sister chromatids are examined in cells undergoing mitosis, they appear paired along their entire length (Cooke et al., 1987; Sumner, 1991). Therefore we favor the idea that the target for ubiquitin-dependent degradation is a protein like INCENP, and CLiP proteins that localize between sister chromatids prior to anaphase (Cooke et al., 1987; Earnshaw and Cooke, 1989; Rattner et al., 1988). Alternatively, it might be one of the numerous proteins associated with centromeres of still associated sister chromatids, whose degradation would allow their separation and poleward migration.

Materials and methods Cycling extracts Cycling extracts were prepared essentially as described by Murray and Kirschner (1989). Briefly, fresh eggs were dejellied in 2% cysteine (pH 7.8), washed three times in 0.25XMMR (100 mM NaCl, 2 mM KCI, 1 mM MgSO4, 2 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES, pH 7.8) and then parthenogenetically activated in a chamber containing 0.25XMMR by electrical stimulation (two 3 s pulses separated by a 7 s pause). Eggs were sorted out, transferred in XB buffer [50 mM sucrose, 0.1 mM CaC12, 1 mM MgCl2, 100 mM KCI, 10 mM K-HEPES (pH 7.7), 50 tg/ml cytocholasin B, 1 mM DTT, 10 ,ug/ml aprotinin and leupeptin] and gently packed at 500 g for 30 s to remove excess buffer. Eggs were then incubated at room temperature until 15 min had elapsed since the electric pulse and were transferred for a further 15 min at 4°C. Eggs were crushed at 12 000 g for 15 min, then the cytoplasmic layer was added to an ATP regenerating system consisting of 10 mM creatine phosphate, 80 gg/ml creatine kinase, 2 mM ATP and 1 mM MgCl2 (final concentrations). Finally the extract was spun at 12 000 g for a further 10 min and the supernatant used immediately for experiments. CSF-arrested extracts Fresh inactivated Xenopus eggs laid in 0.5 XMMR were dejellied, rinsed three times in 0.2XMMR and twice more in XB buffer supplemented with 5 mM EGTA to prevent activation. Eggs were packed at 500 g for 30 s and excess buffer removed. Eggs were immediately crushed at 12 000 g for 15 min and further treated as described for cycling extracts Nuclear and spindle assembly Demembranated frog sperm nuclei, prepared as described previously (Lohka and Masui, 1984), were added (200/pl) simultaneously with rhodamine-labeled tubulin (150 gg/ml, a gift of Dr Didier Job) to either CSF-arrested or cycling extracts. Nuclear formation, nuclear envelope breakdown and spindle assembly were followed by observation with a fluorescent microscope, using Hoechst 33 342 to stain chromosomes and tubulin fluorescence to visualize microtubules.

Induction of anaphase in CSF extracts Metaphase spindles, allowed to assemble for 80 min in CSF extract (Shamu and Murray, 1992), were placed in microfuge tubes and either CaCl2 (0.4 mM) or constitutively active rat brain CaM KII (2 p1/20 p1 of extract) added. The mutant CaM KII used in these experiments

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N.Morin et al. corresponds to fragment 1-290 of CaM Klla, translated in reticulocyte lysate from the corresponding mRNA (Lorca et al., 1993). The CaM KII (281-302) inhibitory peptide (Lorca et al., 1993), when added (as described in the text), was used at 200 gg/ml. Control cycling extracts entered anaphase without further addition. GST-cyclin B, when used, was added to such extracts when nuclear envelope breakdown occurred.

DNA replication DNA replication was assayed by measuring the incorporation of radiolabeled [ac-32P]dCTP into DNA. 10 gl aliquots of extract were removed at the indicated times and incubated with 1 g1 of [a-32P]dCTP (3000 Ci/mmol). Reactions were stopped by the addition of an equal volume of stop buffer (2% SDS, 10 mM EDTA, 10 mM Tris-HCI, pH 7.0), followed by incubation with proteinase K (I mg/ml) for 1 h at 37°C. Samples were loaded and run on a 0.8% agarose gel. Gels were dried and autoradiographed.

Cyclin degradation To follow cyclin degradation in cycling extracts, full-length human cyclin B1 subcloned in the BamHI site of pGEM 4Z (a gift from Dr J.Pines) was transcribed with SP6 polymerase (Lorca et al., 1991). In vitro-translated cyclin B1, made in rabbit reticulocyte lysate in the presence of [35S]methionine, was added (2 g1/30 ,l) once nuclei had been formed (50 min after sperm chromatin). 2 g1 aliquots were removed, transferred in SDS gel sample buffer and run on 12% SDS-PAGE. Gels were treated with an enhancer for low energy radiation, dried and exposed for fluorography at -70°C.

GST-cyclin fusion protein and methylated ubiquitin The GST-cyclin B construct, a gift from Dr J.Gauthier, contains in pGEX2 (Solomon et al., 1990) the NcoI-EcoRI 13-409 fragment of sea urchin cyclin B fused with glutathione transferase. It was expressed essentially as described by Smith and Johnson (1988). The soluble fraction (-0.1%) was purified on glutathione-agarose beads and concentrated to 250 ,g/ml before use. Methylated ubiquitin was prepared according to Hershko and Heller (1985).

Antibodies The polyclonal antibody used to monitor Xenopus c-mos by Western blotting and ECL was purchased from Santa Cruz Biotech, USA (catalog number C237).

Acknowledgements This work was supported by grant number 6241 from Association pour la Recherche sur le Cancer (ARC) to M.D.

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