Distinct roles of PP1 and PP2A‐like phosphatases in control of microtubule dynamics during mitosis

Régis Tournebize; Søren S. L. Andersen; Fulvia Verde; Marcel Dorée; Eric Karsenti; Anthony A. Hyman

doi:10.1093/emboj/16.18.5537

Distinct roles of PP1 and PP2A‐like phosphatases in control of microtubule dynamics during mitosis

Tournebize, Régis; Andersen, Søren S. L.; Verde, Fulvia; Dorée, Marcel; Karsenti, Eric; Hyman, Anthony A. 1997-09-15 00:00:00 The EMBO Journal Vol.16 No.18 pp.5537–5549, 1997 Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis phases, called a catastrophe, and between shrinkage and Re´gis Tournebize , Søren S.L.Andersen, 2 3 growth phases, called a rescue (Walker et al., 1988; Fulvia Verde , Marcel Dore´e , Eric Karsenti Hyman and Karsenti, 1996). During the transition from and Anthony A.Hyman interphase to mitosis, the primary effect on microtubule Cell Biology Program, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, dynamics is an increase in the catastrophe rate (Belmont Germany, Department of Biochemistry and Molecular Biology, et al., 1990; Verde et al., 1992). However, little is known University of Miami School of Medicine, PO Box 016129, Miami, about direct effectors of microtubule dynamics or how FL 33136-1015, USA and CRBM-CNRS, BP 5051, Route de Mende, they are regulated. 34033 Montpellier, France Changes in microtubule dynamics during the transitions Corresponding author into and out of mitosis are controlled by the activity of e-mail: [email protected] cdc2 kinase which phosphorylates a large variety of molecules either directly or indirectly (Belmont et al., Assembly of a mitotic spindle requires the accurate 1990; Verde et al., 1990, 1992). It is likely that the control regulation of microtubule dynamics which is accomp- of microtubule dynamics during these cell cycle transitions lished, at least in part, by phosphorylation–dephos- is determined by the balance between cdc2 kinase and phorylation reactions. Here we have investigated the phosphatases that directly or indirectly oppose its action role of serine-threonine phosphatases in the control on target molecules. Such phosphorylation balances may of microtubule dynamics using speciﬁc inhibitors in also be involved in the stabilization of microtubules by Xenopus egg extracts. Type 2A phosphatases are chromosomes during spindle assembly. required to maintain the short steady-state length of Analysis of mutants in serine-threonine phosphatases microtubules in mitosis by regulating the level of indicates that they participate in ensuring correct chromo- microtubule catastrophes, in part by controlling the the some segregation at mitosis. Some phenotypes suggest microtubule-destabilizing activity and phosphorylation that they do so by regulating cell cycle control. For of Op18/stathmin. Type 1 phosphatases are only instance, type 2A phosphatase (PP2A) negatively regulates required for control of microtubule dynamics during cdc2 kinase at the onset of mitosis in Xenopus (Felix the transitions into and out of mitosis. Thus, although et al., 1990; Clarke et al., 1993; Lee et al., 1994; Lee, both type 2A and type 1 phosphatases are involved in 1995), starﬁsh (Picard et al., 1989, 1991) and both in the regulation of microtubule dynamics, they have ﬁssion and budding yeasts (Kinoshita et al., 1993; Lin distinct, non-overlapping roles. and Arndt, 1995; Evans and Stark, 1997). Inhibition of type Keywords: microtubule dynamics/mitosis/Op18/ 1 phosphatases (PP1) arrests the cells at the metaphase– phosphatase/spindle anaphase transition (Booher and Beach, 1989; Ohkura et al., 1989; Fernandez et al., 1992; Hisamoto et al., 1994; Ishii et al., 1996) because PP1 may be required to activate Introduction the anaphase-promoting complex (APC), required to degrade cyclin B (Ishii et al., 1996). However, other Correct chromosome segregation during mitosis requires phenotypes suggest that phosphatases are also involved in the assembly of a microtubule-based structure called the the control of structural events associated with mitosis. mitotic spindle. This assembly involves dramatic changes Mutants in PP2A genes show defects in spindle assembly in microtubule turnover: from a relatively stable state in and microtubule growth (Kinoshita et al., 1993; Snaith interphase, they become highly dynamic at the onset of et al., 1996; Evans and Stark, 1997), as do some mutants mitosis, their half-life changing from ~5 min to 45 s (Zhai in PP2A regulatory subunit genes (Gomes et al., 1993; et al., 1996). During assembly of the spindle, some mitotic Kinoshita et al., 1996). Mutants in PP1 genes in Saccharo- microtubules appear to be preferentially stabilized around myces cerevisiae (Black et al., 1995), Schizosaccharo- the chromosomes, and this contributes to generating the myces pombe (Ohkura et al., 1988, 1989), Aspergillus bipolar shape of the spindle. Thus, it is important to nidulans (Doonan and Morris, 1989) and Drosophila understand how microtubule dynamics are regulated both (Axton et al., 1990) show complex mitotic phenotypes globally and locally around the chromosomes. with condensed chromosomes, abnormal spindles and Changes in general microtubule stability can be chromosome separation malfunction. Since alterations in described by examining the sum of the dynamic properties the phosphorylation state of proteins involved in spindle of individual microtubules. Microtubules either grow or assembly and function can have effects on cell cycle shrink, the transition from one state to the other being progression and vice versa, the exact roles of phosphatases unpredictable, a property known as dynamic instability. Thus, the behavior of a microtubule is deﬁned by four during mitosis are unclear. parameters: the growth rate, the shrinkage rate and the We have addressed this problem using Xenopus egg frequencies of transitions between growth and shrinkage extracts as they allow study of the role of phosphatases © Oxford University Press 5537 R.Tournebize et al. in the regulation of microtubule dynamics and spindle initiation of spindle assembly, metaphase spindles were assembly independently of their role in cell cycle pro- present. Then 0.4 μM okadaic acid was added and aliquots gression. We have used two speciﬁc inhibitors of of the extract were ﬁxed and observed. In the absence of the serine-threonine phosphatases. Okadaic acid at low con- drug, microtubules were organized in a typical ellipsoidal centrations inhibits PP2A but does not inhibit PP1 activity shape, and chromosomes were aligned on the metaphase (for review, see Cohen, 1989; Shenolikar, 1994). Con- plate (Figure 1A). In contrast, bipolar spindles could not versely, inhibitor 2 (I-2), a 23.8 kDa protein, speciﬁcally be found 20 min after addition of 0.4 μM okadaic acid, inhibits PP1 activity by binding to the catalytic subunit the microtubules were up to 100 μm long, with an average (Cohen, 1989; Shenolikar, 1994). We ﬁnd that PP2A is length of 77 μm, and the chromosomes were scattered required to maintain the short steady-state length of throughout the structure (Figure 1A). Moreover, numerous microtubules during mitosis, in part by regulating Op18/ free microtubules could be observed in the extract. stathmin (Sobel, 1991), a recently identiﬁed molecule This effect of okadaic acid could reﬂect an indirect involved in the control of microtubule dynamics. PP1 has effect on cell cycle progression, for example by inducing a different role, and is involved in control of microtubule the return to interphase, or a direct effect on microtubule dynamics during the transitions between interphase and dynamics. Indeed, this drug is known to activate cdc2 mitosis. kinase in the presence of sub-threshold levels of cyclin (Felix et al., 1990), but also to induce cyclin degradation after some time of incubation (Felix et al., 1990; Lorca Results et al., 1991), which results in ﬁnal cdc2 kinase inactivation. Extracts made from Xenopus eggs can be arrested in In order to study PP2A function independently of any deﬁned states of the cell cycle, either in interphase or in effect on cdc2 kinase activity, we repeated the experiment mitosis, and their position in the cell cycle monitored by in the presence of cyclin Δ90. Because this cyclin construct measuring the activity of cdc2 kinase which is low in is non-degradable, it produces a constitutively active interphase and high in mitosis. Extracts can be arrested kinase when it combines with the endogenous cdc2 cata- in mitosis in two ways. First, extracts made from metaphase lytic subunit of the extract (Glotzer et al., 1991). Under II-arrested oocytes (CSF extracts) contain an activity such conditions, cdc2 kinase activity remained high after which maintains cdc2 kinase in an active form. Second, addition of 0.4 μM okadaic acid. However, microtubules a non-degradable version of cyclin B, cyclin Δ90, can be became long, and bipolar spindles disappeared from the added to extracts in order to stabilize the cdc2 kinase extract, replaced by structures such as that shown in activity. These latter extracts retain much of the mitotic Figure 1A. We conclude that even in the presence of high state even though the APC and the machinery responsible cyclin B–cdc2 kinase activity, microtubules grow long for cyclin degradation are active (Murray et al., 1989). In after inhibition of PP2A activity. Moreover, this result particular, mitotic spindles remain intact with a high also suggests that short microtubules are required to make microtubule turnover in such extracts, although anaphase a spindle. To test this further, we added 0.4 μM okadaic A occurs (Holloway et al., 1993; Surana et al., 1993). acid and sperm nuclei simultaneously to CSF extracts pre- Spindle assembly can be induced in Xenopus extracts incubated with cyclin Δ90. This resulted in the formation by adding sperm nuclei to a CSF extract that is then of asters with long microtubules (data not shown), but no released into interphase in order to allow DNA replication bipolar spindles were assembled. Thus bipolar spindles and centrosome duplication. Then, the extract is sent back may not assemble in the absence of PP2A activity because into metaphase by adding more CSF extract or cyclin of the length of the microtubules formed under such Δ90. This results in the migration of centrosomes around conditions. the nucleus and spindle assembly in a succession of prophase and metaphase ﬁgures (Sawin and Mitchison, PP2A inhibition affects the catastrophe rate of 1991; Shamu and Murray, 1992; Tournebize and Heald, microtubules 1996). Alternatively, spindles can be assembled by incubat- To understand how PP2A activity could control the steady- ing sperm nuclei directly in CSF extracts. Microtubules state length of microtubules during mitosis, we investigated grow from the sperm centrosome towards chromatin, how inhibition of PP2A affected the dynamics of individual forming half-spindles, then two half-spindles fuse, forming microtubules by time-lapse video microscopy. Extracts a bipolar spindle (Sawin and Mitchison, 1991; Tournebize pre-incubated with cyclin Δ90 were treated with 0.4 μM and Heald, 1996). Microtubule dynamics can be examined okadaic acid and supplemented with puriﬁed centrosomes separately by adding centrosomes in the absence of and rhodamine–tubulin. Images of microtubules were chromosomes. Under these conditions, one can see micro- recorded over time and the parameters of microtubule tubule ends better because the microtubule density is dynamics deduced from the analysis of the videos. Figure lower. Additionally, one can determine the changes in 1B shows images taken from two videos, in the presence microtubule dynamics in the absence of the inﬂuence of and absence of 0.4 μM okadaic acid. Microtubules were mitotic chromatin. longer in the presence of okadaic acid and we also noticed a signiﬁcant increase in spontaneous assembly. Analysis A PP2A-like activity keeps microtubules short of individual microtubules in several videos showed that during metaphase inhibition of PP2A activity reduced the level of cata- In order to determine whether PP2A activity was involved strophes by 1.5- to 3-fold, depending on the experiment in the regulation of microtubule length during metaphase, (Table I). The average values from ﬁve independent we tested the effect of okadaic acid on the morphology experiments showed that okadaic acid addition decreased of spindles assembled in extracts. At 60 min after the the catastrophe rate from 2.44 catastrophes/min to 1.22 5538 Phosphatases and mitosis Fig. 1. (A) Addition of okadaic acid to spindles induces microtubule growth. Spindles assembled in cyclin Δ90 Xenopus egg extracts were mock treated (control) or treated with 0.4 μM okadaic acid for 20 min at 20°C. (B) Addition of 0.4 μM okadaic acid increases microtubule steady-state length. Images taken at the same recording time from videos in the presence (bottom) or absence (top) of 0.4 μM okadaic acid. Time is h:min:s. Bars are 10 μm. Table I. Addition of okadaic acid to extracts reduces catastrophe frequencies Growth rate Shrinkage rate Catastrophe frequency Rescue frequency Calculated microtubule (μm/min) (μm/min) (events/min) (events/min) length (μm) Control 11.44 1.21 (105) 13.49 1.90 (91) 2.44 0.54 (91) 0.70 (29) 7.4 Okadaic acid 13.00 1.24 (87) 16.37 2.81 (58) 1.22 0.21 (58) 1.40 (17) 34.4 Okadaic acid (0.4 μM) was added to cyclin Δ90 extracts. After 20 min incubation at 20°C, extracts were frozen and aliquots of these used for microtubule dynamics measurements for 5 min. Growth rate, shrinkage rate and catastrophe frequency were determined after measurements of microtubules over time. Rescue frequencies reported here are not statistically signiﬁcant. Values are means standard error of the mean (number of total events measured). Average steady-state length L was calculated using the measured values. catastrophes/min. Average growth and shrinkage rates acid. We found an average microtubule length L of were not signiﬁcantly affected, being ~12 and 15 μm/min 7.4 μm in control and 34.4 μm in extracts treated with respectively. Rescues were 0.70 event/min in control and 0.4 μM okadaic acid (Table I). Thus, inhibition of PP2A 1.40 events/min in the presence of okadaic acid. Due to results in a 5-fold increase in microtubule steady-state a low number of events observed, this difference is not length L due to only a 2-fold reduction in the cata- statistically signiﬁcant. Okadaic acid addition had no effect strophe rate. on interphase microtubule dynamics (data not shown). Verde et al. (1992) showed that the steady-state length PP2A regulates Op18/stathmin phosphorylation of microtubules is related directly to the parameters of We were interested in ﬁnding targets of PP2A that could microtubule dynamics and that microtubule steady-state regulate microtubule dynamics. Since Op18/stathmin is length can be calculated from the values of microtubule thought to destabilize microtubules in Xenopus egg extracts dynamics using a simple equation (see Materials and (Belmont and Mitchison, 1996), and since phosphorylation methods). Using this formula, we calculated the micro- is known to regulate its activity (Marklund et al., 1996; tubule steady-state length L in the presence and Horwitz et al., 1997), we examined the role of PP2A in absence of 0.4 μM okadaic acid. We assumed that the regulating Op18/stathmin phosphorylation. Okadaic acid rescue frequency was not affected by treatment with was added to an extract containing cyclin Δ90 in order to okadaic acid, taking a value of 1.05 rescues/min, the inhibit PP2A, and [γ- P]ATP was added in order to average of the values measured with and without okadaic label phosphorylated proteins. Op18/stathmin was then 5539 R.Tournebize et al. immunopuriﬁed and the level of P incorporation (Marklund et al., 1996; Horwitz et al., 1997), while monitored. In untreated extracts, Op18/stathmin was phosphorylated Op18/stathmin does not. Thus our experi- poorly phosphorylated. Addition of increasing amounts of ments suggest that, normally, Op18/stathmin in extracts okadaic acid to the extract resulted in a gradual increase is dephosphorylated and active. PP2A inhibition promotes in the level of P incorporated into Op18/stathmin protein phosphorylation of OP18/stathmin, thus microtubules (Figure 2B). Quantiﬁcation of the level of P incorporated become more stable. If PP2A inhibition promoted micro- showed that at 0.5 μM okadaic acid, Op18/stathmin tubule growth by promoting hyperphosphorylation of contained four times more P than the control. For Op18/stathmin, then a mutant Op18/stathmin which cannot comparison, addition of 3 μM I-2, which speciﬁcally be phosphorylated should overcome the effect of PP2A inhibits PP1, only induced a 1.5-fold increase in the level inhibition on microtubule steady-state length. In order to of P incorporated. Taken together, these observations test this idea, we used puriﬁed Op18/stathmin protein in show that PP2A activity maintains Op18/stathmin in a which the four known phosphorylated serines had been low phosphorylation state in metaphase extracts. mutated to alanine. Okadaic acid (0.4 μM) was added together with puriﬁed Op18/stathmin to pre-assembled PP2A regulates microtubule dynamics by spindles that were then ﬁxed and observed. Twenty minutes controlling the phosphorylation state of after addition of the mutant Op18/stathmin protein together Op18/stathmin with okadaic acid, most spindles were bipolar (Figure 3), Previous experiments had suggested that, when unphos- but ~20% of them had short microtubules uniformly phorylated, Op18/stathmin destabilizes microtubules wrapped around chromatin without clear poles, forming round ‘spindles’ (data not shown). Addition of okadaic acid together with wild-type Op18/stathmin generated the same structures as okadaic acid alone (Figure 3). Thus, addition of unphosphorylatable Op18/stathmin rescues the effect of okadaic acid on microtubule growth while wild- type Op18/stathmin does not. Op18/stathmin depletion decreases the catastrophe rate The previous results strongly suggested that PP2A controls microtubule dynamics by regulating the phosphorylation of Op18/stathmin, but did not rule out the possibility that exogenously added mutant Op18/stathmin depolymerized microtubules independently of PP2A. If PP2A acted through Op18/stathmin to control the catastrophe rate of microtubules, then depletion of Op18/stathmin should decrease the catastrophe rate as well. We therefore depleted Op18/stathmin from extracts and measured microtubule Fig. 2. The Op18/stathmin phosphorylation state is regulated by PP2A. dynamics. Results from four independent depletion experi- Different concentrations of okadaic acid and I-2 were added to cyclin ments are shown in Figure 4. Upon removal of ~95% of Δ90 extracts containing [γ- P]ATP. After 20 min, Op18/stathmin was immunoprecipitated and run on a 15% acrylamide gel. (A) Silver Op18/stathmin, the catastrophe frequency decreased 1.5- staining of the gel; (B) an autoradiogram. Phosphorylation of to 5-fold, depending on the experiments. On average, Op18/stathmin increased four times in the presence of 0.5 μM okadaic catastrophes decreased from 1.46 events/min in control acid and 1.5 times in the presence of 3 μM I-2. The arrow points to treated extracts to 0.86 events/min in Op18/stathmin- Op18/stathmin, which resolves as a triplet. Molecular weight markers are indicated. depleted extracts. Thus Op18/stathmin is required to Fig. 3. Non-phosphorylatable Op18/stathmin induces microtubule depolymerization. Spindles were assembled in cyclin Δ90 extracts and okadaic acid was added alone (left panel), together with 250 μg/ml of human Op18/stathmin protein with its four phosphorylation sites mutated to alanine (middle panel) or with 250 μg/ml wild-type human Op18/stathmin (right panel). Spindles were ﬁxed 20 min after incubation at 20°C. Bar is 10 μm. 5540 Phosphatases and mitosis Fig. 4. Catastrophe frequency decreases after Op18/stathmin depletion and okadaic acid addition. Op18/stathmin was depleted from extracts containing cyclin Δ90, and microtubule dynamics were measured. Catastrophe frequency decreases after Op18/stathmin depletion Fig. 5. PP1 is not required for spindle assembly. Spindle assembly was (ΔOp18) and after 0.4 μM okadaic acid addition (MockOA). No monitored in the presence or the absence of I-2. Extracts containing signiﬁcant decrease in catastrophe is observed after addition of 0.4 μM nuclei were released into interphase for 80 min before mitosis was okadaic acid to Op18/stathmin-depleted extract (ΔOp18OA). Values triggered by addition of CSF extract together with 3 μM I-2 (1 μM I-2 are means standard error of the mean. was then added every 15 min). Spindles were ﬁxed every 10 min and the percentage of spindles in metaphase scored. No statistical difference was observed upon PP1 inhibition compared with the maintain a high level of catastrophes during metaphase. control. We also analyzed the effect of Op18/stathmin depletion on the growth and shrinkage rates of microtubules. These length was not affected over 60 min of observation (data rates were not statistically changed by Op18/stathmin not shown). These results suggested that once the extract depletion. Using these values, we calculated the average contains a high level of cdc2 kinase activity, PP1 is not length L of microtubules. We found that they are required to regulate microtubule dynamics and spindle 7.9 μm long in mock-depleted extracts and 100 μmin assembly. Op18/stathmin-depleted extracts. These values show that Op18/stathmin is a key regulator of microtubule length PP1 and cdc2 kinase act antagonistically to control in vitro. the steady-state length of microtubules at the In order to test whether Op18/stathmin could account onset of mitosis for all the changes in catastrophes observed upon PP2A Although inhibition of PP1 had no effect on spindle inhibition, we added 0.4 μM okadaic acid to Op18/ assembly, we wondered whether it could participate in the stathmin- or mock-depleted extracts. The catastrophe fre- regulation of microtubule dynamics during the early phase quency dropped from 0.85 to 0.59 in Op18/stathmin- of mitosis when cdc2 kinase begins to act on microtubule depleted extracts. However, this difference was not found dynamics, in prophase. To test this hypothesis, we made to be statistically signiﬁcant (Figure 4). Growth and use of puriﬁed cdc2 kinase which, when added to an shrinkage rates were not signiﬁcantly changed. The calcu- interphase extract, triggers mitotic microtubule dynamics lated microtubule length L after addition of 0.4 μM (Verde et al., 1990). We reasoned that if PP1 is opposing okadaic acid was 61.2 μm in mock-treated extracts and cdc2 kinase activity during the transition of microtubule 100 μm in Op18/stathmin-depleted extracts. These dynamics from an interphase to a metaphase state, then results show that Op18/stathmin accounts for most of the inhibition of PP1 should speed up the rate at which cdc2 catastrophes regulated by PP2A in Xenopus extracts. In kinase phosphorylates substrates and microtubules become conclusion, PP2A maintains the short steady-state length dynamic. Cdc2 kinase was added to interphase extracts of microtubules by regulating the catastrophe rate, mainly with or without I-2. Extracts were complemented with through regulation of the activity of Op18/stathmin. centrosomes and rhodamine–tubulin, ﬁxed 15 min later and spun down onto coverslips, and the length of microtub- PP1 is required for the control of microtubule ules were measured. As shown in Figure 6, microtubules dynamics during the transitions into and out of are long in interphase extracts (25 μm) and become shorter mitosis upon addition of cdc2 kinase (Figure 6A). In the presence To examine the role of PP1 in the regulation of microtubule of PP1 inhibitor, 9–10 pmol/μl/min of cdc2 kinase activity dynamics, we used a speciﬁc inhibitor of PP1, I-2, a were required to convert the average microtubule length 23.8 kDa protein which binds speciﬁcally to the catalytic to a mitotic state (Figure 6B and C) while 20 pmol/μl/ site of PP1 to inhibit its activity. We added I-2 to min were necessary in the absence of inhibitor. This result interphasic and mitotic extracts and observed microtubule suggests that PP1 and cdc2 kinase have opposing effects length and dynamics. Inhibition of PP1 had no effect on the in controlling microtubule dynamics during the interphase parameters of microtubule dynamics in either interphase or to metaphase transition. metaphase (data not shown). To test whether PP1 is required for spindle assembly, extracts containing sperm PP1 participates in the regulation of global nuclei were released into interphase and I-2 was added at changes in microtubule dynamics during the the same time as CSF extracts. The kinetics of spindle metaphase to anaphase transition assembly were statistically similar in the presence and The previous results suggested that PP1 opposed cdc2 absence of I-2 (Figure 5), and maintenance of spindle kinase during the onset of metaphase. We wondered 5541 R.Tournebize et al. events/min. In control extracts, the calculated microtubule length L was 8.7 μm in metaphase and 100 μm30 min after anaphase induction. In the absence of PP1 activity, the calculated microtubule length L remained unchanged at ~5–8 μm (Figure 7F). Therefore, PP1 is required in anaphase to increase the steady-state length of microtubules by increasing the growth rate and decreasing the catastrophe rate. PP1 is also required for spindle microtubule elongation and chromosome separation in anaphase The previous results indicated that PP1 was required for microtubule elongation in anaphase in the absence of chromosomes. We wanted to examine what would happen to spindle microtubules. As shown in Figure 8A, under normal conditions, chromosomes start to separate and move towards the poles 10 min after calcium addition. By 25 min, anaphase is completed. When PP1 was inhibited, spindles gradually lost microtubules and the poles moved towards chromatin and eventually fell apart, forming half-spindles (Figure 8B). No clear chromosome segregation was observed. Similar observations were made using CaMKII to trigger anaphase. We quantiﬁed spindle length during anaphase with and without PP1. At the end of anaphase, spindles were 42 μm long in the control and 22 μm in the absence of PP1 activity (Figure 8C). We looked at chromosome separation under such conditions. As shown in Figure 8D, chromosomes moved towards poles in the control while they stayed in the middle of the spindle when I-2 was used. One hour after CaMKII addition, the microtubules had not returned to their Fig. 6. PP1 opposes the effect of cdc2 kinase on microtubule length. interphase length and, in addition, chromatin was still Increasing amounts of puriﬁed starﬁsh cdc2 kinase were added to condensed as previously reported (Axton et al., 1990). interphase extract in the absence (A) or the presence (B) of I-2. Less These results show that PP1 is necessary both for spindle cdc2 kinase is required to obtain mitotic microtubule length in the presence of I-2 (C). Numbers in the bottom right of the pictures stability and elongation, and for chromatin decondensation indicate cdc2 kinase activity (pmol/μl/min). during anaphase, and that this is not due to an effect of PP1 inhibition on the inactivation of cdc2 kinase. whether PP1 was also required to increase the steady- state length of microtubules during the onset of anaphase. Discussion To measure these changes, we used a downstream effector of calcium in the cascade triggering cyclin degradation at Since the initial demonstration that microtubule length is anaphase onset: the calcium/calmodulin-dependent kinase governed during metaphase by phosphorylation– II (CaMKII) (Lorca et al., 1993; Morin et al., 1994). We dephosphorylation reactions under the control of cdc2 looked at cdc2 kinase activity after induction of anaphase kinase (Verde et al., 1990) and that this was mediated by in the presence and absence of I-2. In both cases, cdc2 an increase in catastrophe rate (Belmont et al., 1990; kinase activity dropped to interphase levels by 30 min Verde et al., 1992), little progress has been made in the (Figure 7A) with identical kinetics, showing that, at least characterization of the biochemical pathways or signaling in Xenopus extracts, PP1 is not required for the inactivation mechanisms involved. Here, we undertook a study of the of cdc2 kinase. role of type 1 and type 2A-like phosphatases in these Microtubule dynamics were recorded at 15 min intervals processes during mitosis. We show that PP2A is required after anaphase induction by CaMKII. In control extracts, continuously in mitosis to control the short steady-state chromosome to pole movement was completed 30 min length of microtubules, primarily by regulating the after CAMKII addition (Figure 7B and see below). At phosphorylation state of the microtubule-destabilizing this time, the catastrophe frequency decreased from 2 to factor Op18/stathmin. In contrast, PP1 is required to 1 event/min, and the growth rate increased from 9.9 to stabilize microtubules during the transitions between 16.55 μm/min (Figure 7C and E). The shrinkage rate was interphase and mitosis. Thus these two phosphatases have not changed during this transition (Figure 7D). In contrast, distinct roles in the control of microtubule dynamics in extracts treated with I-2, catastrophe frequency, growth during mitotic progression. and shrinkage rates were identical for the ﬁrst 30 min We have characterized these activities using inhibitors after induction of anaphase (Figure 7C–E). We calculated of serine-threonine phosphatases. In vitro, the IC of the average length L of microtubules using the values okadaic acid is 0.2 nM for PP2A and 20 nM for PP1 (for measured and assuming a constant rescue rate of 1.05 review, see Cohen, 1989). These concentrations are much 5542 Phosphatases and mitosis Fig. 7. PP1 is required for returning from metaphase to interphase microtubule dynamics. (A) Cdc2 kinase inactivation in anaphase induced by CaMKII is not affected by PP1 inhibition. (B) The distance from chromosomes to the pole in control extract decreases as chromosomes segregate. (C–E) PP1 prevents changes in microtubule dynamics in anaphase. In these experiments, CSF extracts were pre-incubated for 15 min with (gray bars) or without (black bars) 3 μM I-2 before anaphase was induced with CaMKII. Microtubule dynamics were recorded at 0, 15 and 30 min for 5–7 min. Growth rate (C), shrinkage rate (D) and catastrophe frequency (E) were determined after measurements of individual microtubules over time. Rescue frequency is omitted here because too few events were observed and values are not statistically signiﬁcant. (F) Microtubule length L does not increase in the presence of I-2 during anaphase. Calculated microtubule lengths were determined using the measured values. Rescues were assumed to be 1.05 events/min. Data in (B–E) are means standard error of the mean. lower than that used in our study, but it is known that the in PP2A regulatory subunits also display defects in micro- inhibitory activity of these inhibitors is altered by high tubule organization (Gomes et al., 1993; Kinoshita et al., protein concentration (Ingebritsen et al., 1983) and our 1996). Furthermore, associated subunits of PP2A bind to extracts have a protein concentration of ~60–80 mg/ml. microtubules and PP2A is associated with microtubules We have measured phosphatase activities in concentrated in tissue culture cells (Sontag et al., 1995). This suggests extracts under the same conditions as used to assay that PP2A is indeed the phosphatase catalytic subunit microtubule dynamics and spindle assembly. We found involved in controlling microtubule length. that under these conditions, 0.4 μM okadaic acid is Previous results had also shown that inhibition of required to produce a 50% inhibition of PP2A-like activi- phosphatases using okadaic acid results in the formation ties, whereas 3 μM I-2 is required to inhibit PP1 fully. of long microtubules. In starﬁsh (Picard et al., 1991), Low concentrations of okadaic acid may affect the activity mouse oocytes (de Pennart et al., 1993) and GH4 rat of catalytic subunits other than the typical PP2A. Indeed, pituitary cells (Van Dolah and Ramsdell, 1992), spindles four phosphatases, PP3 (Honkanen et al., 1991), PP4 were shown to be destroyed and replaced by extremely (Brewis et al., 1993) (previously named PPX), PP5 (Chen long microtubule networks. This is consistent with our et al., 1994) and PPV in Drosophila (Armstrong et al., results but, in some of these previous experiments, it was 1995), have been shown recently to be highly sensitive to not possible to know whether a type 1 or 2A phosphatase okadaic acid and insensitive to I-2. Therefore, we cannot was inhibited. Interestingly, in clariﬁed interphasic extracts rule out the possibility that one of these is the inhibited from sea urchin, addition of high concentrations of okadaic phosphatase in our experiments. However, PP2A mutants acid (1–2.5 μM) resulted in a decrease in rescues and a in Drosophila, S.pombe and S.cerevisiae show abnormal slight increase in catastrophes (Gliksman et al., 1992). microtubule morphology (Kinoshita et al., 1993; Snaith However, these extracts had a lower protein concentration et al., 1996; Evans and Stark, 1997), with an extensive (20–25 mg/ml) and it is likely that both PP1 and PP2A microtubule network similar to the one seen here. Mutants were inhibited. The effects observed are probably the 5543 R.Tournebize et al. Fig. 8. PP1 is required for spindle stability and chromosome segregation in anaphase. Spindles were assembled and PP1 was inhibited for 15 min before anaphase was induced by CaMKII. Spindles were ﬁxed at various time points. (A) Normal metaphase and anaphase spindles. (B) Collapsed and half-spindles observed in anaphase in the presence of I-2. Graphs show the percentage of spindles in metaphase (d) and anaphase (s)inthe absence (A) or presence (B) of I-2 during anaphase. (C) Spindle length in the absence (j) or presence (u) of I-2. (D) Chromosome position in the spindle during anaphase in the absence (j) or presence (u) of I-2. Chromosome position is determined by the chromosome to pole distance divided by the pole to pole distance. Bar is 10 μm. consequence of a complex regulation by many phos- and fungi? One major difference is the lack of checkpoint phatases which may act in many different ways. In controls in Xenopus extracts under our conditions. Thus clariﬁed Xenopus interphase extracts, addition of high PP1 mutants may cause a metaphase delay in fungi by concentrations of okadaic acid results in a decrease in activating the spindle assembly checkpoint (for review, growth and shrinkage rates, while it has little effect on see Wells, 1996). Indeed, in S.cerevisiae, the PP1 encoded these rates in mitotic extracts (Parsons and Salmon, 1997). by the GLC7 gene has been shown to regulate chromosome We also noticed that catastrophes were decreased in mitotic segregation (Francisco and Chan, 1994; Hisamoto et al., clariﬁed extracts (R.Tournebize and R.Heald, unpublished 1994; Black et al., 1995). Recent genetic data in S.pombe observations). One possibility is that most of PP2A is suggest that PP1 may regulate the APC and, therefore, depleted from clariﬁed extracts (A.A.Hyman, unpublished exit from mitosis (Ishii et al., 1996). However, since the observation), thus resulting in Op18/stathmin inactivation. APC regulates cyclin destruction and, therefore, cdc2 Because PP1 is the only phosphatase known to be kinase inactivation, we would expect to see a mitotic inhibited efﬁciently by I-2, this small polypeptide can be delay in Xenopus if PP1 inhibition down-regulates the used to determine what is regulated by PP1-like enzymes. APC. As this is not observed in Xenopus extracts, it is Again, we determined that adding 3 μM I-2 followed by possible that PP1 acts upstream of the APC through a regular addition of 1 μM produced a complete inhibition spindle checkpoint rather than directly in cyclin of PP1 in our concentrated extracts. Our results show that degradation. PP1 has a particular role in the control of microtubule turnover in Xenopus extracts, during the switches between Regulation of microtubule dynamics and interphase and mitosis. In addition, PP1 is required for steady-state length during metaphase by PP2A chromosome decondensation, conﬁrming previous results and Op18/stathmin in Drosophila (Axton et al., 1990). However, PP1 does Our results suggest that Op18/stathmin is a major down- not seem to be required for cell cycle changes necessary stream substrate of PP2A in the regulation of the steady- for exit from mitosis in Xenopus egg extracts, while state length of microtubules. This protein regulates the inhibition of PP1 in fungi causes a metaphase arrest or length of microtubules both in Xenopus extracts (Belmont delay. Why should there be a difference between Xenopus and Mitchison, 1996; this study) and in tissue culture cells 5544 Phosphatases and mitosis (Marklund et al., 1996; Horwitz et al., 1997), and this during spindle assembly and disassembly (Andersen effect is regulated by phosphorylation. The phosphorylated et al., 1997). form of Op18/stathmin loses its microtubule-destabilizing activity (Marklund et al., 1996; Horwitz et al., 1997). Our Regulation of microtubule dynamics by PP1 during results in Xenopus conﬁrm these observations. Moreover, the transitions from interphase to mitosis and they suggest that the phosphatase which dephosphorylates back to interphase Op18/stathmin is PP2A. Although the kinase that opposes We have shown that PP1 is required for reorganization of PP2A is unidentiﬁed, MAP kinase and the cdc2 kinase the microtubule network during the transitions between are likely candidates (Larsson et al., 1995). This would interphase and mitosis. It is hard to say whether PP1 is make sense since PP2A opposes cdc2 kinase and some regulating microtubule-stabilizing or -destabilizing factors MAP kinase activities on many substrates like the brain or both during entry into mitosis and during anaphase. microtubule-associated protein tau (Goedert et al., 1992; One clue comes from the analysis of the dynamic para- Ferrigno et al., 1993). Although it is formally possible meters during exit from mitosis. We have shown that the that PP2A regulates Op18/stathmin by increasing MAP growth rate increases during exit from mitosis, but that kinase activity (Shibuya et al., 1992; Nebreda and Hunt, this increase in growth rate is blocked in the absence of 1993), this is unlikely because, in extracts treated with PP1. Since MAPs are generally thought to stimulate cyclin Δ90, MAP kinase activity is already high (Shibuya microtubule growth, this strongly suggests that one of the et al., 1992; Minshull et al., 1994) although Op18/stathmin roles of PP1 is to activate MAPs in order to stabilize is poorly phosphorylated (Figure 3). The fact that Op18/ microtubules during anaphase. stathmin is poorly phosphorylated in metaphase extracts During metaphase, inhibiting PP1 has no effect on the means that it is active in these extracts. This ﬁnding is in stability of the mitotic spindle, whereas, during anaphase, apparent contradiction to results obtained in vivo where it results in a highly unstable spindle. This instability Op18/stathmin is phosphorylated during metaphase. This manifests itself by a loss of microtubules from the spindles contradiction can be resolved by the observation that the and a movement of the poles towards the chromatin. This phosphorylation of Op18 is stimulated by chromatin is reminiscent of the effect of nocodazole, a drug which which is present at low levels in our extracts (Andersen inhibits microtubule assembly (Ault et al., 1991). It may et al., 1997). seem strange that spindle microtubules only become Catastrophes in mitosis are 8–10 times more frequent unstable during anaphase following PP1 inhibition. How- than in interphase. Following depletion of Op18/stathmin ever, two things must be considered. First, during meta- from a mitotic extract, the catastrophe rate drops to a phase, the chromosomes play an important role in rate only ~4–5 times more frequent than in interphase. stabilizing microtubules (Nicklas and Gordon, 1985; Therefore, other microtubule-depolymerizing factors regu- Zhang and Nicklas, 1995; Dogterom et al., 1996; Heald lated independently of PP2A must account for the et al., 1996). This activity may be lost during anaphase remaining catastrophes in mitosis. Such a factor can be and this may have to be substituted by a global PP1 XKCM1 (Walczak et al., 1996) and other as yet unidenti- activity acting to dephosphorylate factors required to ﬁed catastrophe factors. In interphase, Op18/stathmin is stabilize microtubules again at the exit from metaphase. also dephosphorylated (Brattsand et al., 1994; Larsson Secondly, there may be a large increase in length during et al., 1995) and active, but catastrophes are low. This anaphase B, which requires microtubule polymerization strongly suggests that although microtubules could be and stabilization. Thus, many mutants in PP1 probably destabilized by the non-phosphorylated form of Op18/ manifest themselves in anaphase due to a lack of micro- stathmin in interphase, they are not because of the presence tubule stabilization. In other words, PP1 may act to reverse of stabilizing factors like MAPs. We suggest that, during the phosphorylation state of microtubule-stabilizing fac- mitosis, the catastrophe rate decreases because Op18/ tors, like MAPs, that are inactivated by phosphorylation stathmin remains active globally whereas MAPs do not during metaphase. stabilize microtubules efﬁciently. We observed that PP2A inhibition resulted in an increase Switches and steady-state balances in microtubule spontaneous assembly in metaphase One of the main conclusions of our studies is that extracts. This could be due to regulation of the activity PP1 and PP2A have distinct functions in the control of of microtubule nucleating sites. Alternatively, the high microtubule dynamics. PP1 is required for the switches catastrophe rate observed in metaphase may normally between mitosis and interphase while PP2A is required to prevent short microtubules from reaching a visible size. maintain the short steady-state length of microtubules We were not able to determine whether the decrease in during mitosis. This illustrates two types of regulation catastrophe rate produced by inhibition of PP2A may be involved in spindle assembly: global regulation during the sufﬁcient to explain the increase in spontaneous assembly transitions between cell cycle states and local regulation observed under these conditions. during spindle formation. Thus, PP2A seems to act as In conclusion, PP2A controls the steady-state length of part of a phosphorylation balance that maintains the microtubules at least in part by controlling the activity of steady-state length of microtubules during metaphase. We Op18/stathmin. Thus the level of stability of metaphase imagine that the balance works in the following way. PP2A microtubules seems to depend upon a phosphorylation– and its opposing kinase are both active with comparable dephosphorylation balance involving PP2A and cdc2 activities. Subtle regulation of the activity of either of the kinase acting directly or indirectly on Op18/stathmin. enzymes would allow precise adjustment of the steady- Subtle spatial regulation of this balance could participate state length of microtubules. Adjustment of such a balance in the regulation of microtubule dynamics and organization would provide a means by which chromosomes locally 5545 R.Tournebize et al. and inactive, with the consequence that microtubules will stay short during the progression into interphase. Evidence for such a model comes from our studies that show that PP1 opposes cdc2 at the onset of mitosis and in anaphase. It is well known that the activity of cdc2 ﬂuctuates in the cell cycle, and it appears that the enzymatic activities of some PP1 catalytic subunits are inactivated by cdc2 phosphorylation during mitosis (Dohadwala et al., 1994; Yamano et al., 1994; Kwon et al., 1997). It is likely that PP1 and PP2A affect microtubule dynamics by acting on different substrates. Some may stabilize, others may destabilize microtubules. We have shown how one of these substrates, Op18/stathmin, is regulated by PP2A. Other microtubule regulators under the control of this enzyme and PP1 now need to be identiﬁed. Materials and methods Enzymatic assays Unless speciﬁed, all reagents were from Sigma. Cdc2 kinase activity assays were performed as described by Felix et al. (1989). PP2A activities were measured using phosphorylated casein prepared as described by McGowan and Cohen (1988), and PP1 activities using phosphorylated phosphorylase (phosphatase assay kit, Gibco BRL). Phosphatase activi- ties were measured in crude concentrated extracts similar to those used by Felix et al. (1989). We ﬁrst characterized inhibitor concentrations necessary to inhibit phosphatases in crude 10 000 g extracts. Okadaic Fig. 9. Model for a switch regulation between cdc2 kinase and PP1. acid (Gibco BRL) was used to inhibit PP2A, and I-2 (a gift from Phil (A) A substrate X which stabilizes microtubules is regulated by cdc2 Cohen) used to inhibit PP1. Typically, after incubation of extracts with kinase and PP1. When phosphorylated (X-P), X is inactive, but it is either okadaic acid or I-2 for 15 min at room temperature, 5 μlof active in its unphosphorylated form (X). (B) In a normal cell cycle, substrate were added to 10 μl of extracts for 30–45 s. The reaction was cdc2 kinase increases during mitosis (M) and decreases at anaphase. stopped by adding 100 μl of 10 mM EDTA, EGTA, 100 mM NaF, Concomitantly, PP1 activity decreases in mitosis and is high in β-glycerophosphate, 1 mg/ml bovine serum albumin (BSA), 0.1% interphase (I). In interphase, no cdc2 kinase is present and PP1 is β-mercaptoethanol (BME) and 100 μl of 10% trichloroacetic acid (TCA). active, therefore X is dephosphorylated, active and stabilizes Samples were incubated on ice for 10 min followed by centrifugation microtubules (promoting a long steady-state length). In mitosis, less at 15 000 g for 10 min, and soluble P released accounting for PP1 is active, cdc2 kinase activity is high and most of X is phosphatase activity were counted. Okadaic acid was titrated and found phosphorylated and inactive. Thus microtubules are short. (C)Inthe to inhibit 50% of PP2A at 0.4 μM. Using cyclin Δ90 (Glotzer et al., absence of PP1, at the interphase–metaphase transition, cdc2 kinase 1991), cdc2 kinase activity was not modiﬁed by incubation with okadaic phosphorylates X more quickly, thus inactivating it, such that less cdc2 acid. It was found that 3 μM I-2 was sufﬁcient to inhibit 50–60% of kinase activity is required to obtain a maximal effect. During the total phosphatase activity, showing that PP1 accounts for about half of metaphase–anaphase transition, cdc2 kinase is inactivated. If PP1 is the total serine-threonine phosphatase activity measurable with phos- inactive, X stays in an inactive phosphorylated form for longer and phorylated phosphorylase. Because I-2 can be inactivated by glycogen microtubules stay shorter. synthetase kinase phosphorylation, we added 1 μM I-2 every 15 min to the initial 3 μM used to inhibit PP1. Using such conditions, PP1 was constantly inhibited during 90 min. stabilize microtubules during spindle assembly (Karsenti, 1991; Hyman and Karsenti, 1996). Extract preparation and spindle assembly On the other hand, we believe that PP1 acts as a switch Xenopus extracts were prepared as described in Murray (1991). Meiotic extracts (which we refer to as CSF extracts) and spindles were prepared because the activities of PP1 and its opposing kinase, using a two-step procedure (Shamu and Murray, 1992). Sperm nuclei presumably cdc2, are different in interphase and mitosis. were added to CSF extracts released into interphase by calcium. After A model for the function of PP1 in the interphase–mitosis DNA and centrosomes duplicated, some CSF extract was added back switch is shown in Figure 9. Figure 9A and B shows the and spindles assembled for 60 min. When necessary, cyclin Δ90 was added 45 min after CSF extract addition for 45 min and then okadaic normal situation. A substrate X is dephosphorylated and acid was added. To test the role of PP2A in controlling spindle assembly, active and stabilizes microtubules in interphase. Con- sperm nuclei together with okadaic acid were added directly to extracts versely, it is phosphorylated and inactive in mitosis and (Sawin and Mitchison, 1991) pre-incubated with cyclin Δ90. Okadaic does not stabilize microtubules so that they can undergo acid was added at 0.4 μM ﬁnal concentration and spindles ﬁxed catastrophes and become shorter. At the entry of mitosis, every 10 min. Maximum effect was observed 20 min after okadaic acid addition. cdc2 kinase activity increases and PP1 activity decreases, Anaphase was induced by adding 0.4 mM CaCl (Shamu and Murray, triggering phosphorylation and inactivation of X. Micro- 1992) or 0.1 vol. of a dominant active CaMKII (Morin et al., 1994) tubules are no longer stabilized and become shorter. At subcloned in pCYCΔ13T (Glotzer et al., 1991) and transcribed in the metaphase–anaphase transition, cdc2 activity declines reticulocyte lysates (TNT-coupled reticulocyte lysate systems, Promega). During anaphase, I-2 was ﬁrst added to 3 μM to CSF extracts for 15 min. and PP1 activity increases, dephosphorylating X and Then 1 μM I-2 and CaCl /CaMKII were added to induce anaphase, and activating it. As a result, short microtubules in mitosis spindles were observed during 30–45 min. After anaphase induction,1 μM can be converted to long ones in interphase. Figure 9C I-2 was added every 15 min. shows what happens if PP1 activity is inhibited during the Puriﬁed human Op18/stathmin, either wild-type or with all four metaphase–anaphase transition. X will stay phosphorylated phosphorylatable serines mutated to alanine [kind gift of Andre Sobel, 5546 Phosphatases and mitosis 10 mg/ml in phosphate-buffered saline (PBS)], was added to pre- and a t-test (assuming the variance unknown) and paired t-test were assembled spindles at the same time as 0.4 μM okadaic acid. Microtubule performed. Values from experiments where n was 5 were discarded. growth was dependent on the concentration of mutated Op18/stathmin Differences reported were signiﬁcant at least at the 5% level of added. Images of spindles were taken on a Zeiss Axioskop microscope conﬁdence, except for the catastrophe frequency after I-2 addition in with either a Sony SSC-M370CE black and white camera or a color anaphase and okadaic acid addition to cyclin Δ90 extracts which are Coolview camera (Photonic Science). Images were then processed further signiﬁcant at the 7% level. This reﬂects variations between different with Adobe Photoshop. experiments. Only few rescues could be observed such that proper statistics could be done. Immunodepletions Microtubule steady-state length was calculated using the following Peptide antibodies to Op18 were prepared according to Belmont and equation described in Verde et al. (1992): Mitchison (1996). Immunodepletions were done as previously described V F – V F (Walczak et al., 1996). Typically, 400 μg of antibody were bound to g res s cat 60 μl of Afﬁprep protein A beads (Bio Rad) for 1 h at 4°C . After F F cat res washing, 200 μl of cyclin Δ90 extracts were added to packed beads. Extracts were depleted for 1 h at 4°C on a rotating wheel. Depleted where V , V , F and F are growth rate, shrinkage rate, catastrophe g s cat res extracts were then used for microtubule dynamics. Ninety-ﬁve percent frequency and rescue frequency respectively. As a rescue frequency, we depletion was usually achieved by this procedure, as determined by used the averaged value (1.05 events/min) of the rescue frequencies Western blotting. Mock-treated extracts were depleted using the same from the okadaic acid addition experiment. If rescues had increased in amount of pure rabbit IgG (Dianova). Treatment of the extract with the any of the experiments, we should have noticed. In the absence of any beads affected microtubule polymerization, explaining a decrease in increase, we assumed that the rescue frequency was not changed and catastrophe frequency between untreated extracts and mock-depleted used a value of 1.05 events/min in all microtubule length calculations. extracts. Op18 phosphorylation Acknowledgements Op18 was phosphorylated in a 55 μl extract containing cyclin Δ90, 1 μCi [γ- P]ATP/μl extract and different concentrations of okadaic acid We are grateful to Phil Cohen for his gift of inhibitor 2, to Andre Sobel or I-2. After 15 min, phosphorylation was stopped by adding 1 vol. of for his puriﬁed Op18/stathmin protein, to Thiel Lorca for CamkII 2 stabilization buffer (SB: 50 mM NaF, 40 mM β-glycerophosphate, construct and to Michael Glotzer for cyclin Δ90. We would also like to 10 mM EDTA, 10 mM sodium pyrophosphate, pH 7.2), plus 1 μM ´ ` ¨ thank Helene Defacque, Suzanne Eaton, Michael Glotzer, Pierre Gonczy, microcystin, 10 μg/ml pepstatin, leupeptin, chymostatin containing 7 μl Cayetano Gonzales, Andrew Murray, Angel Nebreda, Ingrid Sasson and of Afﬁprep protein A beads coated with anti-Op18/stathmin antibodies. Mitsuhiro Yanagida for critical reading of the manuscript, and Jo Howard Op18/stathmin was depleted for 30 min at 4°C. Beads were washed for help with the statistical analysis. once with 2 SB, then twice with PBS, 100 mM NaCl, 0.1% Triton X-100, and resuspended in 40 μl of sample buffer. Twenty-ﬁve microlitres were loaded onto a 15% gel and analyzed by silver staining and References PhosphorImager (Molecular Dynamics). Andersen,S.S.L., Ashford,A.J., Tournebize,R., Gavet,O., Sobel,A., Microtubule dynamics Hyman,A.A. and Karsenti,E. (1997) Mitotic chromatin regulates Microtubule dynamics were measured in 10 000 g extracts. Typically, phosphorylation of Stathmin/Op18. Nature, in press. 10 μl of extract were complemented with 1 μl of puriﬁed human Armstrong,C.G., Mann,D.J., Berndt,N. and Cohen,P.T. (1995) Drosophila centrosomes (210 centrosomes/ml), 0.42 μl of rhodamine–tubulin PPY, a novel male speciﬁc protein serine/threonine phosphatase (Hyman et al., 1991) [10 mg/ml, diluted to 2.8 mg/ml in BRB80 (80 mM localised in somatic cells of the testis. J. Cell Sci., 108, 3367–3375. PIPES, 1 mM EGTA, 1 mM MgCl , pH 6.8) and spun for 5 min in an Ault,J.G., DeMarco,A.J., Salmon,E.D. and Rieder,C.L. (1991) Studies Airfuge at 30 p.s.i. at 4°C to remove aggregates], 0.5 μl of saturated on the ejection properties of asters: astral microtubule turnover hemoglobin (bovine, Sigma) (dissolved in water, spun in a microfuge inﬂuences the oscillatory behavior and positioning of mono-oriented for 3 min to pellet undissolved hemoglobin), 0.33 μl of antifade [5 μl chromosomes. J. Cell Sci., 99, 701–710. of catalase (10 mg/ml in BRB80–50% glycerol); 5 μl of glucose oxidase Axton,J.M., Dombradi,V., Cohen,P.T. and Glover,D.M. (1990) One of (10 mg/ml in BRB80–50% glycerol); and 5 μl of glucose (1 M made in the protein phosphatase 1 isoenzymes in Drosophila is essential for water)]. All mixing was done on ice. Then 2.2 μl of this extract mix mitosis. Cell, 63, 33–46. were spotted onto a slide and gently covered by a 2222 mm coverslip Belmont,L.D. and Mitchison,T.J. (1996) Identiﬁcation of a protein that (Fisher Scientiﬁc). All glass used was washed with ethanol and water. interacts with tubulin dimers and increases the catastrophe rate of Images were recorded using a Zeiss Axioskop, a 100 Apochromat lens microtubules. Cell, 84, 623–631. NA1.4 and a long pass rhodamine ﬁlter (Chromatech). Images were Belmont,L.D., Hyman,A.A., Sawin,K.E. and Mitchison,T.J. (1990) Real- taken using an 8-bit black and white camera (Sony SSC-M370CE), time visualization of cell cycle-dependent changes in microtubule processed using an Argus 10 image processor (Hamamatsu) and stored dynamics in cytoplasmic extracts. Cell, 62, 579–589. on a Macintosh using the public domain NIH Image program (developed Black,S., Andrews,P.D., Sneddon,A.A. and Stark,M.J. (1995) A regulated at the US National Institutes of Health and available on the internet at MET3–GLC7 gene fusion provides evidence of a mitotic role for http://rsb.info.nih.gov/nih-image/). Recordings were done for no more Saccharomyces cerevisiae protein phosphatase 1. Yeast, 11, 747–759. than 5 min. Images were recorded every 2 s. Special care was taken to Booher,R. and Beach,D. (1989) Involvement of a type 1 protein avoid photobleaching and photodamage by reducing the illumination phosphatase encoded by bws1 in ﬁssion yeast mitotic control. Cell, time using a computer-controlled shutter (Uniblitz, Vincent Associates). 57, 1009–1016. For experiments using okadaic acid, extracts were frozen 20 min after Brattsand,G., Marklund,U., Nylander,K., Roos,G. and Gullberg,M. addition of 0.4 μM okadaic acid and used for microtubule dynamics. (1994) Cell-cycle-regulated phosphorylation of oncoprotein 18 on Extracts depleted of Op18/stathmin with or without okadaic acid addition Ser16, Ser25 and Ser38. Eur. J. Biochem., 220, 359–368. were treated similarly. For experiments investigating the role of PP1 in Brewis,N.D., Street,A.J., Prescott,A.R. and Cohen,P.T. (1993) PPX, a anaphase, 3 μM I-2 was added to extracts at room temperature for novel protein serine/threonine phosphatase localized to centrosomes. 15 min. Extracts were complemented with 0.1 vol. of CaMKII, an EMBO J., 12, 987–996. additional 1 μM I-2, rhodamine–tubulin, hemoglobin and antifade, and Chen,M.X., McPartlin,A.E., Brown,L., Chen,Y.H., Barker,H.M. and kept at room temperature. Then 1 μM I-2 and centrosomes were added Cohen,P.T. (1994) A novel human protein serine/threonine every 15 min after triggering anaphase. Recordings were done for 5 min phosphatase, which possesses four tetratricopeptide repeat motifs and at 0, 15, 30 and 45 min after CaMKII addition. localizes to the nucleus. EMBO J., 13, 4278–4290. Clarke,P.R., Hoffmann,I., Draetta,G. and Karsenti,E. (1993) Data analysis Dephosphorylation of cdc25-C by a type-2A protein phosphatase: Analysis of microtubule dynamics was done using a home-made Micro- speciﬁc regulation during the cell cycle in Xenopus egg extracts. Mol. soft Excel macro. Differences between experiments were evaluated using Biol. Cell, 4, 397–411. Student’s t-test; t-tests were performed within single experiments to Cohen,P. (1989) The structure and regulation of protein phosphatases. assess signiﬁcant variations. Different experiments were then averaged, Annu. Rev. Biochem., 58, 453–508. 5547 R.Tournebize et al. de Pennart,H., Verlhac,M.H., Cibert,C., Santa Maria,A. and Maro,B. Ishii,K., Kumada,K., Toda,T. and Yanagida,M. (1996) Requirement for (1993) Okadaic acid induces spindle lengthening and disrupts the PP1 phosphatase and 20S cyclosome/APC for the onset of anaphase interaction of microtubules with the kinetochores in metaphase II- is lessened by the dosage increase of a novel gene sds23 . EMBO J., arrested mouse oocytes. Dev. Biol., 157, 170–181. 15, 6629–6640. Dogterom,M., Fe´lix,M.-A., Guet,C.C. and Leibler,S. (1996) Inﬂuence of Karsenti,E. (1991) Mitotic spindle morphogenesis in animal cells. Semin. M-phase chromatin on the anisotropy of microtubule asters. J. Cell Cell Biol., 2, 251–260. Biol., 133, 125–140. Kinoshita,N., Yamano,H., Niwa,H., Yoshida,T. and Yanagida,M. (1993) Dohadwala,M., Da Cruz E Silva,E.F., Hall,F.L., Williams,R.T., Carbonaro- Negative regulation of mitosis by the ﬁssion yeast protein phosphatase Hall,D.A., Nairn,A.C., Greengard,P. and Berndt,N. (1994) ppa2. Genes Dev., 7, 1059–1071. Phosphorylation and inactivation of protein phosphatase 1 by cyclin- Kinoshita,K., Nemoto,T., Nabeshima,K., Kondoh,H., Niwa,H. and dependent kinases. Proc. Natl Acad. Sci. USA, 91, 6408–6412. Yanagida,M. (1996) The regulatory subunits of ﬁssion yeast protein Doonan,J.H. and Morris,N.R. (1989) The bimG gene of Aspergillus phosphatase 2A (PP2A) affect cell morphogenesis, cell wall synthesis nidulans, required for completion of anaphase, encodes a homolog of and cytokinesis. Genes to Cells, 1, 633–644. mammalian phosphoprotein phosphatase 1. Cell, 57, 987–996. Kwon,Y., Lee,S.Y., Choi,Y., Greengard,P. and Nairn,A.C. (1997) Cell Evans,D.R.H. and Stark,M.J.R. (1997) Mutations in the Saccharomyces cycle-dependent phosphorylation of mammalian protein phosphatase cerevisiae type 2A protein phosphatase catalytic subunit reveal roles 1 by cdc2 kinase. Proc. Natl Acad. Sci. USA, 94, 2168–2173. in cell wall integrity, actin cytoskeleton organization and mitosis. Larsson,N., Melander,H., Marklund,U., Osterman,O. and Gullberg,M. Genetics, 145, 227–241. (1995) G /M transition requires multisite phosphorylation of Felix,M.A., Pines,J., Hunt,T. and Karsenti,E. (1989) A post-ribosomal oncoprotein 18 by two distinct protein kinase systems. J. Biol. Chem., supernatant from activated Xenopus eggs that displays post- 270, 14175–14183. translationally regulated oscillation of its cdc2 mitotic kinase activity. Lee,T.H. (1995) The role of protein phosphatase type-2A in the Xenopus cell cycle: initiation of the G EMBO J., 8, 3059–3069. /M transition. Semin. Cancer Biol., 6, Felix,M.A., Cohen,P. and Karsenti,E. (1990) Cdc2 H1 kinase is negatively 203–209. regulated by a type 2A phosphatase in the Xenopus early embryonic Lee,T.H., Turck,C. and Kirschner,M.W. (1994) Inhibition of cdc2 cell cycle: evidence from the effects of okadaic acid. EMBO J., 9, activation by INH/PP2A. Mol. Biol. Cell, 5, 323–338. 675–683. Lin,F.C. and Arndt,K.T. (1995) The role of Saccharomyces cerevisiae Fernandez,A., Brautigan,D.L. and Lamb,N.J. (1992) Protein phosphatase type 2A phosphatase in the actin cytoskeleton and in entry into type 1 in mammalian cell mitosis: chromosomal localization and mitosis. EMBO J., 14, 2745–2759. involvement in mitotic exit. J. Cell Biol., 116, 1421–1430. Lorca,T., Fesquet,D., Zindy,F., Le Bouffant,F., Cerruti,M., Brechot,C., Ferrigno,P., Langan,T.A. and Cohen,P. (1993) Protein phosphatase 2A1 Devauchelle,G. and Dore´e,M. (1991) An okadaic acid-sensitive is the major enzyme in vertebrate cell extracts that dephosphorylates phosphatase negatively controls the cyclin degradation pathway in several physiological substrates for cyclin-dependent protein kinases. amphibian eggs. Mol. Cell. Biol., 11, 1171–1175. Mol. Biol. Cell, 4, 669–677. Lorca,T., Cruzalegui,F.H., Fesquet,D., Cavadore,J.C., Mery,J., Means,A. Francisco,L. and Chan,C.S. (1994) Regulation of yeast chromosome and Dore´e,M. (1993) Calmodulin-dependent protein kinase II mediates segregation by Ipl1 protein kinase and type 1 protein phosphatase. inactivation of MPF and CSF upon fertilization of Xenopus eggs. Cell. Mol. Biol. Res., 40, 207–213. Nature, 366, 270–273. Gliksman,N.R., Parsons,S.F. and Salmon,E.D. (1992) Okadaic acid Marklund,U., Larsson,N., Gradin,H.M., Brattsand,G. and Gullberg,M. induces interphase to mitotic-like microtubule dynamic instability by (1996) Oncoprotein 18 is a phosphorylation-responsive regulator of inactivating rescue. J. Cell Biol., 119, 1271–1276. microtubule dynamics. EMBO J., 15, 5290–5298. Glotzer,M., Murray,A.W. and Kirschner,M.W. (1991) Cyclin is degraded McGowan,C.H. and Cohen,P. (1988) Protein phosphatase-2C from rabbit by the ubiquitin pathway. Nature, 349, 132–138. skeletal muscle and liver: an Mg -dependent enzyme. Methods Goedert,M., Cohen,E.S., Jakes,R. and Cohen,P. (1992) p42 MAP kinase Enzymol., 159, 416–426. phosphorylation sites in microtubule-associated protein tau are Minshull,J., Sun,H., Tonks,N.K. and Murray,A.W. (1994) A MAP kinase- dephosphorylated by protein phosphatase 2A1. Implications for dependent spindle assembly checkpoint in Xenopus egg extracts. Cell, Alzheimer’s disease. FEBS Lett., 312, 95–99. 79, 475–486. Gomes,R., Karess,R.E., Ohkura,H., Glover,D.M. and Sunkel,C.E. (1993) Morin,N., Abrieu,A., Lorca,T., Martin,F. and Dore´e,M. (1994) The Abnormal anaphase resolution (aar): a locus required for progression proteolysis-dependent metaphase to anaphase transition: calcium/ through mitosis in Drosophila. J. Cell Sci., 104, 583–593. calmodulin-dependent protein kinase II mediates onset of anaphase in Heald,R., Tournebize,R., Blank,T., Sandaltzopoulos,R., Becker,P., extracts prepared from unfertilized Xenopus eggs. EMBO J., 13, Hyman,A. and Karsenti,E. (1996) Self-organization of microtubules 4343–4352. into bipolar spindles around artiﬁcial chromosomes in Xenopus egg Murray,A.W. (1991) Cell cycle extracts. In Kay,B.K. and Peng,H.B. extracts. Nature, 382, 420–425. (eds), Cell Cycle Extracts. Academic Press Inc., San Diego, Vol. 36, Hisamoto,N., Sugimoto,K. and Matsumoto,K. (1994) The Glc7 type 1 pp. 581–605. protein phosphatase of Saccharomyces cerevisiae is required for cell Murray,A.W., Solomon,M.J. and Kirschner,M.W. (1989) The role of cycle progression in G /M. Mol. Cell. Biol., 14, 3158–3165. cyclin synthesis and degradation in the control of maturation promoting Holloway,S.L., Glotzer,M., King,R.W. and Murray,A.W. (1993) factor activity. Nature, 339, 280–286. Anaphase is initiated by proteolysis rather than by the inactivation of Nebreda,A.R. and Hunt,T. (1993) The c-mos proto-oncogene protein maturation-promoting factor. Cell, 73, 1393–1402. kinase turns on and maintains the activity of MAP kinase, but not Honkanen,R.E., Zwiller,J., Daily,S.L., Khatra,B.S., Dukelow,M. and MPF, in cell-free extracts of Xenopus oocytes and eggs. EMBO J., Boynton,A.L. (1991) Identiﬁcation, puriﬁcation, and characterization 12, 1979–1993. of a novel serine/threonine protein phosphatase from bovine brain. Nicklas,R.B. and Gordon,G.W. (1985) The total length of spindle J. Biol. Chem., 266, 6614–6619. microtubules depends on the number of chromosomes present. J. Cell Horwitz,S.B., Shen,H.-J., He,L., Dittmar,P., Neef,R., Chen,J. and Biol., 100, 1–7. Schubart,U.K. (1997) The microtubule-destabilizing activity of Ohkura,H., Adachi,Y., Kinoshita,N., Niwa,O., Toda,T. and Yanagida,M. metablastin (p19) is controlled by phosphorylation. J. Biol. Chem., (1988) Cold-sensitive and caffeine-supersensitive mutants of the 272, 8129–8132. Schizosaccharomyces pombe dis genes implicated in sister chromatid Hyman,A.A. and Karsenti,E. (1996) Morphogenetic properties of separation during mitosis. EMBO J., 7, 1465–1473. microtubules and mitotic spindle assembly. Cell, 84, 401–410. Ohkura,H., Kinoshita,N., Miyatani,S., Toda,T. and Yanagida,M. (1989) Hyman,A., Drechsel,D., Kellogg,D., Salser,S., Sawin,K., Steffen,P., The ﬁssion yeast dis2 gene required for chromosome disjoining Wordeman,L. and Mitchison,T. (1991) Preparation of modiﬁed encodes one of two putative type 1 protein phosphatases. Cell, 57, tubulins. Methods Enzymol., 196, 478–485. 997–1007. Ingebritsen,T.S., Stewart,A.A. and Cohen,P. (1983) The protein Parsons,S.F. and Salmon,E.D. (1997) Microtubule assembly in clariﬁed phosphatases involved in cellular regulation; 6. Measurement of type- Xenopus egg extracts. Cell Motil. Cytoskel., 36, 1–11. 1 and type-2 protein phosphatases in extracts of mammalian tissues; Picard,A., Capony,J.P., Brautigan,D.L. and Dore´e,M. (1989) Involvement an assessment of their physiological roles. Eur. J. Biochem., 132, of protein phosphatases 1 and 2A in the control of M phase-promoting 297–307. factor activity in starﬁsh. J. Cell Biol., 109, 3347–3354. 5548 Phosphatases and mitosis Picard,A., Labbe´,J.C., Barakat,H., Cavadore,J.C. and Dore´e,M. (1991) Okadaic acid mimics a nuclear component required for cyclin B–cdc2 kinase microinjection to drive starﬁsh oocytes into M phase. J. Cell Biol., 115, 337–344. Sawin,K.E. and Mitchison,T.J. (1991) Mitotic spindle assembly by two different pathways in vitro. J. Cell Biol., 112, 925–940. Shamu,C.E. and Murray,A.W. (1992) Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase. J. Cell Biol., 117, 921–934. Shenolikar,S. (1994) Protein serine/threonine phosphatases—new avenues for cell regulation. Annu. Rev. Cell Biol., 10, 55–86. Shibuya,E.K., Polverino,A.J., Chang,E., Wigler,M. and Ruderman,J.V. (1992) Oncogenic ras triggers the activation of 42-kDa mitogen- activated protein kinase in extracts of quiescent Xenopus oocytes. Proc. Natl Acad. Sci. USA, 89, 9831–9835. Snaith,H.A., Armstrong,C.G., Guo,Y., Kaiser,K. and Cohen,P.T.W. (1996) Deﬁency of protein phosphatase 2A uncouples the nuclear and centrosome cycle and prevents attachment of microtubules to the kinetochore in Drosophila microtubule star (mts) embryos. J. Cell Sci., 109, 3001–3012. Sobel,A. (1991) Stathmin: a relay phosphoprotein for multiple signal transduction? Trends Biochem. Sci., 16, 301–305. Sontag,E., Nunbhakdi Craig,V., Bloom,G.S. and Mumby,M.C. (1995) A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle. J. Cell Biol., 128, 1131–1144. Surana,U., Amon,A., Dowzer,C., McGrew,J., Byers,B. and Nasmyth,K. (1993) Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. EMBO J., 12, 1969–1978. Tournebize,R. and Heald,R. (1996) Mitotic spindles and microtubules dynamics in Xenopus egg extracts. Semin. Cell Dev. Biol., 7, 467–473. Van Dolah,F.M. and Ramsdell,J.S. (1992) Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells. J. Cell Physiol., 152, 190–198. Verde,F., Labbe´,J.C., Dore´e,M. and Karsenti,E. (1990) Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature, 343, 233–238. Verde,F., Dogterom,M., Stelzer,E., Karsenti,E. and Leibler,S. (1992) Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts. J. Cell Biol., 118, 1097–1108. Walczak,C.E., Mitchison,T.J. and Desai,A. (1996) XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly. Cell, 84, 37–47. Walker,R.A., O’Brien,E.T., Pryer,N.K., Soboeiro,M.F., Voter,W.A., Erickson,H.P. and Salmon,E.D. (1988) Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies. J. Cell Biol., 107, 1437–1448. Wells,W.A.E. (1996) The spindle-assembly checkpoint: aiming for a perfect mitosis, every time. Trends Cell Biol., 6, 228–234. Yamano,H., Ishii,K. and Yanagida,M. (1994) Phosphorylation of dis2 protein phosphatase at the C-terminal cdc2 consensus and its potential role in cell cycle regulation. EMBO J., 13, 5310–5318. Zhai,Y., Kronebusch,P.J., Simon,P.M. and Borisy,G.G. (1996) Microtubule dynamics at the G /M transition: abrupt breakdown of cytoplasmic microtubules at nuclear envelope breakdown and implication for spindle morphogenesis. J. Cell Biol., 135, 201–214. Zhang,D. and Nicklas,R.B. (1995) The impact of chromosomes and centrosomes on spindle assembly as observed in living cells. J. Cell Biol., 129, 1287–1300. Received on April 3, 1997; revised on June 26, 1997 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals http://www.deepdyve.com/lp/springer-journals/distinct-roles-of-pp1-and-pp2a-like-phosphatases-in-control-of-8EHNPKlASZ

Loading next page...

References (90)

U Surana, A Amon, C Dowzer, J McGrew, B Byers, K Nasmyth (1993)
Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast
Semin Cell Dev Biol, 12
S Black, PD Andrews, AA Sneddon, MJ Stark (1995)
A regulated MET3–GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1
Yeast, 11
JM Axton, V Dombradi, PT Cohen, DM Glover (1990)
One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis
Cell, 63
M Dogterom, M‐A Félix, CC Guet, S Leibler (1996)
Influence of M‐phase chromatin on the anisotropy of microtubule asters
J Cell Biol, 133
R Heald, R Tournebize, T Blank, R Sandaltzopoulos, P Becker, A Hyman, E Karsenti (1996)
Self‐organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts
Nature, 382
FM Van Dolah, JS Ramsdell (1992)
Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells
J Cell Physiol, 152
A Sobel (1991)
Stathmin: a relay phosphoprotein for multiple signal transduction?
Trends Biochem Sci, 16
AW Murray, MJ Solomon, MW Kirschner (1989)
The role of cyclin synthesis and degradation in the control of maturation promoting factor activity
Nature, 339
TS Ingebritsen, AA Stewart, P Cohen (1983)
The protein phosphatases involved in cellular regulation; 6. Measurement of type‐1 and type‐2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles
Eur J Biochem, 132
EK Shibuya, AJ Polverino, E Chang, M Wigler, JV Ruderman (1992)
Oncogenic ras triggers the activation of 42‐kDa mitogen‐activated protein kinase in extracts of quiescent Xenopus oocytes
Proc Natl Acad Sci USA, 89
HA Snaith, CG Armstrong, Y Guo, K Kaiser, PTW Cohen (1996)
Defiency of protein phosphatase 2A uncouples the nuclear and centrosome cycle and prevents attachment of microtubules to the kinetochore in Drosophila microtubule star (mts) embryos
J Cell Sci, 109
WAE Wells (1996)
The spindle‐assembly checkpoint: aiming for a perfect mitosis, every time
Trends Cell Biol, 6
E Karsenti (1991)
Mitotic spindle morphogenesis in animal cells
Semin Cell Biol, 2
FC Lin, KT Arndt (1995)
The role of Saccharomyces cerevisiae type 2A phosphatase in the actin cytoskeleton and in entry into mitosis
EMBO J, 14
SB Horwitz, H‐J Shen, L He, P Dittmar, R Neef, J Chen, UK Schubart (1997)
The microtubule‐destabilizing activity of metablastin (p19) is controlled by phosphorylation
J Biol Chem, 272
L Francisco, CS Chan (1994)
Regulation of yeast chromosome segregation by Ipl1 protein kinase and type 1 protein phosphatase
Cell Mol Biol Res, 40
M Goedert, ES Cohen, R Jakes, P Cohen (1992)
p42 MAP kinase phosphorylation sites in microtubule‐associated protein tau are dephosphorylated by protein phosphatase 2A1. Implications for Alzheimer's disease
FEBS Lett, 312
N Larsson, H Melander, U Marklund, O Osterman, M Gullberg (1995)
G2/M transition requires multisite phosphorylation of oncoprotein 18 by two distinct protein kinase systems
J Biol Chem, 270
RA Walker, ET O'Brien, NK Pryer, MF Soboeiro, WA Voter, HP Erickson, ED Salmon (1988)
Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies
Trends Cell Biol, 107
P Ferrigno, TA Langan, P Cohen (1993)
Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin‐dependent protein kinases
Mol Biol Cell, 4
HA Snaith, CG Armstrong, Y Guo, K Kaiser, PTW Cohen (1996)
Defiency of protein phosphatase 2A uncouples the nuclear and centrosome cycle and prevents attachment of microtubules to the kinetochore in Drosophila microtubule star (mts) embryos
Trends Biochem Sci, 109
T Lorca, D Fesquet, F Zindy, F Le Bouffant, M Cerruti, C Brechot, G Devauchelle, M Dorée (1991)
An okadaic acid‐sensitive phosphatase negatively controls the cyclin degradation pathway in amphibian eggs
Mol Cell Biol, 11
CE Shamu, AW Murray (1992)
Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase
J Cell Biol, 117
H Yamano, K Ishii, M Yanagida (1994)
Phosphorylation of dis2 protein phosphatase at the C‐terminal cdc2 consensus and its potential role in cell cycle regulation
J Cell Biol, 13
H Yamano, K Ishii, M Yanagida (1994)
Phosphorylation of dis2 protein phosphatase at the C‐terminal cdc2 consensus and its potential role in cell cycle regulation
EMBO J, 13
WAE Wells (1996)
The spindle‐assembly checkpoint: aiming for a perfect mitosis, every time
EMBO J, 6
R Tournebize, R Heald (1996)
Mitotic spindles and microtubules dynamics in Xenopus egg extracts
J Cell Physiol, 7
H Ohkura, N Kinoshita, S Miyatani, T Toda, M Yanagida (1989)
The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases
Cell, 57
R Booher, D Beach (1989)
Involvement of a type 1 protein phosphatase encoded by bws1+ in fission yeast mitotic control
Cell, 57
CH McGowan, P Cohen (1988)
Protein phosphatase‐2C from rabbit skeletal muscle and liver: an Mg2+‐dependent enzyme
Methods Enzymol, 159
NR Gliksman, SF Parsons, ED Salmon (1992)
Okadaic acid induces interphase to mitotic‐like microtubule dynamic instability by inactivating rescue
J Cell Biol, 119
SSL Andersen, AJ Ashford, R Tournebize, O Gavet, A Sobel, AA Hyman, E Karsenti (1997)
Mitotic chromatin regulates phosphorylation of Stathmin/Op18
Nature
SF Parsons, ED Salmon (1997)
Microtubule assembly in clarified Xenopus egg extracts
Cell Motil Cytoskel, 36
A Fernandez, DL Brautigan, NJ Lamb (1992)
Protein phosphatase type 1 in mammalian cell mitosis: chromosomal localization and involvement in mitotic exit
J Cell Biol, 116
JH Doonan, NR Morris (1989)
The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1
Cell, 57
S Shenolikar (1994)
Protein serine/threonine phosphatases—new avenues for cell regulation
Annu Rev Cell Biol, 10
A Picard, JP Capony, DL Brautigan, M Dorée (1989)
Involvement of protein phosphatases 1 and 2A in the control of M phase‐promoting factor activity in starfish
J Cell Biol, 109
S Shenolikar (1994)
Protein serine/threonine phosphatases—new avenues for cell regulation
Proc Natl Acad Sci USA, 10
M Glotzer, AW Murray, MW Kirschner (1991)
Cyclin is degraded by the ubiquitin pathway
Nature, 349
LD Belmont, TJ Mitchison (1996)
Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules
Cell, 84
E Sontag, V Nunbhakdi Craig, GS Bloom, MC Mumby (1995)
A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle
EMBO J, 128
D Zhang, RB Nicklas (1995)
The impact of chromosomes and centrosomes on spindle assembly as observed in living cells
J Cell Biol, 129
J Minshull, H Sun, NK Tonks, AW Murray (1994)
A MAP kinase‐dependent spindle assembly checkpoint in Xenopus egg extracts
Cell, 79
AW Murray (1991)
Cell cycle extracts
Cell Cycle Extracts
MA Felix, J Pines, T Hunt, E Karsenti (1989)
A post‐ribosomal supernatant from activated Xenopus eggs that displays post‐translationally regulated oscillation of its cdc2+ mitotic kinase activity
EMBO J, 8
LD Belmont, AA Hyman, KE Sawin, TJ Mitchison (1990)
Real‐time visualization of cell cycle‐dependent changes in microtubule dynamics in cytoplasmic extracts
Cell, 62
K Ishii, K Kumada, T Toda, M Yanagida (1996)
Requirement for PP1 phosphatase and 20S cyclosome/APC for the onset of anaphase is lessened by the dosage increase of a novel gene sds23+
EMBO J, 15
RB Nicklas, GW Gordon (1985)
The total length of spindle microtubules depends on the number of chromosomes present
J Cell Biol, 100
TH Lee (1995)
The role of protein phosphatase type‐2A in the Xenopus cell cycle: initiation of the G2/M transition
Semin Cancer Biol, 6
DRH Evans, MJR Stark (1997)
Mutations in the Saccharomyces cerevisiae type 2A protein phosphatase catalytic subunit reveal roles in cell wall integrity, actin cytoskeleton organization and mitosis
Genetics, 145
H Ohkura, Y Adachi, N Kinoshita, O Niwa, T Toda, M Yanagida (1988)
Cold‐sensitive and caffeine‐supersensitive mutants of the Schizosaccharomyces pombe dis genes implicated in sister chromatid separation during mitosis
EMBO J, 7
RE Honkanen, J Zwiller, SL Daily, BS Khatra, M Dukelow, AL Boynton (1991)
Identification, purification, and characterization of a novel serine/threonine protein phosphatase from bovine brain
J Biol Chem, 266
CE Walczak, TJ Mitchison, A Desai (1996)
XKCM1: a Xenopus kinesin‐related protein that regulates microtubule dynamics during mitotic spindle assembly
Cell, 84
MA Felix, P Cohen, E Karsenti (1990)
Cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid
EMBO J, 9
F Verde, JC Labbé, M Dorée, E Karsenti (1990)
Regulation of microtubule dynamics by cdc2 protein kinase in cell‐free extracts of Xenopus eggs
Nature, 343
CG Armstrong, DJ Mann, N Berndt, PT Cohen (1995)
Drosophila PPY, a novel male specific protein serine/threonine phosphatase localised in somatic cells of the testis
J Cell Sci, 108
M Dohadwala, EF Da Cruz E Silva, FL Hall, RT Williams, DA CarbonaroHall, AC Nairn, P Greengard, N Berndt (1994)
Phosphorylation and inactivation of protein phosphatase 1 by cyclin‐dependent kinases
Proc Natl Acad Sci USA, 91
K Kinoshita, T Nemoto, K Nabeshima, H Kondoh, H Niwa, M Yanagida (1996)
The regulatory subunits of fission yeast protein phosphatase 2A (PP2A) affect cell morphogenesis, cell wall synthesis and cytokinesis
Genes to Cells, 1
T Lorca, FH Cruzalegui, D Fesquet, JC Cavadore, J Mery, A Means, M Dorée (1993)
Calmodulin‐dependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs
Nature, 366
Y Zhai, PJ Kronebusch, PM Simon, GG Borisy (1996)
Microtubule dynamics at the G2/M transition: abrupt breakdown of cytoplasmic microtubules at nuclear envelope breakdown and implication for spindle morphogenesis
J Cell Biol, 135
MX Chen, AE McPartlin, L Brown, YH Chen, HM Barker, PT Cohen (1994)
A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus
EMBO J, 13
N Hisamoto, K Sugimoto, K Matsumoto (1994)
The Glc7 type 1 protein phosphatase of Saccharomyces cerevisiae is required for cell cycle progression in G2/M
Mol Cell Biol, 14
SL Holloway, M Glotzer, RW King, AW Murray (1993)
Anaphase is initiated by proteolysis rather than by the inactivation of maturation‐promoting factor
Cell, 73
N Kinoshita, H Yamano, H Niwa, T Yoshida, M Yanagida (1993)
Negative regulation of mitosis by the fission yeast protein phosphatase ppa2
Genes Dev, 7
N Morin, A Abrieu, T Lorca, F Martin, M Dorée (1994)
The proteolysis‐dependent metaphase to anaphase transition: calcium/calmodulin‐dependent protein kinase II mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs
EMBO J, 13
R Gomes, RE Karess, H Ohkura, DM Glover, CE Sunkel (1993)
Abnormal anaphase resolution (aar): a locus required for progression through mitosis in Drosophila
J Cell Sci, 104
A Hyman, D Drechsel, D Kellogg, S Salser, K Sawin, P Steffen, L Wordeman, T Mitchison (1991)
Preparation of modified tubulins
Methods Enzymol, 196
AR Nebreda, T Hunt (1993)
The c‐mos proto‐oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell‐free extracts of Xenopus oocytes and eggs
EMBO J, 12
A Sobel (1991)
Stathmin: a relay phosphoprotein for multiple signal transduction?
J Cell Biol, 16
A Picard, JC Labbé, H Barakat, JC Cavadore, M Dorée (1991)
Okadaic acid mimics a nuclear component required for cyclin B–cdc2 kinase microinjection to drive starfish oocytes into M phase
J Cell Biol, 115
ND Brewis, AJ Street, AR Prescott, PT Cohen (1993)
PPX, a novel protein serine/threonine phosphatase localized to centrosomes
EMBO J, 12
PR Clarke, I Hoffmann, G Draetta, E Karsenti (1993)
Dephosphorylation of cdc25‐C by a type‐2A protein phosphatase: specific regulation during the cell cycle in Xenopus egg extracts
Mol Biol Cell, 4
KE Sawin, TJ Mitchison (1991)
Mitotic spindle assembly by two different pathways in vitro
J Cell Biol, 112
AR Nebreda, T Hunt (1993)
The c‐mos proto‐oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell‐free extracts of Xenopus oocytes and eggs
J Cell Biol, 12
EK Shibuya, AJ Polverino, E Chang, M Wigler, JV Ruderman (1992)
Oncogenic ras triggers the activation of 42‐kDa mitogen‐activated protein kinase in extracts of quiescent Xenopus oocytes
J Cell Sci, 89
TH Lee, C Turck, MW Kirschner (1994)
Inhibition of cdc2 activation by INH/PP2A
Mol Biol Cell, 5
H de Pennart, MH Verlhac, C Cibert, A Santa Maria, B Maro (1993)
Okadaic acid induces spindle lengthening and disrupts the interaction of microtubules with the kinetochores in metaphase II‐arrested mouse oocytes
Dev Biol, 157
P Cohen (1989)
The structure and regulation of protein phosphatases
Annu Rev Biochem, 58
RB Nicklas, GW Gordon (1985)
The total length of spindle microtubules depends on the number of chromosomes present
EMBO J, 100
CE Shamu, AW Murray (1992)
Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase
Annu Rev Cell Biol, 117
U Marklund, N Larsson, HM Gradin, G Brattsand, M Gullberg (1996)
Oncoprotein 18 is a phosphorylation‐responsive regulator of microtubule dynamics
EMBO J, 15
R Tournebize, R Heald (1996)
Mitotic spindles and microtubules dynamics in Xenopus egg extracts
Semin Cell Dev Biol, 7
SF Parsons, ED Salmon (1997)
Microtubule assembly in clarified Xenopus egg extracts
J Cell Biol, 36
G Brattsand, U Marklund, K Nylander, G Roos, M Gullberg (1994)
Cell‐cycle‐regulated phosphorylation of oncoprotein 18 on Ser16, Ser25 and Ser38
Eur J Biochem, 220
AA Hyman, E Karsenti (1996)
Morphogenetic properties of microtubules and mitotic spindle assembly
Cell, 84
U Surana, A Amon, C Dowzer, J McGrew, B Byers, K Nasmyth (1993)
Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast
EMBO J, 12
Y Kwon, SY Lee, Y Choi, P Greengard, AC Nairn (1997)
Cell cycle‐dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase
Proc Natl Acad Sci USA, 94
E Sontag, V Nunbhakdi Craig, GS Bloom, MC Mumby (1995)
A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle
J Cell Biol, 128
F Verde, M Dogterom, E Stelzer, E Karsenti, S Leibler (1992)
Control of microtubule dynamics and length by cyclin A‐ and cyclin B‐dependent kinases in Xenopus egg extracts
J Cell Biol, 118
JG Ault, AJ DeMarco, ED Salmon, CL Rieder (1991)
Studies on the ejection properties of asters: astral microtubule turnover influences the oscillatory behavior and positioning of mono‐oriented chromosomes
J Cell Sci, 99

Publisher: Springer Journals
Copyright: Copyright © European Molecular Biology Organization 1997
ISSN: 0261-4189
eISSN: 1460-2075
DOI: 10.1093/emboj/16.18.5537
Publisher site: See Article on Publisher Site

Abstract

The EMBO Journal Vol.16 No.18 pp.5537–5549, 1997 Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis phases, called a catastrophe, and between shrinkage and Re´gis Tournebize , Søren S.L.Andersen, 2 3 growth phases, called a rescue (Walker et al., 1988; Fulvia Verde , Marcel Dore´e , Eric Karsenti Hyman and Karsenti, 1996). During the transition from and Anthony A.Hyman interphase to mitosis, the primary effect on microtubule Cell Biology Program, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, dynamics is an increase in the catastrophe rate (Belmont Germany, Department of Biochemistry and Molecular Biology, et al., 1990; Verde et al., 1992). However, little is known University of Miami School of Medicine, PO Box 016129, Miami, about direct effectors of microtubule dynamics or how FL 33136-1015, USA and CRBM-CNRS, BP 5051, Route de Mende, they are regulated. 34033 Montpellier, France Changes in microtubule dynamics during the transitions Corresponding author into and out of mitosis are controlled by the activity of e-mail: [email protected] cdc2 kinase which phosphorylates a large variety of molecules either directly or indirectly (Belmont et al., Assembly of a mitotic spindle requires the accurate 1990; Verde et al., 1990, 1992). It is likely that the control regulation of microtubule dynamics which is accomp- of microtubule dynamics during these cell cycle transitions lished, at least in part, by phosphorylation–dephos- is determined by the balance between cdc2 kinase and phorylation reactions. Here we have investigated the phosphatases that directly or indirectly oppose its action role of serine-threonine phosphatases in the control on target molecules. Such phosphorylation balances may of microtubule dynamics using speciﬁc inhibitors in also be involved in the stabilization of microtubules by Xenopus egg extracts. Type 2A phosphatases are chromosomes during spindle assembly. required to maintain the short steady-state length of Analysis of mutants in serine-threonine phosphatases microtubules in mitosis by regulating the level of indicates that they participate in ensuring correct chromo- microtubule catastrophes, in part by controlling the the some segregation at mitosis. Some phenotypes suggest microtubule-destabilizing activity and phosphorylation that they do so by regulating cell cycle control. For of Op18/stathmin. Type 1 phosphatases are only instance, type 2A phosphatase (PP2A) negatively regulates required for control of microtubule dynamics during cdc2 kinase at the onset of mitosis in Xenopus (Felix the transitions into and out of mitosis. Thus, although et al., 1990; Clarke et al., 1993; Lee et al., 1994; Lee, both type 2A and type 1 phosphatases are involved in 1995), starﬁsh (Picard et al., 1989, 1991) and both in the regulation of microtubule dynamics, they have ﬁssion and budding yeasts (Kinoshita et al., 1993; Lin distinct, non-overlapping roles. and Arndt, 1995; Evans and Stark, 1997). Inhibition of type Keywords: microtubule dynamics/mitosis/Op18/ 1 phosphatases (PP1) arrests the cells at the metaphase– phosphatase/spindle anaphase transition (Booher and Beach, 1989; Ohkura et al., 1989; Fernandez et al., 1992; Hisamoto et al., 1994; Ishii et al., 1996) because PP1 may be required to activate Introduction the anaphase-promoting complex (APC), required to degrade cyclin B (Ishii et al., 1996). However, other Correct chromosome segregation during mitosis requires phenotypes suggest that phosphatases are also involved in the assembly of a microtubule-based structure called the the control of structural events associated with mitosis. mitotic spindle. This assembly involves dramatic changes Mutants in PP2A genes show defects in spindle assembly in microtubule turnover: from a relatively stable state in and microtubule growth (Kinoshita et al., 1993; Snaith interphase, they become highly dynamic at the onset of et al., 1996; Evans and Stark, 1997), as do some mutants mitosis, their half-life changing from ~5 min to 45 s (Zhai in PP2A regulatory subunit genes (Gomes et al., 1993; et al., 1996). During assembly of the spindle, some mitotic Kinoshita et al., 1996). Mutants in PP1 genes in Saccharo- microtubules appear to be preferentially stabilized around myces cerevisiae (Black et al., 1995), Schizosaccharo- the chromosomes, and this contributes to generating the myces pombe (Ohkura et al., 1988, 1989), Aspergillus bipolar shape of the spindle. Thus, it is important to nidulans (Doonan and Morris, 1989) and Drosophila understand how microtubule dynamics are regulated both (Axton et al., 1990) show complex mitotic phenotypes globally and locally around the chromosomes. with condensed chromosomes, abnormal spindles and Changes in general microtubule stability can be chromosome separation malfunction. Since alterations in described by examining the sum of the dynamic properties the phosphorylation state of proteins involved in spindle of individual microtubules. Microtubules either grow or assembly and function can have effects on cell cycle shrink, the transition from one state to the other being progression and vice versa, the exact roles of phosphatases unpredictable, a property known as dynamic instability. Thus, the behavior of a microtubule is deﬁned by four during mitosis are unclear. parameters: the growth rate, the shrinkage rate and the We have addressed this problem using Xenopus egg frequencies of transitions between growth and shrinkage extracts as they allow study of the role of phosphatases © Oxford University Press 5537 R.Tournebize et al. in the regulation of microtubule dynamics and spindle initiation of spindle assembly, metaphase spindles were assembly independently of their role in cell cycle pro- present. Then 0.4 μM okadaic acid was added and aliquots gression. We have used two speciﬁc inhibitors of of the extract were ﬁxed and observed. In the absence of the serine-threonine phosphatases. Okadaic acid at low con- drug, microtubules were organized in a typical ellipsoidal centrations inhibits PP2A but does not inhibit PP1 activity shape, and chromosomes were aligned on the metaphase (for review, see Cohen, 1989; Shenolikar, 1994). Con- plate (Figure 1A). In contrast, bipolar spindles could not versely, inhibitor 2 (I-2), a 23.8 kDa protein, speciﬁcally be found 20 min after addition of 0.4 μM okadaic acid, inhibits PP1 activity by binding to the catalytic subunit the microtubules were up to 100 μm long, with an average (Cohen, 1989; Shenolikar, 1994). We ﬁnd that PP2A is length of 77 μm, and the chromosomes were scattered required to maintain the short steady-state length of throughout the structure (Figure 1A). Moreover, numerous microtubules during mitosis, in part by regulating Op18/ free microtubules could be observed in the extract. stathmin (Sobel, 1991), a recently identiﬁed molecule This effect of okadaic acid could reﬂect an indirect involved in the control of microtubule dynamics. PP1 has effect on cell cycle progression, for example by inducing a different role, and is involved in control of microtubule the return to interphase, or a direct effect on microtubule dynamics during the transitions between interphase and dynamics. Indeed, this drug is known to activate cdc2 mitosis. kinase in the presence of sub-threshold levels of cyclin (Felix et al., 1990), but also to induce cyclin degradation after some time of incubation (Felix et al., 1990; Lorca Results et al., 1991), which results in ﬁnal cdc2 kinase inactivation. Extracts made from Xenopus eggs can be arrested in In order to study PP2A function independently of any deﬁned states of the cell cycle, either in interphase or in effect on cdc2 kinase activity, we repeated the experiment mitosis, and their position in the cell cycle monitored by in the presence of cyclin Δ90. Because this cyclin construct measuring the activity of cdc2 kinase which is low in is non-degradable, it produces a constitutively active interphase and high in mitosis. Extracts can be arrested kinase when it combines with the endogenous cdc2 cata- in mitosis in two ways. First, extracts made from metaphase lytic subunit of the extract (Glotzer et al., 1991). Under II-arrested oocytes (CSF extracts) contain an activity such conditions, cdc2 kinase activity remained high after which maintains cdc2 kinase in an active form. Second, addition of 0.4 μM okadaic acid. However, microtubules a non-degradable version of cyclin B, cyclin Δ90, can be became long, and bipolar spindles disappeared from the added to extracts in order to stabilize the cdc2 kinase extract, replaced by structures such as that shown in activity. These latter extracts retain much of the mitotic Figure 1A. We conclude that even in the presence of high state even though the APC and the machinery responsible cyclin B–cdc2 kinase activity, microtubules grow long for cyclin degradation are active (Murray et al., 1989). In after inhibition of PP2A activity. Moreover, this result particular, mitotic spindles remain intact with a high also suggests that short microtubules are required to make microtubule turnover in such extracts, although anaphase a spindle. To test this further, we added 0.4 μM okadaic A occurs (Holloway et al., 1993; Surana et al., 1993). acid and sperm nuclei simultaneously to CSF extracts pre- Spindle assembly can be induced in Xenopus extracts incubated with cyclin Δ90. This resulted in the formation by adding sperm nuclei to a CSF extract that is then of asters with long microtubules (data not shown), but no released into interphase in order to allow DNA replication bipolar spindles were assembled. Thus bipolar spindles and centrosome duplication. Then, the extract is sent back may not assemble in the absence of PP2A activity because into metaphase by adding more CSF extract or cyclin of the length of the microtubules formed under such Δ90. This results in the migration of centrosomes around conditions. the nucleus and spindle assembly in a succession of prophase and metaphase ﬁgures (Sawin and Mitchison, PP2A inhibition affects the catastrophe rate of 1991; Shamu and Murray, 1992; Tournebize and Heald, microtubules 1996). Alternatively, spindles can be assembled by incubat- To understand how PP2A activity could control the steady- ing sperm nuclei directly in CSF extracts. Microtubules state length of microtubules during mitosis, we investigated grow from the sperm centrosome towards chromatin, how inhibition of PP2A affected the dynamics of individual forming half-spindles, then two half-spindles fuse, forming microtubules by time-lapse video microscopy. Extracts a bipolar spindle (Sawin and Mitchison, 1991; Tournebize pre-incubated with cyclin Δ90 were treated with 0.4 μM and Heald, 1996). Microtubule dynamics can be examined okadaic acid and supplemented with puriﬁed centrosomes separately by adding centrosomes in the absence of and rhodamine–tubulin. Images of microtubules were chromosomes. Under these conditions, one can see micro- recorded over time and the parameters of microtubule tubule ends better because the microtubule density is dynamics deduced from the analysis of the videos. Figure lower. Additionally, one can determine the changes in 1B shows images taken from two videos, in the presence microtubule dynamics in the absence of the inﬂuence of and absence of 0.4 μM okadaic acid. Microtubules were mitotic chromatin. longer in the presence of okadaic acid and we also noticed a signiﬁcant increase in spontaneous assembly. Analysis A PP2A-like activity keeps microtubules short of individual microtubules in several videos showed that during metaphase inhibition of PP2A activity reduced the level of cata- In order to determine whether PP2A activity was involved strophes by 1.5- to 3-fold, depending on the experiment in the regulation of microtubule length during metaphase, (Table I). The average values from ﬁve independent we tested the effect of okadaic acid on the morphology experiments showed that okadaic acid addition decreased of spindles assembled in extracts. At 60 min after the the catastrophe rate from 2.44 catastrophes/min to 1.22 5538 Phosphatases and mitosis Fig. 1. (A) Addition of okadaic acid to spindles induces microtubule growth. Spindles assembled in cyclin Δ90 Xenopus egg extracts were mock treated (control) or treated with 0.4 μM okadaic acid for 20 min at 20°C. (B) Addition of 0.4 μM okadaic acid increases microtubule steady-state length. Images taken at the same recording time from videos in the presence (bottom) or absence (top) of 0.4 μM okadaic acid. Time is h:min:s. Bars are 10 μm. Table I. Addition of okadaic acid to extracts reduces catastrophe frequencies Growth rate Shrinkage rate Catastrophe frequency Rescue frequency Calculated microtubule (μm/min) (μm/min) (events/min) (events/min) length (μm) Control 11.44 1.21 (105) 13.49 1.90 (91) 2.44 0.54 (91) 0.70 (29) 7.4 Okadaic acid 13.00 1.24 (87) 16.37 2.81 (58) 1.22 0.21 (58) 1.40 (17) 34.4 Okadaic acid (0.4 μM) was added to cyclin Δ90 extracts. After 20 min incubation at 20°C, extracts were frozen and aliquots of these used for microtubule dynamics measurements for 5 min. Growth rate, shrinkage rate and catastrophe frequency were determined after measurements of microtubules over time. Rescue frequencies reported here are not statistically signiﬁcant. Values are means standard error of the mean (number of total events measured). Average steady-state length L was calculated using the measured values. catastrophes/min. Average growth and shrinkage rates acid. We found an average microtubule length L of were not signiﬁcantly affected, being ~12 and 15 μm/min 7.4 μm in control and 34.4 μm in extracts treated with respectively. Rescues were 0.70 event/min in control and 0.4 μM okadaic acid (Table I). Thus, inhibition of PP2A 1.40 events/min in the presence of okadaic acid. Due to results in a 5-fold increase in microtubule steady-state a low number of events observed, this difference is not length L due to only a 2-fold reduction in the cata- statistically signiﬁcant. Okadaic acid addition had no effect strophe rate. on interphase microtubule dynamics (data not shown). Verde et al. (1992) showed that the steady-state length PP2A regulates Op18/stathmin phosphorylation of microtubules is related directly to the parameters of We were interested in ﬁnding targets of PP2A that could microtubule dynamics and that microtubule steady-state regulate microtubule dynamics. Since Op18/stathmin is length can be calculated from the values of microtubule thought to destabilize microtubules in Xenopus egg extracts dynamics using a simple equation (see Materials and (Belmont and Mitchison, 1996), and since phosphorylation methods). Using this formula, we calculated the micro- is known to regulate its activity (Marklund et al., 1996; tubule steady-state length L in the presence and Horwitz et al., 1997), we examined the role of PP2A in absence of 0.4 μM okadaic acid. We assumed that the regulating Op18/stathmin phosphorylation. Okadaic acid rescue frequency was not affected by treatment with was added to an extract containing cyclin Δ90 in order to okadaic acid, taking a value of 1.05 rescues/min, the inhibit PP2A, and [γ- P]ATP was added in order to average of the values measured with and without okadaic label phosphorylated proteins. Op18/stathmin was then 5539 R.Tournebize et al. immunopuriﬁed and the level of P incorporation (Marklund et al., 1996; Horwitz et al., 1997), while monitored. In untreated extracts, Op18/stathmin was phosphorylated Op18/stathmin does not. Thus our experi- poorly phosphorylated. Addition of increasing amounts of ments suggest that, normally, Op18/stathmin in extracts okadaic acid to the extract resulted in a gradual increase is dephosphorylated and active. PP2A inhibition promotes in the level of P incorporated into Op18/stathmin protein phosphorylation of OP18/stathmin, thus microtubules (Figure 2B). Quantiﬁcation of the level of P incorporated become more stable. If PP2A inhibition promoted micro- showed that at 0.5 μM okadaic acid, Op18/stathmin tubule growth by promoting hyperphosphorylation of contained four times more P than the control. For Op18/stathmin, then a mutant Op18/stathmin which cannot comparison, addition of 3 μM I-2, which speciﬁcally be phosphorylated should overcome the effect of PP2A inhibits PP1, only induced a 1.5-fold increase in the level inhibition on microtubule steady-state length. In order to of P incorporated. Taken together, these observations test this idea, we used puriﬁed Op18/stathmin protein in show that PP2A activity maintains Op18/stathmin in a which the four known phosphorylated serines had been low phosphorylation state in metaphase extracts. mutated to alanine. Okadaic acid (0.4 μM) was added together with puriﬁed Op18/stathmin to pre-assembled PP2A regulates microtubule dynamics by spindles that were then ﬁxed and observed. Twenty minutes controlling the phosphorylation state of after addition of the mutant Op18/stathmin protein together Op18/stathmin with okadaic acid, most spindles were bipolar (Figure 3), Previous experiments had suggested that, when unphos- but ~20% of them had short microtubules uniformly phorylated, Op18/stathmin destabilizes microtubules wrapped around chromatin without clear poles, forming round ‘spindles’ (data not shown). Addition of okadaic acid together with wild-type Op18/stathmin generated the same structures as okadaic acid alone (Figure 3). Thus, addition of unphosphorylatable Op18/stathmin rescues the effect of okadaic acid on microtubule growth while wild- type Op18/stathmin does not. Op18/stathmin depletion decreases the catastrophe rate The previous results strongly suggested that PP2A controls microtubule dynamics by regulating the phosphorylation of Op18/stathmin, but did not rule out the possibility that exogenously added mutant Op18/stathmin depolymerized microtubules independently of PP2A. If PP2A acted through Op18/stathmin to control the catastrophe rate of microtubules, then depletion of Op18/stathmin should decrease the catastrophe rate as well. We therefore depleted Op18/stathmin from extracts and measured microtubule Fig. 2. The Op18/stathmin phosphorylation state is regulated by PP2A. dynamics. Results from four independent depletion experi- Different concentrations of okadaic acid and I-2 were added to cyclin ments are shown in Figure 4. Upon removal of ~95% of Δ90 extracts containing [γ- P]ATP. After 20 min, Op18/stathmin was immunoprecipitated and run on a 15% acrylamide gel. (A) Silver Op18/stathmin, the catastrophe frequency decreased 1.5- staining of the gel; (B) an autoradiogram. Phosphorylation of to 5-fold, depending on the experiments. On average, Op18/stathmin increased four times in the presence of 0.5 μM okadaic catastrophes decreased from 1.46 events/min in control acid and 1.5 times in the presence of 3 μM I-2. The arrow points to treated extracts to 0.86 events/min in Op18/stathmin- Op18/stathmin, which resolves as a triplet. Molecular weight markers are indicated. depleted extracts. Thus Op18/stathmin is required to Fig. 3. Non-phosphorylatable Op18/stathmin induces microtubule depolymerization. Spindles were assembled in cyclin Δ90 extracts and okadaic acid was added alone (left panel), together with 250 μg/ml of human Op18/stathmin protein with its four phosphorylation sites mutated to alanine (middle panel) or with 250 μg/ml wild-type human Op18/stathmin (right panel). Spindles were ﬁxed 20 min after incubation at 20°C. Bar is 10 μm. 5540 Phosphatases and mitosis Fig. 4. Catastrophe frequency decreases after Op18/stathmin depletion and okadaic acid addition. Op18/stathmin was depleted from extracts containing cyclin Δ90, and microtubule dynamics were measured. Catastrophe frequency decreases after Op18/stathmin depletion Fig. 5. PP1 is not required for spindle assembly. Spindle assembly was (ΔOp18) and after 0.4 μM okadaic acid addition (MockOA). No monitored in the presence or the absence of I-2. Extracts containing signiﬁcant decrease in catastrophe is observed after addition of 0.4 μM nuclei were released into interphase for 80 min before mitosis was okadaic acid to Op18/stathmin-depleted extract (ΔOp18OA). Values triggered by addition of CSF extract together with 3 μM I-2 (1 μM I-2 are means standard error of the mean. was then added every 15 min). Spindles were ﬁxed every 10 min and the percentage of spindles in metaphase scored. No statistical difference was observed upon PP1 inhibition compared with the maintain a high level of catastrophes during metaphase. control. We also analyzed the effect of Op18/stathmin depletion on the growth and shrinkage rates of microtubules. These length was not affected over 60 min of observation (data rates were not statistically changed by Op18/stathmin not shown). These results suggested that once the extract depletion. Using these values, we calculated the average contains a high level of cdc2 kinase activity, PP1 is not length L of microtubules. We found that they are required to regulate microtubule dynamics and spindle 7.9 μm long in mock-depleted extracts and 100 μmin assembly. Op18/stathmin-depleted extracts. These values show that Op18/stathmin is a key regulator of microtubule length PP1 and cdc2 kinase act antagonistically to control in vitro. the steady-state length of microtubules at the In order to test whether Op18/stathmin could account onset of mitosis for all the changes in catastrophes observed upon PP2A Although inhibition of PP1 had no effect on spindle inhibition, we added 0.4 μM okadaic acid to Op18/ assembly, we wondered whether it could participate in the stathmin- or mock-depleted extracts. The catastrophe fre- regulation of microtubule dynamics during the early phase quency dropped from 0.85 to 0.59 in Op18/stathmin- of mitosis when cdc2 kinase begins to act on microtubule depleted extracts. However, this difference was not found dynamics, in prophase. To test this hypothesis, we made to be statistically signiﬁcant (Figure 4). Growth and use of puriﬁed cdc2 kinase which, when added to an shrinkage rates were not signiﬁcantly changed. The calcu- interphase extract, triggers mitotic microtubule dynamics lated microtubule length L after addition of 0.4 μM (Verde et al., 1990). We reasoned that if PP1 is opposing okadaic acid was 61.2 μm in mock-treated extracts and cdc2 kinase activity during the transition of microtubule 100 μm in Op18/stathmin-depleted extracts. These dynamics from an interphase to a metaphase state, then results show that Op18/stathmin accounts for most of the inhibition of PP1 should speed up the rate at which cdc2 catastrophes regulated by PP2A in Xenopus extracts. In kinase phosphorylates substrates and microtubules become conclusion, PP2A maintains the short steady-state length dynamic. Cdc2 kinase was added to interphase extracts of microtubules by regulating the catastrophe rate, mainly with or without I-2. Extracts were complemented with through regulation of the activity of Op18/stathmin. centrosomes and rhodamine–tubulin, ﬁxed 15 min later and spun down onto coverslips, and the length of microtub- PP1 is required for the control of microtubule ules were measured. As shown in Figure 6, microtubules dynamics during the transitions into and out of are long in interphase extracts (25 μm) and become shorter mitosis upon addition of cdc2 kinase (Figure 6A). In the presence To examine the role of PP1 in the regulation of microtubule of PP1 inhibitor, 9–10 pmol/μl/min of cdc2 kinase activity dynamics, we used a speciﬁc inhibitor of PP1, I-2, a were required to convert the average microtubule length 23.8 kDa protein which binds speciﬁcally to the catalytic to a mitotic state (Figure 6B and C) while 20 pmol/μl/ site of PP1 to inhibit its activity. We added I-2 to min were necessary in the absence of inhibitor. This result interphasic and mitotic extracts and observed microtubule suggests that PP1 and cdc2 kinase have opposing effects length and dynamics. Inhibition of PP1 had no effect on the in controlling microtubule dynamics during the interphase parameters of microtubule dynamics in either interphase or to metaphase transition. metaphase (data not shown). To test whether PP1 is required for spindle assembly, extracts containing sperm PP1 participates in the regulation of global nuclei were released into interphase and I-2 was added at changes in microtubule dynamics during the the same time as CSF extracts. The kinetics of spindle metaphase to anaphase transition assembly were statistically similar in the presence and The previous results suggested that PP1 opposed cdc2 absence of I-2 (Figure 5), and maintenance of spindle kinase during the onset of metaphase. We wondered 5541 R.Tournebize et al. events/min. In control extracts, the calculated microtubule length L was 8.7 μm in metaphase and 100 μm30 min after anaphase induction. In the absence of PP1 activity, the calculated microtubule length L remained unchanged at ~5–8 μm (Figure 7F). Therefore, PP1 is required in anaphase to increase the steady-state length of microtubules by increasing the growth rate and decreasing the catastrophe rate. PP1 is also required for spindle microtubule elongation and chromosome separation in anaphase The previous results indicated that PP1 was required for microtubule elongation in anaphase in the absence of chromosomes. We wanted to examine what would happen to spindle microtubules. As shown in Figure 8A, under normal conditions, chromosomes start to separate and move towards the poles 10 min after calcium addition. By 25 min, anaphase is completed. When PP1 was inhibited, spindles gradually lost microtubules and the poles moved towards chromatin and eventually fell apart, forming half-spindles (Figure 8B). No clear chromosome segregation was observed. Similar observations were made using CaMKII to trigger anaphase. We quantiﬁed spindle length during anaphase with and without PP1. At the end of anaphase, spindles were 42 μm long in the control and 22 μm in the absence of PP1 activity (Figure 8C). We looked at chromosome separation under such conditions. As shown in Figure 8D, chromosomes moved towards poles in the control while they stayed in the middle of the spindle when I-2 was used. One hour after CaMKII addition, the microtubules had not returned to their Fig. 6. PP1 opposes the effect of cdc2 kinase on microtubule length. interphase length and, in addition, chromatin was still Increasing amounts of puriﬁed starﬁsh cdc2 kinase were added to condensed as previously reported (Axton et al., 1990). interphase extract in the absence (A) or the presence (B) of I-2. Less These results show that PP1 is necessary both for spindle cdc2 kinase is required to obtain mitotic microtubule length in the presence of I-2 (C). Numbers in the bottom right of the pictures stability and elongation, and for chromatin decondensation indicate cdc2 kinase activity (pmol/μl/min). during anaphase, and that this is not due to an effect of PP1 inhibition on the inactivation of cdc2 kinase. whether PP1 was also required to increase the steady- state length of microtubules during the onset of anaphase. Discussion To measure these changes, we used a downstream effector of calcium in the cascade triggering cyclin degradation at Since the initial demonstration that microtubule length is anaphase onset: the calcium/calmodulin-dependent kinase governed during metaphase by phosphorylation– II (CaMKII) (Lorca et al., 1993; Morin et al., 1994). We dephosphorylation reactions under the control of cdc2 looked at cdc2 kinase activity after induction of anaphase kinase (Verde et al., 1990) and that this was mediated by in the presence and absence of I-2. In both cases, cdc2 an increase in catastrophe rate (Belmont et al., 1990; kinase activity dropped to interphase levels by 30 min Verde et al., 1992), little progress has been made in the (Figure 7A) with identical kinetics, showing that, at least characterization of the biochemical pathways or signaling in Xenopus extracts, PP1 is not required for the inactivation mechanisms involved. Here, we undertook a study of the of cdc2 kinase. role of type 1 and type 2A-like phosphatases in these Microtubule dynamics were recorded at 15 min intervals processes during mitosis. We show that PP2A is required after anaphase induction by CaMKII. In control extracts, continuously in mitosis to control the short steady-state chromosome to pole movement was completed 30 min length of microtubules, primarily by regulating the after CAMKII addition (Figure 7B and see below). At phosphorylation state of the microtubule-destabilizing this time, the catastrophe frequency decreased from 2 to factor Op18/stathmin. In contrast, PP1 is required to 1 event/min, and the growth rate increased from 9.9 to stabilize microtubules during the transitions between 16.55 μm/min (Figure 7C and E). The shrinkage rate was interphase and mitosis. Thus these two phosphatases have not changed during this transition (Figure 7D). In contrast, distinct roles in the control of microtubule dynamics in extracts treated with I-2, catastrophe frequency, growth during mitotic progression. and shrinkage rates were identical for the ﬁrst 30 min We have characterized these activities using inhibitors after induction of anaphase (Figure 7C–E). We calculated of serine-threonine phosphatases. In vitro, the IC of the average length L of microtubules using the values okadaic acid is 0.2 nM for PP2A and 20 nM for PP1 (for measured and assuming a constant rescue rate of 1.05 review, see Cohen, 1989). These concentrations are much 5542 Phosphatases and mitosis Fig. 7. PP1 is required for returning from metaphase to interphase microtubule dynamics. (A) Cdc2 kinase inactivation in anaphase induced by CaMKII is not affected by PP1 inhibition. (B) The distance from chromosomes to the pole in control extract decreases as chromosomes segregate. (C–E) PP1 prevents changes in microtubule dynamics in anaphase. In these experiments, CSF extracts were pre-incubated for 15 min with (gray bars) or without (black bars) 3 μM I-2 before anaphase was induced with CaMKII. Microtubule dynamics were recorded at 0, 15 and 30 min for 5–7 min. Growth rate (C), shrinkage rate (D) and catastrophe frequency (E) were determined after measurements of individual microtubules over time. Rescue frequency is omitted here because too few events were observed and values are not statistically signiﬁcant. (F) Microtubule length L does not increase in the presence of I-2 during anaphase. Calculated microtubule lengths were determined using the measured values. Rescues were assumed to be 1.05 events/min. Data in (B–E) are means standard error of the mean. lower than that used in our study, but it is known that the in PP2A regulatory subunits also display defects in micro- inhibitory activity of these inhibitors is altered by high tubule organization (Gomes et al., 1993; Kinoshita et al., protein concentration (Ingebritsen et al., 1983) and our 1996). Furthermore, associated subunits of PP2A bind to extracts have a protein concentration of ~60–80 mg/ml. microtubules and PP2A is associated with microtubules We have measured phosphatase activities in concentrated in tissue culture cells (Sontag et al., 1995). This suggests extracts under the same conditions as used to assay that PP2A is indeed the phosphatase catalytic subunit microtubule dynamics and spindle assembly. We found involved in controlling microtubule length. that under these conditions, 0.4 μM okadaic acid is Previous results had also shown that inhibition of required to produce a 50% inhibition of PP2A-like activi- phosphatases using okadaic acid results in the formation ties, whereas 3 μM I-2 is required to inhibit PP1 fully. of long microtubules. In starﬁsh (Picard et al., 1991), Low concentrations of okadaic acid may affect the activity mouse oocytes (de Pennart et al., 1993) and GH4 rat of catalytic subunits other than the typical PP2A. Indeed, pituitary cells (Van Dolah and Ramsdell, 1992), spindles four phosphatases, PP3 (Honkanen et al., 1991), PP4 were shown to be destroyed and replaced by extremely (Brewis et al., 1993) (previously named PPX), PP5 (Chen long microtubule networks. This is consistent with our et al., 1994) and PPV in Drosophila (Armstrong et al., results but, in some of these previous experiments, it was 1995), have been shown recently to be highly sensitive to not possible to know whether a type 1 or 2A phosphatase okadaic acid and insensitive to I-2. Therefore, we cannot was inhibited. Interestingly, in clariﬁed interphasic extracts rule out the possibility that one of these is the inhibited from sea urchin, addition of high concentrations of okadaic phosphatase in our experiments. However, PP2A mutants acid (1–2.5 μM) resulted in a decrease in rescues and a in Drosophila, S.pombe and S.cerevisiae show abnormal slight increase in catastrophes (Gliksman et al., 1992). microtubule morphology (Kinoshita et al., 1993; Snaith However, these extracts had a lower protein concentration et al., 1996; Evans and Stark, 1997), with an extensive (20–25 mg/ml) and it is likely that both PP1 and PP2A microtubule network similar to the one seen here. Mutants were inhibited. The effects observed are probably the 5543 R.Tournebize et al. Fig. 8. PP1 is required for spindle stability and chromosome segregation in anaphase. Spindles were assembled and PP1 was inhibited for 15 min before anaphase was induced by CaMKII. Spindles were ﬁxed at various time points. (A) Normal metaphase and anaphase spindles. (B) Collapsed and half-spindles observed in anaphase in the presence of I-2. Graphs show the percentage of spindles in metaphase (d) and anaphase (s)inthe absence (A) or presence (B) of I-2 during anaphase. (C) Spindle length in the absence (j) or presence (u) of I-2. (D) Chromosome position in the spindle during anaphase in the absence (j) or presence (u) of I-2. Chromosome position is determined by the chromosome to pole distance divided by the pole to pole distance. Bar is 10 μm. consequence of a complex regulation by many phos- and fungi? One major difference is the lack of checkpoint phatases which may act in many different ways. In controls in Xenopus extracts under our conditions. Thus clariﬁed Xenopus interphase extracts, addition of high PP1 mutants may cause a metaphase delay in fungi by concentrations of okadaic acid results in a decrease in activating the spindle assembly checkpoint (for review, growth and shrinkage rates, while it has little effect on see Wells, 1996). Indeed, in S.cerevisiae, the PP1 encoded these rates in mitotic extracts (Parsons and Salmon, 1997). by the GLC7 gene has been shown to regulate chromosome We also noticed that catastrophes were decreased in mitotic segregation (Francisco and Chan, 1994; Hisamoto et al., clariﬁed extracts (R.Tournebize and R.Heald, unpublished 1994; Black et al., 1995). Recent genetic data in S.pombe observations). One possibility is that most of PP2A is suggest that PP1 may regulate the APC and, therefore, depleted from clariﬁed extracts (A.A.Hyman, unpublished exit from mitosis (Ishii et al., 1996). However, since the observation), thus resulting in Op18/stathmin inactivation. APC regulates cyclin destruction and, therefore, cdc2 Because PP1 is the only phosphatase known to be kinase inactivation, we would expect to see a mitotic inhibited efﬁciently by I-2, this small polypeptide can be delay in Xenopus if PP1 inhibition down-regulates the used to determine what is regulated by PP1-like enzymes. APC. As this is not observed in Xenopus extracts, it is Again, we determined that adding 3 μM I-2 followed by possible that PP1 acts upstream of the APC through a regular addition of 1 μM produced a complete inhibition spindle checkpoint rather than directly in cyclin of PP1 in our concentrated extracts. Our results show that degradation. PP1 has a particular role in the control of microtubule turnover in Xenopus extracts, during the switches between Regulation of microtubule dynamics and interphase and mitosis. In addition, PP1 is required for steady-state length during metaphase by PP2A chromosome decondensation, conﬁrming previous results and Op18/stathmin in Drosophila (Axton et al., 1990). However, PP1 does Our results suggest that Op18/stathmin is a major down- not seem to be required for cell cycle changes necessary stream substrate of PP2A in the regulation of the steady- for exit from mitosis in Xenopus egg extracts, while state length of microtubules. This protein regulates the inhibition of PP1 in fungi causes a metaphase arrest or length of microtubules both in Xenopus extracts (Belmont delay. Why should there be a difference between Xenopus and Mitchison, 1996; this study) and in tissue culture cells 5544 Phosphatases and mitosis (Marklund et al., 1996; Horwitz et al., 1997), and this during spindle assembly and disassembly (Andersen effect is regulated by phosphorylation. The phosphorylated et al., 1997). form of Op18/stathmin loses its microtubule-destabilizing activity (Marklund et al., 1996; Horwitz et al., 1997). Our Regulation of microtubule dynamics by PP1 during results in Xenopus conﬁrm these observations. Moreover, the transitions from interphase to mitosis and they suggest that the phosphatase which dephosphorylates back to interphase Op18/stathmin is PP2A. Although the kinase that opposes We have shown that PP1 is required for reorganization of PP2A is unidentiﬁed, MAP kinase and the cdc2 kinase the microtubule network during the transitions between are likely candidates (Larsson et al., 1995). This would interphase and mitosis. It is hard to say whether PP1 is make sense since PP2A opposes cdc2 kinase and some regulating microtubule-stabilizing or -destabilizing factors MAP kinase activities on many substrates like the brain or both during entry into mitosis and during anaphase. microtubule-associated protein tau (Goedert et al., 1992; One clue comes from the analysis of the dynamic para- Ferrigno et al., 1993). Although it is formally possible meters during exit from mitosis. We have shown that the that PP2A regulates Op18/stathmin by increasing MAP growth rate increases during exit from mitosis, but that kinase activity (Shibuya et al., 1992; Nebreda and Hunt, this increase in growth rate is blocked in the absence of 1993), this is unlikely because, in extracts treated with PP1. Since MAPs are generally thought to stimulate cyclin Δ90, MAP kinase activity is already high (Shibuya microtubule growth, this strongly suggests that one of the et al., 1992; Minshull et al., 1994) although Op18/stathmin roles of PP1 is to activate MAPs in order to stabilize is poorly phosphorylated (Figure 3). The fact that Op18/ microtubules during anaphase. stathmin is poorly phosphorylated in metaphase extracts During metaphase, inhibiting PP1 has no effect on the means that it is active in these extracts. This ﬁnding is in stability of the mitotic spindle, whereas, during anaphase, apparent contradiction to results obtained in vivo where it results in a highly unstable spindle. This instability Op18/stathmin is phosphorylated during metaphase. This manifests itself by a loss of microtubules from the spindles contradiction can be resolved by the observation that the and a movement of the poles towards the chromatin. This phosphorylation of Op18 is stimulated by chromatin is reminiscent of the effect of nocodazole, a drug which which is present at low levels in our extracts (Andersen inhibits microtubule assembly (Ault et al., 1991). It may et al., 1997). seem strange that spindle microtubules only become Catastrophes in mitosis are 8–10 times more frequent unstable during anaphase following PP1 inhibition. How- than in interphase. Following depletion of Op18/stathmin ever, two things must be considered. First, during meta- from a mitotic extract, the catastrophe rate drops to a phase, the chromosomes play an important role in rate only ~4–5 times more frequent than in interphase. stabilizing microtubules (Nicklas and Gordon, 1985; Therefore, other microtubule-depolymerizing factors regu- Zhang and Nicklas, 1995; Dogterom et al., 1996; Heald lated independently of PP2A must account for the et al., 1996). This activity may be lost during anaphase remaining catastrophes in mitosis. Such a factor can be and this may have to be substituted by a global PP1 XKCM1 (Walczak et al., 1996) and other as yet unidenti- activity acting to dephosphorylate factors required to ﬁed catastrophe factors. In interphase, Op18/stathmin is stabilize microtubules again at the exit from metaphase. also dephosphorylated (Brattsand et al., 1994; Larsson Secondly, there may be a large increase in length during et al., 1995) and active, but catastrophes are low. This anaphase B, which requires microtubule polymerization strongly suggests that although microtubules could be and stabilization. Thus, many mutants in PP1 probably destabilized by the non-phosphorylated form of Op18/ manifest themselves in anaphase due to a lack of micro- stathmin in interphase, they are not because of the presence tubule stabilization. In other words, PP1 may act to reverse of stabilizing factors like MAPs. We suggest that, during the phosphorylation state of microtubule-stabilizing fac- mitosis, the catastrophe rate decreases because Op18/ tors, like MAPs, that are inactivated by phosphorylation stathmin remains active globally whereas MAPs do not during metaphase. stabilize microtubules efﬁciently. We observed that PP2A inhibition resulted in an increase Switches and steady-state balances in microtubule spontaneous assembly in metaphase One of the main conclusions of our studies is that extracts. This could be due to regulation of the activity PP1 and PP2A have distinct functions in the control of of microtubule nucleating sites. Alternatively, the high microtubule dynamics. PP1 is required for the switches catastrophe rate observed in metaphase may normally between mitosis and interphase while PP2A is required to prevent short microtubules from reaching a visible size. maintain the short steady-state length of microtubules We were not able to determine whether the decrease in during mitosis. This illustrates two types of regulation catastrophe rate produced by inhibition of PP2A may be involved in spindle assembly: global regulation during the sufﬁcient to explain the increase in spontaneous assembly transitions between cell cycle states and local regulation observed under these conditions. during spindle formation. Thus, PP2A seems to act as In conclusion, PP2A controls the steady-state length of part of a phosphorylation balance that maintains the microtubules at least in part by controlling the activity of steady-state length of microtubules during metaphase. We Op18/stathmin. Thus the level of stability of metaphase imagine that the balance works in the following way. PP2A microtubules seems to depend upon a phosphorylation– and its opposing kinase are both active with comparable dephosphorylation balance involving PP2A and cdc2 activities. Subtle regulation of the activity of either of the kinase acting directly or indirectly on Op18/stathmin. enzymes would allow precise adjustment of the steady- Subtle spatial regulation of this balance could participate state length of microtubules. Adjustment of such a balance in the regulation of microtubule dynamics and organization would provide a means by which chromosomes locally 5545 R.Tournebize et al. and inactive, with the consequence that microtubules will stay short during the progression into interphase. Evidence for such a model comes from our studies that show that PP1 opposes cdc2 at the onset of mitosis and in anaphase. It is well known that the activity of cdc2 ﬂuctuates in the cell cycle, and it appears that the enzymatic activities of some PP1 catalytic subunits are inactivated by cdc2 phosphorylation during mitosis (Dohadwala et al., 1994; Yamano et al., 1994; Kwon et al., 1997). It is likely that PP1 and PP2A affect microtubule dynamics by acting on different substrates. Some may stabilize, others may destabilize microtubules. We have shown how one of these substrates, Op18/stathmin, is regulated by PP2A. Other microtubule regulators under the control of this enzyme and PP1 now need to be identiﬁed. Materials and methods Enzymatic assays Unless speciﬁed, all reagents were from Sigma. Cdc2 kinase activity assays were performed as described by Felix et al. (1989). PP2A activities were measured using phosphorylated casein prepared as described by McGowan and Cohen (1988), and PP1 activities using phosphorylated phosphorylase (phosphatase assay kit, Gibco BRL). Phosphatase activi- ties were measured in crude concentrated extracts similar to those used by Felix et al. (1989). We ﬁrst characterized inhibitor concentrations necessary to inhibit phosphatases in crude 10 000 g extracts. Okadaic Fig. 9. Model for a switch regulation between cdc2 kinase and PP1. acid (Gibco BRL) was used to inhibit PP2A, and I-2 (a gift from Phil (A) A substrate X which stabilizes microtubules is regulated by cdc2 Cohen) used to inhibit PP1. Typically, after incubation of extracts with kinase and PP1. When phosphorylated (X-P), X is inactive, but it is either okadaic acid or I-2 for 15 min at room temperature, 5 μlof active in its unphosphorylated form (X). (B) In a normal cell cycle, substrate were added to 10 μl of extracts for 30–45 s. The reaction was cdc2 kinase increases during mitosis (M) and decreases at anaphase. stopped by adding 100 μl of 10 mM EDTA, EGTA, 100 mM NaF, Concomitantly, PP1 activity decreases in mitosis and is high in β-glycerophosphate, 1 mg/ml bovine serum albumin (BSA), 0.1% interphase (I). In interphase, no cdc2 kinase is present and PP1 is β-mercaptoethanol (BME) and 100 μl of 10% trichloroacetic acid (TCA). active, therefore X is dephosphorylated, active and stabilizes Samples were incubated on ice for 10 min followed by centrifugation microtubules (promoting a long steady-state length). In mitosis, less at 15 000 g for 10 min, and soluble P released accounting for PP1 is active, cdc2 kinase activity is high and most of X is phosphatase activity were counted. Okadaic acid was titrated and found phosphorylated and inactive. Thus microtubules are short. (C)Inthe to inhibit 50% of PP2A at 0.4 μM. Using cyclin Δ90 (Glotzer et al., absence of PP1, at the interphase–metaphase transition, cdc2 kinase 1991), cdc2 kinase activity was not modiﬁed by incubation with okadaic phosphorylates X more quickly, thus inactivating it, such that less cdc2 acid. It was found that 3 μM I-2 was sufﬁcient to inhibit 50–60% of kinase activity is required to obtain a maximal effect. During the total phosphatase activity, showing that PP1 accounts for about half of metaphase–anaphase transition, cdc2 kinase is inactivated. If PP1 is the total serine-threonine phosphatase activity measurable with phos- inactive, X stays in an inactive phosphorylated form for longer and phorylated phosphorylase. Because I-2 can be inactivated by glycogen microtubules stay shorter. synthetase kinase phosphorylation, we added 1 μM I-2 every 15 min to the initial 3 μM used to inhibit PP1. Using such conditions, PP1 was constantly inhibited during 90 min. stabilize microtubules during spindle assembly (Karsenti, 1991; Hyman and Karsenti, 1996). Extract preparation and spindle assembly On the other hand, we believe that PP1 acts as a switch Xenopus extracts were prepared as described in Murray (1991). Meiotic extracts (which we refer to as CSF extracts) and spindles were prepared because the activities of PP1 and its opposing kinase, using a two-step procedure (Shamu and Murray, 1992). Sperm nuclei presumably cdc2, are different in interphase and mitosis. were added to CSF extracts released into interphase by calcium. After A model for the function of PP1 in the interphase–mitosis DNA and centrosomes duplicated, some CSF extract was added back switch is shown in Figure 9. Figure 9A and B shows the and spindles assembled for 60 min. When necessary, cyclin Δ90 was added 45 min after CSF extract addition for 45 min and then okadaic normal situation. A substrate X is dephosphorylated and acid was added. To test the role of PP2A in controlling spindle assembly, active and stabilizes microtubules in interphase. Con- sperm nuclei together with okadaic acid were added directly to extracts versely, it is phosphorylated and inactive in mitosis and (Sawin and Mitchison, 1991) pre-incubated with cyclin Δ90. Okadaic does not stabilize microtubules so that they can undergo acid was added at 0.4 μM ﬁnal concentration and spindles ﬁxed catastrophes and become shorter. At the entry of mitosis, every 10 min. Maximum effect was observed 20 min after okadaic acid addition. cdc2 kinase activity increases and PP1 activity decreases, Anaphase was induced by adding 0.4 mM CaCl (Shamu and Murray, triggering phosphorylation and inactivation of X. Micro- 1992) or 0.1 vol. of a dominant active CaMKII (Morin et al., 1994) tubules are no longer stabilized and become shorter. At subcloned in pCYCΔ13T (Glotzer et al., 1991) and transcribed in the metaphase–anaphase transition, cdc2 activity declines reticulocyte lysates (TNT-coupled reticulocyte lysate systems, Promega). During anaphase, I-2 was ﬁrst added to 3 μM to CSF extracts for 15 min. and PP1 activity increases, dephosphorylating X and Then 1 μM I-2 and CaCl /CaMKII were added to induce anaphase, and activating it. As a result, short microtubules in mitosis spindles were observed during 30–45 min. After anaphase induction,1 μM can be converted to long ones in interphase. Figure 9C I-2 was added every 15 min. shows what happens if PP1 activity is inhibited during the Puriﬁed human Op18/stathmin, either wild-type or with all four metaphase–anaphase transition. X will stay phosphorylated phosphorylatable serines mutated to alanine [kind gift of Andre Sobel, 5546 Phosphatases and mitosis 10 mg/ml in phosphate-buffered saline (PBS)], was added to pre- and a t-test (assuming the variance unknown) and paired t-test were assembled spindles at the same time as 0.4 μM okadaic acid. Microtubule performed. Values from experiments where n was 5 were discarded. growth was dependent on the concentration of mutated Op18/stathmin Differences reported were signiﬁcant at least at the 5% level of added. Images of spindles were taken on a Zeiss Axioskop microscope conﬁdence, except for the catastrophe frequency after I-2 addition in with either a Sony SSC-M370CE black and white camera or a color anaphase and okadaic acid addition to cyclin Δ90 extracts which are Coolview camera (Photonic Science). Images were then processed further signiﬁcant at the 7% level. This reﬂects variations between different with Adobe Photoshop. experiments. Only few rescues could be observed such that proper statistics could be done. Immunodepletions Microtubule steady-state length was calculated using the following Peptide antibodies to Op18 were prepared according to Belmont and equation described in Verde et al. (1992): Mitchison (1996). Immunodepletions were done as previously described V F – V F (Walczak et al., 1996). Typically, 400 μg of antibody were bound to g res s cat 60 μl of Afﬁprep protein A beads (Bio Rad) for 1 h at 4°C . After F F cat res washing, 200 μl of cyclin Δ90 extracts were added to packed beads. Extracts were depleted for 1 h at 4°C on a rotating wheel. Depleted where V , V , F and F are growth rate, shrinkage rate, catastrophe g s cat res extracts were then used for microtubule dynamics. Ninety-ﬁve percent frequency and rescue frequency respectively. As a rescue frequency, we depletion was usually achieved by this procedure, as determined by used the averaged value (1.05 events/min) of the rescue frequencies Western blotting. Mock-treated extracts were depleted using the same from the okadaic acid addition experiment. If rescues had increased in amount of pure rabbit IgG (Dianova). Treatment of the extract with the any of the experiments, we should have noticed. In the absence of any beads affected microtubule polymerization, explaining a decrease in increase, we assumed that the rescue frequency was not changed and catastrophe frequency between untreated extracts and mock-depleted used a value of 1.05 events/min in all microtubule length calculations. extracts. Op18 phosphorylation Acknowledgements Op18 was phosphorylated in a 55 μl extract containing cyclin Δ90, 1 μCi [γ- P]ATP/μl extract and different concentrations of okadaic acid We are grateful to Phil Cohen for his gift of inhibitor 2, to Andre Sobel or I-2. After 15 min, phosphorylation was stopped by adding 1 vol. of for his puriﬁed Op18/stathmin protein, to Thiel Lorca for CamkII 2 stabilization buffer (SB: 50 mM NaF, 40 mM β-glycerophosphate, construct and to Michael Glotzer for cyclin Δ90. We would also like to 10 mM EDTA, 10 mM sodium pyrophosphate, pH 7.2), plus 1 μM ´ ` ¨ thank Helene Defacque, Suzanne Eaton, Michael Glotzer, Pierre Gonczy, microcystin, 10 μg/ml pepstatin, leupeptin, chymostatin containing 7 μl Cayetano Gonzales, Andrew Murray, Angel Nebreda, Ingrid Sasson and of Afﬁprep protein A beads coated with anti-Op18/stathmin antibodies. Mitsuhiro Yanagida for critical reading of the manuscript, and Jo Howard Op18/stathmin was depleted for 30 min at 4°C. Beads were washed for help with the statistical analysis. once with 2 SB, then twice with PBS, 100 mM NaCl, 0.1% Triton X-100, and resuspended in 40 μl of sample buffer. Twenty-ﬁve microlitres were loaded onto a 15% gel and analyzed by silver staining and References PhosphorImager (Molecular Dynamics). Andersen,S.S.L., Ashford,A.J., Tournebize,R., Gavet,O., Sobel,A., Microtubule dynamics Hyman,A.A. and Karsenti,E. (1997) Mitotic chromatin regulates Microtubule dynamics were measured in 10 000 g extracts. Typically, phosphorylation of Stathmin/Op18. Nature, in press. 10 μl of extract were complemented with 1 μl of puriﬁed human Armstrong,C.G., Mann,D.J., Berndt,N. and Cohen,P.T. (1995) Drosophila centrosomes (210 centrosomes/ml), 0.42 μl of rhodamine–tubulin PPY, a novel male speciﬁc protein serine/threonine phosphatase (Hyman et al., 1991) [10 mg/ml, diluted to 2.8 mg/ml in BRB80 (80 mM localised in somatic cells of the testis. J. Cell Sci., 108, 3367–3375. PIPES, 1 mM EGTA, 1 mM MgCl , pH 6.8) and spun for 5 min in an Ault,J.G., DeMarco,A.J., Salmon,E.D. and Rieder,C.L. (1991) Studies Airfuge at 30 p.s.i. at 4°C to remove aggregates], 0.5 μl of saturated on the ejection properties of asters: astral microtubule turnover hemoglobin (bovine, Sigma) (dissolved in water, spun in a microfuge inﬂuences the oscillatory behavior and positioning of mono-oriented for 3 min to pellet undissolved hemoglobin), 0.33 μl of antifade [5 μl chromosomes. J. Cell Sci., 99, 701–710. of catalase (10 mg/ml in BRB80–50% glycerol); 5 μl of glucose oxidase Axton,J.M., Dombradi,V., Cohen,P.T. and Glover,D.M. (1990) One of (10 mg/ml in BRB80–50% glycerol); and 5 μl of glucose (1 M made in the protein phosphatase 1 isoenzymes in Drosophila is essential for water)]. All mixing was done on ice. Then 2.2 μl of this extract mix mitosis. Cell, 63, 33–46. were spotted onto a slide and gently covered by a 2222 mm coverslip Belmont,L.D. and Mitchison,T.J. (1996) Identiﬁcation of a protein that (Fisher Scientiﬁc). All glass used was washed with ethanol and water. interacts with tubulin dimers and increases the catastrophe rate of Images were recorded using a Zeiss Axioskop, a 100 Apochromat lens microtubules. Cell, 84, 623–631. NA1.4 and a long pass rhodamine ﬁlter (Chromatech). Images were Belmont,L.D., Hyman,A.A., Sawin,K.E. and Mitchison,T.J. (1990) Real- taken using an 8-bit black and white camera (Sony SSC-M370CE), time visualization of cell cycle-dependent changes in microtubule processed using an Argus 10 image processor (Hamamatsu) and stored dynamics in cytoplasmic extracts. Cell, 62, 579–589. on a Macintosh using the public domain NIH Image program (developed Black,S., Andrews,P.D., Sneddon,A.A. and Stark,M.J. (1995) A regulated at the US National Institutes of Health and available on the internet at MET3–GLC7 gene fusion provides evidence of a mitotic role for http://rsb.info.nih.gov/nih-image/). Recordings were done for no more Saccharomyces cerevisiae protein phosphatase 1. Yeast, 11, 747–759. than 5 min. Images were recorded every 2 s. Special care was taken to Booher,R. and Beach,D. (1989) Involvement of a type 1 protein avoid photobleaching and photodamage by reducing the illumination phosphatase encoded by bws1 in ﬁssion yeast mitotic control. Cell, time using a computer-controlled shutter (Uniblitz, Vincent Associates). 57, 1009–1016. For experiments using okadaic acid, extracts were frozen 20 min after Brattsand,G., Marklund,U., Nylander,K., Roos,G. and Gullberg,M. addition of 0.4 μM okadaic acid and used for microtubule dynamics. (1994) Cell-cycle-regulated phosphorylation of oncoprotein 18 on Extracts depleted of Op18/stathmin with or without okadaic acid addition Ser16, Ser25 and Ser38. Eur. J. Biochem., 220, 359–368. were treated similarly. For experiments investigating the role of PP1 in Brewis,N.D., Street,A.J., Prescott,A.R. and Cohen,P.T. (1993) PPX, a anaphase, 3 μM I-2 was added to extracts at room temperature for novel protein serine/threonine phosphatase localized to centrosomes. 15 min. Extracts were complemented with 0.1 vol. of CaMKII, an EMBO J., 12, 987–996. additional 1 μM I-2, rhodamine–tubulin, hemoglobin and antifade, and Chen,M.X., McPartlin,A.E., Brown,L., Chen,Y.H., Barker,H.M. and kept at room temperature. Then 1 μM I-2 and centrosomes were added Cohen,P.T. (1994) A novel human protein serine/threonine every 15 min after triggering anaphase. Recordings were done for 5 min phosphatase, which possesses four tetratricopeptide repeat motifs and at 0, 15, 30 and 45 min after CaMKII addition. localizes to the nucleus. EMBO J., 13, 4278–4290. Clarke,P.R., Hoffmann,I., Draetta,G. and Karsenti,E. (1993) Data analysis Dephosphorylation of cdc25-C by a type-2A protein phosphatase: Analysis of microtubule dynamics was done using a home-made Micro- speciﬁc regulation during the cell cycle in Xenopus egg extracts. Mol. soft Excel macro. Differences between experiments were evaluated using Biol. Cell, 4, 397–411. Student’s t-test; t-tests were performed within single experiments to Cohen,P. (1989) The structure and regulation of protein phosphatases. assess signiﬁcant variations. Different experiments were then averaged, Annu. Rev. Biochem., 58, 453–508. 5547 R.Tournebize et al. de Pennart,H., Verlhac,M.H., Cibert,C., Santa Maria,A. and Maro,B. Ishii,K., Kumada,K., Toda,T. and Yanagida,M. (1996) Requirement for (1993) Okadaic acid induces spindle lengthening and disrupts the PP1 phosphatase and 20S cyclosome/APC for the onset of anaphase interaction of microtubules with the kinetochores in metaphase II- is lessened by the dosage increase of a novel gene sds23 . EMBO J., arrested mouse oocytes. Dev. Biol., 157, 170–181. 15, 6629–6640. Dogterom,M., Fe´lix,M.-A., Guet,C.C. and Leibler,S. (1996) Inﬂuence of Karsenti,E. (1991) Mitotic spindle morphogenesis in animal cells. Semin. M-phase chromatin on the anisotropy of microtubule asters. J. Cell Cell Biol., 2, 251–260. Biol., 133, 125–140. Kinoshita,N., Yamano,H., Niwa,H., Yoshida,T. and Yanagida,M. (1993) Dohadwala,M., Da Cruz E Silva,E.F., Hall,F.L., Williams,R.T., Carbonaro- Negative regulation of mitosis by the ﬁssion yeast protein phosphatase Hall,D.A., Nairn,A.C., Greengard,P. and Berndt,N. (1994) ppa2. Genes Dev., 7, 1059–1071. Phosphorylation and inactivation of protein phosphatase 1 by cyclin- Kinoshita,K., Nemoto,T., Nabeshima,K., Kondoh,H., Niwa,H. and dependent kinases. Proc. Natl Acad. Sci. USA, 91, 6408–6412. Yanagida,M. (1996) The regulatory subunits of ﬁssion yeast protein Doonan,J.H. and Morris,N.R. (1989) The bimG gene of Aspergillus phosphatase 2A (PP2A) affect cell morphogenesis, cell wall synthesis nidulans, required for completion of anaphase, encodes a homolog of and cytokinesis. Genes to Cells, 1, 633–644. mammalian phosphoprotein phosphatase 1. Cell, 57, 987–996. Kwon,Y., Lee,S.Y., Choi,Y., Greengard,P. and Nairn,A.C. (1997) Cell Evans,D.R.H. and Stark,M.J.R. (1997) Mutations in the Saccharomyces cycle-dependent phosphorylation of mammalian protein phosphatase cerevisiae type 2A protein phosphatase catalytic subunit reveal roles 1 by cdc2 kinase. Proc. Natl Acad. Sci. USA, 94, 2168–2173. in cell wall integrity, actin cytoskeleton organization and mitosis. Larsson,N., Melander,H., Marklund,U., Osterman,O. and Gullberg,M. Genetics, 145, 227–241. (1995) G /M transition requires multisite phosphorylation of Felix,M.A., Pines,J., Hunt,T. and Karsenti,E. (1989) A post-ribosomal oncoprotein 18 by two distinct protein kinase systems. J. Biol. Chem., supernatant from activated Xenopus eggs that displays post- 270, 14175–14183. translationally regulated oscillation of its cdc2 mitotic kinase activity. Lee,T.H. (1995) The role of protein phosphatase type-2A in the Xenopus cell cycle: initiation of the G EMBO J., 8, 3059–3069. /M transition. Semin. Cancer Biol., 6, Felix,M.A., Cohen,P. and Karsenti,E. (1990) Cdc2 H1 kinase is negatively 203–209. regulated by a type 2A phosphatase in the Xenopus early embryonic Lee,T.H., Turck,C. and Kirschner,M.W. (1994) Inhibition of cdc2 cell cycle: evidence from the effects of okadaic acid. EMBO J., 9, activation by INH/PP2A. Mol. Biol. Cell, 5, 323–338. 675–683. Lin,F.C. and Arndt,K.T. (1995) The role of Saccharomyces cerevisiae Fernandez,A., Brautigan,D.L. and Lamb,N.J. (1992) Protein phosphatase type 2A phosphatase in the actin cytoskeleton and in entry into type 1 in mammalian cell mitosis: chromosomal localization and mitosis. EMBO J., 14, 2745–2759. involvement in mitotic exit. J. Cell Biol., 116, 1421–1430. Lorca,T., Fesquet,D., Zindy,F., Le Bouffant,F., Cerruti,M., Brechot,C., Ferrigno,P., Langan,T.A. and Cohen,P. (1993) Protein phosphatase 2A1 Devauchelle,G. and Dore´e,M. (1991) An okadaic acid-sensitive is the major enzyme in vertebrate cell extracts that dephosphorylates phosphatase negatively controls the cyclin degradation pathway in several physiological substrates for cyclin-dependent protein kinases. amphibian eggs. Mol. Cell. Biol., 11, 1171–1175. Mol. Biol. Cell, 4, 669–677. Lorca,T., Cruzalegui,F.H., Fesquet,D., Cavadore,J.C., Mery,J., Means,A. Francisco,L. and Chan,C.S. (1994) Regulation of yeast chromosome and Dore´e,M. (1993) Calmodulin-dependent protein kinase II mediates segregation by Ipl1 protein kinase and type 1 protein phosphatase. inactivation of MPF and CSF upon fertilization of Xenopus eggs. Cell. Mol. Biol. Res., 40, 207–213. Nature, 366, 270–273. Gliksman,N.R., Parsons,S.F. and Salmon,E.D. (1992) Okadaic acid Marklund,U., Larsson,N., Gradin,H.M., Brattsand,G. and Gullberg,M. induces interphase to mitotic-like microtubule dynamic instability by (1996) Oncoprotein 18 is a phosphorylation-responsive regulator of inactivating rescue. J. Cell Biol., 119, 1271–1276. microtubule dynamics. EMBO J., 15, 5290–5298. Glotzer,M., Murray,A.W. and Kirschner,M.W. (1991) Cyclin is degraded McGowan,C.H. and Cohen,P. (1988) Protein phosphatase-2C from rabbit by the ubiquitin pathway. Nature, 349, 132–138. skeletal muscle and liver: an Mg -dependent enzyme. Methods Goedert,M., Cohen,E.S., Jakes,R. and Cohen,P. (1992) p42 MAP kinase Enzymol., 159, 416–426. phosphorylation sites in microtubule-associated protein tau are Minshull,J., Sun,H., Tonks,N.K. and Murray,A.W. (1994) A MAP kinase- dephosphorylated by protein phosphatase 2A1. Implications for dependent spindle assembly checkpoint in Xenopus egg extracts. Cell, Alzheimer’s disease. FEBS Lett., 312, 95–99. 79, 475–486. Gomes,R., Karess,R.E., Ohkura,H., Glover,D.M. and Sunkel,C.E. (1993) Morin,N., Abrieu,A., Lorca,T., Martin,F. and Dore´e,M. (1994) The Abnormal anaphase resolution (aar): a locus required for progression proteolysis-dependent metaphase to anaphase transition: calcium/ through mitosis in Drosophila. J. Cell Sci., 104, 583–593. calmodulin-dependent protein kinase II mediates onset of anaphase in Heald,R., Tournebize,R., Blank,T., Sandaltzopoulos,R., Becker,P., extracts prepared from unfertilized Xenopus eggs. EMBO J., 13, Hyman,A. and Karsenti,E. (1996) Self-organization of microtubules 4343–4352. into bipolar spindles around artiﬁcial chromosomes in Xenopus egg Murray,A.W. (1991) Cell cycle extracts. In Kay,B.K. and Peng,H.B. extracts. Nature, 382, 420–425. (eds), Cell Cycle Extracts. Academic Press Inc., San Diego, Vol. 36, Hisamoto,N., Sugimoto,K. and Matsumoto,K. (1994) The Glc7 type 1 pp. 581–605. protein phosphatase of Saccharomyces cerevisiae is required for cell Murray,A.W., Solomon,M.J. and Kirschner,M.W. (1989) The role of cycle progression in G /M. Mol. Cell. Biol., 14, 3158–3165. cyclin synthesis and degradation in the control of maturation promoting Holloway,S.L., Glotzer,M., King,R.W. and Murray,A.W. (1993) factor activity. Nature, 339, 280–286. Anaphase is initiated by proteolysis rather than by the inactivation of Nebreda,A.R. and Hunt,T. (1993) The c-mos proto-oncogene protein maturation-promoting factor. Cell, 73, 1393–1402. kinase turns on and maintains the activity of MAP kinase, but not Honkanen,R.E., Zwiller,J., Daily,S.L., Khatra,B.S., Dukelow,M. and MPF, in cell-free extracts of Xenopus oocytes and eggs. EMBO J., Boynton,A.L. (1991) Identiﬁcation, puriﬁcation, and characterization 12, 1979–1993. of a novel serine/threonine protein phosphatase from bovine brain. Nicklas,R.B. and Gordon,G.W. (1985) The total length of spindle J. Biol. Chem., 266, 6614–6619. microtubules depends on the number of chromosomes present. J. Cell Horwitz,S.B., Shen,H.-J., He,L., Dittmar,P., Neef,R., Chen,J. and Biol., 100, 1–7. Schubart,U.K. (1997) The microtubule-destabilizing activity of Ohkura,H., Adachi,Y., Kinoshita,N., Niwa,O., Toda,T. and Yanagida,M. metablastin (p19) is controlled by phosphorylation. J. Biol. Chem., (1988) Cold-sensitive and caffeine-supersensitive mutants of the 272, 8129–8132. Schizosaccharomyces pombe dis genes implicated in sister chromatid Hyman,A.A. and Karsenti,E. (1996) Morphogenetic properties of separation during mitosis. EMBO J., 7, 1465–1473. microtubules and mitotic spindle assembly. Cell, 84, 401–410. Ohkura,H., Kinoshita,N., Miyatani,S., Toda,T. and Yanagida,M. (1989) Hyman,A., Drechsel,D., Kellogg,D., Salser,S., Sawin,K., Steffen,P., The ﬁssion yeast dis2 gene required for chromosome disjoining Wordeman,L. and Mitchison,T. (1991) Preparation of modiﬁed encodes one of two putative type 1 protein phosphatases. Cell, 57, tubulins. Methods Enzymol., 196, 478–485. 997–1007. Ingebritsen,T.S., Stewart,A.A. and Cohen,P. (1983) The protein Parsons,S.F. and Salmon,E.D. (1997) Microtubule assembly in clariﬁed phosphatases involved in cellular regulation; 6. Measurement of type- Xenopus egg extracts. Cell Motil. Cytoskel., 36, 1–11. 1 and type-2 protein phosphatases in extracts of mammalian tissues; Picard,A., Capony,J.P., Brautigan,D.L. and Dore´e,M. (1989) Involvement an assessment of their physiological roles. Eur. J. Biochem., 132, of protein phosphatases 1 and 2A in the control of M phase-promoting 297–307. factor activity in starﬁsh. J. Cell Biol., 109, 3347–3354. 5548 Phosphatases and mitosis Picard,A., Labbe´,J.C., Barakat,H., Cavadore,J.C. and Dore´e,M. (1991) Okadaic acid mimics a nuclear component required for cyclin B–cdc2 kinase microinjection to drive starﬁsh oocytes into M phase. J. Cell Biol., 115, 337–344. Sawin,K.E. and Mitchison,T.J. (1991) Mitotic spindle assembly by two different pathways in vitro. J. Cell Biol., 112, 925–940. Shamu,C.E. and Murray,A.W. (1992) Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase. J. Cell Biol., 117, 921–934. Shenolikar,S. (1994) Protein serine/threonine phosphatases—new avenues for cell regulation. Annu. Rev. Cell Biol., 10, 55–86. Shibuya,E.K., Polverino,A.J., Chang,E., Wigler,M. and Ruderman,J.V. (1992) Oncogenic ras triggers the activation of 42-kDa mitogen- activated protein kinase in extracts of quiescent Xenopus oocytes. Proc. Natl Acad. Sci. USA, 89, 9831–9835. Snaith,H.A., Armstrong,C.G., Guo,Y., Kaiser,K. and Cohen,P.T.W. (1996) Deﬁency of protein phosphatase 2A uncouples the nuclear and centrosome cycle and prevents attachment of microtubules to the kinetochore in Drosophila microtubule star (mts) embryos. J. Cell Sci., 109, 3001–3012. Sobel,A. (1991) Stathmin: a relay phosphoprotein for multiple signal transduction? Trends Biochem. Sci., 16, 301–305. Sontag,E., Nunbhakdi Craig,V., Bloom,G.S. and Mumby,M.C. (1995) A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle. J. Cell Biol., 128, 1131–1144. Surana,U., Amon,A., Dowzer,C., McGrew,J., Byers,B. and Nasmyth,K. (1993) Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. EMBO J., 12, 1969–1978. Tournebize,R. and Heald,R. (1996) Mitotic spindles and microtubules dynamics in Xenopus egg extracts. Semin. Cell Dev. Biol., 7, 467–473. Van Dolah,F.M. and Ramsdell,J.S. (1992) Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells. J. Cell Physiol., 152, 190–198. Verde,F., Labbe´,J.C., Dore´e,M. and Karsenti,E. (1990) Regulation of microtubule dynamics by cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature, 343, 233–238. Verde,F., Dogterom,M., Stelzer,E., Karsenti,E. and Leibler,S. (1992) Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts. J. Cell Biol., 118, 1097–1108. Walczak,C.E., Mitchison,T.J. and Desai,A. (1996) XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly. Cell, 84, 37–47. Walker,R.A., O’Brien,E.T., Pryer,N.K., Soboeiro,M.F., Voter,W.A., Erickson,H.P. and Salmon,E.D. (1988) Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies. J. Cell Biol., 107, 1437–1448. Wells,W.A.E. (1996) The spindle-assembly checkpoint: aiming for a perfect mitosis, every time. Trends Cell Biol., 6, 228–234. Yamano,H., Ishii,K. and Yanagida,M. (1994) Phosphorylation of dis2 protein phosphatase at the C-terminal cdc2 consensus and its potential role in cell cycle regulation. EMBO J., 13, 5310–5318. Zhai,Y., Kronebusch,P.J., Simon,P.M. and Borisy,G.G. (1996) Microtubule dynamics at the G /M transition: abrupt breakdown of cytoplasmic microtubules at nuclear envelope breakdown and implication for spindle morphogenesis. J. Cell Biol., 135, 201–214. Zhang,D. and Nicklas,R.B. (1995) The impact of chromosomes and centrosomes on spindle assembly as observed in living cells. J. Cell Biol., 129, 1287–1300. Received on April 3, 1997; revised on June 26, 1997

Journal

The EMBO Journal – Springer Journals

Published: Sep 15, 1997

Keywords: microtubule dynamics; mitosis; Op18; phosphatase; spindle

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Distinct roles of PP1 and PP2A‐like phosphatases in control of microtubule dynamics during mitosis

Distinct roles of PP1 and PP2A‐like phosphatases in control of microtubule dynamics during mitosis

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Distinct roles of PP1 and PP2A‐like phosphatases in control of microtubule dynamics during mitosis

Distinct roles of PP1 and PP2A‐like phosphatases in control of microtubule dynamics during mitosis

References (90)

Abstract

Journal

Recommended Articles

There are no references for this article.

Our policy towards the use of cookies