The EMBO Journal vol. 1 1 no. 5 pp. 1 763 - 1772, 1992

Relocation and distinct subcellular localization ofp34cdc2- cyclin B complex at meiosis reinitiation instarfish oocytes

Kayoko Ookata, Shin-ichi Hisanaga,Takayuki Okano, Kazunori Tachibanaand Takeo KishimotolLaboratory of Cell and Developmental Biology, Department ofBiological Science, Tokyo Institute of Technology, Nagatsuta,Midori-ku, Yokohama 227, Japan'Corresponding author

Communicated by T.Hunt

M phase promoting factor (MPF) is a major elementcontrolling entry into the M phase of the eukaryotic cellcycle. MPF is composed of two subunits, p34C&2 andcyclin B. Using indirect immunofluorescence saining with

specific antibody against starfish cyclin B, we monitoredthe dynamics of the subcellular distribution of MPFduring meiosis reinitiation in starfish oocytes. We foundthat all of the cyclin B is already associated with p34cdc2in immature oocytes arrested at the G2/M border andthat this inactive complex is present exclusively in thecytoplasm. After its activation, part of thep34cdc2 -cyclin B complex moves into the germinalvesicle before nuclear envelope breakdown,independently of either microtubules or actin filaments.Thereafter, some part of the complex accumulates in thenucleolus and condensed chromosomes. Another portionof the complex accumulates on meiotic asters andspindles, while the rest is still present throughout thecytoplasm. As these patterns of localization are detectedin the detergent-extracted oocytes, we propose at leastfour distinct subcellular states of the p34cdc2-cyclin Bcomplex: freely soluble, microtubule-associated,detergent-resistant cytoskeleton-associated andchromosome-associated. Thus, in addition to theintramolecular modification of p34cdc2- cyclin Bcomplex, its intracellular relocation plays a key role inpromoting the M phase.Key words: cdc2 -cyclin B complex/cell cycle control/meiosis reinitiation/starfish oocytes/subcellular localization

IntroductionEntry into mitosis and resumption of meiosis in all eukaryoticcells involves the activation of a serine/threonine proteinkinase named maturation promoting factor (MPF) (reviewsin Kishimoto, 1988; Hunt, 1989; Lohka, 1989; Nurse, 1990;Maller, 1991). This MPF consists of at least two subunits,a catalytic subunit homologous to p34cdc2 in fission yeastand a regulatory cyclin B subunit homologous to the yeastcdc13 gene product (Arion et al., 1988; Dunphy et al.,1988; Gautier et al., 1988; Labbe et al., 1988, 1989; Lohkaet al., 1988; Draetta et al., 1989; Meijer et al., 1989; Pinesand Hunter, 1989; Westendorf et al., 1989; Gautier et al.,1990). After the accumulation of newly synthesized cyclin

(C Oxford University Press

B and its association with p34cdc2 during interphase, post-translational modification of the inactive p34cdc2-cyclin Bcomplex is required for the activation of MPF (Minshullet al., 1989; Murray and Kirschner, 1989; Minshull et al.,1990; Gautier and Maller, 1991; Kobayashi et al., 1991;Minshull et al., 1991). This modification involves thesequential phosphorylation and dephosphorylation of tyrosineand threonine residues within the ATP-binding-site of thep34cdc2 subunit (Dunphy and Newport, 1989; Gautier et al.,1989; Gould and Nurse, 1989; Morla et al., 1989; Pondavenet al., 1990; Solomon et al., 1990; Meijer et al., 1991). Thephosphorylation was recently shown to depend on the fissionyeast wee] gene product (Parker et al., 1991) and thedephosphorylation to be performed by a phosphatasehomologous to the fission yeast cdc25 gene product (Dunphyand Kumagai, 1991; Gautier et al., 1991; Kumagai andDunphy, 1991; Millar et al., 1991b; Strausfeld et al., 1991).

In addition to the intramolecular modification of thep34cdc2 -cyclin B complex, its subcellular localizationmight play a key role in its function in vivo. In fact, partof p34cdc2 is associated with centrosomes, kinetochores andkinetochore-to-pole microtubules in mammalian cells (Baillyet al., 1989; Riabowol et al., 1989; Rattner et al., 1990)and cyclin B with spindle pole bodies in fission yeast (Alfaet al., 1990). These observations support the possiblefunction of p34cdc2-cyclin B protein kinase in generatinga mitotic spindle. In contrast to the metaphase, however,several conflicting observations have been reported on thedistribution of p34cdc2 and cyclin B in the G2 or interphasecells; in mammalian cells p34cdc2 is located only in thenucleus (Riabowol et al., 1989), only in the cytoplasm(Akhurst et al., 1989), or in both the nucleus and cytoplasm(Bailly et al., 1989); cyclin B is located in the cytoplasmalone in amphibian oocytes (Gautier and Maller, 1991), inthe cytoplasm and in the intranuclear dots in Drosophila earlyembryo (Lehner and O'Farrell, 1990a), or in the nucleusin fission yeast (Booher et al., 1989). Thus, it appears thatfurther clarification is required on the intracellulardistribution of p34cdc2-cyclin B at the G2 to M phasetransition.MPF was first recognized as a cytoplasmic activity

mediating the hormonal stimulation at the oocyte surface tothe nuclei of immature oocytes (Masui and Markert, 1971;Kishimoto and Kanatani, 1976). Consistently, all detectablecyclin B is present in the cytoplasm but not in the germinalvesicle in immature oocytes ofXenopus (Gautier and Maller,1991). Recent studies revealed, however, that MPF directlyphosphorylates and depolymerizes lamin polymers to causenuclear membrane disassembly (Peter et al., 1990b, 1991;Ward and Kirschner, 1990). Considering that lamin proteinsoccur inside the nucleus and scaffold the inner nuclearmembrane, it can be presumed that the subcellulardistribution of MPF may change from the cytoplasm to thenucleus before nuclear breakdown.Here we studied the subcellular localization of the

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p34cdc2-cyclin B complex during meiosis reinitiation ofstarfish oocytes with a probe of anti-cyclin B antibody. Theresults indicated that inactive MPF is present exclusively inthe cytoplasm of immature oocytes, and after its activationone part of MPF relocates into the germinal vesicle andaccumulates in the nucleolus and condensed chromosomes,while some other part of MPF associates with meiotic astersand spindle.

ResultsAll of cyclin B associates with p34cdc2 in immatureand maturing starfish oocytesThe cDNA of starfish cyclin B (Tachibana et al., 1990) wasinserted into a T7 polymerase expression system (Studierand Moffat, 1986). A serum raised against bacteriallyproduced starfish cyclin B was further affinity purified (seeMaterials and methods). The specificity of the antibody wastested by Western blots of inclusion bodies from Escherichiacoli expressing starfish cyclin B and of whole lysates fromimmature starfish oocytes. Figure IA shows that theantibody was specific for the cyclin B and recognized a singleband in oocyte lysate with an apparent molecular size of 48kDa. In addition, the antibody reacted with the 48 kDasubunit of purified MPF which consisted primarily of twobands of 34 kDa and 48 kDa on SDS - PAGE after p1 3SUCIaffinity chromatography (Okumura,E. and Kishimoto,T., inpreparation). The specificity of the anti-cyclin B antibodywas confirmed by its pre-incubation with the bacteriallyproduced starfish cyclin B which completely eliminated thereaction with the 48 kDa band in oocyte lysate (data notshown).The Western blot analysis of Figure lA also implies that

A B r

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1 2 3 4- 1 2 2

Fig. 1. All of cyclin B forms a complex with p34((1C2 and is localized inthe cytoplasm but not in the germinal vesicle of immature starfish oocytes.A. The specificity of antibody against starfish cyclin B. Lane 1, CBB stainingafter SDS-PAGE of E. coli inclusion bodies containing bacterially expressedstarfish cyclin B protein. Lane 2, Western blot of cyclin B-containinginclusion bodies probed with anti-cyclin B antibody. Approximately one-thousandth of the amount of protein in lane 1 was loaded on the gel. Lane 3,CBB staining after SDS -PAGE of whole lysates from immature starfishoocytes. Lane 4, Western blot of lane 3 probed with anti-cyclin B antibody.B. Western blots of flow through fraction (lane 1) and pl3SYcI beads-adsorbed fraction (lane 2) probed with both anti-cyclin B antibody and anti-PSTAIR antibody. C. Western blots of germinal vesicle contents (lane 1)and the residual cytoplasm (lane 2) probed with both anti-cyclin B antibodyand anti-PSTAIR antibody. Germinal vesicle contents and its membranewere sucked up into a micropipette from 30 immature oocytes and thendissolved directly into SDS sample buffer to serve as the germinal vesiclefraction. The residual 10 enucleated oocytes were also dissolved into SDSsample buffer to serve as the cytoplasm fraction. The anti-PSTAIR antibodyrecognized two distinct bands near 34 kDa, the upper (U) and middle (M)in enucleated immature oocytes, while M alone in germinal vesicles (seeFigure 4B and C). The acrylamide concentration was 10% in A and B and12% in C.

cyclin B is already present in immature oocytes of thestarfish. Then, we examined whether the occurrence ofcyclin B is unassociated with p34cdc2 in immature starfishoocytes. The crude extracts of immature oocytes wereincubated with p 13sc1 beads which adsorb p34cdc2 and itsrelated proteins (Brizuela et al., 1987; Fang and Newport,1991) and separated by brief centrifugation into the beadedprecipitate and the unadsorbed supernatant. When theWestern blots of each fraction were probed with anti-cyclinB antibody (see Materials and methods), cyclin B wasdetected only in the precipitate and not in the supernatant,indicating that all of cyclin B is present as associated form(s)in immature oocytes (Figure lB).

In contrast to anti-cyclin B antibody, the anti-PSTAIRantibody recognized three distinct bands near 34 kDadepending on the stages of oocyte maturation (see Figure 4Bupper); they are designated here as the upper (U), middle(M) and lower (L). Although both U and M were presentin immature oocytes, U and a small amount of M wererecovered in the pl3sucJ beads (Figure lB and 4B upper).To determine which band of U and M associates with cyclinB, immunoprecipitation was performed with anti-cyclin Bantibody. As shown in Figure 4B (lower), U alone wascontained in the anti-cyclin B immunoprecipitate of theextracts from immature oocytes. This was confirmed in theanti-cyclin B immunoprecipitate of the eluate from thepl3sucJ beads (Okumura,E. and Kishimoto,T., unpublishedresults). Thus, among the PSTAIR-containing proteins, Ualone was complexed with cyclin B in immature starfishoocytes.

After the triggering of meiosis reinitiation by1-methyladenine, a natural maturation-inducing hormone instarfish oocytes (Kanatani et al., 1969), U which had beenpresent in immature oocytes completely disappearedcoincidently with the activation of histone H1 kinase, andL which had not been present in immature oocytes appearedreciprocally, while M was present continuously fromimmature to GVBD stage (Figure 4A and B).Immunoprecipitation with the anti-cyclin B antibody revealedthat both U and L, but not M, associated with cyclin B(Figure 4B). These facts suggest that U and L are p34cd(2and that U changed to L by dephosphorylation of p34cdc2during meiosis reinitiation; the U-cyclin B complexrepresents inactive preMPF and the L-cyclin B complexactive MPF.Taken together, although the anti-PSTAIR antibody may

recognize and the pl3suc1 beads may bind several differentbut related 34 kDa proteins including p34cdc2 andCDK2/Eg1 (see Discussion), it is most probable that thePSTAIR-containing protein that associates with starfishcyclin B is p34cdc2. In fact, at present no evidence has beenobtained to suggest that B-type cyclins associate with anyPSTAIR-containing protein other than p34cdc2.

After the triggering of meiosis reinitiation, the levels ofboth cyclin B and p34cdc2-associated cyclin B are almostconstant until the degradation of cyclin B which occurs atthe end of first meiosis (Okano,T. and Kishimoto,T., inpreparation). These findings, together with the cyclin Bassociation to p34cdc2 in immature oocytes, indicate that inmaturing oocytes also, all of the cyclin B associates withp34cdc2 until the end of first meiosis. Accordingly, it canbe concluded that the detection of cyclin B by probing withanti-cyclin B antibody solely demonstrates the occurrence

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of all the p34cd(2-cyclin B complex in both immature andmaturing starfish oocytes at the first meiotic cycle.

p34cdc2 - cyclin B complex is a cytoplasmic protein inprophase-arrested starfish oocytesTo test the distribution of p34cdc2-cyclin B complex inimmature starfish oocytes, germinal vesicle contents fromimmature oocytes and, separately, the residual enucleatedoocytes (see Materials and methods) were directly dissolvedin SDS sample buffer. When Western blots of each fractionwere probed with anti-cyclin B antibody and anti-PSTAIRantibody, cyclin B and the U band were detected in thecytoplasm alone, while the M band was detected in both thecytoplasm and germinal vesicle (Figure IC). Thus, thep34cdc2 - cyclin B complex is present exclusively in thecytoplasm of prophase-arrested immature starfish oocytes.

Further evidence for the cytoplasmic localization of thep34cdc2 - cyclin B complex was obtained from indirectimmunofluorescence staining with anti-cyclin B antibody.In frozen sections of unextracted immature oocytes, the anti-cyclin B staining was observed almost uniformly throughoutthe cytoplasm but not in the germinal vesicle (Figure 2A andC). Similar cytoplasmic staining of cyclin B was alsoobtained in the whole mount of extracted immature oocyteswhich had been permeabilized with a detergent-containingand microtubule-stabilizing buffer before fixation(Figure 3A; see also Figure 5A). In both preparations ofimmature oocytes, no specific signal was observed withpreimmune serum (Figure 2E and 3a).

Relocation of p34cdC2 - cyclin B complex duringreinitiation of first meiotic cycleIn immature oocytes, the p34cdc2-cyclin B complex occursin an inactive form, which represents the precursor ofMPF(preMPF). Hence, the foregoing results indicate that preMPFlocalizes in the cytoplasm. Considering that nuclear lamins,one of the direct targets of active MPF, are associated withthe inner nuclear membrane, we can suppose that thep34cdc2 - cyclin B complex moves into the germinal vesicleeither before or after its activation. Then, we examined thedistribution of the p34cdc2 -cyclin B complex at the onsetof germinal vesicle breakdown.

In the frozen section of oocytes which proceeded to thestage just after the onset of germinal vesicle breakdown, anti-cyclin B staining was detected at high intensity in thegerminal vesicle, while less staining was still observedthroughout the cytoplasm (Figure 2B and D). No specificsignal was observed with preimmune serum (Figure 2F). Asimilar pattern of cyclin B staining was observed in the wholemount (Figure 3E) or the confocal section (Figure 5C) ofthe detergent-extracted oocytes, although - 60% of the totalprotein had already been removed by the extractionprocedure (data not shown). These observations indicate thatpart of the p34cdc2 -cyclin B complex relocates into thegerminal vesicle at the reinitiation of meiosis and that at leastpart of the relocation can be detected even in the extractedoocytes.Although cytoplasmic staining for cyclin B was greater

in Figure 3E than Figure 2B, this might be caused by theprocedure for oocyte preparation; the frozen section ofoocytes (Figure 2B) and the whole mount of extractedoocytes (Figure 3E). Considering that the diameter ofgerminal vesicle is -25% of that of oocytes at this stage,

cytoplasmic staining might be relatively higher in the wholemount than in the section when compared to the germinalvesicle staining.

Next, we monitored the timing in the extracted oocyteswhen the relocation occurs during the reinitiation of firstmeiosis. At various time points after the addition of1-methyladenine, oocytes were transferred into the extractionbuffer and then processed for anti-cyclin Bimmunofluorescence staining. Although cyclin B occurredexclusively throughout the cytoplasm at 10 min (Figure 3B),its staining became detectable along the inner side of thegerminal vesicle membrane at 13 min (Figure 3C; see alsoFigure SB). Then, while cyclin B was still present throughoutthe cytoplasm, the staining was seen to extend throughoutthe germinal vesicle at 16 min (Figure 3D), when no

D

Fig. 2. The p34cd(2-cyclin B complex is cytoplasmic at prophase arrest,but nuclear and cytoplasmic at germinal vesicle breakdown in starfishoocytes. Immature oocyte (A, C and E) and maturing oocyte (B, D andF) 20 min after the addition of 1-methyladenine were fixed withparaformaldehyde. Frozen sections were then processed forimmunofluorescence staining with anti-cyclin B antibody (A and B) or withpreimmune serum (E and F). C and D are the same sections as in A andB, respectively, observed with differential interference optics. In D thegerminal vesicle is at the onset of breakdown. Bar represents 50 /Lm.

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observable change of the membrane had yet been detected;2 min later the onset of its disassembly was observed underdifferential interference optics. At 20 min, when undulationsformed along the previously smooth periphery of the

germinal vesicle, the intensity of the anti-cyclin Bimmunofluorescence increased in the germinal vesicle areathan in the cytoplasm (Figure 3E; see also Figures 2B andD and 5C). After the breakdown of the germinal vesicle,

I

I

Fig. 3. Time course of p34cdc2-cyclin B complex relocation into the geminal vesicle during meiosis reinitiation in starfish oocytes. At various times afterl-methyladenine was added, oocytes were extracted with a detergent-containing buffer and then stained with anti-cyclin B antibody (A-F), with preimmuneserum (a) and with DAPI (b-f) (see Materials and methods). Whole mount specimens were observed. A, a, immature oocytes; B, 10 min; C, 13 min;D, 16 min; E, 20 min; and F 30 min after the addition of 1-methyladenine, respectively. b-f are the same oocytes as in B-F, respectively. Arrows andarrowhead point to condensing chromosomes and the nucleolus, respectively. Bar represents 50 Itm.

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a higher intensity of staining persisof the cytoplasm, possibly where thoriginally present, while less stthroughout the rest of the cytoplasm (of the p34cdc2 -cyclin B complex reliin the germinal vesicle before tbreakdown, while the rest of the coicytoplasm.

Active p34Cdc2 - cyclin B complexvesicleAfter the addition of 1-methyladeniiB complex is converted from an inawhich has histone HI kinase activity.the active or inactive form of the p34relocates into the germinal vesicle, Mof activation of histone HI kinase ibatch as used in Figure 3. Al

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0

U',m: CL'w !!y wr0

A

Time after 1-MeAde addition (min'

Fig. 4. Time course of activation of hidephosphorylation of p34X"1'2 after the ad(I-MeAde) in starfish oocytes. A. Histone Fextracts obtained various times after the additioiindicates the onset of germinal vesicle breakdowith the anti-PSTAIR antibody of oocyteimmunoprecipitates by the anti-cyclin B antibbands near 34 kDa, the upper (U), middlerecognized in oocyte extracts by the anti-PSTthe stages of meiosis reinitiation (upper). Bothimmunoprecipitated with the anti-cyclin B anblots probed with the anti-PSTAIR antibody ofoocytes. In B and C, the number at the top of(min) after the addition of 1-methyladenine.

,ted in a restricted area 1-methyladenine, histone HI kinase activity had alreadyte germinal vesicle was begun to increase at 7 min and reached almost two-thirdstaining was observed of maximum level at 10 min, when the relocation of the(Figure 3F). Thus, part p34cdc2-cyclin B complex had not yet been detectedocates and accumulates (Figure 4A; also see Figure 3B and C). The histone HIthe completion of its kinase activity had already reached a maximum level ofmplex still exists in the -75% at 13 min, when the p34cdc2-cyclin B complex was

first observed within the germinal vesicle.At the same time as the histone HI kinase activation,

moves into germinal p34cdc2 was dephosphorylated from the U band to the Lband both in oocyte lysates and in their anti-cyclin B

ne, the p34cdc2-cyclin immunoprecipitates (Figure 4B). This precise coincidence,ctive to an active form together with the fact that the anti-cyclin B antibodyTo determine whether precipitates almost all the histone HI kinase activity

tcdc2-cyclin B complex (Okano,T. and Kishimoto,T., in preparation), indicates that{e monitored the timing almost all the observed activity of histone HI kinase is duein oocytes of the same to p34cdc2-cyclin B complex. In addition, the U bandfter the addition of changed to the L band even in enucleated oocytes after the

addition of 1-methyladenine (Figure 4C), suggesting that thedephosphorylation of p34cdc2 occurred in the cytoplasm.Thus, the p34cdc2 -cyclin B complex appears to be activated

15 20 (min, in the cytoplasm and then relocated into the germinal vesicle.UM Confocal observation clearly confirmed the completeL absence of cyclin B in the germinal vesicle of prophase-u arrested immature oocytes (Figure SA). In contrast,L densitometric measurement of Figure SC revealed that just

after the onset of germinal vesicle breakdown, the intensityof the anti-cyclin B immunofluorescence was - 2-fold higher

2 in) in the germinal vesicle than in the cytoplasm. Since theL diameter of the germinal vesicle at this stage measures one-

third of that of the oocyte, we estimate the amount of thep34cdc2-cyclin B complex that is present in the germinalvesicle to be - 8% of its total amount in the whole extractedoocyte. Thus, the concentration gradient of the

istone HI kinase and the p34cdc2 - cyclin B complex between the cytoplasm andidition of 1-methyladenine germinal vesicle was reversed during the reinitiation ofi1 kinase activity in oocyte meiosis, indicating the active translocation of the complexn of 1-methyladenine. Arrow into the germinal vesicle, not merely its passive diffusion.wn. B. Western blots probed How does the active p34cdc2-cyclin B complex moveextracts (upper) and their)ody (lower). Three distinct into the germinal vesicle? As can be seen in Figure SB, the(M) and lower (L), were anti-cyclin B staining appears to be associated with fibrousAIR antibody depending on elements both in the cytoplasm and in the germinal vesicle.i U and L, but not M, were Since it is conceivable that these filamentous structures maywtibody (lower). C. Wested be involved in the translocation of the p34cdc2 -cyclin Beach lane indicates the time complex, we examined the effect of cytoskeleton drugs on

the movement of the complex. After the addition of either

IFig. 5. Confocal observation of p34"'1"2-cyclin B complex relocation in starfish oocytes. At 0 min (A), 13 min (B) or 20 min (C) after 1-methyladeninewas added, oocytes were extracted with a detergent-containing buffer, stained with anti-cyclin B antibody and then processed for confocal microscopy.The arrow points to anti-cyclin B staining associated with fibrous element. Bar represents 50 Am.

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Fig. 6. Relocation of the p34(d(2-cyclin B complex into the germinal vesicle is not prevented by cytoskeleton drugs. Immature starfish oocytes were placedin either 1 AM cytochalasin D or 3 tiM nocodazole-containing seawater. Before or 20 min after the addition of I-methyladenine, oocytes were extractedwith a detergent-containing buffer and then processed for immunofluorescence with anti-cyclin B antibody. A. Cytochalasin D-treated immature oocytes.B. Cytochalasin D-treated oocytes undergoing germinal vesicle breakdown. C. Nocodazole-treated oocytes undergoing germinal vesicle breakdown. Bar

represents 50 Am.

t:

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.,.

A

Fig. 7. The p34C'dI2-cyclin B complex associates with meiotic asters and meiotic spindle in starfish oocytes. 22 min (A) or 45 min (B-C) after

1-methyladenine was added, oocytes were extracted with detergent-containing buffer and then processed for immunofluorescence with anti-cyclin B antibody.C. Higher magnification of oocyte in B, showing meiotic spindle. D. Same oocyte as in C, observed with differential interference optics. Arrows and arrowheads

point to meiotic asters and metaphase chromosomes, respectively. Bars represent 50 zm (B) and 10 tim (D).

cytochalasin D or nocodazole to immature oocytes, thelocalization of p34cdc2-cyclin B complex was stillcytoplasmic, although indirect immunofluorescenceobservations with anti-actin antibody or anti-tubulin antibodyconfirmed that these drugs affected the cytoskeletal structure(Figure 6A; data not shown). In the presence of either ofthese cytoskeleton drugs, both the accumulation of anti-cyclinB immunofluorescence to the germinal vesicle and the onsetof germinal vesicle breakdown occurred normally afterl-methyladenine was added (Figure 6B and C). Also, Taxolhad no effect on the relocation of the p34cdc2 -cyclin Bcomplex into the germinal vesicle (data not shown). Thus,neither microtubules nor actin filaments appear to beinvolved in the movement of the complex into the germinalvesicle, although this does not rule out the possibility of theassociation of the complex with these filaments.

Accumulation of p34cdc2 - cyclin B complex onchromosomes and meiotic spindleRegarding the relocation of the p34cdc2 -cyclin B complexinto the germinal vesicles during meiosis reinitiation, partof the complex was observed to accumulate specifically inthe nucleolus (Figures 3D and 5B; see also Figure 6B). Inaddition, several bright spots of anti-cyclin Bimmunofluorescence in the germinal vesicle were noticedwhich were not visible before the relocation of the complexinto the germinal vesicle (Figure 3A-E). When compared

with DAPI staining in the same preparations (Figure 3b -e),these spots were superimposed on the chromosomes thatbegan to condense. After the dissolution of the germinalvesicle membrane, we observed a mass of bright spots ofthe anti-cyclin B immunofluorescence, corresponding to themass of condensed chromosomes (Figure 3F and f). At thefirst meiotic metaphase, the anti-cyclin B staining was stilldetected in the equatorial region of the meiotic spindle, whichcorresponded to the metaphase chromosomes aligning on thespindle (Figure 7B -D). These observations indicate that partof the p34cdc2 - cyclin B complex accumulated on thecondensed chromosomes after its translocation into thegerminal vesicle.While p34cdc2-cyclin B accumulated in the germinal

vesicle at the onset of its breakdown, prominent anti-cyclinB immunofluorescence was noticed in two separate spots juston the boundary of the germinal vesicle (Figure 7A; see alsoFigure 6B). We presume these to be meiotic asters whichwere now growing after the triggering of meiosis reinitiation(Otto and Schroeder, 1984; Shirai et al., 1990). Thereafter,at the first meiotic metaphase, the whole meiotic spindle washighly stained with anti-cyclin B immunofluorescence, whileless staining was still observed throughout the cytoplasm(Figure 7B - D). These findings demonstrate that some ofthe p34cdc2-cyclin B complex accumulates in the meioticapparatus including spindle poles, half spindles andmetaphase chromosomes.

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Discussion

The present results clearly demonstrate that (i) all of cyclinB is already complexed with p34cdc2 in immature starfishoocytes; (ii) this inactive complex, which representspreMPF, is localized exclusively in the cytoplasm ofimmature oocytes; (iii) after the conversion to active form,part of the active p34cdc2-cyclin B complex moves fromthe cytoplasm into the germinal vesicle before the onset ofdisassembly of the nuclear membrane, and then some portionsettles in the nucleolus and condensed chromosomes; and(iv) another part of the complex accumulates in meiotic astersand the meiotic spindle, while the rest is still presentthroughout the cytoplasm. These detailed dynamics of thesubcellular distribution of the p34cdc2-cyclin B complexduring transition from G2 to M phase are in goodagreement with the possible functions of MPF includinglamin phosphorylation, mitosis-specific phosphorylation ofnucleolar proteins (Peter et al., 1990a), chromosomecondensation and spindle formation.

In support of our observations, during the preparation ofthis manuscript, Pines and Hunter (1991) reported similarrelocation of cyclin B1 at the G2-M phase transition andits association with the mitotic apparatus in human somaticcells. However, they suggested that almost all of cyclin B 1is translocated into the nucleus, after which p34cdc2-cyclinBi is activated. We suppose that these differences betweenstarfish oocytes and HeLa cells may be partially due to thecytoplasm:nucleus volume ratio and the localization of thecdc25 protein (see below).

In contrast to our observations and those of Pines andHunter (1991) that the inactive p34cdc2-cyclin B complex islocated exclusively in the cytoplasm until the transition to theM phase, Bailly et al. (1989) reported that p34cdc2 is locatedin both the nucleus and cytoplasm of human interphase cellsand Riabowol et al. (1989) reported that p34cdc2 localizesexclusively in the nucleus during the interphase of mammaliancultured cells. These discrepancies probably stem from thedifferences in probes used for immunocytochernistry; whilewe used the anti-cyclin B antibodies, both reports are basedon antibodies against either the bacterially produced p34cdc2of fission yeast (Riabowol et al., 1989) or oligopeptidescoding the partial sequences of human CDC2 protein (Baillyet al., 1989). In fact, if we had used the anti-PSTAIR antibodyas a probe for immunocytochemistry, we might haveconcluded that MPF is distributed in both the cytoplasm andgerminal vesicle of immature oocytes in spite of the fact thatcyclin B does not form a complex with the PSTAIR-containingprotein (the M band) contained in the germinal vesicle (seeFigure 1 and 4B).

In addition, recent studies (reviewed in Hunter and Pines,1991) have revealed the occurrence of various types of cdc2related proteins including Eg 1 or CDK2, regardless whetherthe PSTAIR sequence is completely conserved or not(Lehner and O'Farrell, 1990b; Elledge and Spottswood,1991; Fang and Newport, 1991; Paris et al., 1991; Tsaiet al., 1991). At present, each of these cdc2 related proteinsis presumed to form a complex with each of the variouscyclins including A, C, D and E, although it is not clearwith which proteins cyclins C and D associate (Draetta et al.,1989; Gautier et al., 1990; Koff et al., 1991; Lew et al.,1991; Matsushime et al., 1991; Motokura et al., 1991;Xiong et al., 1991). Based on the antibody specific for each

of cyclin A and B 1, Pines and Hunter (1991) have recentlyfound that at the G2 phase of human somatic cells, cyclinB 1 is exclusively cytoplasmic while all of cyclin A is alreadynuclear, indicating the differential localization of cyclin Aand Bi. Accordingly, previous reports on the subcellularlocalization of p34cdc2 should be reconsidered with aspecific probe for each of the cdc2 related proteins andcyclins.Pines and Hunter (1991) suggested that the

p34cdc2-cyclin B1 complex is activated after translocationinto the nucleus because of the nuclear localization of cdc25protein in HeLa cells (Millar et al., 1991a). Our presentresults have demonstrated, however, that after conversionto the active form, the p34cdc2-cyclin B complex movesinto the germinal vesicle. In support of this timing of therelocation in starfish oocytes, histone HI kinase has beenshown to be fully activated even in enucleated oocytes ofstarfish (Picard et al., 1988; see also Figure 4C) and ofXenopus (Dabauvalle et al., 1988), suggesting that theputative cdc25 protein would be cytoplasmically located inoocytes of starfish and Xenopus. Thus, although Booheret al. (1989) suggested that cyclin acts to regulate both thecatalytic properties and the nuclear localization of cdc2kinase, the complex formation of the cdc2 protein with cyclinB appears not to be sufficient for nuclear localization andadditional modification of the complex might be requiredfor the translocation.How does the translocation of the p34cdc2-cyclin B

complex occur? In contrast to the observation in fission yeastwhere mitotic microtubule function is required for theactivation of cdc2 kinase (Alfa et al., 1990), our presentstudy, together with that of Pines and Hunter (1991),demonstrates that the activation and movement of thep34cdc2-cyclin B complex from the cytoplasm to thenucleus depend on neither microtubules nor actin filaments.Furthermore, the occurrence of the complex could bedetected both before and after its relocation even in thenocodazole- or cytochalasin-treated and extracted oocytesin winch detergent-resistant cytoskeletons constitute the mainresiduals. In fact, cyclin B staining appears to occur onfilamentous structures (see Figure 5B). Thus, it is likely thatpart of the p34cdc2-cyclin B complex anchors thedetergent-resistant cytoskeletons at least before and after itsrelocation. This notion is further supported by the previousreport that cdc2 kinase is extractable from detergent-resistantcytoskeletons (Chou et al., 1990; Kusubata et al.,submitted).After the nuclear translocation, a portion of

p34cdc2 - cyclin B complex accumulated on condensedchromosomes in starfish oocytes. Similar association ofcyclin B with condensed chromosomes is reported inDrosophila embryo (Lehner and O'Farrell, 1990a) and inHeLa cells (Pines and Hunter, 1991). These observationsare consistent with the report that cdc2 kinase is saltextractable from chromatin of rat hepatoma cells (Chambersand Langan, 1990).

Apart from the translocation into the nucleus, accumulationof cyclin B on growing meiotic asters and the metaphasespindle in starfish oocytes coincides with the previouslyreported localization of p34cdc2 in centrosomes or spindlepole bodies by Bailly et al. (1989), Riabowol et al. (1989)and Alfa et al. (1990), along kinetochore-to-polemicrotubules by Rattner et al. (1990) and in the mitotic

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apparatus by Pines and Hunter (1991). In accordance withthese observations, microtubules prepared from bothimmature and maturing starfish oocytes containp34cdc2-cyclin B complex and the purified starfishp34cdc2-cyclin B complex is able to associate with isolatedmicrotubules in vitro (Ookata et al., submitted). Thus, it islikely that part of the p34cdc2-cyclin B complex associateswith microtubules in vivo.

In conclusion, although MPF was at first assumed to bea soluble protein, our present results and the aboveconsiderations suggest at least four distinct subcellular statesof the p34cdc2-cyclin B complex: freely soluble,microtubule-associated, detergent-resistant cytoskeleton-associated and chromosome-associated. In addition to theregulatory mechanism of intramolecular modification for theactivation of the p34cdc2-cyclin B complex, elucidation ofthe molecular basis for these distinct intracellular states ofthe complex will be required for achieving an understandingof how MPF brings about various M phase events in vivo.

Materials and methodsAnimals and oocyte preparationStarfish, Asterina pectinifera, were collected during the breeding seasonand kept in laboratory aquaria supplied with circulating seawater at 14°C.Immature oocytes without follicles were released from isolated ovaries aftertreatment with Ca-free seawater (Tachibana et al., 1990). For cytologicalanalysis, the isolated immature oocytes were treated with 0.02% pronase(Kaken Kagaku, Kyoto) for 30 min to digest jelly and vitelline coats, followedby extensive washing with Ca-free seawater. Meiosis reinitiation of starfishoocytes was induced by the addition of 1-methyladenine to seawater at afinal concentration of 1 ltM at 22°C.An enucleated oocyte was prepared by sucking up the whole germinal

vesicle content and its membrane into a micropipette under microscopy(Kishimoto et al., 1981; Kishimoto, 1986). The germinal vesicle materialobtained from 30 immature oocytes and the residual 10 enucleated oocytes,respectively, were directly dissolved in SDS sample buffer and processedfor Western blotting.

Preparation and use of p 13s" -Sepharose beadsThe plasmid pRK172 containing the fission yeast sucl gene (kindly providedby Dr D.Beach, Cold Spring Harbor) was transformed intoBL21(DE3)pLysS (kindly provided by Dr F.W.Studier, Brookhaven; Studieret al., 1990). pl3s5"'/ was prepared and purified according to Brizuela et al.(1987) with slight modifications (Okumura,E. and Kishimoto,T., inpreparation). pl3s5cI was then conjugated with CNBr-activated Sepharose4B (Pharmacia) according to the instructions of the manufacturer at aconcentration of 5 mg/ml of gel. Unreacted groups on the resin werequenched with 1 M ethanolamine, pH 8.0.

Immature starfish oocytes (packed volume - 100s1) were suspended with3 vol of extraction buffer containing 80 mM Na-,(-glycerophosphate, 20mM EGTA, 15 mM MgCl2, 1 mM DTT, 20 Ag/ml leupeptin and 0.3 mMPMSF, pH 7.3 (buffer A) supplemented with 500 mM NaCl, sonicatedfor 30 s at 0°C and immediately frozen in liquid N2. After thawing andcentrifugation at 12 000 g for 10 min at 2°C, the supernatant, whichrecovered almost all the cyclin B and PSTAIR-containing peptides presentin whole immature oocytes (data not shown), was immediately loaded onan equal volume of p13S"Il beads, followed by constant rotation at 2°Cfor 20 min. After brief centrifugation, the supernatant was recovered andthe pl3sucl beads were washed twice with the same buffer as used forextraction. The supernatant was then added with 1/5 vol of 4 xconcentratedSDS sample buffer, while the pl3S"C' beads were suspended in2 xconcentrated SDS sample buffer of the same volume as the supernatant.This ensured a similar level of detection. After boiling for 3 min, eachpreparation was processed for SDS-PAGE and Western blotting.

Histone Hi kinase assayAt various times after the addition of 1-methyladenine, 0.5 ml aliquots ofthe oocyte suspension were rapidly centrifuged for 10 s. The supernatantwas removed by aspiration and the oocyte pellet (packed volume - 30 il)was added with 2 vol of 2 x concentrated buffer A supplemented with 200mM sucrose and 200 mM KCI. The tube was frozen immediately in liquidN2. After thawing, the oocytes were homogenized by a 3 s sonication.

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After centrifugation at 12 000 g for 10 min at 2°C, the supernatant wasrecovered and processed for histone HI kinase assay.The kinase assays, in a final volume of 10 itl, contained 2 pA of oocyte

extract, 5 LI of buffer A and 3 pl1 of histone HI -ATP mixture consistingof 3 mg/ml histone HI (Boehringer Mannheim), 0.9 mM ATP and 1 mCi/mI[Y-32P]ATP. After the incubation for 20min at 20°C, kinase reactions wereterminated by spotting 8 pl aliquots onto Whatman P81 phosphocellulosepaper and after 3 min, the filters were washed five times (5 min each time)in a 200ml solution of1 % phosphoric acid. The wet filters were transferredinto scintillation vials, 5 ml of ACSII (Amersham) scintillation fluid wasadded and the samples were counted in an Aloka scintillation counter.

Raising and purifying antibodiesPolyclonal antibodies for starfish cyclin B were raised in mice against thestarfish cyclin B protein overexpressed in E.coli. A 1.4 kb HindIll -XhoIfragment of the starfish cyclin B cDNA clone (Tachibana et al., 1990),containing all of the coding region except for 114 nt at the 5' end, wasfilled in with Klenow polymerase (Takara, Kyoto). The blunt-ended fragmentwas ligated with BamHI linkers and inserted into a BamHI site of pET-3avector kindly provided by Dr F.W.Studier, Brookhaven. This plasmidpET3a-cyclOHX was transformed into E.coli strain, BL21(DE3)pLysS(kindly provided by Dr F.W.Studier), which contains a chromosomal copyof T7RNA polymerase under the control of the lacUV5 promoter. Whenthe bacteria had grown to an OD6W of 0.6 at 37°C in LB containing 5014g/mi ampicillin and 30 Ag/ml chloramphenicol, IPTG was added to a finalconcentration of 1 mM to induce cyclin B synthesis. Cyclin B formedinsoluble inclusion bodies, which were prepared according to Marston (1988).Briefly, the bacteria were pelleted 3 h after the induction, frozen and thenthawed in 20 ml of a lysis buffer (50mM Tris-HCI, pH 8.0, 2 mM EDTA,1 mM DTT, 1 mM PMSF and 5% glycerin), followed by the addition of4 mg of lysozyme. After standing on ice for 30 min, the cells were lysedwith a polytron six times, 15 s each time, with intervals for cooling. Thesuspension was added with 1/10 vol of 10% Triton X-100, stood on icefor 30 min and then centrifuged at 10 000 g for 30 min at 4°C. Afterrepeating the above procedure again, the final pellet containing the inclusionbodies was dissolved in the sample buffer and then run on an SDS -PAGEof 10% gel. After staining briefly with Coomassie Blue, the cyclin B bandwas excised, homogenized in PBS and then injected subcutaneously intoBALB/c mice. 50 1l of gel suspension containing cyclin B at a concentrationof -0.5- 1.0 mg/ml was used for a total of five injections per mouse attwo-week intervals. The mice were bled one week after the last injectionand the sera were kept at -200C.The anti-cyclin B antibody was further affinity purified using nitrocellulose

strips onto which the bacterially produced starfish cyclin B was transferred(Talian et al., 1983). After Western blotting of inclusion body proteinsincluding cyclin B to an Immobilon P sheet (Millipore), a part of themembrane was cut and cyclin B was visualized with 0.1 % amide black in45% methanol and 10% acetic acid. Then the part of the membrane thathad been blotted with cyclin B was excised and blocked with Tris-bufferedsaline (TBS) containing 3% skimmed milk for 1 h at room temperature.After washing with TBS, the membrane was incubated with 100 11 of2 xdiluted anti-serum overnight at 4°C, followed by three washes of 5 mineach with TBS. The anti-cyclin B antibodies were eluted from the membraneby the addition of 200 ,ul of 0.2 M glycine, pH 2.8, at 4°C for 2 min.After the removal of the membrane, 40 Al of 1.0 M Tris-HCI, pH 8.0,was added to the antibody solution to get neutral pH, followed by the furtheraddition of 2.0 ml of 0.1% BSA in TBS. Then the anti-cyclin B antibody-containing solution was concentrated to 1/5 vol with a centricon (Amicon).The affinity purified anti-cyclin B antibody was used forimmunocytochemistry at 1:4 dilution and for immunoblotting at 1:50 dilution.Preimmune serum was purified similarly by using the part of membranewhere had not been blotted with cyclin.The monoclonal antibody against the conserved PSTAIR region of the

cdc2 protein was a kind gift from Dr Y.Nagahama (Okazaki, Japan). Theanti-PSTAIR antibody was used at a dilution of 1:10.

Electrophoresis, Western blotting and immunoprecipitationSDS-PAGE analysis was performed according to Laemmli (1970). Thegel contained 10% or 12% acrylamide and was stained with CoomassieBlue. For immunoblotting, proteins were transferred onto nitrocelluloseaccording to Towbin et al. (1979). The membrane was then blocked withTBS containing 3% skimmed milk and subsequently incubated with theantibody for 1 h at room temperature. After washing with TBS, themembrane was incubated with alkaline phosphatase-conjugated rabbit anti-mouse IgG (DAKO) at a dilution of 1:400 for 30 min. After washing withTBS, the antibodies were located with a BCIP/NBT phosphatase substratesystem (KPL).For immunoprecipitation, 40 IAl of oocyte extracts was precleared for 60

p34cdc2 - cyclin B relocation

min at4°C with 20 gl of a 50% (v/v) slurry of Protein A-Sepharose CL4B(Pharmacia) which had previously been suspended in an immunoprecipitationbuffer containing 50 mM Tris-HCI (pH 7.5), 150 mM NaCi, I % TritonX-100, 0.1 % SDS, 50 mM NaF, 10 mM Na-pyrophosphate, 0.1 mM Na-orthovanadate, 10 mM p-nitrophenylphosphate and 0.1 mM ZnC12. Aftercentrifugation at 12 000 g for 3 min, 40 gl of the supernatant was addedwith 10 Al of anti-cyclin B antiserum diluted 10-fold with TBS containing0.1 % BSA, followed by 60 min incubation at 4°C. Thirty g1 of a 50%slurry of Protein A-Sepharose CL4B was added and the mixture was

incubated for 90min at4°C. The antibody-containing pellet was recoveredby centrifugation at 12 000 g for 3 min at 4°C, washed three times in 1

ml of the immunoprecipitation buffer and then added with 40 y1 of2 xconcentrated SDS sample buffer. After boiling for 5 min, 10 11 of thesupematant was processed for SDS - PAGE and Western blotting with theanti-PSTAIR antibody.

Immunofluorescence microscopyFor frozen sections, starfish oocytes were fixed at room temperature with1 % paraformaldehyde in 90% Ca-free seawater for 30-60 min, followedby sequential washing with Ca-free seawater and 0.1 M phosphate buffer,pH 7.8, containing 10% sucrose. The oocytes were then embedded in Tissue-Tek OCT compound, frozen in dry ice-acetone and sectioned with a

cryostat.Extracted oocytes for the whole mount were prepared according to

Hashimoto and Kishimoto (1988) and Shirai et al. (1990) under microtubule-stabilizing conditions. Starfish oocytes were transferred to an extraction buffercontaining 25 mM imidazole, 10 mM KCI, 10 mM EGTA, 1% TritonX-100, 0.2 mM PMSF and 20% glycerol, pH 6.9 and left to stand for 30min at room temperature. The oocytes were then attached to a 1 mg/mlpoly-L-lysine (MW 100 kDa) coated coverslip and washed with theextraction buffer lacking glycerol. The oocytes were subsequently fixed byimmersion in absolute methanol at -20°C for 10 min, followed by washingwith TBS.

After blocking with 3% skimmed milk in TBS and then washing withTBS containing 0.01 % Triton X-100, each preparation of either frozensections or extracted oocytes was incubated for 1 h at room temperature

with the affinity purified anti-cyclin B antibody, followed by washing withthe Triton-containing TBS. The oocytes were further incubated for 1 h withFITC-conjugated goat anti-mouse IgG (Cappel) diluted to 1:100 in PBSand finally washed with TBS. The DNA in the extracted oocytes was thenstained with 1 itg/mn DAPI. The oocytes were mounted with TBS containing20% glycerol and 100 ig/ml 1,4-diazabicyclo[2,2,2]-octan (DABCO,Aldrich). The covership was fixed to a glass slide using nail polish.Specimens were observed under an epifluorescence microscope (Olympus).Confocal observation was performed using a Zeiss laser scan microscope.

AcknowledgementsWe thank Drs D.Beach, F.W.Studier, M.Yamashita and Y.Nagahama forsupplying sucl containing plasmid pRK172, E.coli strain BL21(DE3)pLysSand pET-3a vector and the anti-PSTAIR antibody, respectively; Ms Y.Suzukifor preparing pl3si"' beads; and Carl Zeiss, Tokyo for the use of theconfocal laser microscope. This work was supported by grants from theToray Science Foundation, the Mitsubishi Science Foundation and theMinistry of Education, Science and Culture, Japan to T.K.

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Received on December 9, 1991; revised on Februarv 12, 1992

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