RESEARCH ARTICLE 887 Evidence for a role of the α-tubulin C terminus in the regulation of cyclin B synthesis in developing oocytes

Sophie Vée1, Laurence Lafanechère2, Daniel Fisher3, Jürgen Wehland4, Didier Job2 and André Picard1,* 1Laboratoire Arago, BP 44, Banyuls sur mer F-66651 cedex, France 2Laboratoire DBMS/CS, U366, CEA, 17 rue des martyrs, Grenoble F-38054 cedex, France 3CRBM, CNRS 1919 route de Mende, Montpellier F-34033 cedex, France 4GBF Mascheroder Weg 1, D-3300 Braunschweig, Germany *Author for correspondence (e-mail: [email protected])

Accepted 21 December 2000 Journal of Cell Science 114, 887-898 © The Company of Biologists Ltd

SUMMARY

Microinjected mAb YL1/2, an α-tubulin antibody specific microtubules and was mimicked by injected synthetic for the tyrosinated form of the protein, blocks the cell cycle peptides corresponding to the tyrosinated α-tubulin C in developing oocytes. Here, we have investigated the terminus, whereas peptides lacking the terminal tyrosine mechanism involved in the mAb effect. Both developing were ineffective. These results indicate that tyrosinated α- starfish and Xenopus oocytes were injected with two tubulin, or another protein sharing the same C-terminal different α-tubulin C terminus antibodies. The injected epitope, is involved in specific regulation of cyclin B antibodies blocked cell entry into mitosis through specific synthesis in developing oocytes. inhibition of cyclin B synthesis. The antibody effect was independent of the presence or absence of polymerized Key words: Tubulin, Cyclin B, Meiosis, Cleavage, Cell cycle

INTRODUCTION cyclin B being massively synthesized whereas other maternal mRNAs are little or not translated. This feature of developing The cell cycle progression is controlled through periodic oocytes may favor the identification of molecules specifically activation and inactivation of cyclin dependent kinases (cdks: involved in the translation of mitotic cyclins: such molecules see Pines, 1995, for a review). These kinases form dimeric remain largely unknown, with the exception of ERK2, which complexes with different cyclins. The various complexes have is emerging as an important regulator of the control of cyclin different functions, principally determined by the nature of the B translation (de Moor et al., 1997; Ballantyne et al., 1997; cyclin. The mitotic cyclins, such as cyclins A and B, control Fisher et al., 1999). the G2/M transition and thereby cell entry in mitosis. The Here we have investigated the role of the tubulin molecule correct control of the expression of mitotic cyclins is central as one possible factor involved in the control of cyclin B for the control of cell division. Deregulation of these cyclins is synthesis in developing oocytes. Our interest in tubulin a frequent occurrence in tumor cells where cyclins A and B stemmed from two lines of observations. Firstly, previous are generally both prematurely expressed and overexpressed observations showed that antibodies directed against the (Keyomarsi and Pardee, 1993). Therefore, understanding the tyrosinated form of the α-tubulin C terminus block cell entry regulation of the mitotic cyclin expression is an important into mitosis in developing sea urchin eggs (Oka et al., 1990). challenge for cell biologists and pathologists. The molecular mechanisms involved in the cell cycle blockage Mitotic cyclins are expressed during a phase of the cell cycle remain unknown, but may clearly involve an inhibition of when there is little gene transcription (White et al., 1995; cyclin B accumulation since this is the major factor responsible Yonaha et al., 1995; Leresche et al., 1996; Klein and Grummt, for cell entry into mitosis in developing eggs. Secondly, recent 1999). Furthermore they have a complex pattern of expression observations have shown that the tyrosination cycle is often (Trembley et al., 1994; Hwang et al., 1998). For these reasons, suppressed during tumor progression, leading to the it is very likely that mitotic cyclin expression is also controlled accumulation of detyrosinated tubulin variants in tumor cells at the translational level. Deciphering the regulation of mRNA (Lafanechère et al., 1998). Such observations also indicate a translation is a difficult task in somatic cells where mRNAs possible regulatory role of the α-tubulin C terminus in the are synthesized, used and degraded in a continuous way. The regulation of the cell cycle. We used blocking antibodies and regulation of mRNA translation is much easier to study in competing peptides to probe the role of the α-tubulin C oocytes and early embryos, where maternal mRNAs are terminus in both starfish and Xenopus oocytes. Our data stocked in a nontranslatable form and are subsequently indicate a direct influence of the recognized epitope on Cdk1 activated to yield protein synthesis (Vassali and Stutz, 1995). activation. We provide evidence that both antibodies directed With regard to the regulation of cyclin B synthesis, developing against the tyrosinated form of the α-tubulin C terminus and oocytes have the additional advantage that at some stages of their epitope peptides inhibit cyclin B accumulation and entry development, the cyclin B mRNA is preferentially translated, into mitosis. 888 JOURNAL OF CELL SCIENCE 114 (5)

MATERIALS AND METHODS Xenopus oocytes Pieces of ovaries were manually dissected with watchmaker forceps, Antibodies then follicle-free oocytes were kept in Ringer’s Modified Medium YL1/2 is a rat monoclonal antibody directed against the C terminus (MMR: 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 of yeast α-tubulin (Kilmartin et al., 1982). It has been biochemically mM EDTA, 5 mM Hepes, pH 7.8). Progesterone (Sigma-Aldrich characterized (Wehland et al., 1983; Wehland et al., 1984). 20C6 is a SARL), was dissolved to 2 mM in methanol, then adjusted to 2 µM mouse monoclonal antibody directed against a C-terminal peptide of in MMR. tyrosinated α-tubulin (Wehland and Weber, 1987). L3 and L7 are For western blots and in-gel MBP kinase activity, one oocyte was rabbit polyclonal antibodies, specific for detyrosinated α-tubulin and resuspended in 20 µl of EB, homogenized and frozen in liquid ∆2-α-tubulin, respectively. These antibodies were obtained using nitrogen. After thawing, the homogenate was centrifuged at 13,000 synthetic peptides as immunogens and total IgGs were purified rpm at 4°C for 15 minutes, and 15 µl of the supernatant were added according to McKinney and Parkinson (McKinney and Parkinson, to 15 µl of 2× loading buffer, then boiled for 3 minutes. 10 µl were 1987), as described (Paturle-Lafanechère et al., 1994). For loaded for each lane. microinjections, antibodies were concentrated to 20 mg/ml, free of For synchronization and microinjection, oocytes in 2 µM sodium azide. L3 IgGs were affinity-purified on the corresponding progesterone were collected just at the beginning of germinal vesicle peptide and further concentrated to a protein concentration of 10 breakdown (GVBD: appearance of a dark circle near the animal pole), mg/ml by ultrafiltration. α-tubulin antibody was a commercial usually 2-6 hours after hormone addition (p.h.a.), every 15 minutes. monoclonal mouse antibody (N 356; Amersham France SA). β- Oocytes collected within 15 minutes were considered as synchronous; tubulin (clone TUB2.1) mouse monoclonal antibody was from Sigma half of the collected oocytes were microinjected and kept apart until (Sigma-Aldrich Chimie SARL, St Quentin Fallavier, France). IgGs homogenization. While several experiments were performed, all the were purified from ascitic fluid on a protein A-Sepharose column results given in this paper came frome the same complete experiment, (Pharmacia), according to Ey et al. (Ey et al., 1978). Purified IgGs except for those in Fig. 7D. were concentrated to 10 mg/ml by ultrafiltration. Anti-mos antibodies and anti-active-ERK antibodies were from Santa Cruz (sc-94 and sc- Kinase activity 7383, respectively; Santa Cruz Biotechnology INC, Santa Cruz, CA, Histone H1 kinase activity was measured from homogenates in USA). Anti-Xenopus cyclin B1 antibodies were rabbit anti- phosphorylation mix (1 mM MgCl2, 50 µM ATP, 200 µg/ml histone recombinant Xenopus cyclin B1, affinity purified (Abrieu et al., H1, 40 mM Hepes-NaOH, pH 7.4) plus 1 µl [γ-32P]dATP at 110 1997b). Anti-starfish cyclin B were polyclonal antibodies raised TBq/mmol, 370 MBq/ml (AA 0068, Amersham France), in a final against recombinant starfish cyclin B (Labbé et al., 1989). volume of 100 µl. Secondary antibodies: sheep anti-mouse Ig, fluorescein linked, In-gel MBP kinase detection was performed exactly according to were from Amersham France (ref. N 1031); goat anti-rat, alkaline Shibuya et al. (Shibuya et al., 1992). phosphatase-coupled (A 8438), goat anti-mouse (A 9917) and goat 35S-labelled amino acids were from Amersham-France (redivue anti-rabbit (A 1949) peroxidase-coupled, were from Sigma. Pro-mix L [35S] in vitro cell labelling mix, 37 TBq/mmol, 530 All antibody concentrations indicated in the text are final molar MBq/ml). concentrations in the cell, assuming that the molar mass of IgG is 150,000 Da. RESULTS Peptides Peptides were synthesized by Neosystem company (Strasbourg, France) Microinjection of mAb YL1/2 after meiosis induces at over 95% purity. The sequences were the last 17 amino acids of interphase arrest bovine tyrosinated α-tubulin (peptide Y: VGVDSVEGEGEEEGEEY) α Previous work indicated that the inactivation of the tyrosinated and the last 16 amino acids of detyrosinated -tubulin (control peptide α E: VGVDSVEGEGEEEGEE). We cloned and sequenced the starfish -tubulin C terminus by mAb YL1/2 could block sand dollar α-tubulin, and found the C-terminal sequence is almost identical: eggs either in interphase or in mitosis (Oka et al., 1990). We VGIDSVDGEGEDEGEEY. tested whether such a blockage also occurred in starfish and/or Concentrations indicated are final molar concentrations in injected in Xenopus cells. cells. First, we wanted to identify the proteins recognized by mAb YL1/2 in starfish and Xenopus oocytes. The reactivity of the Starfish oocytes antibody was tested on immunoblots of oocyte extracts. The The starfish Astropecten aranciacus was collected by diving from YL1/2 antibody reacted with a single band migrating at the October to May in the ‘Baie de Banyuls’, near the laboratoire Arago α (France), and kept in running sea water (SW). Pieces of ovaries and expected molecular mass of -tubulin (Fig. 1). The intensity testes were taken through an incision in the dorsal wall of the animals. of the signal remained constant during meiosis and mitosis (not Prophase-arrested oocytes were prepared free of follicle cells by shown). Immunofluorescence examination of oocytes stained several rinses in artificial calcium-free SW (475 mM NaCl, 10 mM with mAb YL1/2 showed bright staining of both interphasic KCl, 50 mM MgCl2, 10 mM Hepes-NaOH, pH 8.2). The hormone 1- and mitotic microtubular networks (Fig. 2), confirming that the methyladenine (1-MeAde) was from Sigma. Microinjection was major reactive protein was indeed tubulin. Furthermore, we performed according to Hiramoto (Hiramoto, 1974). confirmed by western blotting that α-tubulin was the only For western blots, ten A. aranciacus oocytes in 5 µl SW were added protein recognized by mAb YL1/2 in mAb YL1/2 µ to 15 l loading buffer (Laemmli, 1970). Synchronization was not immunoprecipitates (not shown). These data indicate that necessary, since starfish oocytes reinitiate meiosis and enter mitosis tyrosinated tubulin is present in starfish oocytes and that it is with a naturally high synchronism. For in-gel MBP kinase activity, ten oocytes in 5 µl SW were homogenized in 15 µl EB (80 mM β- the only protein strongly recognized by the YL1/2 antibody. glycerophosphate, 20 mM EGTA, 15 mM MgCl2, pH 7.3) and frozen We then microinjected fertilized starfish eggs with mAb in liquid nitrogen. After thawing, homogenates were centrifuged for YL1/2 (3.3 µM final concentration) following the second polar 15 minutes at 13,000 rpm (10,000 g), and 15 µl of the supernatant body emission. Injected eggs never entered M phase (Fig. were added to 5 µl of 4× loading buffer and boiled for 3 minutes. 3Aa), whereas control eggs showed a normal cleavage pattern α-tubulin and cyclin B synthesis 889

Fig. 1. Specificity of mAb YL1/2 and mAb 20C6 for α- tubulin. Equal amounts (10 µg protein per lane) of either Xenopus (Xen) or starfish (Sf) oocytes were loaded and analyzed on western blots using mAb YL1/2 (lanes 1 and 2) and a commercial mAb directed against α- tubulin (lanes 3 and 4), as probes. Molar mass markers on left are (from top to bottom, in kDa): 97, 64, 43, 30, 20 and 14. In both extracts, mAb YL1/2 stains only the same prominent band at 55 kDa, which is also recognized by the commercial anti α-tubulin antibody (arrowhead).

Fig. 3. Microinjection of mAb YL1/2 or 20C6 specifically induces interphase arrest in fertilized eggs. (A) Biological effects. (a) A fertilized egg was microinjected with mAb YL1/2 after completion of meiosis (120 minutes p.h.a. in this experiment), and photographed at 300 minutes p.h.a.; the cell cycle arrested with a prominent pronucleus. (b) A control fertilized egg of the same batch, left uninjected, taken at 300 minutes, had already completed three cell divisions. (c) One blastomere at the 4-cell stage (260 minutes p.h.a.) was microinjected with mAb 20C6; the microinjected blastomere (recognized by the two oil droplets microinjected with the sample) arrested with a permanent nucleus, while uninjected control blastomeres had divided 3 times more at 400 minutes p.h.a. (d-f) Control oocytes were microinjected at 120 minutes with antibodies L3 (d) and L7 (e), which recognize only nontyrosinated α- tubulin C terminus, or left uninjected (f), and photographed at 350 minutes p.h.a. Neither L3 nor L7 microinjection induced cell cycle arrest, nor even a delay in cleavage progression. Bar, 100 µm. (B) The biologically significant target of mAb YL1/2 is not the ribonucleotide reductase (rnr) small subunit: [35S]methionine- labelled sea urchin fertilized eggs were homogenized, and the Fig. 2. mAb YL1/2 stains the microtubular network in prophase as supernatant was subjected to immunoprecipitation with mAbYL1/2 well as in M-phase starfish oocytes. Prophase (A,B) and (lanes 1 and 4) or 20C6 (lanes 2 and 6). Lanes 1 and 2, prometaphase (C,D) oocytes of A. aranciacus were processed for autoradiography of immunoprecipitated material; lanes 3 and 5, immunofluorescence with a commercial anti α-tubulin antibody autoradiography of the whole supernatant before (A,C) or with mAb YL1/2 (B,D). Both antibodies stained essentially immunoprecipitation; lanes 4 and 6, autoradiography of the same microtubular structures: the cortical network and prophase immunodepleted supernatant. Sea urchin p41 has previously been asters (arrows) in prophase, and prometaphase spindle (arrowheads). identified as rnr small subunit (Standart et al., 1985). Note the accumulation of tyrosinated α-tubulin in the nuclear area in prometaphase, not observed in prophase. Bar, 100 µm. and Table 1 show that the saturation of mAb YL1/2 by its epitope peptide suppressed the effect of the antibody on the cell cycle. This result strongly suggests that the interaction of (Fig. 3Ab). A similar cell cycle arrest was observed in mAb mAb YL1/2 with specific epitopes is indeed essential for YL1/2 injected blastomeres (not shown). Injection of eggs with interphase arrest and does not reflect a nonspecific side effect antibody concentrations lower than 3.3 µM did not induce of the antibody. complete cell cycle arrest (Table 1). A series of control experiments was run to further assess the To test whether the interaction of mAb YL1/2 with a cellular specificity of the observed cell cycle blockage induced by mAb epitope was indeed essential to the observed effects of the YL1/2. antibody we microinjected a mixture containing 5 mg/ml We microinjected eggs with tubulin C terminus antibodies peptide Y (a synthetic peptide which mimics the sequence of having different epitope specificities from YL1/2. Eggs were the last 17 amino acids of bovine tyrosinated α-tubulin) and 10 injected either with L3 polyclonal antibody (an antibody mg/ml mAb YL1/2 (final concentration 6.7 µM). Fig. 4A,B selective for the detyrosinated α-tubulin), or with L7 890 JOURNAL OF CELL SCIENCE 114 (5)

Table 1. Dose-response of fertilized starfish eggs to various antibodies and peptides Concentration of antibody/peptide (µM) mAb YL1/2 mAb 20C6 peptide Y mAb YL1/2 (6.7 µM) 6.7 3.3 1.7 8 4 2 1000 500 250 +peptide Y (250 µM) Arrested/microinjected after 180 minutes 25/25 40/40 0/15 15/15 22/22 2/25 20/20 1/24 0/25 0/10

Fertilized maturing oocytes were microinjected with the final indicated concentrations of antibodies or peptides, and observed until 500 minutes p.h.a. An egg was considered as arrested when no cytokinesis occurred and the nuclear envelope was permanent. Dead or injured oocytes were not recorded. polyclonal antibody (an antibody selective for the ∆2 α- tubulin, lacking both tyrosine and the last glutamate), at similar IgG concentrations to those used for mAb YL1/2. These antibodies were ineffective in arresting cell cycles (Fig. 3Ad- f). In another series of experiments, we microinjected affinity- purified L3 antibody IgGs at the same final concentration with similar results (not shown). Starfish fertilized eggs were then injected with a commercial antibody directed against the β- tubulin C terminus (mAb TUB 2.1) at a final concentration of 13.3 µM, which was unable to arrest the cell cycle (Fig. 4C). In previous work, the only protein that has been found to cross-react significantly with mAb YL1/2, besides α-tubulin, is the small subunit of ribonucleotide reductase (rnr: Standart et al., 1985). As this subunit is abundant in early embryogenesis, we wanted to test the hypothesis that the observed effect of mAb YL1/2 microinjection was due to the activation of some Fig. 4. Cell cycle progression is specifically regulated by mAb checkpoint mechanism, as a consequence of rnr inhibition. YL1/2 epitope. (A) A fertilized egg was microinjected during its first We confimed that mAb YL1/2 immunoprecipitates from meiotic cell cycle with mAb YL1/2 (6.7 µM final concentration) and homogenates of sea urchin embryo the [35S]methionine- photographed at 400 minutes p.h.a.; the cell cycle did not progress labeled 41 kDa protein identified as rnr small subunit (Fig. 3B, later. (B) Eggs of the same batch were microinjected with the same µ lanes 1, 3 and 4). Interestingly, another monoclonal antibody amount of antibody saturated with the epitope peptide Y (250 M) that recognizes the tyrosinated α-tubulin C terminus, mAb and photographed at the same time: five mitotic cell cycles occurred, showing the antibody was neutralized by the peptide. (C) A fertilized 20C6, was much less efficient in immunoprecipitating rnr egg microinjected with the commercial mAb TUB 2.1 (13.3 µM under the same conditions (Fig. 3B, lanes 2, 5 and 6), probably final), which recognizes the C-terminal part of native β-tubulin, and reflecting a difference in epitope recognition. Yet, mAb 20C6 photographed at 320 minutes p.h.a.; this antibody has no effect on microinjection resulted in interphase arrest, as shown in Fig. the cell cycle. (D,E) Fertilized eggs were microinjected with 1 mM 3Ac, with similar sensitivity to mAb YL1/2 (Table 1). This final peptide Y (D) or control peptide E (E), which shares the same observation means that the biological effect of mAbs YL1/2 sequence as peptide Y except that it lacks the terminal tyrosine (see and 20C6 microinjection is unlikely to be the result of Materials and Methods), and were photographed at 350 minutes antibody-triggered rnr inhibition. p.h.a.; only peptide Y was able to arrest cell cycle at interphase. Taken together, these results suggested a specific blockage (F) This embryo was microinjected before cleavage with peptide E of entry into M phase in eggs and blastomeres injected with and photographed 28 hours p.h.a.: it developed normally, and α reached the early gastrula stage. White arrowheads: microinjected oil antibodies that bind to the tyrosinated -tubulin C terminus. droplets. Bars, 50 µm (in A, for A-E). Most of the experiments reported below were done both with mAb YL1/2 and with mAb 20C6 with identical results. We chose to show the mAb YL1/2 experiments in the following peptide E, differing from peptide Y by deletion of the C- sections of this paper. terminal tyrosyl residue and corresponding to the detyrosinated α-tubulin C terminus, was microinjected, eggs developed at Interphase arrest is also induced by synthetic least until the swimming gastrula stage (Fig. 4E,F). We also peptides mimicking the tyrosinated α-tubulin C injected eggs with several non α-tubulin peptides having a terminus terminal tyrosine residue. Such peptides were unable to mimic The results reported above indicate that the tyrosinated α- the mAb YL1/2 effect (data not shown). tubulin C terminus epitope has a crucial role in cell cycle In addition to the results observed with mAb YL1/2 progression in starfish oocytes. This raised the possibility that injection, these results provide independent evidence for a peptide Y may function as a competitive inhibitor of the specific involvement of the α-tubulin C terminus epitope in cell relevant epitope in this system. A test of this possibility is cycle regulation in starfish oocytes. Furthermore, the peptide shown in Fig. 4D. Peptide Y was injected in starfish eggs at a microinjection experiments also strongly suggest that only final concentration of 1 mM. The injected peptide had the same tyrosinated epitopes are involved in the observed cell cycle blocking effect as mAb YL1/2 or mAb 20C6. A dose-effect blockage whereas detyrosinated tubulin molecules are not study for peptide Y is shown in Table 1. Interestingly, when concerned. α-tubulin and cyclin B synthesis 891

Fig. 5. mAb YL1/2 microinjection in meiotic oocytes resulted in inhibition of the next M-phase entry. (A) Maturing fertilized A. aranciacus oocytes were microinjected with mAb YL1/2 at the time of GVBD (a-c) or during meiosis II (d), and kept in the continuous presence of 1 mM BrdU. Antitubulin immunofluorescence (a) showed that microinjection induced formation of microtubule bundles, and that the array resembles the GV array, with a prominent aster at the animal pole and a dense cortical network (compare with Fig. 2A,B). Chromosomes remained dispersed at animal pole, as shown by Hoechst DNA staining (b). Even long after the end of meiosis I (150 minutes p.h.a.), BrdU was not incorporated into DNA, showing that DNA replication did not occur (c). When the oocyte was microinjected during meiosis II, however, DNA replication had already occurred at the time of fixation (d), showing that mAb YL1/2 microinjection did not interfere with cell cycle phases other than M phase. Bar, 100 µm. (B) mAb YL1/2 microinjection at GVBD inhibited H1 kinase reactivation after meiosis I in both starfish (a) and Xenopus (b) maturing oocytes. (a) Starfish oocytes were allowed to mature with 1 µM 1-MeAde, and were microinjected with mAb YL1/2 at GVBD. Then, samples of two oocytes microinjected (red line) or not (controls in green) were fixed for determination of whole histone H1 kinase activity. In this experiment emission of the first polar body occurred at 130 minutes p.h.a., coinciding with the B minimal H1 kinase activity in controls. (b) Xenopus oocytes were allowed to mature with 2 µM progesterone, then synchronized. Half a. starfish of the synchronized oocytes were microinjected with mAb YL1/2 (red line) and half were left uninjected (green). The time course of 3000 H1 kinase activity showed that injected oocytes did not enter the second meiotic cell cycle, while the kinase rose again in controls at )

m 120 minutes post-GVBD. p (c 2000 se a in k activation. Furthermore, meiotic oocytes are easier to 1 H 1000 synchronize and yield faster results than mitotic eggs. Fertilized oocytes were injected with mAb YL1/2 either during the first meiotic division (M1), or after emission of the first 0 polar body. Cytological observations showed that starfish 60 80 100 120 140 160 180 oocytes microinjected with mAb YL1/2 during M1 arrested in min pha interphase, with an interphasic microtubule array and dispersed b. Xenopus procaryons (Fig. 5Aa,b). Furthermore, oocytes were arrested in interphase without DNA replication (Fig. 5Ac). mAb 5000 injection per se did not induce inactivation of H1 kinase

4000 activity (Fig. 5Ba), which was at least equal, and often slightly

) higher in microinjected oocytes than in controls. H1 kinase m p (c 3000 dropped in schedule with uninjected oocytes, but it did not rise se

a again for the second meiotic cell cycle and this explained why in k 2000 oocytes were blocked in interphase. The same experiment was 1 H performed in synchronized Xenopus maturing oocytes, with 1000 essentially the same results (Fig. 5Bb). When fertilized starfish oocytes were microinjected after the first polar body emission, 0 they were also blocked in interphase but the cell cycle arrested 0 30 60 90 120 150 180 210 after DNA replication (Fig. 5Ad). min post-GVBD These results indicate a specific effect of mAb YL1/2 injection on Cdk1 activation at the interphase to mitosis YL1/2 microinjection prevents the reactivation of transition. Apparently, the antibody does not affect other histone H1 kinase activity after M-phase exit phases of the cell cycle: the occurrence or nonoccurrence of M phase entry is under the control of a major cycling histone DNA synthesis in microinjected oocytes remains in schedule H1 kinase, which has been identified in starfish as MPF (M- with the normal meiotic cell cycle. phase promoting factor), the active cyclin B-Cdk1 dimer (Labbé et al., 1989). An obvious possibility was that the YL1/2 microinjection does not inhibit pro-MPF inactivation of the tyrosinated α-tubulin C terminus by mAb activation YL1/2 interfered with MPF activation. We used meiotic The activation of Cdk1 depends both on Cdk1 association with oocytes to test this possibility. Meiosis and mitosis differ in the mitotic cyclin B and on several post-translational many ways but have in common a requirement for proper MPF regulations. To be active, Cdk1 must be phosphorylated on 892 JOURNAL OF CELL SCIENCE 114 (5) A Xenopus response to 1-MeAde was not displaced by microinjection into 60 starfish oocytes (Fig. 6B). These results suggest that mAb YL1/2 interferes with the 50 Cdk1 activation cascade upstream of Cdc25 activation and that 40 mAb YL1/2 effect does not involve overactivation of either Wee1 or Myt 1. 30 GVBD

% 20 mAb YL1/2 microinjection impedes cyclin B accumulation 10 One possibility therefore was that mAb YL1/2 injection 0 interfered with cyclin B accumulation. The variations of the 0 2 4 6 8 cyclin B concentration in starfish oocytes stimulated with hours post-progesterone the natural hormone are shown in Fig. 7A. The Fig. shows immunoblots of whole extracts of oocytes fixed at various time B starfish points following hormonal stimulation, probed with cyclin B 100 antibody. Cyclin B concentration is high in GV oocytes (lane 90 1). The cyclin is degraded in prometaphase and the cyclin 80 concentration reaches a minimum at the time of the first polar 70 body emission (80 minutes after hormone treatment, lane 3). 60 50 Then the cyclin accumulates again to reach a new maximum GVBD during meiosis II (90 minutes after hormonal treatment, lane % 40 30 4). Following the completion of meiosis, the cyclin is degraded 20 to reach a new minimum (150 minutes after hormonal 10 treatment, lane 6) and then accumulates again (360 minutes 0 after hormonal treatment, lane 7). 00.5 11.5 To test the effect of YL1/2 injection, GV oocytes were 1-MeAde concentration (µM) stimulated with the physiological hormone and injected during Fig. 6. YL1/2 microinjection does not inhibit activation of pro-MPF. meiosis I, 40 minutes after hormonal stimulation. The injected (A) Xenopus oocytes were microinjected (red) or not (green) with oocytes were then fixed for immunoblot analysis 90 minutes mAb YL1/2. 1 hour later progesterone (2 µM) was added to the (lane 5) or 360 minutes (lane 8) after hormonal stimulation. batch (time 0), then the percentage of maturing oocytes (as judged by The 90 minute time point corresponds approximately to the appearance of the dark ring at the animal pole) was recorded: the maximum cyclin B concentration reached during meiosis 2. In time course of maturation was the same in microinjected oocytes as injected oocytes such a maximum was completely suppressed in controls. (B) A. aranciacus oocytes were microinjected (red) or (lane 5): the cyclin B concentration at 90 minutes was similar not (green) with mAb YL1/2. 1 hour after the last microinjection, to that observed at 80 minutes in controls. This suppression samples of ten microinjected and ten control uninjected oocytes were of cyclin B accumulation was apparently irreversible: the treated with varying concentrations of 1-MeAde. 1 hour later the accumulation of cyclin B normally observed 360 minutes after percentage of GVBD was recorded in each sample: mAb YL1/2 microinjection did not change the sensitivity of oocytes to 1-MeAde. hormonal treatment was completely absent in injected oocytes These two experiments show that inactive pro-MPF, already present (lane 8). In another series of experiments we found that YL1/2 in both Xenopus and starfish oocytes, is normally activated despite microinjection also strongly inhibits cyclin B1 accumulation in the presence of mAb YL1/2. maturing Xenopus oocytes (Fig. 7B). The lack of cyclin B accumulation in injected oocytes could be due either to an inhibition of cyclin B synthesis or to a threonine 161 by Cdk activating kinase (CAK). Furthermore, stimulation of the cyclin B degradation. To test which threonine 14 (T14) and tyrosine 15 (Y15) must be possibility was correct, oocytes injected with mAb YL1/2 330 dephosphorylated by the dual-specificity phosphatase Cdc25. minutes after hormonal treatment (a time point at which the Thus, the observed inhibition of H1 kinase activity in fertilized cyclin B concentration is high) were assayed for cyclin B oocytes injected with mAb YL1/2 could be due to several concentration at time point 360 minutes (Fig. 7A, lane 9). different factors. Cyclin B accumulation could be inhibited, Injected oocytes were indistinguishable from controls with Cdk1-cyclin B association could be inhibited (or reversed); regard to cyclin B concentration. Thus the mAb injection did finally, the inhibitory phosphorylation of T14 and Y15 could not trigger cyclin B degradation. be maintained by activation of the kinases Wee1 or Myt1, and These results strongly suggest that mAb YL1/2 injection inhibition of the phosphatase Cdc25. blocks cyclin B synthesis without affecting cyclin B During the prophase of the first meiotic division (GV, or degradation. To further assess this possibility we tested whether germinal vesicle stage), Cdk1 is already associated with the new synthesis of cyclin B was indeed inhibited in injected cyclin B in a complex maintained inactive by T14-Y15 eggs. For this, control and mAb YL1/2 injected oocytes were phosphorylation. We microinjected YL1/2 in starfish and pulse labeled with 35S-labelled amino acids and assayed for Xenopus GV-stage oocytes, and induced meiosis reinitiation 1 content in labeled proteins at various time points. Cyclin B was hour later with the natural hormones. Fig. 6 shows that the time prominently synthesized at 90 minutes in control oocytes (Fig. course of meiosis reinitiation in Xenopus oocytes was not 7C, 90 minutes, lane 2) and labeling was elevated also after altered by YL1/2 microinjection (Fig. 6A), and that the dose- meiosis completion (180 minutes, lane 3; note that at that time, α-tubulin and cyclin B synthesis 893

Fig. 7. mAb YL1/2 microinjection selectively inhibits cyclin B accumulation. (A) Western blot analysis with anti-cyclin B antibody of samples of ten starfish oocytes. Control oocytes (C) were fixed at various times after hormone addition (minutes, shown on top). Note the slower migrating band of phosphorylated cyclin B in M-phase samples (C, 20; C, 90). GVBD occurred at 20 minutes p.h.a., first and second polar body emissions at 80 and 120 minutes p.h.a., respectively. 20 oocytes were microinjected with mAb YL1/2 at GVBD. Ten were fixed at 90 minutes (lane 5) and ten at 360 minutes (lane 8): cyclin B never accumulated in these oocytes. Ten oocytes were microinjected at 330 minutes and fixed at 360 minutes (lane 9): the accumulated cyclin B was not degraded as a consequence of microinjection. (B) Xenopus oocytes were allowed to mature with 2 µM progesterone, synchronized at GVBD and microinjected with mAb YL1/2. Samples of microinjected (YL1/2) or uninjected (control) oocytes were fixed and processed for western blotting (see Materials and Methods) with anti-cyclin B1 antibodies. In controls, as already observed, cyclin B1 was synthesized during meiosis reinitiation, then progressively degraded and resynthesized for the second meiotic cell cycle. In contrast, in microinjected oocytes, inhibition of cyclin B1 accumulation resulted in the complete disappearance of the corresponding band at 90 minutes post-GVBD. The samples for this experiment were the same as for Fig. 5Bb. (C) A. aranciacus oocytes were microinjected with mAbYL1/2 (lane 4, +YL1/2) or not (lanes 1-3) and committed to mature with 1- MeAde. Then, at various times (minutes, shown on top), five oocytes were microinjected with 35S-labelled amino acids, fixed 10 minutes later and processed for SDS-PAGE and autoradiography of 35S incorporated into proteins. In controls cyclin B (arrow) is almost not synthesized at the GV stage (0), is strongly synthesized during meiosis (90 minutes, lane 2) and becomes the only protein actively synthesized after meiosis, at the pronucleus stage (180 minutes, lane 3. The identity of the only strongly labelled band was cyclin B, as assessed by immunoprecipitation; not shown). However, when mAb YL1/2 was microinjected, cyclin B synthesis was completely abolished (lane 4). (D) Xenopus oocytes were microinjected with mAb YL1/2 (left) or control antibodies (L3: right), then incubated µ 35 cyclin B was by far the major newly translated protein. This for 3 hours with 100 Ci/ml [ S]methionine, followed by processing for SDS-PAGE. No significant difference in incorporation of result was also observed in fully mature oocytes of the starfish methionine into proteins was seen between both samples, indicating Marthasterias glacialis). In injected oocytes, cyclin B labeling that mAb YL1/2 does not inhibit protein synthesis unspecifically. remained low (lane 4), as in GV oocytes (lane 1). We also tested whether the inhibition of protein synthesis was at least in part specific for cyclin B synthesis. In general, undergo meiotic and mitotic cycles almost in schedule with cyclin B-dependent H1 kinase activity may regulate mitotic control oocytes (Picard et al., 1988). Furthermore, the periodic protein synthesis (Kanki and Newport, 1991; Galas et al., oscillations of H1 kinase activity occur in the absence of cyclin 1993). To test the effect of mAb YL1/2 microinjection on B degradation. This is shown in Fig. 8A for A. aranciacus mitotic protein synthesis independently of cyclin B synthesis, oocytes, from which the whole germinal vesicle content had we used Xenopus prophase-arrested oocytes, in which been removed. Such oocytes still showed periodic oscillations H1 kinase is totally repressed. Fig. 7D shows that overall of H1 kinase activity. However, the drop of H1 kinase was mitotic protein synthesis was not inhibited by mAbYL1/2 delayed by 10-20 minutes compared to controls (Fig. 8A, red microinjection. line). In addition, the levels of H1 kinase activity were by far higher before the drop and after the rise in enucleated than in Maintaining a high cyclin B concentration rescues whole oocytes (compare red and black lines). These high levels oocytes from interphase arrest of H1 kinase activity are accounted for by an inhibition of The observed inhibition of cyclin B accumulation in eggs and cyclin B degradation both during meiosis I and meiosis II: the oocytes injected with mAb YL1/2 could be a major factor cyclin B levels remained constant during meiosis, as shown by responsible for the concomitant cell cycle arrest or it could be immunoblot assay of cyclin B levels in the cycling enucleated one perturbation among many. To test which of these possibilities oocytes (Fig. 8B). Interestingly, mAb YL1/2 microinjection was true we used a system in which cyclin B proteolysis does not into maturing enucleated oocytes did not inhibit H1 kinase occur and assessed whether or not such inhibition was sufficient reactivation after its drop at the end of meiosis I (Fig. 8A, blue to suppress the mAb YL1/2 effects on cell cycle. line), whereas a normal inhibitory effect of the antibody was When starfish oocytes are enucleated, meiotic and mitotic observed in control oocytes (green line). Thus mAbYL1/2 does cell cycles can still be induced by hormonal treatment: we have not affect H1 kinase activity in the presence of maintained high previously shown that maturing enucleated starfish oocytes cyclin B concentrations. 894 JOURNAL OF CELL SCIENCE 114 (5) A

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Fig. 8. In A. aranciacus oocytes, failure of cyclin B degradation due to removal of nuclear material results in the failure of YL1/2 to inhibit H1 kinase re-activation. (A) H1 kinase activity in enucleated (red, blue) or not (controls; green, black), hormone-triggered starfish oocytes, microinjected (green, blue) or not (black, red) with mAb YL1/2. (B) Western blots of samples of 10 intact (control) or enucleated maturing starfish oocytes, fixed at various times (minutes p.h.a.), probed with anticyclin B antibody; in absence of nuclear material cyclin B concentration remains as high as in controls taken Fig. 9. Interphase arrest induced by mAb YL1/2 microinjection is not just at GVBD (30 minutes p.h.a., lane 1), thus before the beginning mediated by the microtubular network. (Top) 7 pictures of the same of cyclin B degradation (see Galas et al., 1996). fertilized oocyte kept in the continuous presence of 20 µg/ml Nocodazole since 100 minutes p.h.a. (thus after first polar body These experiments indicate that the observed inhibition of emission), taken at various times p.h.a. (right). Mitotic cycles occurred, as shown by the periodic appearance/disappearance of cyclin B synthesis in starfish oocytes injected by mAb YL1/2 nuclear envelopes. (Bottom) This fertilized oocyte was microinjected is the major factor responsible for the concomitant cell cycle at 80 minutes p.h.a. and kept in the continuous presence of arrest. nocodazole thereafter; disorganization of microtubular network did not rescue cell cycling, as shown by the permanent presence of the Interphase arrest induced by antibody blockage of nuclear envelope around procaryons (arrow points to an oil drop the α-tubulin C-terminal tyrosine is not mediated by microinjected with the antibody). Bar, 50 µm. disorganization of the microtubular network Microinjection of mAb YL1/2 into cells can induce a disorganization of the microtubule network (Wehland and appearance and disappearance of the nuclear envelope (Fig. 9, Willingham, 1983; Warn et al., 1987) and this raised an upper panel) and cycles of H1 kinase activity (not shown) in important question: is the C terminus of α-tubulin directly the presence of 20 µg/ml nocodazole. In contrast, nocodazole- involved in the regulation of the cell cycle or are the observed treated oocytes microinjected with mAb YL1/2 undergo effects of mAb YL1/2 injection secondary to the activation of complete cell cycle arrest, as shown by the persistent presence microtubule-dependent checkpoints? of prokaryons (Fig. 9, lower panel). This observation indicates We used nocodazole-treated oocytes to test whether or not that the interphase arrest induced by YL1/2 microinjection is the mAb YL1/2 effects on the cell cycle were microtubule microtubule-independent. Further support for this conclusion dependent. We and others have previously shown that the came from microinjection experiments with mAb TUB2.1. succession of meiotic and cleavage cell cycles is not inhibited mAb TUB2.1 has the same effect as mAb YL1/2 on by complete depolymerization of microtubules (Picard et al., microtubule organization in cycling cells (data not shown) and 1987a; Picard et al., 1987b; Gerhart et al., 1984). Thus, is nevertheless ineffective in blocking the cell cycle in starfish maturing starfish oocytes still show both successive cycles of oocytes (see above). These data strongly suggest that α-tubulin and cyclin B synthesis 895 could account for the observed inhibition of cyclin B synthesis by mAb YL1/2 in injected cells. In all models, both invertebrate and vertebrate, ERK2 is active during the prometaphase and metaphase of the first meiotic division. ERK2 activation is thought to be implicated in the regulation of microtubule dynamic instability (Gotoh et al., 1991) and in the inhibition of DNA replication between meiosis I and meiosis II (Furuno et al., 1994; Ohsumi et al., 1994). In starfish, ERK2 is activated after GVBD. In starfish and in Xenopus, ERK2 remains active until the second meiotic cell cycle (Sagata et al., 1989; Picard et al., 1996), as shown by probing in-gel MBP kinase activity (Fig. 10A,B). In the present study, when YL1/2 was microinjected after GVBD in either starfish or Xenopus oocytes, ERK2 activity dropped rapidly and remained low at subsequent time points (Fig. 10). Additionally, Fig. 10 shows that there is also a strong inhibition of c-mos accumulation in injected Xenopus oocytes (Fig. 10B), correlative to loss of MAP kinase activity. These results may suggest that mAb YL1/2 injection primarily affects c-mos translation, with subsequent effects on cyclin B synthesis. However, a careful comparison of Figs 7B and 10B suggests a different sequence of events: apparently cyclin B1 disappears rapidly after mAb YL1/2 microinjection in maturing Xenopus oocytes, well before ERK2 inactivation. Therefore, the effect of mAb YL1/2 on cyclin B synthesis is probably not due either to the observed inhibition of c-mos accumulation or to ERK2 inactivation. Fig. 10. mAb YL1/2 microinjection triggers inactivation of ERK2/MAP kinase. (A) A. aranciacus maturing oocytes were microinjected at GVBD (YL1/2) or not (control) by mAb YL1/2. DISCUSSION Samples of ten oocytes were taken at various times (minutes p.h.a.) and processed for in-gel MBP kinase determination: MBP kinase The present work establishes that microinjection of antibodies activity was almost undetectable in microinjected oocytes. specific for the tyrosinated C terminus of α-tubulin induces (B) Synchronized maturing Xenopus oocytes of the same experiment interphase arrest in meiotic starfish and Xenopus oocytes, and shown in Figs 5Bb and 7B, either microinjected (YL1/2) or not (control), were processed for in-gel MBP kinase determination in mitotic cells of starfish early embryos. These observations (upper panel), for western blot probed with anti-active MAP kinase fit well with previous experiments reporting that mAb YL1/2 (middle panel) or for western blot probed with anti-mos antibody microinjection into fertilized sea urchin eggs either impedes (bottom panel). While in controls MBP kinase remained high, ERK2 the G2-M transition or induces a return to interphase, remained phosphorylated and mos accumulated, in microinjected depending on the time of microinjection (Oka et al., 1990). oocytes MBP kinase dropped rapidly (90 minutes post GVBD), and Furthermore, our observation of similar mAb YL1/2 effects in correlatively, mos no longer accumulated and ERK was no longer Xenopus oocytes to those in starfish and sea urchin oocytes recognized by anti-active ERK antibody. excludes the possibility that the interphase arrest caused by YL1/2 microinjection is the result of a singularity in microtubules are not involved in mAbYL1/2-induced cell cycle echinoderm cells. However, in Drosophila early embryos and blockade, although free tubulin dimers remain likely targets for in some somatic mammalian cells, mAb YL1/2 injection YL1/2. induces a mitotic arrest instead of an interphase arrest (Warn et al., 1987; Wehland and Willingham, 1983). Apparently, in Effects of mAb YL1/2 microinjection on c-mos Drosophila, the antibody does not diffuse far from the injection accumulation and activation of the ERK2/MAP area. The discrepancy between the responses of early embryos kinase compared to mitotic cells may be accounted for by prominent Besides Cdk1, another major player in the orchestration of the differences in regulation of cyclin B synthesis, as discussed cell cycle is ERK2/MAP kinase. ERK2 is the major MAP below. kinase activated in meiosis. Furthermore, there is strong evidence that ERK2 activity is involved in the regulation of Interphase arrest is due to inhibition of cyclin B cyclin B synthesis (De Moor and Richter, 1997; Ballantyne et accumulation al., 1997; Fisher et al., 1999). In oocytes of both vertebrates mAb YL1/2 microinjection during the first meiotic cell cycle (Sagata et al., 1988) and invertebrates (Tachibana et al., 2000), induces interphase arrest between meiosis I and meiosis II, ERK2 activity is regulated by c-mos, a protein whose synthesis without DNA replication in the reformed prokaryons. In and accumulation could conceivably be affected by mAb contrast, microinjection during the second meiotic cell cycle YL1/2 injection. Therefore we tested whether or not or just after second polar body emission, both in fertilized perturbations of c-mos accumulation and of ERK2 activation (Fig. 5Ad) and unfertilized (not shown) eggs, induces arrest 896 JOURNAL OF CELL SCIENCE 114 (5) in G2 after DNA replication. This indicates that the arrest is To what extent inhibition of translation by YL1/2 not an unspecific arrest of the cell cycle progression, but microinjection is specific for cyclin B mRNAs? rather corresponds to an inhibition of the mitotic H1 Our results show that mAbYL1/2 inhibits translation of at least kinase activation. mAb YL1/2 microinjection does not one additional protein, c-mos or its starfish homolog (see inhibit meiosis reinitiation in either starfish or in Fig. 10). On the other hand, Fig. 7D shows that mAbYL1/2 Xenopus. Therefore the possibility that it counteracts the injection does not trigger general, nonspecific, inhibition of post-translational activation of Cdk1 caused by protein synthesis, at least in systems where protein synthesis dephosphorylation of T14 and Y15 is excluded. Thus, the does not depend on H1 kinase activity. This shows that the observed inhibition of H1 kinase can be due either to translational regulation of cyclin B, and accumulation of a few inhibition of cyclin B accumulation, or to inhibition of cyclin additional proteins is oversensitive to inhibition induced by B binding to Cdk1. We show that mAb YL1/2 microinjection mAbYL1/2 microinjection: presumably, the binding of the results in an inhibition of cyclin B accumulation. Moreover, antibody to its cellular epitope disrupts a multimolecular it has no detectable effect in enucleated maturing oocytes, complex involved in the specific translation of these molecules. which fail to degrade cyclin B. These data strongly suggest that keeping a low concentration of cyclin B is the only way Does the observed effects of mAb YL1/2 reflect a by which mAb YL1/2 induces interphase arrest. This low specific role of the tyrosinated α-tubulin C cyclin B concentration can be obtained by inhibiting cyclin terminus? B synthesis, or by maintaining active the machinery In previous works, the observed effects of mAb YL1/2 on the responsible for cyclin B degradation. We show by pulse 35S cell cycle have been interpreted as a result of interaction of the incorporation into proteins, that mAb YL1/2 strongly antibody with the α-tubulin C terminus (Wehland and inhibits cyclin B synthesis (Fig. 7C), and by western blotting Willingham, 1983; Warn et al., 1987; Oka et al., 1990), not that it is unable by itself to open the cyclin B degradation with a crossreacting protein. Our data strongly support this window (Fig. 7A). view. We find that both mAb YL1/2 and another independent A peculiarity of early embryogenesis is that RNA and highly specific α-tubulin C terminus antibody have similar transcription is not required for cell cycles to succeed: while effects on the cell cycle and that these effects are observed in progression in G1 phase is essentially under transcriptional widely divergent species (starfish and Xenopus). Further control in somatic cells, complete inhibition of ARN indication that mAb YL1/2 action results from interaction production by Actinomycin D or α-amanitin inhibits only the with tubulin, not with a less abundant crossreacting protein, transition from early to late embryogenesis (MBT: Mid comes from the examination of dose-effect curves. The tubulin Blastula Transition) after several unperturbed cell cycles concentration in the cytoplasm of non-neuronal cells is thought (Newport and Kirschner, 1984). In starfish, as well as in sea to be about 20 µM (Hiller and Weber, 1978). Cell cycle urchin, cleavage cycles occur normally despite complete blockage occurs when mAb YL1/2 concentration in eggs removal of genetic material by enucleation or aphidicolin reaches approx. 3.3 µM. As there are two tubulin binding sites inhibition of DNA-polymerase α (Nagano et al., 1981; Picard per molecule of antibody, this corresponds to a concentration et al., 1988). This shows that during early embryogenesis, of tubulin binding sites of approx. 6.6 µM. Since a sizeable protein synthesis is regulated mainly at the translational level, part of oocytes is occupied by the nucleus and other not the transcriptional level. The proteins whose synthesis is compartments like the vitellus, from which the antibody is required for progression into M phase are translated from a pre- excluded, the antibody concentration in the soluble fraction of existing pool of stable maternal messenger RNAs. oocytes is higher than 6.6 µM. In routine experiments, the The use of Xenopus acellular extracts has allowed a more volume of the soluble fraction obtained following high speed precise identification of the proteins required for the cycling of centrifugation of Xenopus oocytes corresponds to about one early embryonic cells. Cycling extracts are able to reproduce third of the total egg volume. Applying this conservative with a correct schedule some biochemical and cytological estimate to starfish oocytes one obtains an estimate for the mAb features of cell cycling: cycling of H1 kinase activity, cycling YL1/2 concentration in the cytoplasm of about 20 µM, of nuclear envelope breakdown and re-formation, building and corresponding to saturating levels with regard to tubulin. Thus, disappearance of mitotic spindle. In such extracts, Murray and our results are compatible with tubulin being the target of the Kirschner showed that synthesis of cyclin B alone is sufficient antibody. In previous works, the only protein that has been for cycling (Murray and Kirschner, 1989). shown to be efficiently immunoprecipitated by mAb YL1/2 is Taken together, these results show that the whole cell cycle the small subunit of ribonucleotide reductase (rnr: Standart et oscillation of early embryogenesis is regulated essentially al., 1985; Thelander et al., 1985). We confirmed this result with through modulation of the translation of stockpiled cyclin B 35S-methionine-labelled sea urchin extracts in the conditions messenger RNAs and through periodic destruction of cyclin B. described by Standart et al., but we showed additionally that In contrast, in somatic cells, cyclin B mRNAs are not mAb20C6, another monoclonal antibody that also recognizes stockpiled, and even the periodic production of cyclin B the tyrosinated α-tubulin C-terminal peptide, and protein is also under transcriptional control. This probably immunoprecipitates α-tubulin with a similar efficiency, failed explains the discrepancy between the effects of mAb YL1/2 to immunoprecipitate labelled rnr (Fig. 3B). Yet, mAb20C6 microinjection that we observed in meiotic or cleavage cells microinjection was found to inhibit cell cycle progression and and the results obtained in somatic cells. This also strongly cyclin B synthesis exactly like mAbYL1/2 mincroinjection, suggests that the tubulin C-terminal epitope is mainly which is an unexpected result if the biological effect of mAb implicated in the particular regulation of cyclin B translation 20C6 is due to rnr inhibition. Moreover we confirmed in our during early embryogenesis. two usual starfish species that 1 mM hydroxyurea, a potent α-tubulin and cyclin B synthesis 897 inhibitor of rnr, fails to induce cell cycle arrest (not shown), as cyclin B mRNAs and a periodic destruction of cyclin B protein, previously published (Yamada et al., 1988). itself under the control of cyclin B-cdk1 activity. While the Additionally we find that, when injected in oocytes, regulation of the ubiquitin-dependent cyclin B proteolysis is synthetic peptides corresponding to the tyrosinated α-tubulin now well documented, very little is known on how the C terminus have a similar effect on cell division to mAb YL1/2, translation machinery favors cyclin B production. Tubulin whereas detyrosinated peptides are ineffective. The simplest is already known to regulate translation, at least its own explanation is that tyrosinated peptides act as competitive translation by an autoregulatory mechanism. This complex inhibitors of tyrosinated-free tubulin dimers in the regulation mechanism involves both translation-related mRNA of cyclin B synthesis. While they cannot completely rule out destruction (Cleveland, 1989) and repression of the translation the possibility of a crossreaction with another regulatory itself (Gonzalez-Garay and Cabral, 1996). A stimulation of α- protein, such observations strongly support a specific tubulin translation by β-tubulin overexpression has also been involvement of the tyrosinated α-tubulin C terminus in the cell described (Gonzalez-Garay and Cabral, 1995). Our present cycle regulation. results suggest an extension of the function of α-tubulin in the The mechanisms by which tyrosinated α-tubulin could regulation of translation beyond the autoregulatory processes, regulate cyclin B synthesis remain elusive. It may be the case at least during early embryogenesis. Additionally, our results that specific interactions of the tyrosinated α-tubulin C indicate that the tyrosinated form of α-tubulin may play an terminus with effectors of protein translation are involved. essential role in this process and that this role involves the free Grallert and coworkers have recently shown that the partial nonpolymerized form of the protein. inactivation of a yeast general translation factor, the RNA helicase Ded1, was able to arrest cell cycle by impeding We thank warmly Yasmina Saoudi, for originating the story, and specifically cyclin B synthesis (Grallert et al., 2000). These Marcel Dorée, for giving us the opportunity of this fruitful authors showed additionally that only the full-length cyclin B collaboration. This work was supported by grants from ARC mRNA, containing both 5′ and 3′ UTRs, required Ded1 for (Association pour la Recherche sur le Cancer), la Ligue Nationale contre le Cancer, and the Ligues Départementales de l’Aude, de being translated, suggesting that Ded1 relieves an inhibition l’Ardèche et des Pyrénées Orientales. that is mediated by 5′ and 3′ cis-elements. Interestingly, in preliminary experiments, we affinity-purified an RNA helicase belonging to the same family on immobilized peptide Y, REFERENCES whereas the same protein did not bind peptide E (not shown). The ineffectiveness of detyrosinated peptides indicates a Abrieu, A., Fisher, D., Simon, M. N., Dorée, M. and Picard, A. (1997). crucial importance of the presence or absence of the C-terminal MAPK inactivation is required for the G2 to M phase transition of the first tyrosyl residue on the properties of the α tubulin C terminus. mitotic cell cycle. EMBO J. 16, 6407-6413. Ballantyne, S., Daniel, D. R. Jr. and Wickens, M. (1997). A dependent This is compatible with an important role of the tubulin pathway of cytoplasmic polyadenylation reactions linked to cell cycle tyrosination-detyrosination cycle in the regulation of cell control by c-mos and cdk1 activation. Mol. Biol. Cell 8, 1633-1648. division during oocyte maturation. However, molecular tools Cleveland, D. W. (1989). Autoregulated control of tubulin synthesis in animal are presently lacking with which to test directly the role of the cells. Curr. Opin. Cell Biol. 1, 10-14. tubulin tyrosine ligase, the enzyme that catalyzes α-tubulin de Moor, C. H. and Richter, J. D. (1997). The mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes. Mol. Cell. Biol. 17, 6419- tyrosination (Ersfeld et al., 1993), and of the tubulin 6426. carboxypeptidase, in starfish. The mammalian tubulin tyrosine Dorée, M., Peaucellier, G. and Picard, A. (1983). Activity of the maturation- ligase can be inhibited by mAb ID3. However, we have injected promoting factor and the extent of protein phosphorylation oscillate this antibody into starfish eggs without detectable alteration of simultaneously during meiotic maturation of starfish oocytes. Dev. Biol. 99, 489-501. tubulin tyrosination (data not shown). It will obviously be of Ersfeld, K., Wehland, J., Plessmann, U., Dodemont, H., Gerke, V. and great interest to test whether or not the tubulin tyrosination Weber, K. (1993). Characterization of the tubulin-tyrosine ligase. J. Cell cycle is truly essential to cell cycle regulation in developing Biol. 120, 725-732. oocytes. Finally, a series of recent results indicate an important Ey, P. L., Prowse, S. J. and Jenkin, C. R. (1978). Isolation of pure IgG1, role for the tubulin C terminus. Tubulin tyrosination is IgG2a and IgG2b immunoglobulins from mouse serum using protein A- Sepharose. Immunochemistry 15, 429-436. systematically suppressed during tumor growth in animal Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D. and Hunt, T. (1983). models, tyrosinated tubulin is apparently required for cyclin B Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is synthesis in developing oocytes and recent results show that destroyed at each cleavage division. Cell 33, 389-396. invalidation of the ligase gene has fatal consequences in mice Fisher, D. L., Brassac, T., Galas, S. and Dorée, M. (1999). Dissociation of MAP kinase activation and MPF activation in hormone-stimulated (C. Erck, unpublished results). These results cannot be put maturation of Xenopus oocytes. Development 126, 4537-4546. together in a single simple figure at this time but should Furuno, N., Nishizawa, M., Okazaki, K., Tanaka, H., Iwashita, J., Nakajo, eventually unravel unsuspected functions of the tubulin N., Ogawa, Y. and Sagata, N. (1994). Suppression of DNA replication via molecule, namely of the α-tubulin C terminus. Mos function during meiotic divisions in Xenopus oocytes. EMBO J. 13, Our results may also help shed light on poorly understood 2399-2410. Galas, S., Barakat, H., Dorée, M. and Picard, A. (1993). A nuclear factor regulations concerning mRNA translation in meiotic and required for specific translation of cyclin B may control the timing of first cleavage cell cycles. The progression of the meiotic and meiotic cleavage in starfish oocytes. Mol. Biol. Cell 4, 1295-1306. cleavage cell cycles is essentially ensured by the regulated Gerhart, J., Wu M. and Kirshner, M. (1984). Cell cycle dynamics of an M- translation of maternal messenger RNAs. Among them, cyclin phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs. J. Cell Biol. 98, 1247-1255. B mRNAs have been shown to play a leading role, and previous Gonzalez-Garay, M. L. and Cabral, F. (1995). Overexpression of an epitope- works have popularized the view that early embryogenesis cell tagged beta-tubulin in Chinese Hamster Ovary cells causes an increase in cycle oscillation was governed by a sustained translation of endogeneous alpha-tubulin synthesis. Cell Motil. Cytoskel. 31, 259-272. 898 JOURNAL OF CELL SCIENCE 114 (5)

Gonzalez-Garay, M. L. and Cabral, F. (1996). alpha-Tubulin limits its own Picard, A., Karsenti, E., Dabauvalle, M. C. and Dorée, M. (1987b). Release synthesis: evidence for a mechanism involving translational repression. J. of mature starfish oocytes from interphase arrest by microinjection of human Cell Biol. 135, 1525-1534. centrosomes. Nature 327, 170-172. Gotoh, Y., Nishida, E., Matsuda, S., Shiina, N., Kosako, H., Shiokawa, K., Picard, A., Harricane, M. C., Labbé, J. C. and Dorée, M. (1988). Germinal Akiyama, T., Ohta, K. and Sakai, H. (1991). In vitro effects of microtubule vesicle components are not required for the cell cycle oscillator of the early dynamics of purified Xenopus M phase-activated MAP kinase. Nature 349, starfish embryo. Dev. Biol. 128, 121-128. 251-254. Picard, A., Galas, S., Peaucellier, G. and Dorée, M. (1996). Newly Grallert, B., Kearsey, S. E., Lenhard, M., Carlson, C. R., Nurse, P., Boye, assembled cyclin B-cdc2 kinase is required to suppress DNA replication E. and Labib, K. (2000). A fission yeast general translation factor reveals between meiosis I and meiosis II, in starfish oocytes. EMBO J. 15, 3590- links between protein synthesis and cell cycle controls. J. Cell Sci. 113, 3598. 1447-1458. Pines, J. (1995). Cyclins and cyclin-dependent kinases: themes and variations. Hiller, G. and Weber, K. (1978). Radioimmunoassay for tubulin: a Adv Cancer Res. 66, 181-212. quantitative comparison of the tubulin content of different established tissue Sagata, N., Watanabe, N., Vande Woude, G. F. and Ikawa, Y. (1989). The culture cells and tissues. Cell 14, 795-804. c-mos protooncogene product is a cytostatic factor responsible for meiotic Hiramoto, Y. (1974). A method of microinjection. Exp. Cell. Res. 87, 403-406. arrest in vertebrate eggs. Nature 342, 512-518. Hwang, A., McKenna, W. G and Muschel, R. J. (1998). Cell cycle- Shibuya, E. K., Boulton, T. G., Cobb, M. H. and Ruderman, J. V. (1992). dependent usage of transcriptional start sites. A novel mechanism for Activation of p42 MAP kinase and the release of oocytes from cell cycle regulation of cyclin B1. J. Biol. Chem. 273, 31505-31509. arrest. EMBO J. 11, 3963-3975. Kanki, J. P. and Newport, J. W. (1991). The cell cycle dependence of protein Standart, N. M., Bray, S. J., George, E. L., Hunt, T. and Ruderman, J. synthesis during Xenopus laevis development. Dev. Biol. 146, 198-213. (1985). The small subunit of ribonucleotide reductase is encoded by one of Keyomarsi, K. and Pardee, A. B. (1993). Redundant cyclin overexpression the most abundant translationally regulated maternal RNAs in clam and sea and gene amplification in breast cancer cells. Proc. Natl. Acad. Sci. USA 90, urchin eggs. J. Cell Biol. 100, 1968-1976. 1112-1116. Tachibana, K., Tanaka, D., Isobe, T. and Kishimoto, T. (2000). c-Mos forces Kilmartin, J. V., Wright, B. and Milstein, C. (1982). Rat monoclonal the mitotic cell cycle to undergo meiosis II to produce haploid gametes. antitubulin antibody derived by using a new non secreting rat cell line. J. Proc. Natl. Acad. Sci. USA 97, 14301-14306. Cell Biol. 93, 576-582. Thelander, M., Graslund, A. and Thelander, L. (1985). Subunit M2 of Klein, J. and Grummt, I. (1999). Cell cycle-dependent regulation of RNA mammalian ribonucleotide reductase. Characterisation of a homogeneous polymerase I transcription: the nucleolar transcription factor UBF is inactive protein isolated from M2-overproducing mouse cells. J. Biol. Chem. 260, in mitosis and early G12. Proc. Natl. Acad. Sci. USA 96, 6096-6101. 2737-2741. Labbé, J. C., Capony, J. P., Caput, D., Cavadore, J. C., Derancourt, J., Trembley, J. H., Kren, B. T. and Steer, C. J. (1994). Posttranscriptional Kaghad, M., Lelias, J. M., Picard, A. and Dorée, M. (1989). MPF from regulation of cyclin B messenger expression in the regenerating rat liver. starfish oocytes at first meiotic metaphase is a heterodimer containing one Cell Growth Differ. 5, 99-108. molecule of cdc2 and one molecule of cyclin B. EMBO J. 8, 3055-3058. Vassali, J. D. and Stutz, A. (1995). Translational control. Awakening dormant Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly mRNAs. Curr. Biol. 5, 476-479. of the head of bacteriophage T4. Nature 227, 680-685. Walter, S. A., Guadagno, T. M. and Ferrell, J. E. Jr, (1997). Induction of a Lafanechère, L., Courtay-Cahen, C., Kawakami, T., Jacrot, M., Rudiger, G2 phase arrest in Xenopus egg extracts by activation of p42 MAP kinase. M., Wehland, J., Job, D. and Margolis, R. (1998). Suppression of tubulin Mol. Biol. Cell 8, 2157-2169. tyrosine ligase during tumor growth. J. Cell Sci. 111, 171-181. Warn, R. M., Flegg, L. and Warn, A. (1987). An investigation of microtubule Leresche, A., Wolf, V. J. and Gottsfeld, J. M. (1996). Repression of RNA organization and functions in living Drosophila embryos by injection of a polymerases II and III transcription during M phase of the cell cycle. Exp. fluorescently labeled antibody against tyrosylated α-tubulin. J. Cell Biol. Cell Res. 229, 282-288. 105, 1721-1730. McKinney, M. M. and Parkinson, A. (1987). A simple, non-chromatographic Wehland, J., Willingham, M. C. and Sandoval, I. V. (1983). A rat procedure to purify immunoglobulins from serum and ascites fluid. J. monoclonal antibody reacting specifically with the tyrosylated form of Immunol. Methods 96, 271-278. alpha-tubulin. I. Biochemical characterization, effects on microtubule Murray, A. W. and Kirschner, M. W. (1989). Cyclin synthesis drives the polymerization in vitro, and microtubule polymerization and organization early embryonic cell cycle. Nature 339, 275-280. in vivo. J. Cell Biol. 97, 1467-1475. Nagano, H., Hirai, S., Okano, K. and Ikegami, S. (1981). Achromosomal Wehland, J. and Willingham, M. C. (1983). A rat monoclonal antibody cleavage of fertilized starfish eggs in the presence of aphidicolin. Dev. Biol. reacting specifically with the tyrosylated form of α-tubulin. II. Effects on 85, 409-415. cell movement, organization of microtubules and intermediate filaments, and Newport J. W. and M. W. Kirschner. (1984). Regulation of cell cycle during arrangement of Golgi elements. J. Cell Biol. 97, 1476-1490. early Xenopus development. Cell 37, 731-742. Wehland, J., Schroder, H. C. and Weber, K. (1984). Aminoacid sequence Ohsumi, K., Sawada, W. and Kishimoto, T. (1994). Meiosis-specific requirements in the epitope recognized by the alpha-tubulin-specific rat cell cycle regulation in maturing Xenopus oocytes. J. Cell Sci. 107, 3005- monoclonal antibody YL1/2. EMBO J. 3, 1295-1300. 3013. Wehland, J. and Weber, K. (1987). Tubulin-tyrosine ligase has a binding site Oka, M. T., Arai, T. and Hamaguchi, Y. (1990). Microinjection of the on β-tubulin: a two domain structure of the enzyme. J. Cell Biol. 104, 1059- monoclonal antibody YL1/2 inhibits the cleavage of sand dollar eggs. Cell 1067. Struct. Funct. 15, 373-378. White, R. J., Gottlieb, T. M., Downes, C. S. and Jackson, S. P. (1995). Cell Pardee, A. B, (1974). A restriction point for control of normal animal cell cycle regulation of RNA polymerase III transcription. Mol. Cell. Biol. 15, proliferation. Proc. Natl. Acad. Sci. USA 71, 1286-1290. 6653-6662. Paturle-Lafanechère, L., Manier, M., Trigault, N., Pirollet, F., Mazarguil, Yamada, H., Kuraishi, R., HIRAI, S., Katoh, Y., Fusetani, N., Amikura, H. and Job, D. (1994). Accumulation of delta 2-tubulin, a major tubulin R., Okano, K. and Nagano, H. (1988). Induction of achromosomal variant that cannot be tyrosinated, in neuronal tissues and in stable cleavage by hydroxyurea in starfish embryos and the reversal by a microtubule assemblies. J. Cell Sci. 107, 1529-1543. combination of deoxyadenosine and deoxycytidine: possible involvement of Picard, A., Labbé, J. C., Peaucellier, G., Le Bouffant, F., Le Peuch, C. and salvage pathway for deoxynucleoside triphosphate biosynthesis in the Dorée, M. (1987a). Changes in the activity of the maturation promoting presence of hydroxyurea. J. Cell Physiol. 136 326-332. factor are correlated with those of a major cyclic-AMP and calcium Yonaha, M., Chitazakura, T., Kitajima, S. and Yasukochi, Y. (1995). Cell independent protein kinase during the first cell cycles in the early starfish cycle-dependent regulation of RNA polymerase II basal transcription embryo. Dev. Growth Differ. 29, 93-103. activity. Nucl. Acid Res. 23, 4050-4054.