A predominant basic α-tubulin isoform present in prophase Xenopus oocyte decreases during meiotic...

Biol Cell (1992) 75, 173-180 0 Elsevier, Paris

173

Original article

A predominant basic a-tubulin isoform present in prophase Xenopus oocyte decreases during meiotic maturation

Catherine Thibier I, Philippe Denoulet 2, Catherine Jesus I, Rent Ozon l

’ Laboratoire de Physiologie de la Reproduction, URA CNRWINRA 1449, Universitk P et A4 Curie, 4, Place Jussieu, 75252 Paris Cedex 05; z Laboratoire de Biochimie Cellulaire, Colkge de France, 75005 Paris, France

(Received 1 June 1992; accepted 9 July 1992)

Summary - Xenopus oocytes are blocked in prophase of the first meiotic division. During the G2/M transition drastic changes occur both in the cytoskeletal organization and in the capacity of tubulin to polymerize. Posttranslational modification of tubulin isoforms might be one of the factors that control the dynamic properties of microtubules. We have therefore analysed, by two-dimensional polyacrylamide gel electrophoresis, the isotubulins purified from Xenopus oocytes, and we show that tubulin is resolved into at least four a-isoforms and four /3-isoforms. We have identified a basic a (a,)-tubulin isoform which is specific to prophase arrested oocyte and that progressively disappears during meiotic maturation; its decrease is initiated when the nuclear envelope breaks down and is controlled by the nucleus. Using 3’s methionine labelled oocytes we demonstrate that the disappearance of the at, isotubulin results from both an arrest of its biosynthesis after maturation, and from posttranslational modification which induces a shift of this a- isoform to a more acidic pl. Moreover, in vitro experiments using )?S prelabelled tubulin purified from prophase oocytes show that metaphase extracts containing MPF activity are able to induce the acidification of the a,-isoform, suggesting that the observed posttranslational modification might be regulated by p34 cdc2. However, the nature of this modification remains to be elucidated.

tubulin I isoforms I oocyte I meiotic maturation I Xenopus

Introduction

The diversity of both a-and /?-tubulin isoforms in a cell results from the expression of multiple tubulin genes and from several posttranslational modifications. The func- tional significance of tubulin isoform diversity is believed to be an adaptation to specific cellular functions.

It is known that during the cell cycle, when cells enter metaphase, the turnover of microtubules is accelerated [ 11. This has recently been studied in vitro using Xenopus oocyte extracts obtained either from metaphase or interphase state [2 - 81.

Fully grown Xenopus oocytes are blocked in prophase (G2 phase) of the first meiotic division. They enter metaphase (M-phase) under the stimulation of progesterone. Entry into M-phase is controlled by maturation promoting factor (MPF). During the G2/M-transition, the microtubular cytoskeleton of the oocyte is remodelled [9 - 121. This reorganization of the spatial distribution of microtubules is a consequence of both a shortening and an increase in dynamic instability of the microtubules.

The factors involved in controlling this reorganization are poorly understood; several mechanisms may explain the dynamic properties of microtubules such as: posttranslational modifications of tubulin isotypes; interactions with microtubule associated proteins (MAP,) [13]; activation of M-phase regulated protein kinases such as the ~34~~~~ kinase [7] or the MAP, kinase [14] that may change the phosphorylation state of specific proteins involved in the regulation of microtubule dynamic.

Here we show that a basic a-tubulin isoform present in prophase-arrested Xenopus oocytes disappears during meiotic maturation. In vitro experiments with extracts

from M-phase oocytes indicate that a posttranslational mechanism may be involved in the modification of this a-tubulin isoform.

Materials and methods

Animals

Adults Xenopus laevis (Centre de Recherche de Biochimie Mac- romoleculaire du Centre National de la Recherche Scientifique, Montpellier, France) were bred and maintained under laboratory conditions.

Chemicals

[35S]methionine (1000 Ci/mmol) was obtained from Amersham. Tax01 was a gift from Dr M Suffness (NIH, Bethesda, MD, USA).

Antibodies

Anti a-tubulin (N 356 ascites fluid) and anti/%tubulin (N 357 ascites fluid) monoclonal mouse antibodies were purchased from Amersham; monoclonal mouse antibody specific for Tyr-tubulin (designated YL l/2) was kindly provided by JV Kilmartin (Cam- bridge). Antiglutamylated a-tubulin (T335) and anti-acetylated a-tubulin (6-l lB-1) antibodies were a kind gift of A Wolff (Paris) and G Piperno (New York), respectively.

Oocytes preparation

Animals were anaesthetited with MS222 (Sandoz) at 1 g 1-t. Ovaries were removed and transferred to medium A composed

174 C Thibier er al

of 88 mM NaCI, 0.33 mM Ca(NO,),, I mM KCI, 0.41 mM CaC$ 0.82 mM MgSO,, 2 mM Tris, pH 7.4 (Merriam medtum).

After dispase (0.4 mg ml t) digestion for 4 h at laboratory temperature and collagenase (0.8 mg ml -‘) digestion for I h with continuous stirring, stage VI oocytes (I .3 mm diameter) [ 141 were collected. Oocytes were induced to mature by addition of I FM progesterone to the external medium. The criterion for maturation was the appearance of a white spot surrounded by pigment on the coloured animal pole of the oocyte. Matured oocytes are referred to as ‘metaphase oocytes’ or M II, and immature oocytes as ‘prophase oocytes’. In experiments involving enucleated oocytes, cells were manually enucleated using a procedure adapted from Ford and Gurdon [ 151 and described by Jes- sus et a/ [l6].

3’S Methionine protein-lobelling

Oocytes were incubated at laboratory temperature (22°C) with gentle continuous shaking in medium A containing ?S methionine (Amersham) (166 yCi ml I). Oocytes were then washed eight times in medium A at room temperature.

Tubulin purifcation

As reported by Collins and Vallee [ 171 tasol-prepared microtubules can be induced to disassemble when treated by cold in the presence of high concentrations of Ca’*. This provides a mean for obtaining purified tubulin by repeated cycles of reversible assembly and disassembly from oocyte supernatant.

Oocytes were homogenized at 4°C in Iysis buffer (0. I M Pipes, pH 6.6, 5 mM EGTA, 1 mM MgSO,, 0.9 M glycerol, I mM dithiothreitol, 1 mM benzamidine. 2 mM PMSF, 0.5 g/l soybe- an trypsin inhibitor, 2 mg/l leupeptin and 2 mg/l aprotinin). and centrifuged at 30000 g for 30 min at 4°C. The supernatant was recovered and centrifuged at 165000 g for 90 min at 2°C. The supernatant obtained was termed ‘soluble estract’ (Sl). After addition of 20,uM taxol and 1 mM GTP, this soluble extract was incubated at 37°C for I5 min to assemble microtubules and then centrifuged at 30000 g for 30 min at 2°C: the pellet obtained (P,) was resuspended in lysis buffer containing IO mM CaCI, and incubated on ice for 45 min to depolymerize microtubules. After incubation the extract was centrifuged at 30000 g for 30 min at 2°C. The resulting supernatant (S2) was again incubat-

94-

67-

ed with 20 FM taxol and I mM GTP plus EGTA (30 mM) and centrifuged at 30000g for 30 min at 20°C. The final pellet was termed P, or purified tubulin. The purification product steps are illustrated in figure I; tubulin is the major protein in the first pellet (P,): in the third pellet (P,) tubulin is the only detectable protein by Coomassie blue staining.

Preparation of Xertoplrs oocyte exfrocts

Extracts were prepared according to Felix e/ a/ [18]. Stage VI or MI1 oocytes were crushed bv centrifugation at 10000 g for IO min (SW 50. I rotor) in a minimum volume of a buffer containing 100 mM potassium acetate, 2.5 mM magnesium acetate. 60 mM EGTA. 5c(g ml -I cytochalasin D, 1 mM DTT (pH 7.2). An ATP generating system (1 mM ATP. IO mM creatine phosphate, 80 pg ml ’ creatine phosphokinase, final concentrations) was added IO the extract before further centrifugation at 165000 g for 60 min in TLA 100 rotor.

Hectrophoresis and autoradiographv

SDS-solubilized fractions (20- 50 ~1) were submitted to electrophoresis, according to Laemmli [I91 adapted for 7 cm x 8.5cm slab gels (I mm thick) of 0.1% SDS (sodium dodecyl sul- fate) 7.5% polyacrplamide; slab gels were run 2 h at room temperature at a constant voltage of I80 V and were stained with Coomassie blue.

IEF-SDS-PAGE was performed according to O’Farrell [20] modified by Denoulet era/ [21], except that only pH 5 - 5.5 Ser- valytes were used in the IEF gel. The second dimension was car- ried out on 13-cm long, 8% polyacrylamide SDS slab gels as described above.

After the run. gels were stained with Coomassie blue and dried under vacuum. Radioactive polypeptides were detected by es- posing gels to hyperfilm-,3 mas (Amersham).

Western blotting

After 2-D-PAGE, proteins were electrophoretically transferred from SDS slab gels nitrocellulose sheets as described by Towbin et ol [22]. Anti a-tubulin and anti $tubulin were used at a l/5000 dilution; antibody specific for Tyr tubulin (YL 112) was used at a I/300 dilution. Interactions between transferred proteins and

-

Fig, 1. Coomassie-blue-stained gel after SDS-PAGE of tubulin extracts corresponding to different purification steps. Two ovaries (23 ml of tissue) were homogenized in 30 ml lysis buffer; tubulin was purified as described in Materials and methods. P,, PZ and P, refer to pellets. S,. S, and SJ refer to supernatants. Samples accounting for 0.2% of the total extract were loaded in each slot (except S,: O.lslo). The position and molecular masses (kDa) of protein standard are shown.

Tubulin isororms during meiotic maturation of Xenopus oocytes 175

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+

f •

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Fig 2. Tubulin isoforms in stage VI oocytes. Electrophoresis was performed either on a 105 000 g supernatant (a) or on purified fractions as described in Materials and methods. 10/~g of purified tubulin was loaded per gel (b, c, d, e). a, c. Coomassie blue staining of 2-D-PAGE; in c only the tubulin actin areas of the gel are shown, b. Coomassie blue staining of IEF gels, d, e. Immunological identification of the tubulin isoforms: Western blots probed with a mixture of anti~ and anti-,~ (Amersham) (d) or with a specific antibody for the tyrosinated 0¢-tubuLin (YL 1/2) (e). The solid arrows indicate the position of the =b-isoform and the open arrow denotes actin.

b O I , • *

~, ¢ '

O ~ . J

C

d

e

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a

b

Fig 3. IEF analysis of stage gl oocytes tubulin (a) or brain tubulin (b). Tubulin was purified as described in Materials and methods; in both cases L0/~g of purified tubulin (P3 pellet) were loaded per gel. The arrow indicates the position of the ab-isoform.

antibodies were detected using peroxidase-conjugated anti-mouse immuno-globulin (diluted 1/500), which in turn was visualized using 4-chloro-l-naphthol and H,O, as substrates.

Resu l t s

The 165 000 g supernatant (soluble extract) prepared from immature stage VI Xenopus oocytes was analyzed by two- dimensional I E F - S D S - P A G E (2-D-PAGE); at least four ~x-tubulin isoforms and four/3-tubulin isoforms were detected after Coomassie blue staining (fig 2a). When oocyte tubulin was purified by a taxol procedure adapted

f rom Collins and Vallee [17] and then submitted to 2-D- P A G E , the same tubulin isoforms were detected (fig 2c). This result indicates that no segregation, at least for major isoforms detectable by Coomassie blue, has occurred during purification by this purif icat ion procedure. A major basic ~- isoform (pl - 5.6) (fig 2a, b, c, solid arrow) was heavily stained by two antibodies, one specific for e- tubulin and one specific for tyrosinated ~-tubulin (fig 2d, e). This isoform, absent f rom the brain of adult Xenopus (fig 3), appears to be specific for the oocyte and will be subsequently referred to as eb"

When tubulin was purified from M II oocytes and analyzed by IEF, Coomassie blue staining o f the gel shows that the basic e - i soform is absent or present at a very low level (fig 4a, b). In contrast , neither qualitative nor quan- titative changes in/3-tubulin isoforms were detected. The decrease in the amount of the eb-isoform parallels the increase in staining of a more acidic e-isoform. These modifi- cat ions were also observed after 2 - D - P A G E and subsequent immunosta in ing with anti- tubulin antibodies (fig 4c, d).

The timing of disappearance o f the %- isoform was analyzed during progesterone-induced maturat ion. Tubu- lin was purified from oocytes at the time o f germinal vesicle breakdown (GVBD; about 4 h after addition o f progesterone) and subjected to 2-D-PAGE. At that time only a small decrease in the Ocb-isoform was observed; this result indicates that the disappearance o f this isoform is a long- lasting event that is initiated by the time of GVBD and clearly detectable only in MII oocytes.

During the course o f matura t ion, the oocyte nucleus

176 C Thibier et al

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Fig 5. Tubulin in immature, mature or enucleated mature oocytes. 2-D-PAGE was performed on partially purified tubulin (PI pellet as described in fig I). Coomassie blue staining is shown, a. Tubulin isoforms from immatures oocytes (prophase oocyte), b. Tubulin isoforms from mature oocytes (metaphase oocyte), c. Tubulin isoforms from mature enucleated oocytes. Arrows are as in figure 4.

d

Fig 4. Tubulin isoforms in stage VI and metaphase II oocytes. 2-D-PAGE or IEF were performed on purified tubulin (P3 pellet) from metaphase II oocytes except for a (stage VI oocytes); other experimental conditions are as those described in figure 2. a, b. Coomassie blue staining of IEF gels. c. Coomassie blue staining of 2-D-PAGE gels. d. Immunodetection of tubulin with a mixture of anti-a- and anti p-tubulin antibodies (Amersham). The short arrow indicates the position of the %-isoform and the long one indicates the position of the shifted isoform,

breaks down and the nucleoplasm mixes with the cytoplasm. This event has been shown to modify the dynamics of the microtubules since enucleation prior to the induction of maturat ion suppresses the ability of the mature cytoplasm to initiate aster formation [I 1, 23, 24]. In- terestingly, when enucleated oocytes were induced to mature by progesterone, the disappearance of the %- isoform does not occur (fig 5c) suggesting that the nucleus is required to mediate the observed modification (fig 5b).

The disappearance of the %-isoform during oocyte maturat ion may be due to proteolysis, to a posttranslational modification or to an arrest of its synthesis. The possible involvement of a posttranslational event shifting the %-tubulin towards a more acidic p I was tested in experiments using 3~S methionine labelled oocytes. Oocytes were labelled overnight with radioactive methionine then chased in an excess of unlabelled methionine for 7 h, either in a progesterone-containing medium (MII oocytes) or in a progesterone-free medium (control stage VI oocytes).

Autoradiography of two-dimensional gels shows that in matured oocytes there is a strong decrease of the radioactivity associated with %-tubulin isoform and a corresponding increase in the labelling of acidic ~-tubulin isoforms (fig 6a, b). To quantify these results, Coomassie stained spots corresponding to %-, o~- and /~-isoforms were excised from the gels and the amount of 35S radioactivity determined. Given that total amount of radioactivity recovered in ~-isoforms was unchanged in MII oocytes, this value was chosen as a reference. Radioactivity recovered in the %-isoform and in other ~-isoforms was therefore expressed as the percent of radioactivity associated with these isoforms versus the total radioactivity recovered in ~-isoforms (table I). About 50% of the 35S radioactivity that comigrates with the %-isoform in stage VI oocytes has disappeared in MII oocytes. There is a concomitant increase in the radioactivity associated with more acidic a-isoforms (table I). Since these experiments were performed in the presence of an excess of unlabelled methionine, the corresponding increase observed in acidic g-isotubulin is likely due to a shift o f the %-isoform towards a more acidic one rather than to an increase in acidic tubulin biosynthesis rate.

To strengthen the above hypothesis it was of interest to study the biosynthesis of tubulin isoforms in mature oocytes. For that purpose oocytes were first induced to mature in v i t ro and then incubated in 35S methionine for 7 h; although the uptake of 35S methionine is strongly decreased in matured oocytes, figure 6c, d clearly shows that the synthesis of the %-isoform has dropped in metaphase II oocytes; moreover, the acidic isoform that was shown to accumulate after staining with Coomassie blue was only slightly labelled.

Altogether these experiments indicate that the decrease in the amount of a basic a-tubulin isoform during X e n o - p u s oocyte maturat ion involves both an arrest of its biosynthesis and a posttranslational modification.

Tubulin isoforms during meiotic maturation of Xenopus oocytes 177

e

b

C

d

Fig 6. Autoradiographs of two dimensional gels of isotubulins prepared from oocytes labelled with 35S methionine, a, b. Oocytes were radiolabelled with 35S methionine overnight, then washed and incubated in Merriam medium containing unlabelled methionine (10-3M) with (b) or without (a) progesterone (1/~M). Oocytes were homogenized after 7 h incubation (ie 2 h after GVBD has occurred in progesterone treated oocytes). Extracts corresponding respectively to 70 (a) and 40 oocytes (b) were loaded on the corresponding gels. c, d. Oocytes were incubated overnight in a progesterone free medium (c) or in a medium containing progesterone (I /~M) in order to obtain metaphase II oocytes (d). Then oocytes were rinsed and labelled with 35S methionine for 5 h. Extracts corresponding respectively to 100 (e) and 180 oocytes (d) were loaded on the corresponding gels. Tubulin was partially purified (PI pellet as described in fig 1) and 2-D-PAGE was performed as already described. Only tubulin regions are presented.

Table !. Effect of progesterone on the radioactivity associated to a-tubulin isoforms after prelabelling of oocytes with 35S methionine. Oocytes were labelled with 35S methionine overnight. After extensive washings, oocytes were either incubated in medium A containing unlabelled methionine (10 -3 M) without (VI oocytes) or with 1 /~M progesterone for 7 h (MII oocytes) and then homogenized. After purification of tubulin (P~ pellet) and 2-D-PAGE, Coomassie stained spots corresponding to ~b-isoform or to other ~-isoforms were excised from the gel and the amount of radioactivity determined. The radioactivity associated with/3 isoforms was counted and used as a reference. The results are expressed as the mean + SD of percent radioactivity associated to c~-isoforms versus the total radioactivity recovered in/3-isoforms, n = four experiments.

assoform Other a-isoforms (% radioactivity) (% radioactivity)

VI oocytes 17 _+ 5 31 _+ 3 MII oocytes 9 _+ 2 45 _+ 8

To identify the nature of the modification that causes the pI shift of the ab-isoform we have tested for posttranslational modifications which are known to occur with tubulin. The acidic shift of the %-isotubulin does not result from an acetylation reaction since an antibody (6-1 l-B1) raised against acetylated ~t-tubulin does not react with any of the a-isotubulins isolated from MII oocytes. This antibody only recognizes minor acidic isoforms (ie not detectable by Coomassie blue staining) when assayed on tubulin purified from stage VI oocytes (results not shown). A possible posttranslational glutamylation of the ab-isoform was also ruled out by experiments showing that no a-isotubulin (prepared either from stage VI oocytes or from MII oocytes) was recognized by a specific anti a- glutamylated tubulin antibody.

It was reported that phosphorylation of tubulin may represent an in vivo posttranslational modification of/3- tubulin [25]. To test the possibility that a phosphorylation of the ab-isoform may induce the observed acidic shift, we performed two types of experiments. First we purified

tubulin from 32p prelabelled oocytes (stage VI or MII); under these conditions and despite the presence of phos- phatase inhibitors in the extraction medium (orthovana- date,/3-glycerophosphate, NaF, P-nitrophenyl-phosphate) analysis by 2-D-PAGE did not allow to detect any labelling of c¢-tubulin. In a second series of experiments, using the same purification procedure, tubulin was first purified from stage VI oocytes and then incubated in the presence of 32p 7"ATP with different purified kinases. p34 cdc2 and MAP kinase (purified from Xenopus oocyte [26]) do not phosphorylate tubulin at all, whereas the cata- lytic subunit of cAMP dependent protein kinase or a casein kinase II purified from Xenopus oocyte [27] phosphorylate minor ~-tubulin acidic isoforms and to a lesser extent p-tubulin. In both cases only acidic ~-isoforms were labelled and no change in the amount of the ab-isoform was ever detected (not shown). This indicates that the ab- isoform is not a substrate for any kinase tested and thus demonstrates that a phosphorylation reaction may not ac- count for the observed shift.

Mature oocytes become arrested in metaphase of the second meiotic division by the calcium-sensitive cytostatic factor (CSF). Crude extracts prepared from such oocytes in the presence of EGTA maintain high levels of p34 ~dc2 kinase activity as assayed by histone H 1 phosphorylation [28] and exhibit MPF activity as tested by microinjection in a recipient oocyte [2]. It was of major interest to know if the observed in vivo modification of the ~b-isoform could be induced in vitro by metaphase extracts. To test this possibility, radioactive tubulin was purified from stage VI oocytes prelabelled with 35S methionine, and the purified radioactive tubulin was incubated for 2 h with: i) a prophase extract prepared from prophase oocytes; ii) a metaphase extract prepared from MII oocytes; iii) or a metaphase extract plus an excess of Ca 2÷ known to in- activate the CSF and to convert a metaphase extract to an interphase extract devoided of histone H1 kinase and MPF activities. As shown in figure 7 only metaphase extracts containing MPF activity and histone H 1 kinase activity are able to provoke the acidification of the radioactive %-isoform. Therefore, the enzyme complex that causes the posttranslational modification appears to be directly or indirectly regulated by p34 cdc2.

178 C Thibier et a/

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Fig 7. In vitro effects of mitotic extracts on purified tubulin. Tubulin was purified (P3 pellet) from oocytes prelabelled with ~-~S methionine; the pellet was resuspended in the buffer used to prepare the extracts. Prophase or metaphase extracts were prepared as described in Materials and methods; incubations were performed at 30°C for 3 h as follows: 10 ~l of purified tubulin (corresponding to about 40 oocytes) was incubated with 30 ~1 of extracts (corresponding to about 200 oocytes), a,d, Prophase extract, h, e. Metaphase extract. c, I. Metaphase extract + 40 mM CaCI 2. a, b, c. Coomassie blue staining of 2-D-PAGE. tl, e, f. Autoradiograph of the corresponding electrophoresis. The arrow indicates the position of the ab-isoform.

Tubulin isoforms during meiotic maturation of Xenopus oocytes 179

Discussion

The full grown oocyte contains a population of stable microtubules [16, 29], which are believed to play a crucial role in the spatial distribution of developmentally impor- tant molecules. The presence of a microtubule stabilizing activity in the cytoplasm of the prophase oocyte may be responsible for the long ( > 300/zm) and stable microtubules described in this giant cell during oogenesis [10, 12, 30]. Numerous biochemical mechanisms may explain the stabilization of microtubules in oocytes as in somatic cells. Among them two mechanisms have been studied in detail: i) microtubules may associate with stabilizing proteins such as X MAP [13, 31] or with proteins that cap the end of microtubules [32]; and ii) alternatively posttranslational modifications of tubulin, such as acetylation of ~-tubulin, may have a stabilizing effect as these modified tubulins are mainly associated with a subpopulat ion of non- dynamic microtubules in many cell types [33, 34] includ- ing Xenopus oocytes [12].

In this paper we demonstrate that a basic ~-tubulin isoform is present in prophase Xenopus oocyte. This unusual ~b-tubulin is synthetized in the growing oocyte from stage II to stage VI oocyte (results not shown), and its synthesis is arrested after oocyte maturat ion. It remains to be determined whether the ~b-tUbulin isoform is the primary product of a specific ~-tubulin gene expressed only in prophase oocytes or whether it results f rom a posttranslational modification specific to the growing oocyte, in- creasing the pI of a preexisting 0t-tubulin.

During oocyte maturat ion, when the oocyte enters M- phase the %-tubul in i soform slowly disappears. In metaphase II oocytes it is barely detectable (fig 4). Our results show that two biochemical events control the drop in the concentration of this %-isoform: the arrest of its biosynthesis and a posttranslationai modification leading to a decrease in the p! of the protein. The finding that MPF extracts contain an enzymatic activity that induces the pH i shift of the ~b-tubulin isoform in vitro is of special interest. This is in fact the first demonstrat ion of a posttranslational modification of a tubulin isoform that may be regulated by a mechanism involving the activity of MPF. It is known that MPF extracts contain at least two M phase-activated ser/ thr kinases, p34 cdc2 and MAP kinase. However, the kinase activity of p34 cdc2 or of MAP kinase is not sufficient by itself to induce the observed modification since this posttranslational modification does not occur in enucleated mature oocytes, which are known to contain active MPF and active p34 cdc2 and MAP kinases. It may therefore be assumed that one of the components required for the p ! shift of the ~b-isoform is located in the prophase oocyte nucleus. This component, perhaps one of the enzymes catalyzing the reaction itself, could be a substrate of the p34 coo2 kinase or of the MAP kinase. To further analyze this possibility we need to identify the nature of the posttranslational modification that controls the shift of the ~b-tubulin isoform. It will also be of interest to know whether the modifying radical is added cotranslationally in growing prophase oocytes and then removed during maturat ion, or whether it is added during maturat ion, causing the observed p I shift of the %-isoform.

The biological role of the ~b-isoform is not known. This specific isoform could be involved in the stabilization of microtubules during oogenesis. Its disappearance correlates with changes in the dynamics of microtubules at the onset of M-phase in Xenopus oocyte [7, 9, 10] and

with the format ion of a metaphase spindle [24, 37]. Re- cently Vale [37] reported that M-phase extracts of Xenopus oocytes contain an activity that rapidly severs stable microtubules along their length. An attractive hypothesis would be that the observed biochemical modification of the ~b-isoform may be required for this severing activity.

Acknowledgments

We wish to thank Dr Scot Davey for reading the manuscript. This research was supported by grants from INRA, CNRS, and INSERM: CRE 910 713 and CRE 89-6005.

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A predominant basic α-tubulin isoform present in prophase Xenopus oocyte decreases during meiotic...

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