Proc. Nati. Acad. Sci. USA Vol. 88, pp. 9929-9933, November 1991 Cell Biology competent for parthenogenesis in Xenopus eggs support procentriole budding in cell-free extracts (/protein synthesis) FRE9DERIC TOURNIER*, MAREK CYRKLAFFt, ERIC KARSENTIt, AND MICHEL BORNENS* *Centre de Gdn6tique Mol6culaire, Centre National de la Recherche Scientifique, 2 Avenue de la terrasse, 91198 Gif/Yvette, France; and tEuropean Molecular Biology Laboratory, Postfach 102209, Meyerhofstrasse 1, D-6900 Heidelberg, Federal Republic of Germany Communicated by Andre Lwoff, August 1, 1991 (received for review April 22, 1991)

ABSTRACT Heterologous centrosomes from diversed spe- cell cycle from which they have been isolated: centrosomes cies including humans promote egg cleavage when i iected into isolated from G1 or G2 human lymphoid cells (KE37 cell line) metaphase-arrested Xenopus eggs. We have recently isolated or from quiescent cells (peripheral human lymphocytes in Go) centrosomes from calf thymocytes and shown that they were were shown to possess a similar parthenogenetic activity (8). unable to induce egg cleavage, an inability that was apparently We have isolated (10) centrosomes from calf thymocytes correlated with the peculiar structure of these centrosomes (CTs) and shown that the two were linearly asso- rather than with a lack of microtubule-nucleating activity: the ciated by their proximal ends through a mass of dense two centrioles were associated in a colinear orientation by their material. Furthermore, they were unable to induce egg cleav- proximal ends. To promote cleavage, a heterologous cen- age, although centrosomes isolated from other bovine cells trosome probably is required to duplicate, although this has not and from thymocytes of other species could induce egg yet been demonstrated. Therefore, we designed an in vitro assay cleavage (6). These results suggest that the cycle that would enable us to directly observe the duplication pro- can be blocked when the centrioles are prevented from cess. We show that competent centrosomes from KE37 cells separating into a nonlinear configuration, a step that might be synchronized in initiate procentriole budding in critical for the initiation of procentriole budding. interphasic extracts from Xenopus eggs in the absence of Therefore, we have prepared cell-free extracts from Xe- protein synthesis, whereas calf thymocyte centrosomes do not. nopus eggs in the mitotic or interphasic stage, in which we Since calf thymocyte centrosomes do not support parthenogen- could directly compare the duplicative capacity of cen- esis, the present results suggest that duplication of the foreign trosomes that are competent and incompetent (such as CT centrosome is required for centrosome-induced parthenogen- centrosomes) to induce parthenogenesis. We show that com- esis. Furthermore, procentriole budding takes place in the petent centrosomes from human lymphoblasts (KE37 cell absence of protein synthesis in egg extracts arrested in S phase. line), synchronized in G1 phase, initiate procentriole budding This in vitro assay should contribute to the identification of in interphasic extracts from Xenopus eggs in the absence of molecular mechanisms involved in the initiation of centrosome protein synthesis, whereas incompetent (CT) centrosomes do duplication. not. Since CT centrosomes do not support parthenogenesis, the present results strongly suggest that duplication of the In somatic cells, centrosome duplication encompasses the foreign centrosome is required for centrosome-induced par- whole cell cycle and progression in the centrosome cycle thenogenesis. appears to be a marker of progression in the cell cycle. The importance of centrosomes in establishing the spatial orga- MATERIALS AND METHODS nization ofthe mitotic apparatus mandates that the cell tightly controls the reproduction of its centrosome, which must Animals. Xenopus laevis females were obtained from the occur only once in each cell cycle, prior to . The Service d'Elevage d'Amphibiens du Centre National de la morphological events of the centrosome duplication cycle in Recherche Scientifique (France). Eggs were obtained from somatic cells have been described and involve budding of females injected 3-8 days before use with 100 international daughter centrioles offthe wall ofthe parent structures in late units of pregnant-mare-serum gonadotropin (Intervet, An- G1 phase, elongation of the daughter centrioles during S and gers, France) and 1 day before with 1000 international units G2 phases, redistribution of , and sep- of human chorionic gonadotropin (Sigma). The eggs were aration of duplicated centrosomes in prophase (1-3). How- collected in 100 mM NaCl to prevent activation. ever, the molecular mechanisms that govern centrosome Preparation of Extracts. Egg extracts were prepared ac- duplication are not known. cording to Felix et al. (11). Egg jelly was removed in 2% As amphibian eggs apparently lack a functional cen- (wt/vol) cysteine hydrochloride (pH 7.8), and eggs were trosome, which can be replaced by the injection of foreign washed four times in 0.25 x MMR (lx MMR = 0.1 M NaCl/2 centrosomes (parthenogenetic development), they represent mM KCl/1 mM MgSO4/2 mM CaCl2/5 mM Hepes/0.1 mM a favorable system to study the duplication of centrosomes. EDTA, pH 7.4) containing cycloheximide (150 pug/ml; Sig- In this system, no species specificity is apparently required ma), and then activated by an electric shock (11) in the same for the centrosomes: heterologous centrosomes isolated from buffer for 90 min at 20'C. Activated eggs were transferred to sea urchin (4), rodent (5, 6), or human (7-9) cells can induce Beckman SW 50.1 rotor tubes filled with ice-cold acetate cleavage of Xenopus eggs. In this parthenogenetic test, the buffer [AB = 100 mM potassium acetate/2.5 mM magnesium heterologous centrosomes are believed to duplicate, but acetate/cytochalasin D (10 pug/ml; Sigma)/1 mM dithiothrei- direct experimental evidence is lacking. The parthenogenetic tol/250 mM sucrose/leupeptin (10 pug/ml)/pepstatin (10 kkg/ activity of the centrosome is independent of the stage of the ml)/aprotinin (10 ,g/ml), pH 7.2]. Excess acetate buffer was removed from the tubes containing packed eggs prior to The eggs were crushed by centrifugation at The publication costs of this article were defrayed in part by page charge centrifugation. payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: CT, calf thymocyte.

9929 Downloaded by guest on October 1, 2021 9930 Cell Biology: Toumier et al. Proc. Natl. Acad. Sci. USA 88 (1991) 10,000 x g (with maximum acceleration rate) for 10 min at 40C room temperature for 0-3 hr. After incubation, nocodazole was in a L5-65 Beckman centrifuge. The cytoplasmic material added to 30 ,&g/ml and the tubes were kept at 4°C for 30 min. between the upper lipid layer and the yolk pellet was col- Then 0.8 ml of lysis buffer [1 mM Tris'HCl, pH 7.5/0.1% lected (10,000 x g supernatant) and an ATP regenerating 2-mercaptoethanol/0.5% Nonidet P-40/0.5 mM MgClJ1 mM system (10 mM creatine phosphate, creatine phosphokinase phenylmethylsulfonyl fluoride/aprotinin (100pg/ml)/leupeptin (80 jig/ml), and 1 mM ATP (Boehringer Mannheim)] and (1 ,ug/ml)/pepstatin (1 ,ug/ml)] at 4°C was added to each tube protease inhibitors as above were added. A sample was kept and the material in each tube was layered onto 25% (vol/vol) in ice during further steps. The 100,000 x g extracts were glycerol in RG2 buffer (80 mM Pipes.KOH/1 mM MgCl2/1 mM obtained by further centrifugation of the 10,000 x g super- EGTA, pH 6.8) in a 15-ml modified Corex tube with a special natant at 100,000 X g for 60 min at 40C in the SW 50.1 rotor adaptor containing a 12-mm round coverslip. The material was with adaptors for 0.6-ml tubes. The yellow cytoplasmic layer centrifuged at 12,000 rpm (20,000 x g) in a JS-13.1 rotor (100,000 x g supernatant) was collected and kept at 4TC, (Beckman) at 4°C for 15 min to apply the centrosomes to the because freezing the extracts gave inconsistent results in the coverslips. The coverslips were removed, fixed in methanol at centrosome duplication assay. -20°C for 5 min, and processed for immunofluorescence stain- Cell Synchronization and Isolation of Centrosomes. KE37 is ing. a human T-lymphoblastic cell line. KE37 cells, cultured in Immunofluorescence Study. Centrosome duplication was RPMI 1640 medium (Eurobio, Les Ulis, France) containing monitored by measuring the numbers of centrioles and cen- 7% (vol/vol) calf serum, were first synchronized at the trosomes by double immunofluorescence (7). A monoclonal G1/S-phase border with 2.5 mM thymidine (Sigma) for 20 hr antibody against a-tubulin (Amersham) stained centrioles (12) and then separated in a cell-cycle-dependent manner and a human autoimmune serum stained the pericentriolar using centrifugal elutriation according to Tournier et al. (8). material ofhuman centrosomes (J. E. Dominguez, E.K., and Centrifugal elutriation led to an enriched fraction containing J. Avila, unpublished data). >80% G1 phase cells. Centrosomes were prepared from Negative Staining. For negative staining ofelectron micros- KE37 cells in G1 phase according to Tournier et al. (8) and copy preparations, 15 ,ul of G0 centrosomes (1 x 104 cen- from CTs according to Komesli et al. (10). trosomes per ,l) was mixed at 4°C with 300 ,l of extract. Duplication Assay. Typically, 10 ,ul of KE37 G1 centrosomes Further steps were identical to the immunofluorescence (1 x 104 centrosomes per ul) was mixed with 100,ul ofextract protocol until the lysis step. For each point (at 0, 2, and 3 hr (either 10,000 x g or 100,000 X g supernatant) at 4°C. The ofincubation), 100 ,l ofextract was mixed with 200 Al oflysis mixture was equally distributed into fourtubes and incubated at buffer and incubated 2 min at 4°C. The material was loaded

FIG. 1. Double immunofluorescence staining of G1 centrosomes (KE37) in Xenopus extracts arrested in S phase at 0 time (a and a') and after 3 hr of incubation (b and b'). Centrosomes stained with the human autoimmune serum (a) appear to be doublets of tubulin-containing dots corresponding to centrioles (a'). In this experiment, the two centrioles in a centrosome do not split (see Table 1) and after 3 hr of incubation in the extract, the centrosomes (b) contain three or four tubulin-containing dots as judged by the anti-tubulin staining (b'). In the lower parts of each panel, other examples at 0 time (a and a') and after 3 hr of incubation (b and b') are shown. The variable intensities of the centrioles probably depend on their orientation on the coverslip. (x 16,900.) Downloaded by guest on October 1, 2021 Cell Biology: Toumier et al. Proc. Natl. Acad. Sci. USA 88 (1991) 9931 onto 200 Al of 25% glycerol in RG2 buffer in a 0.6-ml tube centrosomes has been described (13-15). In the present containing a Teflon adaptator supporting a carbon grid. The study, visualization of the centrosomes by immunofluores- material was centrifuged at 14,000 rpm (25,000 x g) in a SW cent staining was done after nocodazole-induced disassembly 50.1 rotor (Beckman) for 20 min (40C) to apply centrosomes of the microtubules, which had been nucleated in the extract to the grid. Grids were then removed, rinsed in RG2 buffer, by the added centrosomes. Double staining was used to stained with 2% (wt/vol) phosphotungstate (pH 7 with identify both the pericentriolar domain and the centrioles. KOH), and dried. This allowed us to identify the centrosomes unambiguously by distinguishing them from tubulin aggregates. More repro- ducible results were obtained from cytoplasmic extracts of RESULTS Xenopus eggs prepared from activated oocytes in the pres- Duplication of G, Centrosomes in Cell-Free Extracts: Im- ence of cycloheximide (interphasic extracts). munofluorescence Study. Competent centrosomes were iso- G1 centrosomes from KE37 cells were incubated in 100,000 lated from KE37 cells synchronized in G1 phase. We have x g Xenopus egg extracts for 0-3 hr. At 0 time, centrosomes shown (8) that these centrosomes were capable of inducing were observed as pairs of centrioles (Fig. 1 a and a'), and parthenogenesis when injected into unfertilized Xenopus after 3 hr of incubation, centrosomes contained three or four eggs. The use of cell-free extracts and immunofluorescence tubulin dots (Fig. 1 b and b'). We interpreted these dots as to study microtubule polymerization nucleated by isolated duplicative forms of centrosomes in which the centrioles did not separate. The numbers of centrosomes and centrioles were recorded at various incubation times by double immu- nofluorescence. After 3 hr of incubation, the number of centrosomes remained constant but the number of centrioles -J 0 increased from 1 (reference value at incubation time) to 1.95 °0 FE2 _2 ~~~~~~00l (Fig. 2a) and the number of centrosomes containing 3 or 4 identifiable centrioles increased from 1% to 48% (Fig. 2b). 'U z~~~~~~~~~~~~~L Typically, the number of KE37 centrioles doubled after 2-3 010Q C.)_1 hr of incubation. We noted, however, that the efficiency of LM. centrosome duplication depended largely on the batch of M0 0U extract used. z 0 1 00( 200 In other experiments, the two centrioles in a given cen- z TIME (min) D trosome often separated from each other over large distances (Table 1) during the first hour of incubation as judged by the increase in the proportion of single centrioles counted at this 2cn 60 ~~~~~~~~~~~~~1 time. Duplicated forms observed after 3 hr ofincubation were 0 A. identified as a pair oftubulin-containing dots associated with o centrosome labeling. 40 When the same kinetic experiments were done using low-speed extracts (10,000 x g), no increase in >20 number was detected after 2 or 3 hr of incubation (Fig. 2c) or up to 6 hr of incubation (data not shown), suggesting that the IL3 10,000 x g supernatant contained an activity that inhibited 0 0 centrosome duplication. .10 Duplicative Forms of Centrosomes Observed by Negative TIME (min) Staining. To validate our scoring method by an independent approach (electron microscopy), we examined negatively C,, 'U stained centrosomes that had been incubated in the extract. -j c 0 Table 1. Centriole splitting in interphasic extracts during the first I- 2- z hour of incubation Lou 0 Centriole form, no. Cells Exp. n Time, hr Single Double o KE37 I 5 0 388 (44) 488 (66) 1 952 (84) 189 (16) z 0 1 00 200 II 2 0 49 (25) 148 (75) TIME (min) 1 67 (27) 180 (73) CT 2 0 42 (19) 183 (81) FIG. 2. Duplication of KE37 centrosomes in Xenopus extracts 1 45 152 (77) arrested in S phase. (a) Numbers of centrosomes (u) and centrioles (23) (m) were recorded at 0 time and after 1, 2, and 3 hr ofincubation. After n represents the number of experiments. Centrosomes were re- 3 hr of incubation, the number of centrosomes is constant (as the two corded as pairs of tubulin-containing dots at 0 time and after 1 hr of centrioles in a given centrosome did not split in the extract) whereas incubation. For KE37 centrioles (experiments I), a splitting of the the number of centrioles doubles. Approximately 1 x 105 cen- two centrioles in a given centrosome was observed (44% of single trosomes were added at 0 time in 100 1.l of extract. Ten fields (>100 centrioles at 0 time and 84% after 1 hr of incubation). In five centrosomes) were recorded for each point after incubation. The experiments (I), duplicated forms observed after 3 hr of incubation number of centrioles at 0 time was standardized (1 on the Y axis). (b) in the extract were identified as pairs of tubulin-containing dots The number of duplicated forms (three or four centrioles per cen- associated with centrosome labeling. In two experiments (II), a trosome) increases from 1% to 48%. (c) The number of KE37 splitting of the two centrioles was not observed (25% of single centrioles remains constant with the 10,000 x g supernatant (e) centrioles at 0 time and 27% after 1 hr of incubation). The duplicated incubated for 0-3 hr. In the corresponding 100,000 x g supernatant forms observed after 3 hr in the latter case (II) were recorded as three (o), the number of centrioles increases after 2 hr of incubation. This or four tubulin-containing dots. For CT centrosomes, in all cases, we suggests that the high-speed (100,000 x g) pellet contains an inhib- did not observe any significant splitting of the centrioles after 1 hr of itory activity for centrosome duplication. incubation. Numbers in parentheses are percent of total centrioles. Downloaded by guest on October 1, 2021 9932 Cell Biology: Tournier et al. Proc. Natl. Acad Sci. USA 88 (1991)

2 a CD LU q .r, ..P:..t,

. ....I I.- 't z LU

LI-U 0C.) mLU z t z 0 1 00 200 TIME (min) FIG. 4. Centrosomes isolated from CTs do not duplicate in 100,000 x g supernatant, whereas KE37 centrosomes initiate pro- centriole budding, as shown by the increase of the number of c centrioles. o, CT centrioles; *, KE37 centrioles. of incubation, an orthogonal bud (procentriole) was detected on 60% of the scored centrioles. CT Centrosomes Do Not Support Procentriole Budding. CT centrosomes, which display linearly associated centrioles (10), do not support cleavage when injected into unfertilized Xenopus eggs (6). This suggested that these centrosomes were incompetent to initiate bud formation in an adequate cytoplasmic environment. As shown in Fig. 4, we did not observe an increase in centriole number in an extract incu- bated with CT centrosomes. Since, in the same extract, KE37 centrosomes do form buds, this result shows that CT cen- i r trosomes are incompetent for duplication and interestingly, CT centrioles do not separate (Table 1). .3. '1 . :, .8 DISCUSSION FIG. 3. Duplicating centrosomes observed by negative staining. We have compared the capacity of centrosomes to initiate a Centrioles were in pairs (a and c) or were single (b and d), without centrosome duplication cycle in cell-free extracts by using an (a and b) or with procentriole buds (arrows) (c and d). (Bar = 0.1 immunofluorescence-based assay. Centrosomes competent AM.) to induce parthenogenesis (G1 centrosomes from KE37 cells) initiate procentriole budding whereas incompetent cen- Centrioles were scored either as single centrioles or as pairs trosomes (CT centrosomes) do not. The electron microscopy and with or without an associated orthogonal procentriole study supported the conclusion that the increase in tubulin- (Fig. 3). A representative experiment is shown in Table 2. At containing dots observed by immunofluorescence during 0 time, 46% ofthe centrioles were pairs and 52% were single. incubation of the centrosomes in extracts corresponds to an After 2 hr ofincubation, 12% ofcentrioles were pairs and 82% increase in centriole or procentriole number. But it also of centrioles were single. These results were in agreement indicated that only procentrioles were formed. Indeed, we with immunofluorescence observations and indicated exten- never observed pairs of centrioles tightly associated in an sive splitting of the centrioles during incubation in the ex- orthogonal configuration in which the two centrioles had the tracts. Duplicated forms were only 4% of the total centrioles same length. Further incubation (up to 6 hr) did not increase at 0 time (Table 2), which is consistent with the fact that the number of centrioles (data not shown). In particular, we centrosomes were prepared from G1 cells. After 2 hr and 3 hr never obtained evidence for successive rounds of centriolar duplication. This suggests that the duplication process is not Table 2. Centriole splitting and duplicated forms by electron completed under our in vitro conditions. microscopy after negative staining This may have several causes. First, the high-speed su- pernatant (100,000 x g) may lack components necessary for Time, Centriole form, no. Duplicated bud elongation. Such components may or may not be in the hr n Single Double Triple forms, no. high-speed pellet, because the pellet apparently also contains x 0 97 34(52) 30(46) 1 (2) 4 (4) an activity from the 10,000 g supernatant that was shown 2 21 14 (82) 2 (12) 1 (6) 13 (62) to inhibit the initiation of centriole duplication (Fig. 2c). The 5 most likely explanation is that the components necessary for 3 36 20 (74) (19) 2 (7) 21 (58) assembling full-length centrioles are limiting in comparison to n represents the number ofcentrioles. Note that, at 0 time, 52% of the number of centrosomes incubated in the extract. We centrioles were observed as single centrioles and 46% were observed added between 500 and 1500 centrosomes per ,ul of extract, as centriole pairs. After 2 hr of incubation, single centrioles repre- the immu- sented 82% of the total centrioles observed. Centrioles were some- which is the minimum number required to make times observed (2-7%) as triplets. Duplicated forms correspond to nofluorescence study feasible. This figure is close to the centrioles observed with a centriole bud. For duplicated forms, 4% number ofcentrosomes produced per embryo at the midblas- were observed at 0 time, 62% were observed after 2 hr ofincubation, tula transition (16) (one embryo has a volume of about 1 "I). and 58% were observed after 3 hr of incubation. Numbers in From such a figure, we may conclude that a single cen- parentheses are percent of total centrioles. trosome duplication cycle at most could take place under our Downloaded by guest on October 1, 2021 Cell Biology: Toumier et al. Proc. Natl. Acad. Sci. USA 88 (1991) 9933

conditions. If this were the case, adding centrosomes at a In conclusion, the present data demonstrate the potential lower concentration should allow multiple rounds of dupli- of our in vitro assay to describe in molecular terms the cation in the extract, but we have not succeeded in designing orthogonal budding ofprocentrioles from the proximal end of an experiment to test this hypothesis. parental centrioles. Another implication ofthese results is that the first steps of centrosome duplication are independent of the cytoplasmic We thank Dr. Spencer Brown, Dr. Pedro Santamaria, and Eric oscillator that drives the cell cycle, since procentriole bud- Bailly for their helpful comments and English corrections on the ding takes place in cycloheximide-treated interphasic ex- manuscript. We also thank J. E. Dominguez for the gift ofthe human tracts. However, the incompleteness of the duplication cycle autoimmune serum and Daniel Meur for the art work. This work has could suggest that a signal from the "mitotic clock" is been supported by Centre National de la Recherche Scientifique and necessary for the late steps of the process. This is unlikely by Grant 87.C.0555 from Ministere de la Recherche et de la Tech- nologie to M.B. and by the Association pour la Recherche sur le since multiple cycles of centrosome duplication occurred in Cancer. F.T. is a recipient of a fellowship from the Ministere de la cycloheximide-treated Xenopus blastomeres (16) and a sim- Recherche et de la Technologie. ilar result was also reported for sea urchin eggs (17). Centrosome duplication can be experimentally uncoupled 1. Kuriyama, R. & Borisy, G. G. (1981) J. Cell Biol. 91, 822-826. from the synthesis of mitotic cyclins (refs. 16 and 17 and this 2. Rieder, C. R. & Borisy, G. G. (1982) Biol. Cell. 44, 117-132. report). Our experimental system should, allow investigation 3. Kochanski, R. S. & Borisy, G. G. (1990) J. Cell Biol. 110, ofthe coupling between the centrosome duplication cycle and 1599-1605. the cytoplasmic oscillator. The asynchrony of centrosome 4. Maller, J., Poccia, D., Nishioka, D., Kido, P., Gerhart, J. & reproduction in the absence of protein synthesis (16, 17) Hartman, H. (1976) Exp. Cell Res. 99, 285-294. suggests indeed that phasing ofthe cytoplasmic oscillator and 5. Karsenti, E., Newport, J., Hubble, R. & Kirschner, M. W. (1984) J. Cell Biol. 98, 1730-1745. the centrosome cycle occurs in normal development. p34cdc2, 6. Tournier, F., Komesli, S., Paintrand, M., Job, D. & Bornens, a suci homologue (18), and cyclin B (E. Bailly, J. Pines, T. M. (1991) J. Cell Biol. 113, 1361-1369. Hunter, and M.B., unpublished data), the major components 7. Bornens, M., Paintrand, M., Berges, J., Marty, M. & Karsenti, of the cytoplasmic oscillator, are associated with the cen- E. (1987) Cell Motil. Cytoskeleton 8, 238-249. trosome in G2 and M phases. It will be of interest to study 8. Tournier, F., Karsenti, E. & Bornens, M. (1989) Dev. Biol. 136, centrosome duplication in cycling extracts and the role of 321-329. protein phosphorylation controlled by various cyclins in 9. Klotz, C., Dabauvalle, M. C., Paintrand, M., Weber, H., synchronization of centrosome duplication with the mitotic Bornens, M. & Karsenti, E. (1990) J. Cell Biol. 110, 405-415. cycle. Preliminary experiments in mitotic extracts suggest 10. Komesli, S., Tournier, F., Paintrand, M., Margolis, R., Job, D. & Bornens, M. (1989) J. Cell Biol. 109, 2869-2878. that procentriole budding cannot occur during mitosis (data 11. Felix, M. A., Pines, J., Hunt, T. & Karsenti, E. (1989) EMBO not shown). J. 8, 3059-3069. The inability of CT centrosomes to initiate a centriole bud 12. Bostock, C. J., Prescott, D. M. & Kirkpatrick, J. B. (1971) in vitro strongly suggests that, in parthenogenetic Xenopus Exp. Cell Res. 68, 163-168. eggs, cleavage requires the duplication of the injected cen- 13. Verde, F., Labbe, J. C., Dorde, M. & Karsenti, E. (1990) trosome. This also indicates that the structural configuration Nature (London) 343, 233-238. of the centrosome is essential to initiate budding in a per- 14. Belmont, L. D., Hyman, A. A., Sawin, K. E. & Mitchison, missive cytoplasmic environment. Specific egg proteins are T. J. (1990) Cell 62, 579-589. able to assemble on the two centrioles of the heterologous 15. Sawin, K. E. & Mitchison, T. J. (1991) J. Cell Biol. 112, 925-940. centrosome to form the two poles of the first mitotic spindle, 16. Gard, D. L., Hafezi, S., Zhang, T. & Doxsey, S. J. (1990) J. in which each pole should contain a chimeric centrosome. Cell Biol. 110, 2033-2042. The incompetence ofCT centrosomes is not simply due to the 17. Sluder, G., Miller, F. J., Cole, R. & Rieder, C. L. (1990) J. Cell species or the tissue origin, because the linear arrangement of Biol. 110, 2025-2032. the CT centrosomes apparently represents a locked config- 18. Bailly, E., Doree, M., Nurse, P. & Bornens, M. (1989) EMBO uration. J. 8, 3985-3995. Downloaded by guest on October 1, 2021