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Centrosomes and parthenogenesis
CHAPTER
14
Centrosomes and Parthenogenesis Fr6d6ric Tournier* and Michel B o r n e n s t *Laboratoire de Cytophysiologieet ToxicologieCellulaire Universit6Paris 7 75251 Paris Cedex 05, France ?InstitutCurie, Sectionde Recherche 75248 Paris Cedex 05, France
I. Introduction II. Parthenogenetic Test in Xenopus A. Parthenogenesis versus Fertilization B. Materials C. Procedure D. Comments III. Couphng between Egg Cell Cycle and Centrosome Duplication Pathway A. Temporal Coupling between Egg Activation and Centrosome Injection B. Activity of Centrosomes from Synchronized or Quiescent Cells IV. Species Barrier Specificity A. Procedure B. Comments V. Active or Inactive Centrosomes from the Same Species A. Procedure B. Comments VI. Prospects A. In Vitro Assays B. Centriole Assembly Pathways References
I. I n t r o d u c t i o n In animal cells, two centrioles act as a core structure on which a fibrogranular network assembles to form the centrosome (Bobinnec et al., 1998a,b; Tassin and Bornens, 1999). The centrosome is not only capable of nucleating microtubules in interphase and mitotic M E T H O D S IN CELL BIOLOGY, VOL. 67 Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 0091-679X/01 $35.00
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Fr6d6ric Tournier and Michel Bornens animal cells, but it is also likely to be able to control the release of microtubules (Piel et al., 2000). The bipolarity of the cell division process rests on the reproductive capacity of this organelle. Its duplication cycle occurs throughout the cell cycle and several distinct steps have been described in somatic cells (for a review, see Tournier and Bornens, 1994). A G1 cell inherits a pair of dissociated centrioles from previous mitosis and initiation of orthogonal procentriole budding precedes S-phase entry. After procentriole elongation during S and G2 phases, separation of the duplicated centrosomes is coincident with the onset of the next mitosis. Few centrosomal components, either having a structural role within the organelle or participating in the regulation of its activity, have been described during cell cycle progression (for reviews, see Kimble and Kuryama, 1992; Kalt and Schliwa, 1993). Moreover, several lines of evidence suggest a coupling between centrosome duplication and the progression of the cell division cycle (Bailly etal., 1989; Hinchcliffe et al., 1999; Meraldi et al., 1999). Isolation procedures have provided an efficient way for biochemical, structural, and functional characterization of the centrosome. The parthenogenetic assay in Xenopus eggs is an efficient system to study centrosome duplication. Mature frog eggs are unable to assemble a centrosome from its elements in the absence of a preexisting centrosome. Centrosome assembly from egg precursor components can be triggered by a centrosome from heterologous species, emphasizing the conservation of the centrosome duplication mechanisms among divergent species. This chapter describes the parthenogenetic assay. It also reviews the main established or still enigmatic results concerning the capacity of centrosomes isolated from different cell types in different species to induce frog egg cleavage. These results and the limitation of this approach to study centrosome duplication are discussed, then it briefly discusses the potential of current in vitro systems to identify important molecular events of the pathway and also indicates how mammalian ciliated epithelial cells can be used in parallel to characterize specific or common centrosomal components involved in centriolar or acentriolar pathways.
II. P a r t h e n o g e n e t i c
Test in
Xenopus
A. Parthenogenesis versus Fertilization
At fertilization, the sperm cell triggers the exit of egg meiosis and the resumption of cell cycle progression. The resumption of the egg cell cycle can be triggered by pricking the egg with a needle, leading to cytoplasmic reorganization, as shown by the displacement of cortical pigment, but no cleavage occurs in the absence of the sperm centrosome (Hara et al., 1980; Fig. 1). Once into the egg cytoplasm, the sperm centrosome forms a microtubule aster, probably due to the activation of key centrosomal proteins, and further to the recruitment of maternal proteins. The sperm aster migrates toward the center of the animal hemisphere and duplicates to form the first mitotic spindle. In order to induce a parthenogenetic development, the injected centrosome has to nucleate microtubules from the egg cytoplasm, to interact properly with the nuclear material and to duplicate. Heterologous centrosomes
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• development ~, (diploid)
fertilization/ • • •
/ mature ooeyte (M II arrest)
activation
2 I*
•
centrosome~ 3 injection • • •
•
no cleavage
parthenogenetic • haploid blastula • ' 1 development
diploidization • parthenogenetictadpole Fig. 1 Fertilization and normal development,egg activation, and centrosome-inducedparthenogenetic developmentin Xenopus.(1) Mature oocytes are blocked in metaphaseof the secondmeioticdivision.Fertilization restores diploidy and triggers development. (2) Resumption of the cell cycle can be triggered by pricking the oocyte with a glass needle, leading to sequentialembryoniccell cycles, to cytoplasmicreorganizationas shown by the displacementof cortical pigment, but not to egg cleavage.(3) Cleavagecan be restored if a centrosome is injected into the egg cytoplasm at the pricking step. This leads to the parthenogeneticdevelopment of haploid embryos until the gastrula stage. Haploid embryos display atypical gastrulation and developmental a~rest, leading to embryonic death. Diploidization may occur in an erratic way during the first mitotic cell cycle, leading, in few cases, to parthenogenetic tadpoles and frogs.
from somatic cells apparently meet these criteria, as they are able to provoke egg cleavage and to induce a complete development.
B. Materials
1. Mature unfertilized eggs are obtained from Xenopus laevis females after hormonal challenge. Five to 10 days before laying, females are injected with 500 IU pregnant mare serum gonadotropin to induce oocyte maturation. The day before, females are then injected with 100 IU human chorionic gonadotropin and are maintained overnight at 16-18°C in M M R (5 m M HEPES, pH 7.4, 0.1 M NaC1, 2 m M KCI, 1 m M MgSO4, 2 m M CaC12, 0.1 m M EDTA) buffer to prevent egg activation (Newport and Kirschner, 1984). 2. Isolation of centrosomes from animal cells is carried out as described in Bornens and Moudjou (1999).
C. P r o c e d u r e 1. Fresh layered eggs are dejellied with 2% cysteine (pH 7.8) and washed three times in MMR. Eggs are incubated in M M R containing 5% Ficoll 400 (Sigma) and injected using a nanoinject system (Drummond).
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2. Mature unfertilized eggs are maintained with fine tweezers, and centrosomes are injected near the animal pole. 3. The number ofcentrosomesis scored afterimmunofluorescence analysis of sucrose gradient fractions (Bornens and Moudjou, 1999). 4. The purified centrosomes are injected in a volume of 50 nl after dilution in K-PIPES buffer (10 mM PIPES, pH 7.2 with KOH). In a typical experiment, three dilutions are tested on 50 eggs per dilution: <1, 1, and 10 centrosomes are injected per egg. Theoretically, optimal results are obtained for 1 centrosome injected per egg. Blastulas are scored 3 and 5 h after injection. For further development, blastulas are immersed in 0.25% MMR and further in water.
D. Comments Fresh oocytes can be obtained by squeezing the frog female. This yields eggs of higher quality than those laid overnight. Centrosomes isolated from somatic cells are able to mimic the effect of the sperm centrosome. This activity is not restricted to somatic centrosomes from Xenopus (see later). Blastula development can be obtained with mammalian (human, rat, mouse, bovine) centrosomes [mean value of 62% of parthenogenetic blastulas in 24 independent experiments (seeTournier et al., 1994)]. In contrast, gastrulation and further development are only observed in a few cases (10% of tadpoles out of 80 injected oocytes in 6 independent experiments). This is due to the fact that cellularization upon injection of a centrosome leads to haploid embryos in which gastrulation fails (Fig. 1). Late parthenogenetic development may be possible due to diploidization either by the nonejection of the second polar body or by a segmentation defect during the first mitotic cycle following centrosome injection (Tournier et al., 1994). For two tadpoles and one frog, the karyotype was checked and shown to be diploid. We did not attempt, however, to do a systematic karyotypic analysis of the animals. Three frogs have been living for more than 6 years. Their size was dramatically smaller than the expected size of normally fertilized frogs of the same age. "
III. C o u p l i n g b e t w e e n Egg C e l l C y c l e and C e n t r o s o m e Duplication Pathway When injected into the frog egg, a somatic centrosome is subject to the egg cytoplasmic oscillator, which triggers the succession of S and M phases. Like the sperm centrosome, it has to organize an aster and migrate rapidly to the egg center. After duplication, it organizes the first mitotic spindle, which eventually leads to bipartition of the egg into two equal blastomeres. All these events are likely to be tightly controlled in time and space. The parthenogenetic assay raises two basic questions: (1) How long after egg activation can a centrosome be injected and trigger egg cleavage and (2) does the
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injected centrosome need to be in a particular stage of its own duplication process to be active as a parthenogenetic agent?
A. Temporal Coupling between Egg Activation and Centrosome Injection The parthenogenetic test most often leads to complete blastulas. Nevertheless, approximately 25% of blastulas are incomplete, i.e., a large volume of the egg is uncleaved (Fig. 2). One possibility could be that the injected centrosome does not reach the egg center. Experimental support for this interpretation can be demonstrated by uncoupling egg activation and centrosome injection.
1. Procedure 1. Mature eggs are dejellied, rinsed in MMR, set onto an agarose gel (1%) in an electric activation chamber, and activated by an electric shock (2 s, 12 V). 2. The G1 centrosomes suspension is diluted in K-PIPES (10 mM, pH 7.2) and is injected between 0 and 100 min following oocyte activation. 3. Blastulas are scored 3 h after injection, each of them being examined for total or partial segmentation.
2. C o m m e n t s When a centrosome was injected 10 to 25 min after egg activation, less than 30% of the cleaving eggs produced total blastulas (Fig, 2). After 25 min, cleaving eggs produced exclusively incomplete blastulas. The incorrect migration of the aster formed around the heterologous centrosome could lead to the formation of two unequal blastomeres and further incomplete blastulas (Fig. 2). Moreover, during the first half of the first cell cycle (0-50 min), the number of blastulas decreased linearly with the time elapsed between egg activation and centrosome injection (Fig. 2). These experiments highlight the role of the centrosome as a morphogenetic determinant of the cell division process.
B. Activity o f Centrosomes from Synchronized or Quiescent Cells One can address the question of whether the heterologous centrosome has to be in a particular stage in its own duplication cycle to induce egg cleavage. In other words, is the egg cytoplasmic environment able to initiate the duplication cycle of unduplicated centrosomes? To address this question, the parthenogenetic activity of (1) centrosomes isolated from cells synchonized in G1 and G2 and (2) centrosomes from GO cells was tested.
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a
b 70
"" ~
~
~
60 50 40 30 20 10
0
10
20
|
i
30 40 50 TIME (min)
i
60
i
70
Activation/Injection
Activation
Injection
Fig. 2 The centrosome is required at the time of egg activation to obtain complete blastulas (Tournier et al., 1994). (a) Frog oocytes were activated by an electric shock, and an average of one centrosome per oocyte was injected at different times following egg activation. The capacity to induce cleavage decreased progressively with the time elapsed between egg activation and centrosome injection (white bars). When a centrosome is injected at the time of egg activation (time 0), the majority of blastulas are complete (71%, black bar). When a centrosome is injected l0 to 20 min after egg activation, less than 30% of cleaving eggs produced total blastulas. After 20 min, cleaving eggs produced exclusively incomplete blastulas. (b) A four-cell stage showing four equal blastomeres (arrows) leading to total blastulas. Unequal cleavages (arrowheads) lead to incomplete blastulas in which a large area remains uncleaved. (c) Hypothetical scheme for incomplete blastula formation. When the centrosome is injected at the time of egg activation, it forms a microtubule aster, migrates to the center of the animal hemisphere, and duplicates, leading to two equal blastomeres. When the centrosome is injected after egg activation, it forms a microtubule aster and duplicates but does not migrate to the egg center, leading to two unequal blastomeres. Further asymmetrical divisions would form partial blastulas.
1. P r o c e d u r e a. C e l l s f r o m the K E 3 7 cell line o f T l y m p h o b l a s t i c o r i g i n are s y n c h r o n i z e d in G 1 / S or G 2 / M w i t h t h y m i d i n e or e p i p o d o p h y l o t o x i n , r e s p e c t i v e l y ( T o u r n i e r e t aL, 1989). b. Cells are f u r t h e r e l u t r i a t e d u s i n g a c e n t r i f u g a l e l u t r i a t i o n r o t o r ( B e c k m a n ) . T h i s d o u b l e s y n c h r o n i z a t i o n p r o c e d u r e allows o n e to o b t a i n e n r i c h e d ( > 8 5 % ) G l a n d G 2 cell p o p u l a t i o n s . c. L y m p h o c y t e s are purified o n F i c o l l - P a q u e f r o m h u m a n b l o o d , w h e r e m o r e t h a n 9 0 % o f the cells are in G 0 / G 1 phase.
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d. Centrosome isolation is carried out as described in Tournier et al. (1991b) and Bornens and Moudjou (1999). 2. General C o m m e n t s Centrosomes isolated from human lymphoblastic cells synchronized in G1 or G2 and centrosomes from peripheral human lymphocytes that are arrested in GO were shown to possess similar activities. The capacity of heterologous centrosomes to induce egg cleavage is independent of the stage in the cell cycle that the cells were in at the time of centrosome isolation. Therefore, true initiation of centrosome duplication can be triggered from the components stored in the egg cytoplasm during oogenesis (Tournier et al., 1989). Conversely, the most efficient way to trigger parthenogenetic development is when oocyte activation is provoked by centrosome injection. The success rate of blastula formation declines linearly with the time elapsing between oocyte activation and centrosome injection, in parallel with the increased percentage of incomplete blastulas, which do not lead to further development. These results are compatible with the idea that among divergent species, the centrosome is functionally conserved and acts as a structural template in the initiation of centrosome assembly.
IV. Species Barrier Specificity All together, it has been demonstrated convincingly that centrosomes from inverteb r a t e s - s e a urchin (Mailer et al., 1976) and clam (Spisula; R. E. Palazzo and M. Bornens, unpublished observations)--and from various vertebrates (Toumier et al., 1991b) were competent in the parthenogenetic test. Centrosome activity as a parthenogenetic agent is associated with an insoluble proteinacious structure that is not significantly simpler than the native centrosome (Klotz et al., 1990). Disruption of the centriolar triplets of microtubules was correlated with the loss of parthenogenetic activity. Thus, one may assume that centriole-containing centrosomes are necessary and sufficient for parthenogenesis. In agreement with this view, centrosomes devoid of centrioles, such as yeast spindle pole bodies (SPBs), do not induce egg cleavage. However, centrosomes possessing short centrioles with the typical ninefold radial symmetry, such as Drosophila centrosomes, are also inactive.
A. Procedure SPBs prepared according to Rout and Kilmartin (1990) were kindly provided by J. Kilmartin. 1. The content of Drosophila syncitial embryos is aspirated using a fine glass needle and is deposited in K-PIPES buffer.
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Fr6d6ric Tournier and Michel Bornens 2. The number of centrosomes is estimated from the number of embryos. A mean number of 100 embryos (nuclear cycles 11-13) is used for each experiment. 3. In parallel, part of the collected Drosophila cytoplasm is tested in a microtubule nucleation assay. The number of Drosophila centrosomes is confirmed by counting the number of nuclei in this assay. Drosophila centrosomes were shown to nucleate microtubule asters in Xenopus egg cytoplasmic extracts (Tournier et al., 1999). 4. Centrosome-nucleus complexes are prepared from the asynchronous Kc23 cell line. Cells are rinsed in TMN buffer (10 mM Tris, pH 7.4, 3 mM MgC12, 10 mM NaC1,300 mM sucrose) and resuspended with 1% Nonidet P-40 in TMN buffer. 5. Nuclei are pelleted, washed in TMN buffer, and counted on a microscope using 4',6-diamidino-2-phenylindole staining and phase contrast. Both frozen nuclei (in 30 mM sucrose) and unfrozen nuclei have been tested for microtubule nucleation and parthenogenesis.
B. Comments
Centrosomes devoid of centrioles, such as SPBs isolated from budding yeast, do not form microtubule asters in egg extracts and are inactive in the parthenogenetic test (Tournier et al., 1999). Drosophila centrosomes from both syncitial embryos and cultured cells nucleate microtubule asters in egg extracts (Toumier et aL, 1999). Nevertheless, both isolates are incompetent in the parthenogenetic assay. Drosophila centrosomes possess centrioles with the typical ninefold symmetry organization, but these are shorter in length compared to vertebrate ones. Thus the centriole structure per se is not sufficient to provoke centriole assembly in the Xenopus egg cytoplasm. This inability to trigger parthenogenesis may be due to a pattern of widely divergent structural or regulatory features of the centrosome in both species, i.e., specific molecular regulation of centriole duplication could be controlled differently in the two cases. Alternatively, due to the short length of their centrioles, Drosophila centrosomes could be unable to template the docking of Xenopus components necessary for the nucleation or growth of new centrioles. In conclusion, the parthenogenetic activity of a centrosome is not linked only to its capacity to nucleate microtubules from the egg tubulin. The evolutionary conserved ninefold symetrical structure of the centriole cannot be considered as sufficient for triggering procentriole assembly.
V. A c t i v e o r I n a c t i v e C e n t r o s o m e s
from the Same Species
Centrosome-induced parthenogenesis does not apparently require species specificity, at least in vertebrates. Thus, it was very intriguing to discover that centrosomes from calf thymus, displaying linearly arranged centrioles (Komesli etal., 1989), were incompetent in the parthenogenetic assay.
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A. Procedure
The thymus is the most favorable tissue used to isolate centrosomes, providing a very large number of thymocytes that possess a low cytoplasm-to-nucleus ratio (Komesli et al., 1989; Tournier et al., 1991b; Lange and Gull, 1996). The procedure to obtain large amounts of calf thymus centrosomes was adapted from Bornens et al. (1987) and described in Komesli et al. (1989).
B. C o m m e n t s
The ultrastructure of thymus centrioles is apparently similar to that of other centrosomes. Nevertheless, calf thymus centrosomes display a collinear orientation of their two centrioles both in situ and after isolation (Tournier etal., 1991b). The two centrioles are linearly associated through a mass of electron-dense material that could correspond to a folded form of pericentriolar matrix observed in other cell types. These centrosomes are unable to induce a parthenogenetic development. We tested different hypotheses that could explain the inactivity of calf thymus centrosomes. Neither the species nor the thymic origin could explain their inactivity: thymus centrosomes from other species (human, mouse, rat) are competent (Tournier et al., 1991b). However, these centrosomes do not display the collinear organization of centrioles, suggesting that the main difference is structural. In vitro assays suggest that these centrosomes are unable to initiate centriole budding under conditions in which other centrosomes that are competent to induce egg cleavage are able to do so (Tournier et al., 1991a). We do not know if this peculiar structure represents an irreversibly locked configuration due to the physiological development of thymus or if it is a reversible process along the centrosome duplication cycle. Preliminary experiments suggest that calf thymus centrosomes preincubated in egg cytoplasmic extracts can induce egg cleavage (E Tournier and M. Bornens, unpublished results), suggesting that the centrosomal structure could be modified in the cytoplasmic egg environment.
VI. Prospects A . In Vitro Assays
The parthenogenetic assay represents a first approach to decipher the mechanism of centrosome duplication, as a heterologous centrosome must duplicate to promote egg cleavage. However, the duplication of the injected centrosome cannot be observed directly, as the amphibian egg is very large and is not transparent to visible light. A first alternative approach is the use of animal caps or fixed embryos and confocal microscopic analysis (Gard, 1991; Paoletti et al., 1996). The use of in vitro systems is, however, required to identify the factors controlling the initiation of centriole budding. An assay has been developed using high-speed Xenopus cytoplasmic extracts (Tournier et al., 1991a). Centrosomes isolated from the human KE37 cell line synchronized in G1 initiated procentriole budding in interphase extracts in the absence of protein
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Frad6ric Tournier and Michel Bornens synthesis, whereas calf thymus centrosomes, incompetent in the parthenogenetic test, did not. Procentriole budding was observed and quantified using both immunofluorescence and electronic microscopy techniques. However, this assay remained difficult to develop as the components necessary for assembling full-length centrioles and, afortiori, for obtaining several rounds of centriole duplication, were likely limiting in comparison to the number of centrosomes incubated in the extract. Another strategy, bypassing the technical limitations highlighted in this previous assay, in particular using freshly prepared and stably attached centrosomes and living fluorescent tags, is a promising approach to further analyze the different steps occurring during centrosome duplication (see Chapter 19).
B. Centriole A s s e m b l y Pathways
The centrosome duplication cycle involves the semiconservative duplication of the two centrioles present in G1 phase, each daughter cell inheriting a preexisting and a newly assembled centriole (for a review, see Paoletti and Bornens, 1997). The orthogonal budding of new centrioles in close proximity of parent centrioles suggests that some structure is associated to the proximal wall of each centriole. Other situations requiring an apparent structural discontinuity have been described in embryonic or somatic cells. During terminal differentiation of ciliated epithelial cells, the formation of 100-200 cilia requires the assembly of the same number of centriole/basal bodies, according to a pathway distinct from classical duplication (for a review, see Dirksen, 1991). Numerous centrioles are assembled around dense cytoplasmic granules near the Golgi area. The two pathways probably involve some common mechanisms, as the majority of anticentrosome antibodies recognize fully mature centriole/basal bodies (Muresan et al., 1993; Tassin et aL, 1998; Laoukili et al., 2000; E Tournier, unpublished results). This suggests that common centriolar and pericentriolar proteins may be regulated differentially to promote two cellular functions of the centrioles. Moreover, centrin proteins have been shown to be associated within the precursor structures of centriole/basal bodies, suggesting their implication in the early process of centriole assembly (Laoukili et al., 2000). The development of epithelial cell cultures where the process of mucociliary differentiation can be obtained and quantified in parallel with the development of in vitro assays for centrosome duplication should contribute to the identification of molecular mechanisms involved in the centriole assembly process.
References
Bailly,E., Dorre, M., Nurse,E, and Bornens,M. (1989). p34cdc2is locatedin boththe nucleus and cytoplasm; part is centrosomallyassociatedat G2/M and enters vesiclesat anaphase.EMBO J. 8, 3985-3995. Bobinnec, Y., Khodjakov,A., Mir, L. M., Rieder, C. L., Edde, B., and Bornens, M. (1998a). Centriole disassemblyin vivo and its effecton centrosomestructure and function in vertebratecells. J. Cell Biol. 14, 1575-1589. Bobinnec, Y., Moudjou,M., Fouquet,J. E, Desbruy~res,E., Eddr, B., and Bornens,M. (1998b).Glutamylation of centriole and cytoplasmictubulin in proliferatingnon-neuronalcells. Cell Motil. Cytoskel. 39, 223-232.
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Bomens, M., and Moudjou, M. (1999). Studying the composition and function of centrosomes in vertebrates. Methods Cell Biol. 61, 13-34. Bornens, M., Paintrand, M., Berges, J., Marty, M. C., and Karsenti, E. (1987). Structural and chemical characterization of isolated centrosomes. Cell Motil. Cytoskel. 8, 238-249. Dirksen, E. R. (1991). Centriole and basal body formation during ciliogenesis revisited. Biol. Cell 72, 31-38. Gard, D. L. (1991). Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: A study by confocal immunofluorescence microscopy. Dev. Biol. 143, 346-362. Hara, K., Tydeman, P., and Kirschner, M. (1980). A cytoplasmic clock with the same period as the division cycle in Xenopus eggs. Proc. Natl. Acad. Sci. USA 77, 462-466. Hinchcliffe, E. H., Li, C., Thompson, E. A., Mailer, J. L., and Sluder, G. (1999). Requirement of cdk2-cyclin activity for repeated centrosome reproduction in Xenopus egg extracts. Nature 283, 851-854. Kalt, A., and Schliwa, M. (1993). Molecular components of the centrosome. Trends Cell Biol. 3, 118-128. Kimble, M., and Kuriyama, R. (1992). Functional components of microtbule organizing center. Int. Rev. Cytol. 136, 1-50. Klotz, C., Dabauvale, M. C., Paintrand, M., Weber, V., Bomens, M., and Karsenti, E. (1990). Parthenogenesis in Xenopus eggs requires centrosomal integrity. J. Cell Biol. 110, 405-415. Komesli, S., Tournier, F., Paintrand, M., Margolis, R. L., Job, D., and Bornens, M. (1989). Mass isolation of calf thymus centrosomes: The identification of a specific configuration. J. Cell Biol. 109, 2869-2878. Lange, B. M. H., and Gull, K. (1996). A structural study of isolated mammalian centrioles using negative staining electron microscopy. J. Struct. Biol. 117, 222-226. Laoukili, J., Perret, E., Middendorp, S., Houcine, O., Guennou, C., Marano, F., Bornens, M., and Toumier, F. (2000). Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro. J. Cell Sci. 113, 1355-1364. Mailer, J., Poccia, D., Nishioka, D., Kidd, P., Gerhart, J., and Hartman, H. (1976). Spindle formation and cleavage in Xenopus eggs injected with centriole-containing fractions from sperm. Exp. Cell Res. 99, 285294. Meraldi, P., Lukas, J., Fry, A. M., Bartek, J., and Nigg, E. (1999). Centrosome duplication in mammaJian somatic cells requires E2F and Cdk2-cyclin A. Nature Cell Biol. 1, 88-93. Muresan, V., Joshi, H. C., and Besharse, J. C. (1993). Gamma-tubulin in differentiated cell types: Localization in the vicinity of basal bodies in retinal photoreceptors and ciliated epithelia. J. Cell Sci. 104, 1229-1237. Newport, J. W., and Kirschner, M. W. (1984). Regulation of the cell cycle during early Xenopus development. Cell 37, 731-742. Paoletti, A., and Bornens, M. (1997). Organisation and functional regulation of the centrosome in animal cells. Prog. Cell Cycle Res. 3, 285-299. Paoletti, A., Moudjou, M., Paintrand, M., Samlisbury, J. L., and Bomens, M. (1996). Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles. J. Cell Sci. 109, 3089-3102. Piel, M., Meyer, P., Khodjakov, A., Rieder, C. L., and Bornens, M. (2000). The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells. J. Cell Biol. 149, 317-330. Rout, M. P., and Kilmartin, J. V. (1990). Components of the yeast spindle and spindle pole body. J. Cell Biol. 11, 1913-1927. Tassin, A. M., and Bornens, M. (1999). Centrosome structure and microtubule nucleation in animal cells. Biol. Cell 91,343-354. Tassin, A. M., Celati, C., Moudjou, M., and Bornens, M. (1998). Characterization of the human homologue of the yeast spc98p and its association with gamma-tubulin. J. Cell Biol. 141, 689-701. Tournier, F., Bobinnec, Y., Debec, A., Santamaria, P., and Bornens, M. (1999). Drosophila centrosomes are unable to trigger parthenogenetic development of Xenopus eggs. Biol. Cell 91, 99-108. Tournier, F., and Boruens, M. (1994). Cell cycle regulation of centrosome function. In "Microtubules" (J. Hyams, ed.), pp. 303-324. Wiley-Liss, New York. Toumier, F., Cyrklaff, M., Karsenti, E., and Bomens, M. (1991a). Centrosome competent for parthenogenesis in Xenopus eggs support budding in cell-free extracts. Proc. Natl. Acad. Sci. USA 88, 9929-9933.
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Tournier, E, Karsenti, E., and Bornens, M. (1989). Parthenogenesis in Xenopus eggs injected with centrosomes from synchronized human lymphoid cells. Dev. Biol. 136, 321-329. Tournier, E, Komesli, S., Paintrand, M., Job, D., and Bomens, M. (1991b). The intercentriolar linkage is critical for the ability of heterologous centrosomes to induce parthenogenesis in Xenopus. J. Cell Biol. 113, 1361-1369. Tournier, F., Joly, A., and Bornens, M. (1994). Production of partial blastulas by parthenogenesis in Xenopus. C. R. Acad. Sci. 11I 317, 405-410.
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Centrosomes and Parthenogenesis Fr6d6ric Tournier* and Michel B o r n e n s t *Laboratoire de Cytophysiologieet ToxicologieCellulaire Universit6Paris 7 75251 Paris Cedex 05, France ?InstitutCurie, Sectionde Recherche 75248 Paris Cedex 05, France
I. Introduction II. Parthenogenetic Test in Xenopus A. Parthenogenesis versus Fertilization B. Materials C. Procedure D. Comments III. Couphng between Egg Cell Cycle and Centrosome Duplication Pathway A. Temporal Coupling between Egg Activation and Centrosome Injection B. Activity of Centrosomes from Synchronized or Quiescent Cells IV. Species Barrier Specificity A. Procedure B. Comments V. Active or Inactive Centrosomes from the Same Species A. Procedure B. Comments VI. Prospects A. In Vitro Assays B. Centriole Assembly Pathways References
I. I n t r o d u c t i o n In animal cells, two centrioles act as a core structure on which a fibrogranular network assembles to form the centrosome (Bobinnec et al., 1998a,b; Tassin and Bornens, 1999). The centrosome is not only capable of nucleating microtubules in interphase and mitotic M E T H O D S IN CELL BIOLOGY, VOL. 67 Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 0091-679X/01 $35.00
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Fr6d6ric Tournier and Michel Bornens animal cells, but it is also likely to be able to control the release of microtubules (Piel et al., 2000). The bipolarity of the cell division process rests on the reproductive capacity of this organelle. Its duplication cycle occurs throughout the cell cycle and several distinct steps have been described in somatic cells (for a review, see Tournier and Bornens, 1994). A G1 cell inherits a pair of dissociated centrioles from previous mitosis and initiation of orthogonal procentriole budding precedes S-phase entry. After procentriole elongation during S and G2 phases, separation of the duplicated centrosomes is coincident with the onset of the next mitosis. Few centrosomal components, either having a structural role within the organelle or participating in the regulation of its activity, have been described during cell cycle progression (for reviews, see Kimble and Kuryama, 1992; Kalt and Schliwa, 1993). Moreover, several lines of evidence suggest a coupling between centrosome duplication and the progression of the cell division cycle (Bailly etal., 1989; Hinchcliffe et al., 1999; Meraldi et al., 1999). Isolation procedures have provided an efficient way for biochemical, structural, and functional characterization of the centrosome. The parthenogenetic assay in Xenopus eggs is an efficient system to study centrosome duplication. Mature frog eggs are unable to assemble a centrosome from its elements in the absence of a preexisting centrosome. Centrosome assembly from egg precursor components can be triggered by a centrosome from heterologous species, emphasizing the conservation of the centrosome duplication mechanisms among divergent species. This chapter describes the parthenogenetic assay. It also reviews the main established or still enigmatic results concerning the capacity of centrosomes isolated from different cell types in different species to induce frog egg cleavage. These results and the limitation of this approach to study centrosome duplication are discussed, then it briefly discusses the potential of current in vitro systems to identify important molecular events of the pathway and also indicates how mammalian ciliated epithelial cells can be used in parallel to characterize specific or common centrosomal components involved in centriolar or acentriolar pathways.
II. P a r t h e n o g e n e t i c
Test in
Xenopus
A. Parthenogenesis versus Fertilization
At fertilization, the sperm cell triggers the exit of egg meiosis and the resumption of cell cycle progression. The resumption of the egg cell cycle can be triggered by pricking the egg with a needle, leading to cytoplasmic reorganization, as shown by the displacement of cortical pigment, but no cleavage occurs in the absence of the sperm centrosome (Hara et al., 1980; Fig. 1). Once into the egg cytoplasm, the sperm centrosome forms a microtubule aster, probably due to the activation of key centrosomal proteins, and further to the recruitment of maternal proteins. The sperm aster migrates toward the center of the animal hemisphere and duplicates to form the first mitotic spindle. In order to induce a parthenogenetic development, the injected centrosome has to nucleate microtubules from the egg cytoplasm, to interact properly with the nuclear material and to duplicate. Heterologous centrosomes
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• development ~, (diploid)
fertilization/ • • •
/ mature ooeyte (M II arrest)
activation
2 I*
•
centrosome~ 3 injection • • •
•
no cleavage
parthenogenetic • haploid blastula • ' 1 development
diploidization • parthenogenetictadpole Fig. 1 Fertilization and normal development,egg activation, and centrosome-inducedparthenogenetic developmentin Xenopus.(1) Mature oocytes are blocked in metaphaseof the secondmeioticdivision.Fertilization restores diploidy and triggers development. (2) Resumption of the cell cycle can be triggered by pricking the oocyte with a glass needle, leading to sequentialembryoniccell cycles, to cytoplasmicreorganizationas shown by the displacementof cortical pigment, but not to egg cleavage.(3) Cleavagecan be restored if a centrosome is injected into the egg cytoplasm at the pricking step. This leads to the parthenogeneticdevelopment of haploid embryos until the gastrula stage. Haploid embryos display atypical gastrulation and developmental a~rest, leading to embryonic death. Diploidization may occur in an erratic way during the first mitotic cell cycle, leading, in few cases, to parthenogenetic tadpoles and frogs.
from somatic cells apparently meet these criteria, as they are able to provoke egg cleavage and to induce a complete development.
B. Materials
1. Mature unfertilized eggs are obtained from Xenopus laevis females after hormonal challenge. Five to 10 days before laying, females are injected with 500 IU pregnant mare serum gonadotropin to induce oocyte maturation. The day before, females are then injected with 100 IU human chorionic gonadotropin and are maintained overnight at 16-18°C in M M R (5 m M HEPES, pH 7.4, 0.1 M NaC1, 2 m M KCI, 1 m M MgSO4, 2 m M CaC12, 0.1 m M EDTA) buffer to prevent egg activation (Newport and Kirschner, 1984). 2. Isolation of centrosomes from animal cells is carried out as described in Bornens and Moudjou (1999).
C. P r o c e d u r e 1. Fresh layered eggs are dejellied with 2% cysteine (pH 7.8) and washed three times in MMR. Eggs are incubated in M M R containing 5% Ficoll 400 (Sigma) and injected using a nanoinject system (Drummond).
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2. Mature unfertilized eggs are maintained with fine tweezers, and centrosomes are injected near the animal pole. 3. The number ofcentrosomesis scored afterimmunofluorescence analysis of sucrose gradient fractions (Bornens and Moudjou, 1999). 4. The purified centrosomes are injected in a volume of 50 nl after dilution in K-PIPES buffer (10 mM PIPES, pH 7.2 with KOH). In a typical experiment, three dilutions are tested on 50 eggs per dilution: <1, 1, and 10 centrosomes are injected per egg. Theoretically, optimal results are obtained for 1 centrosome injected per egg. Blastulas are scored 3 and 5 h after injection. For further development, blastulas are immersed in 0.25% MMR and further in water.
D. Comments Fresh oocytes can be obtained by squeezing the frog female. This yields eggs of higher quality than those laid overnight. Centrosomes isolated from somatic cells are able to mimic the effect of the sperm centrosome. This activity is not restricted to somatic centrosomes from Xenopus (see later). Blastula development can be obtained with mammalian (human, rat, mouse, bovine) centrosomes [mean value of 62% of parthenogenetic blastulas in 24 independent experiments (seeTournier et al., 1994)]. In contrast, gastrulation and further development are only observed in a few cases (10% of tadpoles out of 80 injected oocytes in 6 independent experiments). This is due to the fact that cellularization upon injection of a centrosome leads to haploid embryos in which gastrulation fails (Fig. 1). Late parthenogenetic development may be possible due to diploidization either by the nonejection of the second polar body or by a segmentation defect during the first mitotic cycle following centrosome injection (Tournier et al., 1994). For two tadpoles and one frog, the karyotype was checked and shown to be diploid. We did not attempt, however, to do a systematic karyotypic analysis of the animals. Three frogs have been living for more than 6 years. Their size was dramatically smaller than the expected size of normally fertilized frogs of the same age. "
III. C o u p l i n g b e t w e e n Egg C e l l C y c l e and C e n t r o s o m e Duplication Pathway When injected into the frog egg, a somatic centrosome is subject to the egg cytoplasmic oscillator, which triggers the succession of S and M phases. Like the sperm centrosome, it has to organize an aster and migrate rapidly to the egg center. After duplication, it organizes the first mitotic spindle, which eventually leads to bipartition of the egg into two equal blastomeres. All these events are likely to be tightly controlled in time and space. The parthenogenetic assay raises two basic questions: (1) How long after egg activation can a centrosome be injected and trigger egg cleavage and (2) does the
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injected centrosome need to be in a particular stage of its own duplication process to be active as a parthenogenetic agent?
A. Temporal Coupling between Egg Activation and Centrosome Injection The parthenogenetic test most often leads to complete blastulas. Nevertheless, approximately 25% of blastulas are incomplete, i.e., a large volume of the egg is uncleaved (Fig. 2). One possibility could be that the injected centrosome does not reach the egg center. Experimental support for this interpretation can be demonstrated by uncoupling egg activation and centrosome injection.
1. Procedure 1. Mature eggs are dejellied, rinsed in MMR, set onto an agarose gel (1%) in an electric activation chamber, and activated by an electric shock (2 s, 12 V). 2. The G1 centrosomes suspension is diluted in K-PIPES (10 mM, pH 7.2) and is injected between 0 and 100 min following oocyte activation. 3. Blastulas are scored 3 h after injection, each of them being examined for total or partial segmentation.
2. C o m m e n t s When a centrosome was injected 10 to 25 min after egg activation, less than 30% of the cleaving eggs produced total blastulas (Fig, 2). After 25 min, cleaving eggs produced exclusively incomplete blastulas. The incorrect migration of the aster formed around the heterologous centrosome could lead to the formation of two unequal blastomeres and further incomplete blastulas (Fig. 2). Moreover, during the first half of the first cell cycle (0-50 min), the number of blastulas decreased linearly with the time elapsed between egg activation and centrosome injection (Fig. 2). These experiments highlight the role of the centrosome as a morphogenetic determinant of the cell division process.
B. Activity o f Centrosomes from Synchronized or Quiescent Cells One can address the question of whether the heterologous centrosome has to be in a particular stage in its own duplication cycle to induce egg cleavage. In other words, is the egg cytoplasmic environment able to initiate the duplication cycle of unduplicated centrosomes? To address this question, the parthenogenetic activity of (1) centrosomes isolated from cells synchonized in G1 and G2 and (2) centrosomes from GO cells was tested.
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Frederic Tournier and Michel Bornens
a
b 70
"" ~
~
~
60 50 40 30 20 10
0
10
20
|
i
30 40 50 TIME (min)
i
60
i
70
Activation/Injection
Activation
Injection
Fig. 2 The centrosome is required at the time of egg activation to obtain complete blastulas (Tournier et al., 1994). (a) Frog oocytes were activated by an electric shock, and an average of one centrosome per oocyte was injected at different times following egg activation. The capacity to induce cleavage decreased progressively with the time elapsed between egg activation and centrosome injection (white bars). When a centrosome is injected at the time of egg activation (time 0), the majority of blastulas are complete (71%, black bar). When a centrosome is injected l0 to 20 min after egg activation, less than 30% of cleaving eggs produced total blastulas. After 20 min, cleaving eggs produced exclusively incomplete blastulas. (b) A four-cell stage showing four equal blastomeres (arrows) leading to total blastulas. Unequal cleavages (arrowheads) lead to incomplete blastulas in which a large area remains uncleaved. (c) Hypothetical scheme for incomplete blastula formation. When the centrosome is injected at the time of egg activation, it forms a microtubule aster, migrates to the center of the animal hemisphere, and duplicates, leading to two equal blastomeres. When the centrosome is injected after egg activation, it forms a microtubule aster and duplicates but does not migrate to the egg center, leading to two unequal blastomeres. Further asymmetrical divisions would form partial blastulas.
1. P r o c e d u r e a. C e l l s f r o m the K E 3 7 cell line o f T l y m p h o b l a s t i c o r i g i n are s y n c h r o n i z e d in G 1 / S or G 2 / M w i t h t h y m i d i n e or e p i p o d o p h y l o t o x i n , r e s p e c t i v e l y ( T o u r n i e r e t aL, 1989). b. Cells are f u r t h e r e l u t r i a t e d u s i n g a c e n t r i f u g a l e l u t r i a t i o n r o t o r ( B e c k m a n ) . T h i s d o u b l e s y n c h r o n i z a t i o n p r o c e d u r e allows o n e to o b t a i n e n r i c h e d ( > 8 5 % ) G l a n d G 2 cell p o p u l a t i o n s . c. L y m p h o c y t e s are purified o n F i c o l l - P a q u e f r o m h u m a n b l o o d , w h e r e m o r e t h a n 9 0 % o f the cells are in G 0 / G 1 phase.
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d. Centrosome isolation is carried out as described in Tournier et al. (1991b) and Bornens and Moudjou (1999). 2. General C o m m e n t s Centrosomes isolated from human lymphoblastic cells synchronized in G1 or G2 and centrosomes from peripheral human lymphocytes that are arrested in GO were shown to possess similar activities. The capacity of heterologous centrosomes to induce egg cleavage is independent of the stage in the cell cycle that the cells were in at the time of centrosome isolation. Therefore, true initiation of centrosome duplication can be triggered from the components stored in the egg cytoplasm during oogenesis (Tournier et al., 1989). Conversely, the most efficient way to trigger parthenogenetic development is when oocyte activation is provoked by centrosome injection. The success rate of blastula formation declines linearly with the time elapsing between oocyte activation and centrosome injection, in parallel with the increased percentage of incomplete blastulas, which do not lead to further development. These results are compatible with the idea that among divergent species, the centrosome is functionally conserved and acts as a structural template in the initiation of centrosome assembly.
IV. Species Barrier Specificity All together, it has been demonstrated convincingly that centrosomes from inverteb r a t e s - s e a urchin (Mailer et al., 1976) and clam (Spisula; R. E. Palazzo and M. Bornens, unpublished observations)--and from various vertebrates (Toumier et al., 1991b) were competent in the parthenogenetic test. Centrosome activity as a parthenogenetic agent is associated with an insoluble proteinacious structure that is not significantly simpler than the native centrosome (Klotz et al., 1990). Disruption of the centriolar triplets of microtubules was correlated with the loss of parthenogenetic activity. Thus, one may assume that centriole-containing centrosomes are necessary and sufficient for parthenogenesis. In agreement with this view, centrosomes devoid of centrioles, such as yeast spindle pole bodies (SPBs), do not induce egg cleavage. However, centrosomes possessing short centrioles with the typical ninefold radial symmetry, such as Drosophila centrosomes, are also inactive.
A. Procedure SPBs prepared according to Rout and Kilmartin (1990) were kindly provided by J. Kilmartin. 1. The content of Drosophila syncitial embryos is aspirated using a fine glass needle and is deposited in K-PIPES buffer.
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Fr6d6ric Tournier and Michel Bornens 2. The number of centrosomes is estimated from the number of embryos. A mean number of 100 embryos (nuclear cycles 11-13) is used for each experiment. 3. In parallel, part of the collected Drosophila cytoplasm is tested in a microtubule nucleation assay. The number of Drosophila centrosomes is confirmed by counting the number of nuclei in this assay. Drosophila centrosomes were shown to nucleate microtubule asters in Xenopus egg cytoplasmic extracts (Tournier et al., 1999). 4. Centrosome-nucleus complexes are prepared from the asynchronous Kc23 cell line. Cells are rinsed in TMN buffer (10 mM Tris, pH 7.4, 3 mM MgC12, 10 mM NaC1,300 mM sucrose) and resuspended with 1% Nonidet P-40 in TMN buffer. 5. Nuclei are pelleted, washed in TMN buffer, and counted on a microscope using 4',6-diamidino-2-phenylindole staining and phase contrast. Both frozen nuclei (in 30 mM sucrose) and unfrozen nuclei have been tested for microtubule nucleation and parthenogenesis.
B. Comments
Centrosomes devoid of centrioles, such as SPBs isolated from budding yeast, do not form microtubule asters in egg extracts and are inactive in the parthenogenetic test (Tournier et al., 1999). Drosophila centrosomes from both syncitial embryos and cultured cells nucleate microtubule asters in egg extracts (Toumier et aL, 1999). Nevertheless, both isolates are incompetent in the parthenogenetic assay. Drosophila centrosomes possess centrioles with the typical ninefold symmetry organization, but these are shorter in length compared to vertebrate ones. Thus the centriole structure per se is not sufficient to provoke centriole assembly in the Xenopus egg cytoplasm. This inability to trigger parthenogenesis may be due to a pattern of widely divergent structural or regulatory features of the centrosome in both species, i.e., specific molecular regulation of centriole duplication could be controlled differently in the two cases. Alternatively, due to the short length of their centrioles, Drosophila centrosomes could be unable to template the docking of Xenopus components necessary for the nucleation or growth of new centrioles. In conclusion, the parthenogenetic activity of a centrosome is not linked only to its capacity to nucleate microtubules from the egg tubulin. The evolutionary conserved ninefold symetrical structure of the centriole cannot be considered as sufficient for triggering procentriole assembly.
V. A c t i v e o r I n a c t i v e C e n t r o s o m e s
from the Same Species
Centrosome-induced parthenogenesis does not apparently require species specificity, at least in vertebrates. Thus, it was very intriguing to discover that centrosomes from calf thymus, displaying linearly arranged centrioles (Komesli etal., 1989), were incompetent in the parthenogenetic assay.
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A. Procedure
The thymus is the most favorable tissue used to isolate centrosomes, providing a very large number of thymocytes that possess a low cytoplasm-to-nucleus ratio (Komesli et al., 1989; Tournier et al., 1991b; Lange and Gull, 1996). The procedure to obtain large amounts of calf thymus centrosomes was adapted from Bornens et al. (1987) and described in Komesli et al. (1989).
B. C o m m e n t s
The ultrastructure of thymus centrioles is apparently similar to that of other centrosomes. Nevertheless, calf thymus centrosomes display a collinear orientation of their two centrioles both in situ and after isolation (Tournier etal., 1991b). The two centrioles are linearly associated through a mass of electron-dense material that could correspond to a folded form of pericentriolar matrix observed in other cell types. These centrosomes are unable to induce a parthenogenetic development. We tested different hypotheses that could explain the inactivity of calf thymus centrosomes. Neither the species nor the thymic origin could explain their inactivity: thymus centrosomes from other species (human, mouse, rat) are competent (Tournier et al., 1991b). However, these centrosomes do not display the collinear organization of centrioles, suggesting that the main difference is structural. In vitro assays suggest that these centrosomes are unable to initiate centriole budding under conditions in which other centrosomes that are competent to induce egg cleavage are able to do so (Tournier et al., 1991a). We do not know if this peculiar structure represents an irreversibly locked configuration due to the physiological development of thymus or if it is a reversible process along the centrosome duplication cycle. Preliminary experiments suggest that calf thymus centrosomes preincubated in egg cytoplasmic extracts can induce egg cleavage (E Tournier and M. Bornens, unpublished results), suggesting that the centrosomal structure could be modified in the cytoplasmic egg environment.
VI. Prospects A . In Vitro Assays
The parthenogenetic assay represents a first approach to decipher the mechanism of centrosome duplication, as a heterologous centrosome must duplicate to promote egg cleavage. However, the duplication of the injected centrosome cannot be observed directly, as the amphibian egg is very large and is not transparent to visible light. A first alternative approach is the use of animal caps or fixed embryos and confocal microscopic analysis (Gard, 1991; Paoletti et al., 1996). The use of in vitro systems is, however, required to identify the factors controlling the initiation of centriole budding. An assay has been developed using high-speed Xenopus cytoplasmic extracts (Tournier et al., 1991a). Centrosomes isolated from the human KE37 cell line synchronized in G1 initiated procentriole budding in interphase extracts in the absence of protein
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Frad6ric Tournier and Michel Bornens synthesis, whereas calf thymus centrosomes, incompetent in the parthenogenetic test, did not. Procentriole budding was observed and quantified using both immunofluorescence and electronic microscopy techniques. However, this assay remained difficult to develop as the components necessary for assembling full-length centrioles and, afortiori, for obtaining several rounds of centriole duplication, were likely limiting in comparison to the number of centrosomes incubated in the extract. Another strategy, bypassing the technical limitations highlighted in this previous assay, in particular using freshly prepared and stably attached centrosomes and living fluorescent tags, is a promising approach to further analyze the different steps occurring during centrosome duplication (see Chapter 19).
B. Centriole A s s e m b l y Pathways
The centrosome duplication cycle involves the semiconservative duplication of the two centrioles present in G1 phase, each daughter cell inheriting a preexisting and a newly assembled centriole (for a review, see Paoletti and Bornens, 1997). The orthogonal budding of new centrioles in close proximity of parent centrioles suggests that some structure is associated to the proximal wall of each centriole. Other situations requiring an apparent structural discontinuity have been described in embryonic or somatic cells. During terminal differentiation of ciliated epithelial cells, the formation of 100-200 cilia requires the assembly of the same number of centriole/basal bodies, according to a pathway distinct from classical duplication (for a review, see Dirksen, 1991). Numerous centrioles are assembled around dense cytoplasmic granules near the Golgi area. The two pathways probably involve some common mechanisms, as the majority of anticentrosome antibodies recognize fully mature centriole/basal bodies (Muresan et al., 1993; Tassin et aL, 1998; Laoukili et al., 2000; E Tournier, unpublished results). This suggests that common centriolar and pericentriolar proteins may be regulated differentially to promote two cellular functions of the centrioles. Moreover, centrin proteins have been shown to be associated within the precursor structures of centriole/basal bodies, suggesting their implication in the early process of centriole assembly (Laoukili et al., 2000). The development of epithelial cell cultures where the process of mucociliary differentiation can be obtained and quantified in parallel with the development of in vitro assays for centrosome duplication should contribute to the identification of molecular mechanisms involved in the centriole assembly process.
References
Bailly,E., Dorre, M., Nurse,E, and Bornens,M. (1989). p34cdc2is locatedin boththe nucleus and cytoplasm; part is centrosomallyassociatedat G2/M and enters vesiclesat anaphase.EMBO J. 8, 3985-3995. Bobinnec, Y., Khodjakov,A., Mir, L. M., Rieder, C. L., Edde, B., and Bornens, M. (1998a). Centriole disassemblyin vivo and its effecton centrosomestructure and function in vertebratecells. J. Cell Biol. 14, 1575-1589. Bobinnec, Y., Moudjou,M., Fouquet,J. E, Desbruy~res,E., Eddr, B., and Bornens,M. (1998b).Glutamylation of centriole and cytoplasmictubulin in proliferatingnon-neuronalcells. Cell Motil. Cytoskel. 39, 223-232.
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Bomens, M., and Moudjou, M. (1999). Studying the composition and function of centrosomes in vertebrates. Methods Cell Biol. 61, 13-34. Bornens, M., Paintrand, M., Berges, J., Marty, M. C., and Karsenti, E. (1987). Structural and chemical characterization of isolated centrosomes. Cell Motil. Cytoskel. 8, 238-249. Dirksen, E. R. (1991). Centriole and basal body formation during ciliogenesis revisited. Biol. Cell 72, 31-38. Gard, D. L. (1991). Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: A study by confocal immunofluorescence microscopy. Dev. Biol. 143, 346-362. Hara, K., Tydeman, P., and Kirschner, M. (1980). A cytoplasmic clock with the same period as the division cycle in Xenopus eggs. Proc. Natl. Acad. Sci. USA 77, 462-466. Hinchcliffe, E. H., Li, C., Thompson, E. A., Mailer, J. L., and Sluder, G. (1999). Requirement of cdk2-cyclin activity for repeated centrosome reproduction in Xenopus egg extracts. Nature 283, 851-854. Kalt, A., and Schliwa, M. (1993). Molecular components of the centrosome. Trends Cell Biol. 3, 118-128. Kimble, M., and Kuriyama, R. (1992). Functional components of microtbule organizing center. Int. Rev. Cytol. 136, 1-50. Klotz, C., Dabauvale, M. C., Paintrand, M., Weber, V., Bomens, M., and Karsenti, E. (1990). Parthenogenesis in Xenopus eggs requires centrosomal integrity. J. Cell Biol. 110, 405-415. Komesli, S., Tournier, F., Paintrand, M., Margolis, R. L., Job, D., and Bornens, M. (1989). Mass isolation of calf thymus centrosomes: The identification of a specific configuration. J. Cell Biol. 109, 2869-2878. Lange, B. M. H., and Gull, K. (1996). A structural study of isolated mammalian centrioles using negative staining electron microscopy. J. Struct. Biol. 117, 222-226. Laoukili, J., Perret, E., Middendorp, S., Houcine, O., Guennou, C., Marano, F., Bornens, M., and Toumier, F. (2000). Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro. J. Cell Sci. 113, 1355-1364. Mailer, J., Poccia, D., Nishioka, D., Kidd, P., Gerhart, J., and Hartman, H. (1976). Spindle formation and cleavage in Xenopus eggs injected with centriole-containing fractions from sperm. Exp. Cell Res. 99, 285294. Meraldi, P., Lukas, J., Fry, A. M., Bartek, J., and Nigg, E. (1999). Centrosome duplication in mammaJian somatic cells requires E2F and Cdk2-cyclin A. Nature Cell Biol. 1, 88-93. Muresan, V., Joshi, H. C., and Besharse, J. C. (1993). Gamma-tubulin in differentiated cell types: Localization in the vicinity of basal bodies in retinal photoreceptors and ciliated epithelia. J. Cell Sci. 104, 1229-1237. Newport, J. W., and Kirschner, M. W. (1984). Regulation of the cell cycle during early Xenopus development. Cell 37, 731-742. Paoletti, A., and Bornens, M. (1997). Organisation and functional regulation of the centrosome in animal cells. Prog. Cell Cycle Res. 3, 285-299. Paoletti, A., Moudjou, M., Paintrand, M., Samlisbury, J. L., and Bomens, M. (1996). Most of centrin in animal cells is not centrosome-associated and centrosomal centrin is confined to the distal lumen of centrioles. J. Cell Sci. 109, 3089-3102. Piel, M., Meyer, P., Khodjakov, A., Rieder, C. L., and Bornens, M. (2000). The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells. J. Cell Biol. 149, 317-330. Rout, M. P., and Kilmartin, J. V. (1990). Components of the yeast spindle and spindle pole body. J. Cell Biol. 11, 1913-1927. Tassin, A. M., and Bornens, M. (1999). Centrosome structure and microtubule nucleation in animal cells. Biol. Cell 91,343-354. Tassin, A. M., Celati, C., Moudjou, M., and Bornens, M. (1998). Characterization of the human homologue of the yeast spc98p and its association with gamma-tubulin. J. Cell Biol. 141, 689-701. Tournier, F., Bobinnec, Y., Debec, A., Santamaria, P., and Bornens, M. (1999). Drosophila centrosomes are unable to trigger parthenogenetic development of Xenopus eggs. Biol. Cell 91, 99-108. Tournier, F., and Boruens, M. (1994). Cell cycle regulation of centrosome function. In "Microtubules" (J. Hyams, ed.), pp. 303-324. Wiley-Liss, New York. Toumier, F., Cyrklaff, M., Karsenti, E., and Bomens, M. (1991a). Centrosome competent for parthenogenesis in Xenopus eggs support budding in cell-free extracts. Proc. Natl. Acad. Sci. USA 88, 9929-9933.
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Tournier, E, Karsenti, E., and Bornens, M. (1989). Parthenogenesis in Xenopus eggs injected with centrosomes from synchronized human lymphoid cells. Dev. Biol. 136, 321-329. Tournier, E, Komesli, S., Paintrand, M., Job, D., and Bomens, M. (1991b). The intercentriolar linkage is critical for the ability of heterologous centrosomes to induce parthenogenesis in Xenopus. J. Cell Biol. 113, 1361-1369. Tournier, F., Joly, A., and Bornens, M. (1994). Production of partial blastulas by parthenogenesis in Xenopus. C. R. Acad. Sci. 11I 317, 405-410.