Discussion

Gonc^y: In the normally fertilized egg there is a centrosome. However, you are saying that the spindle is assembled using a non-centrosomal pathway in the one-cell stage mouse embryo. This seems surprising to me in light of Rebecca Heald's work, which shows that the centrosomal pathway of spindle assembly is dominant over the non-centrosomal one (Heald et al 1997).

Kubiak: There is a puzzling phenomenon observed in mammalian eggs. The spermatozoon brings a centriole that is disorganized and does not participate in division spindle formation, at least in the mouse embryo, until the fourth or fifth cleavage division. The first cleavages are acentriolar. Entirely maternal centrosomal material is sufficient for normal formation of the spindle. Parthenogenetic mouse embryos cleave normally and develop perfect centrioles and centrosomes.

Gonc^y: What makes the centrosome mature and competent again?

Kubiak: We can only speculate. In different mammals the centrioles appear at different times. We do not know whether pericentriolar material (PCM) foci duplicate like mature centrosomes. Do they preserve any structure? We don't know, although we find them amorphous at the electron microscope level.

Vande Woude: I thought the mouse was different from other mammals. Many of the others use the sperm aster to bring the pronuclei together.

Nasmyth: I was interested by the long mitotic arrest you showed. Perhaps you should get together with Peter Sorger, who has knocked out Mad2 in mouse (Dobles et al 2000). One of the early phenotypes he saw was a failure to arrest when nocadazole was added.

Kubiak: Indeed, but given that it is a checkpoint which is activated at that time, there must be a reason to activate this checkpoint delay in the first mitosis and not in the second one. Whatever the mechanism is, it is maternally determined and it is specific for this particular mitosis. This is the only mitosis that depends entirely on maternal genes in the mouse embryos, so we must look for a mechanism triggering a checkpoint delay.

"Hunt: I thought that the maternal mRNA was degraded even before the first mitosis. Is that not the case?

Kubiak: No, the majority of maternal mRNA is only degraded before the second mitosis. There are some mRNAs, however, that persist until the blastocyst stage.

Mailer: So there was a transient inactivation of CSF. Have you looked to see if MAPK or Rsk are transiently deactivated?

Kubiak: We didn't see any change in the MAPK activity at that moment. Marcel Doree has looked carefully in Xenopus and concluded that MAPK was still active at the moment of inactivation of MPF.

Maiier: What might change that story are the phospho-specific antibodies for Rsk and MAPK, because they can pick up changes of just a few percent. The CSF on kinetochores may require only a tiny pool of those enzymes for regulation.

Reik: Can you comment on the spatial segregation of chromosomes during the first mitosis that you mention? The Mayer et al (2000) study shows this very clearly. We have also carried out the same kind of immunofluorescence studies and we don't see that. This may need some reassessment with other markers. The methyl C antibody may not be the best thing to use.

Kubiak: Recognition of paternal and maternal chromosomes was a nice explanation for the prolongation of the first mitosis in the mouse, but surprisingly we found the first mitosis is also longer in invertebrates such as sea urchins. Pierre Gonczy told me that the first mitosis might be slightly longer in Caenorhabditis elegans, too. In these cases the imprinting hypothesis didn't really fit. What we would like to do now is to find a suitable model in which the first mitosis is exceptional, and to identify mutants. Why not in C. elegans? Then we would look back to the mouse.

Vande Woude: Certainly it is that way in Xenopus. Monica Murakami has published two couple of papers showing that the MAPK activity, prolonged after the p34 kinase destruction, is key to lengthening the first cycle to 60 min instead of 30 min.

Hunt: I don't think she would claim what Jacek Kubiak is claiming, that the first mitosis is a particularly long one. It is a prolongation of the interphase, not the mitosis.

Kubiak: I don't have any data about the differential regulation of duration of mitoses during development except for the 14th and 15th mitosis in Drosophila, where there is a difference (Foe & Alberts 1983).

Edgar: The first meiosis in Drosophila is also very long.

Nasmjth: The chromosomes are all in a twist. Have you attempted to look at Cdc20? Is this taking a while to accumulate?

Kubiak: No, we didn't look at that.

Mailer: Why do you say that the first spindle poles are made of PCM? Is there some reason to think that PCM is needed without a centrosome? In Xenopus, I have never heard anyone argue that there is PCM organizing the meiotic spindles. Ifyou look at the y-tubulin staining pattern, it is totally different in a meiotic spindle than in a mitotic spindle.

Kubiak: In Xenopus it was recently shown by Peter Kalab that there is also the possibility of forming the bipolar structures without chromosomes (Kalab et al

Maller: I don't argue with that, but why do you say 'PCM'? You are saying that one part of the centrosome is still operating in the first cycle, but not the centriole part, which comes from the sperm.

Kubiak: I don't know what happens to the centriole material that is brought by the sperm. What we detect at the spindle pole is a material that is reactive to y-tubulin or MPM2 antibodies. We call it PCM because the same material accompanies centrioles in other cells. All that we know is that there are plenty of these small spots in mouse oocytes also out of the spindle and that they can be gathered to opposite places with respect to the chromosomes. Moreover, as I mentioned, we have shown recently that this also happens in the absence of chromosomes (Brunet et al 1998).

Vande Woude: You see lots of these forming in meiosis I in the mouse.

Kubiak: It varies from experiment to experiment.

References

Brunet S, Polanski Z, Verlhac M-H, Kubiak JZ, Maro B 1998 Bipolar meiotic spindle formation without chromatin. Curr Biol 8:1231—1234 Dobles M, Liberal V, Scott ML, Benezra R, Sorger PK 2000 Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. Cell 101:635—645 Foe VE, Alberts BM 1983 Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. J Cell Sci 61:31—70 Heald R, Tournebize R, Habermann A, Karsenti E, Hyman A 1997 Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization. J Cell Biol 138:615—628

Kalab P, Pu RT, Dasso M 1999 The ran GTPase regulates mitotic spindle assembly. Curr Biol 9:481—484

Mayer W, Niveleau A, Walter J, Fundele R, Haaf T 2000 Demethylation of the zygotic paternal genome. Nature 403:501—502

Novartis 237: The Cell Cycle and Development. Copyright © 2001 John Wiley & Sons Ltd Print ISBN 0-471-49662-6 elSBN 0-470-8 4 6 6 6-6

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