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Nuclei and microtubule asters stimulate maturation/M phase promoting factor (MPF) activation in Xenopus eggs and egg
D Pérez-Mongiovi1, C Beckhelling, P Chang
1UMR 7009, Centre National de la Recherche Scientifique/Université Paris VI, Station Zoologique, 06230 Villefranche-sur-mer, France.
Abstract:
Although maturation/M phase promoting factor (MPF) can activate autonomously in Xenopus egg cytoplasm, indirect evidence suggests that nuclei and centrosomes may focus activation within the cell. We have dissected the contribution of these structures to MPF activation in fertilized eggs and in egg fragments containing different combinations of nuclei, centrosomes, and microtubules by following the behavior of Cdc2 (the kinase component of MPF), the regulatory subunit cyclin B, and the activating phosphatase Cdc25. The absence of the entire nucleus-centrosome complex resulted in a marked delay in MPF activation, whereas the absence of the centrosome alone caused a lesser delay. Nocodazole treatment to depolymerize microtubules through first interphase had an effect equivalent to removing the centrosome. Furthermore, microinjection of isolated centrosomes into anucleate eggs promoted MPF activation and advanced the onset of surface contraction waves, which are close indicators of MPF activation and could be triggered by ectopic MPF injection. Finally, we were able to demonstrate stimulation of MPF activation by the nucleus-centriole complex in vitro, as low concentrations of isolated sperm nuclei advanced MPF activation in cycling cytoplasmic extracts. Together these results indicate that nuclei and microtubule asters can independently stimulate MPF activation and that they cooperate to enhance activation locally.
Insights
Nuclei and centrosomes help activate M phase-promoting factor (MPF) in Xenopus eggs. These structures, along with microtubules, independently stimulate MPF activation and work together for localized enhancement.
Area of Science:
- Cell Biology
- Developmental Biology
Background:
- M phase-promoting factor (MPF) is crucial for cell cycle progression.
- While MPF can activate autonomously in Xenopus egg cytoplasm, its precise regulation by cellular structures is not fully understood.
Purpose of the Study:
- To investigate the roles of nuclei and centrosomes in MPF activation within Xenopus eggs.
- To determine how these components, along with microtubules, contribute to the timing and localization of MPF activation.
Main Methods:
- Studied MPF activation by tracking Cdc2, cyclin B, and Cdc25 in fertilized eggs and egg fragments.
- Utilized experimental manipulations including removal of nucleus-centrosome complexes, centrosomes, and microtubule depolymerization with nocodazole.
- Performed microinjections of centrosomes and analyzed in vitro MPF activation using isolated sperm nuclei.
Main Results:
- Absence of the nucleus-centrosome complex significantly delayed MPF activation.
- Removal of centrosomes or microtubule depolymerization caused a lesser delay.
- Microinjected centrosomes and isolated nuclei stimulated MPF activation, advancing downstream events like surface contraction waves.
Conclusions:
- Both nuclei and microtubule asters independently stimulate MPF activation.
- These structures cooperate to enhance MPF activation locally within the cell.
- This highlights a coordinated mechanism for regulating cell cycle entry in early development.