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.
We have used complementary biochemical and in vivo approaches to study the compartmentalization of M phase-promoting factor (MPF) in prophase Xenopus eggs and oocytes. We first examined the distribution of MPF (Cdc2/CyclinB2) and membranous organelles in high-speed extracts of Xenopus eggs made during mitotic prophase. These extracts were found to lack mitochondria, Golgi membranes, and most endoplasmic reticulum (ER) but to contain the bulk of the pre-MPF pool. This pre-MPF could be pelleted by further centrifugation along with components necessary to activate it. On activation, Cdc2/CyclinB2 moved into the soluble fraction. Electron microscopy and Western blot analysis showed that the pre-MPF pellet contained a specific ER subdomain comprising "annulate lamellae" (AL): stacked ER membranes highly enriched in nuclear pores. Colocalization of pre-MPF with AL was demonstrated by anti-CyclinB2 immunofluorescence in prophase oocytes, in which AL are positioned close to the vegetal surface. Green fluorescent protein-CyclinB2 expressed in oocytes also localized at AL. These data suggest that inactive MPF associates with nuclear envelope components just before activation. This association may explain why nuclei and centrosomes stimulate MPF activation and provide a mechanism for targeting of MPF to some of its key substrates. INTRODUCTIONThe structural changes accompanying mitosis and meiosis are governed in almost all species by M phase-promoting factor (MPF) (Masui and Markert, 1971), a complex of Cyclin B and the kinase Cdc2 (reviewed in Nigg, 1995;Morgan, 1997). Direct and indirect targets for MPF include the nuclear envelope and lamina, chromatin proteins, and regulators of mitotic spindle formation (Moreno and Nurse, 1990;Norbury and Nurse, 1992). MPF activation requires the continuous synthesis and accumulation during interphase of Cyclin B, which binds Cdc2 to form inactive pre-MPF. Pre-MPF is maintained inactive by inhibitory phosphorylation on Cdc2 by the Wee1 and Myt1 kinases. At the onset of mitosis, rapid MPF activation is favored by a positive feedback loop involving Cdc25 phosphatases, MPF itself, and Polo-like kinases. Degradation of Cyclin B at the metaphaseto-anaphase transition by the anaphase promoting complex (APC) destroys the MPF complex and causes exit from mitosis (reviewed in Nurse, 1990; Whitaker and Patel., 1990;Norbury and Nurse, 1992;Nigg, 1995;Morgan, 1997Morgan, , 1999Beckhelling and Ford, 1998;O'Farrell, 2001).Amphibian eggs have proved extremely useful for the study of MPF regulation. Cycles of Cyclin B accumulation and destruction sufficient to drive synchronous cell cycles in the absence of any other protein synthesis occur in both fertilized or activated eggs and in egg extracts (Murray and Kirschner, 1989;Hartley et al., 1996). Although MPF activation cycles in amphibian oocytes and eggs can continue in the absence of nuclei and microtubules (Hara et al., 1980;Shinagawa, 1983;Gerhart et al., 1984;Newport and Kirschner, 1984;Kimelman et al., 1987;Shinagawa, 1992) both in manipulate...
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