Pre-M Phase-promoting Factor Associates with Annulate Lamellae inXenopusOocytes and Egg Extracts

Abstract

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...

Localization and Dynamics of Cdc2-Cyclin B during Meiotic Reinitiation in Starfish Oocytes

“…The agreement of the three methods provides strong evidence that a significant fraction of the inactive Cdc2-cyclin B is not freely soluble in the cytoplasm and that at least some of this fraction is released at the activation. Our results are similar to those of Westendorf et al (1989) who described cyclin B aggregates in clam oocytes that disperse on oocyte activation, and to Beckhelling et al (2003) who found an association of Cdc2-cyclin B with annulate lamellae in frog oocytes that disperses during oocyte maturation.There are several important unresolved issues. The composition of the aggregates in starfish oocytes is not known.…”

“…When the Golgi apparatus was localized by galtransferase-GFP expression, it also shows as spots scattered throughout the cytoplasm (Terasaki, 2000), but double staining by cyclin B immunofluorescence showed that the Golgi apparatus and spots were not colocalized (our unpublished data). Cyclin B is not associated with annulate lamellae as in Xenopus oocytes (Beckhelling et al, 2003) because starfish oocytes do not possess annulate lamellae by electron microscopy or by immunofluorescence labeling (our unpublished data).…”

“…In these conditions, immunofluorescence of cyclin B should show the localization of Cdc2-cyclin B, and it was shown that Cdc2-cyclin B accumulates in the nucleus before germinal vesicle breaks down (GVBD) (Ookata et al, 1992). However, the regulation of nuclear accumulation of Cdc2-cyclin B is not understood and the localization of inactive Cdc2-cyclin B in frog oocytes (Beckhelling et al, 2003) suggests that there may be other features of activation that are unknown. The availability of a GFP chimera of cyclin B (Hagting et al, 1998) led us to reexamine the localization and dynamics of Cdc2-cyclin B during maturation of starfish oocytes.…”

“…Among various reports, Picard and Peaucellier (1998) observed cyclin B in vesiclelike structures in starfish oocytes, Westendorf et al (1989) found evidence for cyclin B aggregates in immature clam oocytes, and Lee and Kirschner (1996) found evidence for a binding inhibitor of Cdc2-cyclin B that is associated with membranes. More recently, inactive Cdc2-cyclin B was found to be associated with annulate lamellae in immature Xenopus oocytes (Beckhelling et al, 2003).Current techniques for localization are subject to limitations of various kinds. The use of green fluorescent protein (GFP) chimeras is promising because localization can be studied in living cells.…”

The dynamics of cyclin B1 distribution during meiosis I in mouse oocytes

“…One possibility is that, despite the reported excess of cyclin B, much of it may be sequestered , de Vantéry et al 1997, Beckhelling et al 2003. A small increase in 'free' exogenous cyclin B1 may provide a sufficient increase in pre-MPF available for cdc25 to initiate the auto-catalytic reaction leading to complete MPF activation (Hoffmann et al 1993, Lincoln et al 2002.…”