Relocation and distinct subcellular localization of p34cdc2-cyclin B complex at meiosis reinitiation in starfish oocytes.

Abstract

M phase promoting factor (MPF) is a major element controlling entry into the M phase of the eukaryotic cell cycle. MPF is composed of two subunits, p34cdc2 and cyclin B. Using indirect immunofluorescence staining with specific antibody against starfish cyclin B, we monitored the dynamics of the subcellular distribution of MPF during meiosis reinitiation in starfish oocytes. We found that all of the cyclin B is already associated with p34cdc2 in immature oocytes arrested at the G2/M border and that this inactive complex is present exclusively in the cytoplasm. After its activation, part of the p34cdc2‐cyclin B complex moves into the germinal vesicle before nuclear envelope breakdown, independently of either microtubules or actin filaments. Thereafter, some part of the complex accumulates in the nucleolus and condensed chromosomes. Another portion of the complex accumulates on meiotic asters and spindles, while the rest is still present throughout the cytoplasm. As these patterns of localization are detected in the detergent‐extracted oocytes, we propose at least four distinct subcellular states of the p34cdc2‐cyclin B complex: freely soluble, microtubule‐associated, detergent‐resistant cytoskeleton‐associated and chromosome‐associated. Thus, in addition to the intramolecular modification of p34cdc2‐cyclin B complex, its intracellular relocation plays a key role in promoting the M phase.

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

“…mRNA coding for this construct was injected into immature starfish oocytes (A. miniata). After overnight incubation, there was fluorescence in the cytoplasm but not the GV (Figure 3), as expected for cyclin B (Ookata et al, 1992). A similar distribution was seen when mRNA coding for the human cyclin B-GFP was injected.…”

“…In salt-and detergent-extracted oocytes, some of the Cdc2-cyclin B moves to the low-salt soluble fraction during maturation. The timing of these changes in localization and extractability corresponds very well with the timing of Cdc2-cyclin B activation as assayed by H1 kinase activity (Ookata et al, 1992).There are uncertainties regarding the results from each of the three methods. The aggregates are seen by immunofluorescence in methanol fixed oocytes, but they are not seen with formaldehyde fixation.…”

“…1-MeAde acts on the surface of the oocyte to activate a G i family G protein, releasing ␤␥ subunits that are the mediators of the downstream effects (Jaffe et al, 1993). Through intermediate steps, Akt/PKB becomes activated, which then phosphorylates and inactivates Myt1 (Okumura et al, 2002 (Ookata et al, 1992). 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).…”

“…Through intermediate steps, Akt/PKB becomes activated, which then phosphorylates and inactivates Myt1 (Okumura et al, 2002 (Ookata et al, 1992). 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 maturation hormone 1-MeAde restarts meiosis, during which the germinal vesicle breaks down (GVBD). Oocytes from the same animal undergo GVBD within a few minutes of each other, but there is variation between different animals so that GVBD normally occurs between ϳ15 and 30 min after application of 1-MeAde.To localize Cdc2-cyclin B by immunofluorescence, we used an antibody to cyclin B similar to that used previously (Ookata et al, 1992). On a Western blot, the rabbit antibody to starfish cyclin B stained a single band of the expected molecular weight.…”