Changes in Organization of the Endoplasmic Reticulum duringXenopusOocyte Maturation and Activation

Abstract

The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releaseability of Ca 2ϩ by IP 3 . The clusters dispersed during the Ca 2ϩ wave at activation. Possible relationships of ER structure and Ca 2ϩ regulation are discussed.

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

“…We were able to confirm that part of the MPF population in the undisturbed cell colocalizes with AL by two independent visualization methods applied to whole oocytes: immunofluorescence on fixed material and live imaging of GFP-CyclinB2. Cyclin B2-GFP expressed in immature oocytes localized to distinctive cigar-shaped structures located close to the vegetal surface with the characteristics defined for AL (Terasaki et al, 2001), whereas an anti-cyclin B2 antibody decorated equivalent structures in fixed eggs, which we showed directly to be enriched in NUP proteins. It would be very hard to demonstrate association of MPF with the nuclear envelope in fertilized eggs because the tiny nucleus is hard to locate, whereas excess nuclear membrane components are dispersed throughout the volume of the egg.…”

“…AL membranes are continuous with and embedded within the ER (this study, Figure 6, A-C;Kessel, 1992;Terasaki et al, 2001), and it is not clear why they separate from the rest of the ER during fractionation, although the high concentration of nuclear pores is likely to significantly affect their density. AL accumulate in interphase Xenopus egg extracts (Dabauvalle et al, 1991;Meier et al, 1995) and disassemble in parallel with the nuclear envelope at mitosis/meiosis (Cordes et al, 1996;Imreh and Hallberg, 2000;Terasaki et al, 2001) and thus will be maximally accumulated at the time we made our extracts, favoring their identification in our study. We were able to confirm that part of the MPF population in the undisturbed cell colocalizes with AL by two independent visualization methods applied to whole oocytes: immunofluorescence on fixed material and live imaging of GFP-CyclinB2.…”

“…In another example, SUMO-1 protease SENP2 associates with NUP153 of the nuclear pore in humans (Hang and Dasso, 2002), as does a related protease with NUP42 in yeast (Takahashi et al, 2000). The presence of AL at the vegetal cortex of the large Xenopus oocyte, and their reorganization at M phase that mirrors that of the nuclear envelope (Terasaki et al, 2001), provides an excellent opportunity to further explore interaction of nuclear membranes with biologically important macromolecules in vivo. In particular a localization study of the various regulators of MPF such as Wee1, Myt1, the CDK inhibitor, Polo kinase, CyclinA-Cdc2, Pin1, CAK, and suc1 may further illuminate the basis of localized MPF regulation in the perinuclear region.…”

“…Unfortunately, the CyclinB antibodies available were not of high enough affinity to allow EM immunolocalization on pelleted material. Instead, we exploited the localization of characteristic AL in the vegetal subcortex of immature (stage VI) Xenopus oocytes (Terasaki et al, 2001) to perform colocalization studies at the light microscope level. The size and opacity of the Xenopus egg is prohibitive for clear visualization of deep structures such as the nucleus; however, the first 10 -20 m below the cell surface where the AL lie is easily accessible to confocal microscopy in both fixed and live eggs.…”

“…AL, which can be clearly detected by the anti-NUP antibody used for Western blot experiments, show up as islands of various shapes, but mostly form long and narrow "cigars" ranging from 10 to 30 m in length and 3-5 m in width and depth (Figures 6, A and D, and 7E;Terasaki et al, 2001). Double labeling for NUP and Grp94 (Figure 6, A and B) confirmed that the NUP-stained islands were embedded within the ER network ( Figure 6C), consistent with the position of AL as an integral part of the ER network (Bal et al, 1968;Kessel, 1992;Terasaki et al, 2001). Strikingly, double labeling with anti-CyclinB2 and anti-NUP (Figure 6, D and E) revealed accumulations of anti-CyclinB2 on the AL as well as in spots distributed throughout the cytoplasm (see Figure 6F for overlay).…”