October 27, 2000;
CPEB, maskin, and cyclin B1 mRNA at the mitotic apparatus: implications for local translational control of cell division.
In Xenopus development, the expression of several maternal mRNAs is regulated by cytoplasmic polyadenylation. CPEB
, two factors that control polyadenylation-induced translation are present on the mitotic apparatus of animal pole blastomeres in embryos. Cyclin B1 protein and mRNA, whose translation is regulated by polyadenylation, are colocalized with CPEB
interacts with microtubules and is involved in the localization of cyclin B1 mRNA to the mitotic apparatus. Agents that disrupt polyadenylation-induced translation inhibit cell division and promote spindle
defects in injected embryos. Two of these agents inhibit the synthesis of cyclin B1 protein and one, which has little effect on this process, disrupts the localization of cyclin B1 mRNA and protein. These data suggest that CPEB
-regulated mRNA translation is important for the integrity of the mitotic apparatus and for cell division.
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Figure 1. Distribution of CPEB and Maskin during Oogenesis(A) Immunohistochemistry of CPEB and maskin using affinity-pure antibodies on stage 1–6 albino oocytes in whole mount (panels 1–3), or on frozen sections of stage 5–6 oocytes (panels 4 and 5). The arrowheads denote concentrated staining of CPEB and maskin in the animal pole of oocytes in whole-mount and intense staining of these proteins in the animal pole cortex of frozen sections. A selected area of the immunostaining for maskin is shown at higher magnification (panel 6).(B) Western analysis of CPEB, maskin, and MAP kinase in stage 1–6 oocytes. The protein from one oocyte was loaded onto each gel lane.(C) Western analysis of CPEB, maskin, and MAP kinase from animal and vegetal sections from stage 3–6 oocytes. Frozen oocytes were cut in half along the equator, and the protein from the animal (a) and vegetal (v) halves was applied to the gel.
Figure 2. CPEB and Maskin Localization during Early Embryogenesis(A) Immunostaining for CPEB and maskin in ovulated eggs, 4 cell, and 16 cell albino embryos. The animal pole is the site of intense staining.(B) Western blot analysis of CPEB, maskin and tubulin in the animal (A) and vegetal (V) halves of stage 6 oocytes, progesterone-matured oocytes, ovulated eggs, and blastula stage embryos. Each lane was loaded with protein from one-half oocyte or embryo equivalent.(C) Embryos that were stained with CPEB and maskin antibodies, or with nonimmune serum, were treated with a benzyl-benzoate:benzyl alcohol (1:2) solution to clear the yolk. CPEB and maskin are associated with structures that resemble mitotic spindles.(D) Embryos were simultaneously immunostained for α-tubulin and either maskin, CPEB, or ElrA.
Figure 5. Localization of Xbub3 and Cyclin B1 mRNAs in Embryos by Whole-Mount In Situ HybridizationRelevant sequence information of the 3′ UTRs of Xbub3 and cyclin B1 mRNAs, denoting the CPEs and polyadenylation hexanucleotides (AAUAAA), is presented. Different embryos were probed with antisense sequences for these two mRNAs as well as for eIF4E mRNA, which does not contain a CPE. Other embryos were probed with α-tubulin antibody. Except for the embryo with the designated animal and vegetal poles, all the embryos are shown with animal facing out. Selected mitotic complexes are shown at higher magnification. DAPI was used to stain DNA.
Figure 7. Effect of CPEB Antibody, CPEB δ4, and Cordycepin Injection on the Localization and Translation of Cyclin B1 mRNA(A) Embryos injected with CPEB δ4, CPEB antibody, or cordycepin were collected following first cleavage (of the controls) and western blotted for cyclin B1 and tubulin (top). Each lane was loaded with protein from one embryo. These western blots were scanned and the relative amount of cyclin B1 at each point was quantified and plotted (bottom).(B) Fertilized eggs, some of which were injected with CPEB δ4 or CPEB antibody, were processed for cyclin B1 mRNA whole-mount in situ hybridization at the same time of embryogenesis.(C) Control and CPEB δ4 injected embryos were analyzed for α-tubulin and cyclin B1 protein by whole mount immunohistochemistry and confocal microscopy.