Carotenuto R et al. (1997), A novel protein cross-reacting with antibodies ...

XB-ART-15621

J Cell Sci 1997 Nov 01;110 ( Pt 21):2683-90. doi: 10.1242/jcs.110.21.2683.

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A novel protein cross-reacting with antibodies against spectrin is localised in the nucleoli of amphibian oocytes.

Carotenuto R , Maturi G , Infante V , Capriglione T , Petrucci TC , Campanella C .

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Cytoskeletal proteins such as actin and myosin are important constituents of the nucleoplasm. Spectrin is an actin binding protein typically related to plasma membrane; recently, it has been found that it is widespread and forms distinct membrane protein domains in such organelles as the Golgi. In this paper, the large germinal vesicle of amphibian oocytes was chosen as a particularly suitable system to investigate the presence and location of spectrin in the nucleus. We manually isolated the germinal vesicles of both Discoglossus pictus and Xenopus laevis oocytes, and processed them for SDS-PAGE, immunoblotting and immunoprecipitation. By the use of an antibody against the general form of brain beta spectrin (betaIIsigma1) and of an anti-alpha brain spectrin (alphaIIsigma*), a band of 230 kDa was identified as a nuclear spectrin-like molecule. Moreover the 230 kDa protein was extracted from the nuclei by 1 M KCl, similarly to spectrin in other systems. In oocyte sections and nuclear spreads incubated with anti-alphaIIsigma* and/or anti-betaIIsigma1 antibodies, the immunostain was localised in the nucleoplasm and in the outer shell of the round bodies abundantly present in the germinal vesicle. Sections of the same oocytes, stained with a monoclonal antibody against nucleolar fibrillarin and anti-alphaIIsigma*, showed co-localisation of the two antibodies. It was concluded that, in the germinal vesicle of amphibian oocytes, a spectrin-like molecule is a part of the outer shell of nucleoli. It is hypothesised that spectrin, together with actin, might be instrumental in keeping nucleoli attached to the inner nuclear membrane, as nucleoli migrate during oogenesis to the inner aspect of the nuclear envelope, where they are stably kept until the end of their growth. Furthermore, these results strongly suggest that the 230 kDa band might comprise both an alpha and a beta chain of the same apparent molecular mass, thus constituting a novel form of a spectrin-like molecule.

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Species referenced: Xenopus laevis
Genes referenced: actl6a fbl
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	Fig. 1. Samples of oocyte GVs and human erythrocyte ghosts were analysed on 5% SDS-PAGE and stained with Coomassie Blue or transferred to nitrocellulose membrane. (A) Gels stained with 1% Coomassie Blue. Molecular mass standards are indicated on the left. Lane a, total GV preparation. The arrow points to a faint molecule of about 230 kDa. Lane b, 240 and 220 kDa bands are indicated, corresponding to a and b erythroid spectrin subunits in human erythrocyte ghosts. (B) GV extracted in 1 M KCl containing buffer. Lane a, soluble extract. The 230 kDa band is indicated. Lane b, pellet; the band is undetectable. Lane c, erythrocyte ghosts. (C) Western blot with anti-bIIS1 spectrin antibodies showing the immunoreactivity of the GV 230 kDa band and of the spectrin b chain (arrow in a), as control. Lane a, erythrocyte ghosts. Lane b, GV. (D) Western blot with anti-aIIS* incubation. Lane a, the 230 kDa band is immunoreactive. Lane b, in human erythrocyte ghosts the spectrin a chain is reactive, while the b spectrin subunit is only slightly positive (arrowhead).
	Fig. 2. GV immunoprecipitation with anti-bIIS1 antibody. Lane a, immunoblots of the immunoprecipitate transferred onto NC and exposed to anti-bIIS1 antibody. The 230 kDa protein is indicated by the arrow. Lane b, human ghost erythrocyte incubated with the same antibody; b spectrin is immunoreactive.
	Fig. 3. (A-C) Immunofluorescence of frozen sections of D. pictus oocytes at an early stage of vitellogenesis (about 320 mm in diameter). (A,B) Anti-bIIS1 antibody incubation. (A) The immunostain is present in the cortex and in the surrounding ovarian tissue (arrow). Furthermore, the fluorescence is located in the GV content, particularly surrounding dark spherical organelles. The nuclear envelope is immunoreactive (double small arrows). ´310. (B) The GV matrix is fluorescent. Round bodies are surrounded by a rim of immunostain (arrows), or fully stained, when tangentially sectioned (double arrows) ´500. (C) Control section incubated with rabbit IgG. Immunostain is absent. ´500. (D) GV Ipreparation spread on a microslide and incubated with anti- bIIS1 antibody. Immunostain is present on the numerous round bodies. ´500. (E) Fluorescence is located around these bodies. ´500. (F) Control GV preparation. Immunostain is absent. ´500. Bars: 50 mm (A,B); 20 mm (C-F).
	Fig. 4. Frozen sections of D. pictus oocytes at early stages of oogenesis. (A) Section of an oocyte of about 300 mm incubated with anti-aIIS. The arrows indicate the rim of immunostain surrounding the round bodies. ´310; (B) Incubation with anti-fibrillarin. Immunostain surrounds spherical bodies thus identified as nucleoli (arrows). ´310. (C) Control section exposed to anti-aIIS preabsorbed with erythroid a and b spectrin; the immunostain is absent. (D) Light microscopy of the same section as in B. The round bodies are indicated (arrows). The section is lightly stained by p-phenylenediamine present in the glycerol-PBS mixture. ´310. (E,F) Serial sections of the same GV from a 500 mm oocyte. (E) Incubated with anti-aIIS* antibody; (F) incubated with anti-fibrillarin. Corresponding labels (arrow, double small arrows, star, small arrow) indicate the same nucleoli immunostained by both antibodies. ´500. Bars: 50 mm (A-C); 20 mm (E,F).
	Fig. 5. Xenopus laevis GV preparation (lane b) and human erythrocyte ghost (lane a) after western blot with anti-bIIS1 antibody. Lane a, immunoreactivity at the 220 kDa b spectrin. Lane b, the GV preparation shows immunoreactivity of the 230 kDa band. Lane c, SDS-PAGE of the GV preparation stained with silver stain. The 230 kDa band is indicated.
	Fig. 6. Frozen oocyte sections of X. laevis. (A) A 350 mm oocyte section incubated with anti-bIIS1 spectrin. Immunostain is in the ovarian tissue in the GV content and surrounds nucleoli (arrows). ´310. (B) Control section incubated with a fraction of rabbit IgG, showing a slight background of positivity. ´310. (C) A 450 mm oocyte section incubated with anti- aIIS*spectrin. In the GV, several nucleoli, as recognised by the corresponding staining with anti-fibrillarin (D), are immunostained. (D) Corresponding labels of C,D (arrows, stars, small arrow) indicate that the same nucleoli are immunostained by this antibody and anti-bIIS1. (C,D) ´500. Bars: 50 mm (A,B); 20 mm (C,D).