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

Abstract

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.

Human α Spectrin II and the FANCA, FANCC, and FANCG Proteins Bind to DNA Containing Psoralen Interstrand Cross-Links

“…The function of spectrin in the nucleus, however, is unclear. R spectrin has been shown to be associated with the nuclear matrix and the nuclear envelope (43,44), and a protein cross-reacting with R and β spectrin antibodies has been detected in nucleoli of amphibian oocytes (45). We have recently identified RSpIIΣ* in the nuclei of normal human lymphoblastoid cells and HeLa cells and have shown that it is a component of a nuclear protein complex involved in repair of DNA interstrand cross-links (13,14) and that it also forms a complex with the FANCA, FANCC, and FANCG proteins (14,Lambert et al,in preparation).…”

Xenopus laevis oocyte as a model for the study of the cytoskeleton

“…In stage-I oocytes, in which the yolk has not yet been deposited, the CSK forms a supple network that gradually spreads throughout the cell and is primarily composed of actin microfilaments, microtubules, vimentin, cytokeratin and spectrin [3,21,34,36]. Actin-binding spectrins of 239 and 100 kDa associate with the cell membrane, the nuclear envelope and the growing mitochondrial cloud (MC) [26,29], similarly to the organization achieved by the intermediate filaments (Fig. 1 and Fig.…”

“…[34,37]. Antibodies to b-spectrin identify a further isoform of about 230 kDa surrounding the nucleoli in the oocyte germinal vesicle (GV) [29]. It is known that protein 4.1 (4.1R), an 80-kDa polypeptide, stabilizes the spectrin-actin network and anchors it to the plasma membrane [38,39].…”

“…1a, b). In addition to actin, several cytoskeletal proteins have been detected in the growing oocyte, including tubulin [4][5][6][7][8][9][10][11][12], vinculin [13], vimentin [14][15][16][17][18][19][20], talin [1], cytocheratin [21][22][23][24][25][26], spectrin [26][27][28][29] and myosin [2,[30][31][32]. When the oocyte reaches final stages of growth (stages 4-6) 1 , it acquires a defined polarization with respect to its virtual animal/vegetal axis (A/V axis).…”

Xenopus laevis (Daudin, 1802) as a Model Organism for Bioscience: A Historic Review and Perspective

“…Here, it is possible to find tubulin and an actin-based cytoskeleton, including vinculin, vimentin, talin, cytokeratin, spectrin and myosin (for a review, see Carotenuto and Tussellino [ 38 ]). Actin-binding spectrins (239 and 100 kDa) are associated with the cell membrane, the nuclear envelope, the growing mitochondrial cloud (MC) and nucleoli [ 39 , 40 ], as well as intermediate filaments [ 41 , 42 ]. Isoforms of protein 4.1 (4.1R) stabilize the spectrin–actin network and anchor it to the plasma membrane [ 43 ].…”