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.