Purification of intact nuclear lamina and identification of novel laminlike proteins in Raji, a cell line devoid of lamins A and C

Abstract

Research on the structure of the nuclear lamina and the nuclear matrix of cells devoid of lamins A and C has been hampered by the fact that intact residual nuclear structures are difficult to isolate from such cells. In this paper, we show that some extraction parameters, such as buffer composition and the nature of the detergent used to remove nuclear membranes, are critical for achieving isolation of whole nuclear residual structures from the lymphoblastic cell line Raji, used as a model for cells without lamins A and C. Electron microscopic analysis shows that the nuclear lamina of Raji cells is formed by a network of intermediate-sized filaments interrupted with circular discontinuities. Both lamins B1 and B2, and lamin D/E, are present in this structure. In addition, a group of 45-kDa proteins or intermediate filament protein - reacting proteins (IFA-RPs), located uniquely in the lamina, were found to exhibit the same immunological and chemical characteristics as lamins. Although they behave like nuclear lamins, microsequencing analysis of the IFA-RPs has revealed no homology with known lamins. These IFA-RPs may contribute to the formation of the nuclear lamina filament network in the absence of lamins A and C. Key words: nuclear lamina, intermediate filaments, lamin.