The isolation of nuclear envelope from peripheral lymphocytes: An ultrastructural study

Abstract

A procedure is presented for the isolation of nuclear envelope from circulatory human lymphocytes. The chromatin of purified lymphocyte nuclei is decondensed by washing with 1 mM sodium bicarbonate buffer (pH 7.2) in the absence of chromatin-stabilizing divalent cations. Following a limited digestion with pancreatic DNAase-I, the nuclear envelope is centrifugally harvested and repeatedly washed in bicarbonate buffer to remove solubilized chromatin. Further purification of the nuclear envelope is achieved by isopycnic banding on sucrose step gradients. The purity and morphology of the isolated nuclear envelope is assessed by negative-staining electron microscopy. All the membranous material present in the purified preparations is found to possess nuclear pore complexes, which are accepted as being morphological ‘markers’ for the nuclear envelope. Relatively intact lympocyte nuclear envelope ‘ghost’ are found to possess a low frequency of nuclear pore complexes together with the presence of nuclear pore complex clustering, in agreement with the literature on freeze-fractured lymphocyte nuclear envelope. Negatively stained fragments of nuclear envelope often contain the nuclear pore complex clusters, indicating that lesions through the outer and inner nuclear membranes have more readily occurred in the regions of envelope devoid of nuclear pores complexes. This observation supports the hypothesis that the nuclear pore complexes act as linking positions between the two nuclear membranes and thereby act as zones of stabilization for the overall nuclear envelope during the isolation of the nuclei and envelope, even though their in vivo function is more likely to be associated with ribonucleoprotein translocation. Electrophoresis of sodium dodecyl sulphate-solubilized lymphocyte nuclear envelope on polyacrylamide gradient gels has revealed the molecular weight range of the principal polypeptides present. Excellent reproducibility of polypeptide content has been achieved for consecutive preparations of lymphocyte nuclear envelope. A comparison of polypeptide content of rat liver and human lymphocyte nuclear envelope is presented. The methodology employed and the data presented is discussed in relation to the available literature on lymphocyte nuclei and nuclear envelope, and also to the existence of a nuclear protein matrix in the transcriptionally inactive nucleus of the lymphocyte.