We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts . Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence . For comparison, lamins and histones were localized using human autoimmune antibodies . At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components . Antibody Pl stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus . Antibody 11 detected an antigen distributed as fine granules throughout the nuclear interior . Monoclonals PI1 and P12 stained both the nuclear periphery and interior, with some characteristic differences . During mitosis, P1 and 11 were chromosome-associated, whereas P11 and P12 dispersed in the cytoplasm . Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order . With antibody 11, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to 11 may be involved in chromatin/ chromosome higher-order organization throughout the cell cycle . Antibodies P11 and P12 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase . Antibody P12 also detected antigen associated with the spindle poles.The nuclear matrix is a complex biochemical fraction consisting of nonhistone nuclear proteins and small quantities of DNA and RNA . It is obtained by sequential extraction of isolated interphase nuclei with low-and high-salt buffers, detergents, and DNAse and RNAse . Structurally, the nuclear matrix comprises the peripheral nuclear pore complex-lamina, an internal fibrogranular network and residual nucleoli . Part of the matrix has been envisaged as an interphase "nuclear skeleton" on which nuclear functions such as DNA replication, RNA transcription and processing, virus replication, and hormone response can be ordered (see references 1-3 for reviews).Several studies have suggested that the nuclear matrix is involved in mitosis (4-7). Recent work (8, 9) has also indicated a potential role for the nuclear matrix in the organization of mitotic chromosomes per se. To examine the distribution of individual nuclear matrix polypeptides throughout the cell cycle, we used monoclonal antibodies against isolated THE JOURNAL OF CELL BIOLOGY " VOLUME 99 AUGUST 1984 661-671 © The Rockefeller University Press -0021-9525/84/08/0661/11 $1 .00 nuclear matrices in an immunofluorescence study of mouse 3T3 fibroblasts . MATERIALS AND METHODSCell Culture: Mouse 3T3 fibroblasts were cultured at 37°C with 5% C02 in Dulbecco's modified Eagle's medium with 10% fetal calf serum, 100 U/ml penicillin, 100 Ag/ml streptomycin, and 0.25 lg/ml fungizone . Mouse myel...

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A major component of nuclear change in concanavalin-A-stimulated bovine lymphocytes is a severalfold increase in interchromatinic volume, which coincides with nuclear swelling and extensive structural remodelling. Large-scale ultrastructural changes in isolated nuclei and nuclear matrices (NM) reflect those occurring within nuclei in situ during mitogenesis. While nonchromatinic nuclear material embedded within nuclease- and salt-extracted whole cells closely resembled in situ interchromatinic matrices, large NM isolated in solution shrank after chromatin was extracted. Numerous perinuclear filaments persisted throughout NM isolation and cytoskeletal proteins were identified in two-dimensional (2-D) gels of such preparations. Taken together these data indicated that the lymphocyte cytoskeleton is likely continuous with the nuclear matrix and could play a role in maintaining nuclear organization. A wide range of lymphocyte NM proteins were resolved in 2-D gels. Significant changes in protein composition coincided with nuclear structural remodelling. Lamin B was prominent at each stage of nuclear development, whereas lamins A and C were only found in stimulated lymphocyte matrices. Lymphoblast NM contained more large basic proteins. Progressively increasing polypeptide complexity of these NM arose by de novo protein synthesis and posttranslational modifications throughout concanavalin A stimulation. NM from stimulated lymphocytes also contained more ribonucleoproteins, possibly indicating the presence of significant amounts of transcriptional material.

SUMMARY The technique of delaying fixation until after freeze‐fracture and thawing, described in an earlier paper (Haggis & Bond, 1979), has been developed further for study of cells in culture, principally mouse lymphocytes stimulated by concanavalin A. Using a thin layer of cells, a cryoprotectant concentration of either 10% glycerol or dimethylsulphoxide, is sufficient to give good structural preservation after rapid freezing and thawing. Nuclear matrices and Triton‐permeabilized cells have been prepared from stimulated lymphocytes for comparative study. Polylysine‐coated fibrin support films have been found to provide a convenient means of handling cells and subcellular preparations during freeze fracture, critical point drying and mounting for high‐resolution scanning electron microscopy.