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...

Nuclear structural changes during fertilization and embryogenesis in mice and in sea urchins have been followed by using antibodies against the nuclear lamins A/C and B and against antigens at the periphery of nuclei and chromosomes. Lamins are found on all pronuclei and nuclei during mouse fertilization, but with a diminished intensity on the second polar body nucleus. On sperm in both systems, lamins are reduced and detected only at the acrosomal and centriolar fossae. In sea urchin eggs, lamins are found on both pronuclei. Unlike in other dividing cells, the mitotic chromosomes of sea urchin eggs and embryos retain an association with lamins. The peripheral antibodies delineate each chromosome and nucleus except the mature mouse sperm nucleus. A dramatic change from the expected lamin distribution occurs during early development. In mouse morulae or blastocysts, lamins A/C are no longer recognized, although lamin B remains. In sea urchins both lamins A/C and lamin B, as detected with polyclonal antibodies, are lost after the blastula stage, although a different lamin A/C epitope emerges as recognized by a monoclonal antibody. These results demonstrate that pronucleus formation in both systems involves a new association or exposure of lamins, that the polar body nucleus is largely restricted from the cytoplasmic pool of lamins, and that mitotic chromosomes in the rapidly proliferating sea urchin egg retain associated lamins. They also suggest that changes in the expression or exposure of different lamins are a common feature of embryogenesis.Fertilization requires several dramatic changes in nuclear organization. The architecture of the nuclear surface (reviewed in refs. 1-4) involves the nuclear lamins, which are typically three proteins subjacent to the inner nuclear membrane (5, 6), and nuclear peripheral proteins referred to as "P1" (7) or "Perichromin" (8), which probably reside between the chromatin and the nuclear lamins. During mitosis in somatic cells, the lamins dissociate from the nuclear envelope at prophase and reappear with the reconstituting envelope at telophase (9). Unlike the behavior of the lamins at mitosis, the peripheral antigens separate from the nuclear periphery and ensheathe the condensing chromosomes before nuclear envelope breakdown and dissolution of the lamins during mitosis (7,8). During spermatogenesis the nuclear lamins are lost or vastly reduced (10-12), whereas during oogenesis the lamina may be comprised of only a single lamin (13-16), which differs from somatic lamins (17).In this study the presence and distribution of nuclear lamins and of the nuclear and chromosomal P1 peripheral antigens are traced during fertilization and embryogenesis in mice and in sea urchins. These two systems represent

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Using a monoclonal antibody (PI1) raised against mouse lymphocyte nuclear matrix fractions we have identified a N-acetylglucosamine (GlcNAc)-containing glycoprotein of Mr 68,000 as a component of the nuclear pore complexes of Xenopus laevis oocytes. The antigenic determinant recognized by antibody PI1 comprises both the sugar moiety and protein sequences since, on the one hand, added GlcNAc competed effectively for antibody binding and, on the other hand, the antibody reacted in immunoblots with only one member of the GlcNAc-containing pore complex glycoprotein family. By using immunogold-electron microscopy we could demonstrate that the Mr 68,000 glycoprotein was located preferentially to the cytoplasmic side of the pore complex channel. When radiolabeled soluble nuclear proteins were injected into the cytoplasm of Xenopus oocytes, their reentry into the nucleus was almost completely inhibited in the presence of antibody PI1 as shown by two-dimensional gel electrophoresis. The results indicate that the evolutionarily conserved Mr 68,000 glycoprotein is involved in transport processes of karyophilic proteins from the cytoplasm into the nucleus.

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