Changes in distribution of nuclear matrix antigens during the mitotic cell cycle.

Abstract

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...

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

“…Murine monoclonal antibody PIl (IgM) was raised against a mouse Iymphocyte nuclear matrix fraction and has been characterized in detail (Chaly et al 1984(Chaly et al , 1986. Experiments were carried out using PIl ascites fluid purified by hydroxylapatite chromatography, concentrated and lyophilized as previously described (Stanker et al 1985).…”

“…As shwon in Figure 2, the antibody reacted specifically with a single polypeptide of Mr 68000 (lane 2', arrow). A poly- peptide band of identical Mr value has been detected with PI1 in immunoblots using isolated nuclear envelopes from Xenopus oocytes (Dabauvalle et al 1988a) and of slightly higher electrophoretic mobility when bovine lymphocyte nuclear fractions were used as the antigen source (Chaly et al 1984).…”

Identification of a soluble precursor complex essential for nuclear pore assembly in vitro

“…Among the WGA-binding pore complex glycoproteins of Xenopus oocytes we have recently identified a major component of Mr 68,000 (p68) which is confined to the pore channel and crucially involved in nuclear protein transport processes (Dabauvalle et al 1988 b; see also Featherstone et al 1988). This pore complex glycoprotein is evolutionarily highly conserved and has been detected in a variety of species ranging from insects to mammals by means ofimmunofluorescence and immunoblotting studies employing the monoclonal antibody Pl1 (Chaly et al 1984(Chaly et al , 1986Benavente et al 1989 a, b;Dabauvalle et al 1988 b). Furthermore, microinjection of antibody PI1 inhibited nuclear protein uptake not only in Xenopus oocytes (Dabauvalle et al 1988 b) but also in mammalian cells (Benavente et al 1989 a, b).…”

Assembly and disassembly of mammalian Chromosome pellicle

“…Up to the present, some building material of chromosome pellicle are known to come from nuclear periphery (PI antigen, M108 antigen, perichromin) and nucleoli (PCN). The assembly disassembly cycles of these nuclear structures are well documented [1,2,3,7,8], Thus in relation to the assembly disassembly of chromosome pellicle, there are 3 such cycles of nuclear structures, namely nuclear periphery, nucleolus and the surface of condensed chromosomes. Therefore, during mitosis, there are all together 4 interrelated cycles, Previous and the present studies showed that the assembly disassembly cycle of chromosome pellicle is approximately in phase with that of the surface of condensed chromosomes and in antiphase with those of nuclear periphery and nucleoli.…”

“…Recently, from the immunofluorescence studies on cells, evidence has been found that the pellicle is a real structure of the condensed chromosomes (metaphase and anaphase chromosomes) [1,2,3,4]. Some of its chemical constituents have been characterized by immuno-blotting.…”