Functional changes of intermediate filaments in fibroblastic cells revealed by a monoclonal antibody.

Abstract

We describe reversible changes of intermediate filaments of fibroblastic cells associated with changes in the functional state of the cells. The changes are revealed by comparing the immunofluorescence patterns given by a monoclonal antibody and a polyclonal serum, both recognizing vimentin. The state of the filaments depends on culture density; this effect cannot be attributed to the nutritional state of the cells, their growth rate, or substances released into the medium. It seems to depend mainly on the aggregation of filaments during strong cell movements. The possible significance of these findings for the functional role of intermediate filaments is discussed.Intermediate filaments, 8-10 mm in diameter, are present in most cells. They are made up of different proteins that share some common epitope (1). In fibroblastic cells, the main constituent of the filaments is vimentin (2, 3), with a molecular weight of 58,000; smaller related peptides (molecular weight, >40,000) are also observed (4, 5) and are thought to be cleavage products of vimentin by a specific protease (6). Vimentin molecules associate to form protofilaments: various models have been proposed for this association (7,8). Protofilaments are associated in bundles; in muscle cells and nucleated erythrocytes, they are crosslinked by another protein, synemin (9, 10); protofilaments are aggregated by cationic proteins (11). Vimentin undergoes phosphorylation, which may have a regulatory significance (12,13). Under the action of colchicine, intermediate filaments collapse into a coiled mass near the nucleus (14); collapse is also caused by intracellular injection of a monoclonal antibody to a common antigen of the intermediate filaments (15) and, in lymphocytes, by capping of surface molecules (16). This and other evidence shows that intermediate filaments are connected to the plasma membrane (10, 17). They also form a cage around the nucleus (18) and appear to be connected to the nuclear membrane (19 for 2 hr; they were then fixed in neutral formalin for 20 min, washed in phosphate-buffered saline, and dried. They were overlaid with photographic emulsion, developed after 1 wk, and counterstained with hematoxylin.Characterization of Material Stained by Hybridoma TllA9e. The material stained by Tl1A9e was characterized by the immunological blotting technique. Cytoplasmic extracts of sparse or confluent NIH 3T3 cultures were prepared (23) by lysing monolayers in 1% (wt/vol) deoxycholate/1% (vol/vol) Nonidet P-40/1 mM phenylmethylsulfonyl fluoride/7 mM NaCI/1 mM MgCl2/100 mM NaF/7 mM Tris-HCI, pH 7.4. The lysates were centrifuged at 4°C for 10 min at 3,000 X g to sediment nuclei. The supernatants were run on 10% NaDodSO4 gels and blotted to nitrocellulose paper (Schleicher & Schuell; BA85, 0.45 ,tm) (24). The nitrocellulose paper was incubated in undiluted culture supernatant of T1lA9e hybridoma or a 1:30 dilution of Tl1A9e mouse serum. Bound TllA9e was located by using either l"I-labeled goat anti-mouse Ig or rabbit anti-mouse IgM followed...

Expression of cell adhesion molecule 1 in malignant pleural mesothelioma as a cause of efficient adhesion and growth on mesothelium

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Alpha-smooth muscle actin-positive fibroblasts promote biliary cell proliferation and correlate with poor survival in cholangiocarcinoma

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