Eight mouse monoclonal antibodies, CH1, CH106, CH291, CL2, CG1, CG3, CG beta 2 and CG beta 6, against chicken tropomyosin isoforms have been prepared and characterized. The antigens recognized by these isoform-specific monoclonal antibodies were identified by both solid-phase radioimmunoassay and protein immunoblotting. To some extent, most antibodies showed isoform-specific, but one (CG3) recognized all isoforms of tropomyosin from chicken materials. The effects of monoclonal antibodies on the binding of cardiac tropomyosin to F-actin were investigated. Antibodies CH1, CH106, and CH291 had the ability to interfere with the binding of tropomyosin to F-actin, whereas others appeared to have no effect. Monoclonal antibody CL2 was able to distinguish the skeletal muscle tropomyosin-enriched microfilaments from the fibroblastic tropomyosin-enriched microfilaments of differentiating muscle cells. This antibody will be most useful for studying the compartmentalization of microfilaments and microfilament-associated proteins, particularly actin and tropomyosin isoforms during muscle differentiation. Immunofluorescence microscopy with CG1 antibody which recognized CEF tropomyosin isoforms 1 and 3 revealed the continuous staining of stress fibers in some populations of CEF cells. On the other hand, both periodic fluorescent staining and continuous staining of stress fibers were observed with CG3 antibody in all CEF cells.

Seven polypeptides (a, b, c, 1, 2, 3a, and 3b) have been previously identified as tropomyosin isoforms in chicken embryo fibroblasts (CEF) (Lin, J. J.-C., Matsumura, F., and Yamashiro-Matsumura, S., 1984, J. Cell. Biol., 98:116-127). Spots a and c had identical mobility on two-dimensional gels with the slow-migrating and fast-migrating components, respectively, of chicken gizzard tropomyosin. However, the remaining isoforms of CEF tropomyosin were distinct from chicken skeletal and cardiac tropomyosins on two-dimensional gels. The mixture of CEF tropomyosin has been isolated by the combination of Triton/glycerol extraction of monolayer cells, heat treatment, and ammonium sulfate fractionation. The yield of tropomyosin was estimated to be 1.4% of total CEF proteins. The identical set of tropomyosin isoforms could be found in the antitropomyosin immunoprecipitates after the cell-free translation products of total poly(A)+ RNAs isolated from CEF cells. This suggested that at least seven mRNAs coding for these tropomyosin isoforms existed in the cell. Purified tropomyosins (particularly 1, 2, and 3) showed different actin-binding abilities in the presence of 100 mM KCl and no divalent cation. Under this condition, the binding of tropomyosin 3 (3a + 3b) to actin filaments was significantly weaker than that of tropomyosin 1 or 2. CEF tropomyosin 1, and probably 3, could be cross-linked to form homodimers by treatment with 5,5'-dithiobis-(2-nitrobenzoate), whereas tropomyosin a and c formed a heterodimer. These dimer species may reflect the in vivo assembly of tropomyosin isoforms, since dimer formation occurred not only with purified tropomyosin but also with microfilament-associated tropomyosin. The expression of these tropomyosin isoforms in Rous sarcoma virus-transformed CEF cells has also been investigated. In agreement with the previous report by Hendricks and Weintraub (Proc. Natl. Acad. Sci. USA., 78:5633-5637), we found that major tropomyosin 1 was greatly reduced in transformed cells. We have also found that the relative amounts of tropomyosin 3a and 3b were increased in both the total cell lysate and the microfilament fraction of transformed cells. Because of the different actin-binding properties observed for CEF tropomyosins, changes in the expression of these isoforms may, in part, be responsible for the reduction of actin cables and the alteration of cell shape found in transformed cells.

No abstract