Reversion from transformed to normal phenotype by inhibition of protein synthesis in rat kidney cells infected with a temperature-sensitive mutant of Rous sarcoma virus.

Abstract

By the use of a rat kidney cell line infected with a temperature-sensitive Rous sarcoma virus, we have shown that, at permissive temperatures where the cells are transformed, concanavalin A induces a clustering of its cell membrane receptors into patches, and the intracellular smooth muscle myosin-like protein is in a disordered state. By contrast, with infected cells grown at nonpermissive temperatures, the addition of concanavalin A does not alter the uniform distribution of its receptors, and the smooth muscle myosin-like protein is arranged in an ordered filamentous structure. These results are consistent with the hypothesis that the myosin protein is part of an intracellular aggregating-disaggregating complex. In the normal cell it is in its aggregated state and inhibits the lateral mobility of the concanavalin A receptors in the membrane; in the transformed cell the complex is relatively disaggregated and permits the concanava in A receptors to be mobile.The addition of protein synthesis inhibitors to infected cells grown at the permissive temperature causes the cells to change from the transformed phenotype to the normal. Removal of the reversible inhibitors causes the cells to revert to the transformed phenotype. These results show that (i) protein synthesis, presumably of an unstable product of the transforming gene of the temperature-sensitive virus, is required to maintain the transformed state in these infected cells at the permissive temperature; and (ii) protein synthesis is not required for the intracellular myosin-containing complex to revert from its disordered transformed state to its ordered normal state. This suggests that the product of the transforming gene directly or indirectly causes the disaggregation of the myosin-containing complex in the process of transformation.A number of different receptor molecules in the cell membranes of cultured fibroblasts show a greater lateral mobility in transformed than in normal cells (1-4). It has been suggested (5, 6) that these differences in receptor mobility are directly connected to the loss of control of normal cell growth which occurs upon transformation. That is, if cell growth requires the mobility of receptors in the cell membrane (to allow membrane expansion and cytokinesis to occur, for example), then normal cell growth can be inhibited by restricting receptor mobility by means of mechanisms that the transformed cell is no longer able to use (5). Several molecular mechanisms have-been proposed to account for these mobility differences, no one of which has yet been generally accepted. We have a working hypothesis (5,7, 8) to explain these mobility differences. (a) There is an intracellular smooth muscle actomyosin-like protein complex present in nonmuscle cells (9) that forms at least part of the microfilament assemblies that have been observed inside these cells (10-12). (b) These actomyosin-like proteins are, at least in part, peripheral proteins (13) bound to the cytoplasmic surfaces of these cell membranes (8), attached noncoval...

Concanavalin-A-induced transmembrane linkage of concanavalin A surface receptors to intracellular myosin-containing filaments.

“…In the latter case, the linear arrays of Con A receptors were superimposable on the linear arrays of the intracellular myosin-containing filaments, strongly suggesting that the receptors and the filaments had become physically linked. These observations are relevant to our recently published comparative study (7) of the distributions of Con A receptors and myosin-containing structures in NRK cells infected with Rous sarcoma virus. MATERIALS (7).…”

“…These observations are relevant to our recently published comparative study (7) of the distributions of Con A receptors and myosin-containing structures in NRK cells infected with Rous sarcoma virus. MATERIALS (7).…”

“…Fl-Con A was prepared as described (7). Succinylated Con A (Suc-Con A) was a single-stage modified product (9) prepared by Dr. Randy Schekman and characterized elsewhere (10).…”

Effect of transformation by Rous sarcoma virus on the character and distribution of actin in Rat-1 fibroblasts: A biochemical and microscopical study

“…Immunofluoroscence microscopy has demonstrated larger microfilament bundle networks within whole cells (Goldman et al, 1975) and a reduction in the number and thickness of the bundles has been demonstrated following viral transformation by RNA viruses (Ash et al, 1976, Wang & Goldberg, 1979, Boschek et al, 1981 and DNA viruses . However, the limited resolution of this technique does not identify the finer detail of these changes.…”

“…Immunofluorescence studies of virally-transformed cells in culture have frequently shown a reduction in the number of actin-containing stress fibres Ash, Vogt & Singer, 1976;Boscheck et al, 1981;Pollack et al, 1975), and the higher resolution of electron microscopy has indicated that, rather than undergoing a gross disassembly after viral transformation, the microfilaments in the large multifilament bundles (stress fibres) reorganise to form looser submembraneous networks (Goldman et al, Schloss, 1976;Wang & Goldberg, 1976).…”