Surface ruffles as markers for studies of cell transformation by Rous sarcoma virus.

Abstract

Confluent chick embryo fibroblasts infected with the Ts68 mutant of Rous sarcoma virus were examined by scanning electron microscopy at the permissive (360) and nonpermissive (410) temperatures for transformation. Infected cells shifted from 410 to 360 undergo a change in shape from elongated to rounded. This process is preceded by the appearance of surface ruffles on the cell. These surface ruffles are not observed on cells maintained at 410, appear as early as 0.5 hr after a shift to 360, and are common on cells maintained at 360. By 3.5 hr after the shift from 410 to 360, cultures appear fully transformed by the criteria of cell roundedness and the presence of surface ruffles. This surface alteration of cells is the earliest event of those so far reported during the transformation process and is not dependent upon protein synthesis and extracellular plasminogen during the period of temperature shift. Transformation of cultured cells by RNA tumor viruses is accompanied by changes in morphology and other biochemical functions. When chick embryo fibroblasts are infected with Kawai and Hanafusa's Ts68 mutant of the SchmidtRuppin strain (subgroup A) of Rous sarcoma virus (RSV), the expression of the transformation phenotype of infected cells is dependent upon temperature, i.e., permissive at 360 and nonpermissive at 410, whereas other viral related functions are reported to be the same (1). Alterations in several transformation-related biochemical functions in Ts68-infected cells have been reported during the time course of a temperature shift from 410 to 360. In brief, there was an increase in glucose uptake from 4 to 6 hr; loss of membrane-associated protein of molecular weight 45,000 from 3 to 6 hr (2); secretion of proteases from 8 to 20 hr (3); and reduction in a surface protein of 250,000 daltons (LETS or Z protein) after 20 hr (4).However, the most remarkable observation has been the rapid change in cell morphology from a flattened and elongated to a more rounded, refractile shape during temperature shift. This significant change can be detected by use of the light microscope as early as 1.5-6 hr (1,4 (5). Cultures were not washed prior to fixation to avoid dislodging poorly attached cells. The medium was sucked off and a glutaraldehyde solution, 2.5% in 0.05 M cacodylate and half-strength phosphate-buffered saline at pH 7.2, was added gently to the culture dish containing the coverslip for 20 min. Cultures were then washed in 0.1 M cacodylate, postfixed for 20 min in 2% osmium tetroxide buffered with 0.1 M cacodylate at pH 7.2, and finally washed with distilled water. The cells were then subjected to sequential dehydration by transfer through a graded series of water/acetone and then acetone/amyl acetate mixtures, and finally dried at the critical point of CO2 in a critical point dryer. Specimens were stored in a desiccator under vacuum until coated with a 200-300 A film of gold. The preparations were viewed in a JSM-U3 scanning electron microscope at 25 kV. Transmission electron micrographs were m...

Transformation by Rous sarcoma virus induces clathrin heavy chain phosphorylation.

“…These results indicate the presence of clathrin heavy chain phosphatase activity in the membranebound fraction. Transformation by RSV is known to cause phenotypic changes, in part through alterations in cytoskeletal organization (Ambros et al, 1975). We compared the distribution of clathrin in normal and transformed cells by immunofluorescent staining using either of two anti-clathrin light chain antibodies.…”

“…Morphological alterations of the cell surface and cytoskeleton are among the earliest manifestations of the src gene product (Ambros et al, 1975;Boschek et al, 1981). Among the cell surface changes, it has been observed that a number of receptors, including those for epidermal growth factor and thrombin, are cleared from the surface by endocytic events that may be altered by pp60 ~-s~ (Decker, 1983;Cooper et al, 1983;Zetter et al, 1977).…”

Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts.

“…The earliest phenotypic manifestation of src activation is membrane ruffling along the cell margins when fibroblasts infected with a temperature-sensitive v-src mutant are shifted from the restrictive to permissive temperature (45). Activation of a temperature-sensitive v-src mutant also leads to association of activated v-src with the detergent-insoluble cytoskeleton, and is accompanied by changes in both actin organization and the distribution of the actin-binding proteins ␣-actinin and vinculin (46,47).…”

Fourier analysis of cell motility: correlation of motility with metastatic potential.

“…These types of motility may be expressed after several types of biological stimulation. For example, oncogenes (1)(2)(3)(4), hormones (5), and various growth factors (6)(7) have been demonstrated to induce membrane ruffling in cultured cells. It is becoming apparent that quantitative information describing these types of cell movement is needed to study and understand better the processes of cell motility.…”