Localization and organization of microfilaments and related proteins in normal and virus‐transformed cells

Abstract

The localization and organization of actin-like microfilaments in normal, SV-40 and adenovirus transformed cells are determined by the coordinated use of light optical, electron optical and biochemical techniques. In adenovirus-type 5 transformed hamster embryo cells, microfilament meshworks appear to be the predominant organizational form of cellular action, while in normal hamster cells, microfilament bundles are prevalent. Differences between 3T3 and SV-40 transformed 3T3 cells are less apparent and may be related to the packing and intracellular distribution of microfilament bundles. Attempts at relating these ultrastructural changes in transformed cells to the images obtained following reaction with fluorescein-labelled myosin fragments and indirect immunofluorescence with smooth muscle myosin antibody are discussed. In several instances the fluorescence microscope images to not correspond to the ultrastructural observations. The results are discussed in terms of the possible relationships between alterations in cytoplasmic contractile elements and the abnormal behavior of transformed cells.

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts.

“…Many types of transformed cells exhibit reduced quantities of microfilament bundles (4,24,48) and are deficient in binding fibronectin fibers to their external surfaces (4,12,38,46) . Recently, p60src protein, the gene product responsible for maintaining the transformed phenotype in Rous sarcoma virustransformed cells (27), also has been localized in the focal contacts (40).…”

Altered distributions of the cytoskeletal proteins vinculin and alpha-actinin in cultured fibroblasts transformed by Rous sarcoma virus.

“…The two types of sites can be distinguished in light microscopy of cell cultures by suitable interference reflection optics (2, 6); focal adhesions appear as black images, whereas close contacts appear gray. Upon transformation of fibroblasts by oncogenic viruses, the intracellular bundles of microfilaments are disrupted and the filaments become disorganized (1,(7)(8)(9)(10)(11)(12), but what happens to the focal adhesion plaques and close contacts is not clear (1, 13).It has recently been shown (14) that a protein of molecular weight 130,000 named vinculin, isolated from chicken gizzard smooth muscle, is in part concentrated at the sites of focal adhesions on the cytoplasmic side of the surface membrane of cultured normal chicken cells. This has been demonstrated by immunofluorescence labeling for vinculin combined with interference reflection microscopy and has been confirmed (15).…”

Morphological evidence for cyclic AMP-induced reverse transformation in vole cells infected with avian sarcoma virus.

“…(i) The cells were grown on glass cover slips and immediately fixed in 2.7% formaldehyde in PBS at pH 7.2 and 37°C for 10 min, washed two to three times in PBS at room temperature, treated with fluorescent phalloidin (1.5 ,ug/ml) in 1% dimethyl sulfoxide for 40 min, and washed two to three times in PBS, and the cover slips were mounted with PBS and glycerol. (ii) Antiactin serum was used as described previously (12,21).…”

“…Studies concerning the status of microfilaments in cultured, transformed cells are more generally in agreement. A shape change occurs in several different cell types infected with sarcoma virus (3,8,42,43,45), simian virus 40 (30,41), or adenovirus (12) that causes these cells to become more rounded as the viral gene is expressed, an event that can be temperature sensitive. Microfilament bundles are lost in most cases (2,8,23,27,30,41,42).…”