Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes

Abstract

Rous sarcoma virus-transformed BHK cells (RSV/B4-BHK) adhere to a fibronectin-coated substratum primarily at specific dot-shaped sites. Such sites contain actin and vinculin and represent close contacts with the substratum as revealed by interference reflection microscopy. Only a few adhesion plaques and actin filament bundles can be detected in these cells as compared to untransformed parental fibroblasts. In thin sections examined with transmission electron microscopy (TEM) these adhesion sites correspond to short protrusions of the ventral cell surface that contact the substratum at their apical portion. These structures, which may represent cellular feet, are therefore called podosomes. By screening a number of different transformed fibroblasts plated on a fibronectin-coated substratum we find that podosomes are common to mammalian and avian cell lines transformed either by Rous sarcoma virus (RSV) or by Fujinami avian sarcoma virus (FSV), whose oncogenes encode specific tyrosine kinases. Using antibodies reacting with phosphotyrosine in immunofluorescence experiments, we show that phosphotyrosine-containing molecules are concentrated in podosomes. Podosomes are not detected in fibroblasts transformed by other retroviruses (Snyder-Theilen sarcoma virus, Abelson leukemia virus and Kirsten sarcoma virus) or by DNA tumor viruses (polyoma, SV40), indicating that podosome-mediated adhesion in transformed fibroblasts is related to the peculiar properties of some oncoproteins and possibly to their tropism for adhesion systems. Podosomes and adhesion plaques, although similar in cytoskeletal protein composition, have different mechanisms and kinetics of formation. Assembly of podosomes, in fact (i) does not require fetal calf serum (FCS) in the adhesion medium, that is necessary for the organization of adhesion plaques; (ii) does not require protein synthesis; and (iii) is insensitive to the ionophore monensin, that prevents adhesion plaque formation. Moreover, during attachment to fibronectin-coated dishes, podosomes appear in the initial phase (60 min) of attachment, while adhesion plaques require a minimum of 180 min. In conclusion podosomes of RSV- and FSV-transformed fibroblasts represent a phenotypic variant of adhesion structures.