Ultrastructure and gold-immunolabelling of cell-substratum adhesions (podosomes) in RSV-transformed BHK cells

ISABELLA GAVAZZI 0 MILAN V. NERMUT 0 PIER CARLO MARCHISIO 0 National Institute for Medical Research , The Ridgeway, Mill Hill, London MV7 1AA , UK 1 Dipartimento di Scienze Biomediche e Oncologia Umana, Universita di Torino , 10126 Torino , Italy - Rous sarcoma virus-transformed BHK (RSV/B4BHK) cells develop peculiar dot-like adhesions, that have been named podosomes, which, in the presence of serum, aggregate into ring- or crescentshaped adhesion sites, the rosettes of podosomes. We have used the lysis-squirting technique and gold-immunolabelling to study the 3D-organisation of podosomes and the location of vinculin, gelsolin, phosphotyrosine-containing proteins and pp60src at an ultrastructural level. Podosomes appear to be conical bodies, 01-0-5/im high, made by a dense aggregation of actin oligomers and several associNeoplastic transformation involves a marked redistribution of the cytoskeleton that affects cell shape, motility and adhesion (for review see Burridge, 1986). The most prominent feature of fibroblasts transformed by oncogenes coding for tyrosine protein kinases in vitro is the disappearance of actin microfilament bundles of the stress fibre type, and an appearance of a wealth of surface protrusions containing F-actin (Boschek et al. 1981). In cells transformed by oncogene9 coding for tyrosine protein kinases, F-actin is also associated with punctate ventral membrane adhesions, which have been classified as close contact adhesion sites (Carley et al. 1981; DavidPfeuty & Singer, 1980; Rohrschneider, 1979; Tarone et al. 1985). Small dot-like adhesions in Rous sarcoma virus (RSV)-transformed cells have been called podosomes (Tarone et al. 1985), because of their possible function as cellular feet. Podosomes can be seen in interference reflection microscopy (IRM) as individual black dots surrounded by a white ring, but tend to form clusters in the shape of rosettes, rings or scrolls in the presence of low concentrations of serum (Tarone et al. 1985). In RSV-transformed BHK cells, along with the reduction of stress fibres, adhesion plaques of the focal contact type are reduced in number or are completely absent. The most frequent form of substratum adhesion ated proteins, connected in the rosette by actin filaments. Gelsolin and some phosphotyrosine-containing proteins are found within the podosomes, often associated with the actin filaments, while vinculin is found predominantly at the podosome periphery, associated with microfilaments, and pp60"rc is located on the adjacent plasma membrane. in cells transformed by viruses that code for a tyrosine kinase are dot-like (punctate) structures containing many cytoskeleton-associated proteins such as vinculin (Carley et al. 1981; David-Pfeuty & Singer, 1980; Geiger, 1979; Marchisio et al. 1987; Shriver & Rohrschneider, 1981; Tarone et al. 1985), talin (Burridge & Connell, 1983a,6; Marchisio et al. 1987), tt-actinin (Carley et al. 1985; David-Pfeuty & Singer, 1980; Lazarides & Burridge, 1975; Shriver & Rohrschneider, 1981), fimbrin (Carley et al. 1985) and gelsolin (Marchisio et al. 1987; Wang et al. 1984), as shown by immunofluorescence. Structures similar in size and morphology to podosomes have also been found in monocytes and monocytederived cells like osteoclasts and macrophages (Marchisio et al. 1984a; Marchisio et al. 1987) and in B-chronic lymphocytic leukemia cells (Caligaris-Cappio et al. 1986; Marchisio et al. 1988a). Moreover, isolated podosomes and rosettes can be induced in normal fibroblasts by treatment with orthovanadate (Marchisio et al. 19886), and this suggests that increasing the level of tyrosinephosphorylated proteins in cells may trigger per se the events leading to microfilament redistribution in podosomes. In this paper we describe the results of an investigation into the ultrastructure of podosomes in RSV-transformed BHK cells, and immunolocalisation of some of the constituent proteins. We find that the major difference from focal adhesions (normal cells) is in the organisation of actin microfilaments, i.e. in the absence of stress fibres, and in a certain dislocation of vinculin. To expose large areas of ventral membrane in adherent cells, preserving at the same time the topography of the membrane-associated structure, the lysis-squirting technique (Nermut, 1982) has proved most successful (Nicol and Nermut, 1987) and has therefore been used here. For ultrastructural studies the exposed cytoplasmic surface of ventral membranes was replicated with platinum-carbon, after freeze-drying or critical-point-drying. Whole mount preparations have also been used. The colloidal gold immunoreplica technique (Nicol et al. 1987) was used in localising some of the important proteins such as vinculin, gelsolin, pp60jrc and phosphotyrosine-containing proteins. Materials and methods Cells Baby Hamster Kidney (BHK) fibroblasts transformed by the Bryan high titre strain of Rous sarcoma virus (RSV/B4-BHK) were a gift from Dr L. Warren. Control untransformed BHK 21-C13 cells were obtained from Dr I. Macpherson. Both cell lines were propagated at 37 C in a water-saturated atmosphere of 95 % air-5 % CO2 in Alpha-modified Eagle's medium (Alpha-MEM) containing antibiotics and supplemented with 10% foetal calf serum (FCS). Cells were harvested from culture dishes by EGTA treatment (lmM-EGTA in PBS) and plated in Alpha-MEM supplemented with 2 % FCS (for RSV/B4-BHK cells) or 10% FCS for untransformed cells, either on glass coverslips or on carbon-coated gold grids (G100 hexagonal mesh). Sulphuric acid-washed glass coverslips or gold grids with Formvar-carbon support film were coated with purified plasma fibronectin (10/igmP1) in ISOmM-sodium chloride, lOmM-sodium phosphate buffer, p H 7 ' 4 (PBS) for 60min at room temperature. Preparation of ventral membranes Normal or transformed cells were seeded on glass coverslips or gold grids coated with Formvar-carbon films 16 or 18 h before the experiments. All subsequent steps were carried out at room temperature. Cells grown on coverslips were rinsed for 10 s in PIPES buffer, p H 6 1 (lOOmM-KCl, SmM-MgCl2, 20mM-PIPES, 3 mM-EGTA) and transferred to 20 % PIPES, pH 6-1 for up to 4 min. Using a syringe equipped with a special nozzle (Nermut, 1986) the coverslip was squirted over with 5 ml PIPES buffer, p H 6 - l , then rinsed in PIPES, pH7-0 and transferred into fixative. Cells grown on grids were incubated for 3 min in PIPES buffer, pH 6-1, transferred to 20 % PIPES for 45 s, and squirted over with 2 ml PIPES buffer using a syringe fitted with a hypodermic needle. The grid was then rinsed in PIPES, pH 7-0 and transferred into the appropriate fixative as described below. Fixation For ultrastructural studies, membranes were fixed in 2-5% glutaraldehyde in PIPES buffer, pH7-0 for 15 min, rinsed in PIPES buffer, pH 7-0, extensively washed in distilled water and treated with 1 % uranyl acetate for 1 min before freeze-drying or with 2 % OsC>4 in lOOmM-sodium cacodyl (...truncated)