Ultrastructure and Gold-Immunolabelling of Cell-Substratum Adhesions (Podosomes) in RSV-Transformed BHK Cells

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

ISABELLA GAVAZZI1*, MILAN V. NERMUT1 and PIER CARLO MARCHISIO2

1 National Institute for Medical Research, The Ridgeway, Mill Hill, London MV7 1AA, UK ^Dipartimento di Scienze Biomediche e Oncologia Umana, Universita di Torino, 10126 Torino, Italy

•Author for correspondence at: Dipartimento di Scienze Biomediche e Oncologia Umana, Universita di Torino, Corso Massimo d'Azeglio 52, 10126 Torino, Italy

Summary

Rous sarcoma virus-transformed BHK (RSV/B4- ated proteins, connected in the rosette by actin BHK) cells develop peculiar dot-like adhesions, that filaments. Gelsolin and some phosphotyrosine-con- have been named podosomes, which, in the pres- taining proteins are found within the podosomes, ence of serum, aggregate into ring- or crescent- often associated with the actin filaments, while shaped adhesion sites, the rosettes of podosomes. vinculin is found predominantly at the podosome We have used the lysis-squirting technique and periphery, associated with microfilaments, and gold-immunolabelling to study the 3D-organisation pp60"rc is located on the adjacent plasma mem- of podosomes and the location of vinculin, gelsolin, brane. phosphotyrosine-containing proteins and pp60src at an ultrastructural level. Podosomes appear to be conical bodies, 01-0-5/im high, made by a dense Key words: electron microscopy, RSV transformation, aggregation of actin oligomers and several associ- adhesion, cytoskeleton, gold-immunolabelling.

Introduction in cells transformed by viruses that code for a tyrosine kinase are dot-like (punctate) structures containing many Neoplastic transformation involves a marked redistri- cytoskeleton-associated proteins such as vinculin (Carley bution of the cytoskeleton that affects cell shape, motility et al. 1981; David-Pfeuty & Singer, 1980; Geiger, 1979; and adhesion (for review see Burridge, 1986). The most Marchisio et al. 1987; Shriver & Rohrschneider, 1981; prominent feature of fibroblasts transformed by onco- Tarone et al. 1985), talin (Burridge & Connell, 1983a,6; genes coding for tyrosine protein kinases in vitro is the Marchisio et al. 1987), tt-actinin (Carley et al. 1985; disappearance of actin microfilament bundles of the stress David-Pfeuty & Singer, 1980; Lazarides & Burridge, fibre type, and an appearance of a wealth of surface 1975; Shriver & Rohrschneider, 1981), fimbrin (Carley et protrusions containing F-actin (Boschek et al. 1981). In al. 1985) and gelsolin (Marchisio et al. 1987; Wang et al. cells transformed by oncogene9 coding for tyrosine pro- 1984), as shown by immunofluorescence. tein kinases, F-actin is also associated with punctate Structures similar in size and morphology to podo- ventral membrane adhesions, which have been classified somes have also been found in monocytes and monocyte- as close contact adhesion sites (Carley et al. 1981; David- derived cells like osteoclasts and macrophages (Marchisio Pfeuty & Singer, 1980; Rohrschneider, 1979; Tarone et et al. 1984a; Marchisio et al. 1987) and in B-chronic al. 1985). Small dot-like adhesions in Rous sarcoma virus lymphocytic leukemia cells (Caligaris-Cappio et al. 1986; (RSV)-transformed cells have been called podosomes Marchisio et al. 1988a). Moreover, isolated podosomes (Tarone et al. 1985), because of their possible function as and rosettes can be induced in normal fibroblasts by cellular feet. Podosomes can be seen in interference treatment with orthovanadate (Marchisio et al. 19886), reflection microscopy (IRM) as individual black dots and this suggests that increasing the level of tyrosine- surrounded by a white ring, but tend to form clusters in phosphorylated proteins in cells may trigger per se the the shape of rosettes, rings or scrolls in the presence of events leading to microfilament redistribution in podo- low concentrations of serum (Tarone et al. 1985). somes. In RSV-transformed BHK cells, along with the re- In this paper we describe the results of an investigation duction of stress fibres, adhesion plaques of the focal into the ultrastructure of podosomes in RSV-transformed contact type are reduced in number or are completely BHK cells, and immunolocalisation of some of the absent. The most frequent form of substratum adhesion constituent proteins. We find that the major difference Journal of Cell Science 94, 85-99 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 85 from focal adhesions (normal cells) is in the organisation For immunolabelling, coverslip- or grid-attached ventral of actin microfilaments, i.e. in the absence of stress membranes were prefixed either in 0-1-0-5 % glutaraldehyde in fibres, and in a certain dislocation of vinculin. PIPES, pH7-0 (for polyclonal antibodies and anti-gelsolin antibody) for 30 min, or in 3 % paraformaldehyde in PBS, To expose large areas of ventral membrane in adherent pH7-0 (for monoclonal antibodies) for 15 min. After immuno- cells, preserving at the same time the topography of the labelling, the specimens were fixed and dried as described membrane-associated structure, the lysis-squirting tech- above. nique (Nermut, 1982) has proved most successful (Nicol and Nermut, 1987) and has therefore been used here. For Critical-point-drying ultrastructural studies the exposed cytoplasmic surface of Fixed ventral membranes were washed several times in distilled ventral membranes was replicated with platinum-car- water, dehydrated in graded ethanol and critical-point-dried in bon, after freeze-drying or critical-point-drying. Whole a SAMDRI-780 apparatus (Tousimis, Rockville, Maryland, mount preparations have also been used. USA). The coverslip-attached ventral membranes were then The colloidal gold immunoreplica technique (Nicol et unidirectionally shadowed with platinum-carbon and repli- al. 1987) was used in localising some of the important cated with carbon. The grid-attached ventral membranes were proteins such as vinculin, gelsolin, pp60jrc and phospho- either directly observed in transmission electron microscopy tyrosine-containing proteins. (TEM) or lightly shadowed prior to observation.

Freeze-drying Materials and methods Coverslips with fixed membranes were rinsed several times in distilled water, touched to blotting paper to remove excess Cells water, plunged quickly into liquid nitrogen (Nermut, 1977) and Baby Hamster Kidney (BHK) fibroblasts transformed by the mounted under liquid nitrogen on a flat specimen stage of the Bryan high titre strain of Rous sarcoma virus (RSV/B4-BHK) freeze-etching unit (Balzers BAF 300). The stage was then were a gift from Dr L. Warren. Control untransformed BHK transferred into the vacuum chamber using the counter-flow 21-C13 cells were obtained from Dr I. Macpherson. Both cell loading system. Freeze-drying was carried out at — 85°C for 6 lines were propagated at 37 °C in a water-saturated atmosphere 20-45 min at a vacuum better than 5xlO~ mbar. This was of 95 % air-5 % CO2 in Alpha-modified Eagle's medium followed by shadowing with platinum-carbon (at 40°) and (Alpha-MEM) containing antibiotics and supplemented with rotary replication with carbon. Replicas were floated off the 10% foetal calf serum (FCS). glass onto 4% hydrofluoric acid and transferred through Cells were harvested from culture dishes by EGTA treatment distilled water to sodium hypochlorite for cleaning. After 2-4 h (lmM-EGTA in PBS) and plated in Alpha-MEM sup- the replicas were washed on three changes of distilled water and plemented with 2% FCS (for RSV/B4-BHK cells) or 10% picked up on G400 or G200 mesh grids. FCS for untransformed cells, either on glass coverslips or on carbon-coated gold grids (G100 hexagonal mesh). Sulphuric Gold-immunolabelling acid-washed glass coverslips or gold grids with Formvar-car- The fixed ventral membranes were: (1) rinsed in PIPES or bon support film were coated with purified plasma fibronectin PBS; (2) incubated with 0-1 M-ethanolamine in PIPES, pH7-0 1 (10/igmP ) in ISOmM-sodium chloride, lOmM-sodium phos- for 15 min (omitted when formaldehyde was used); (3) rinsed in phate buffer, pH7'4 (PBS) for 60min at room temperature. Tris-buffered saline (TBS; 20mM-Tris, 154mM-NaCl, 20mM- NaN3) pH8-2; washed in 0-1 % bovine serum albumin (BSA) Preparation of ventral membranes and 0-2% gelatin in TBS buffer, pH8-2 for 10 min, and (4) Normal or transformed cells were seeded on glass coverslips or incubated with the primary antibody for 45 min, then rinsed in gold grids coated with Formvar-carbon films 16 or 18 h before 0-1 % BSA, 0-2 % gelatin in TBS, pH 8-2 for 15 min. the experiments. All subsequent steps were carried out at room When using monoclonal antibodies the ventral membranes temperature. were then: (1) incubated with rabbit anti-mouse IgG for Cells grown on coverslips were rinsed for 10 s in PIPES 45 min; (2) washed in 0-1 % BSA, 0-2 % gelatin in TBS, pH 8-2 buffer, pH61 (lOOmM-KCl, SmM-MgCl2, 20mM-PIPES, for 15 min; (3) incubated with gold-conjugated goat anti-rabbit 3 mM-EGTA) and transferred to 20 % PIPES, pH 6-1 for up to IgG for 30 min, and (4) washed in 0-1 % BSA, 0-2% gelatin in 4 min. Using a syringe equipped with a special nozzle (Nermut, TBS, pH8-2 for 10 min. 1986) the coverslip was squirted over with 5 ml PIPES buffer, When using polyclonal antibodies, the incubation with rabbit pH6-l, then rinsed in PIPES, pH7-0 and transferred into anti-mouse IgG was omitted. Coverslips and grids were then fixative. rinsed twice in TBS and postfixed in 2 % glutaraldehyde in PBS Cells grown on grids were incubated for 3 min in PIPES for 30-60 min. buffer, pH 6-1, transferred to 20 % PIPES for 45 s, and squirted All steps were performed at room temperature. over with 2 ml PIPES buffer using a syringe fitted with a Control experiments, in which a rabbit pre-immune serum or hypodermic needle. The grid was then rinsed in PIPES, pH 7-0 1 % BSA in TBS (in case of monoclonal antibodies) were and transferred into the appropriate fixative as described below. substituted for the primary antibody, were routinely carried out. Fixation For ultrastructural studies, membranes were fixed in 2-5% Immunoreagents glutaraldehyde in PIPES buffer, pH7-0 for 15 min, rinsed in A monoclonal antibody against chicken gizzard vinculin was PIPES buffer, pH 7-0, extensively washed in distilled water and purchased from Bio Yeda (Rehovot, Israel) and used at 1:10 treated with 1 % uranyl acetate for 1 min before freeze-drying or dilution. A mouse monoclonal antibody against human platelet with 2% OsC>4 in lOOmM-sodium cacodylate buffer, pH7-2 for gelsolin was provided by Dr J. Bryan and used at 1: 10, 1:20 05-1 h followed by 1 % uranyl acetate for 1 min for critical- and 1:30 dilutions of a hybridoma supernatant. A polyclonal point-drying. serum against pp60Ifr expressed in Esdierichia coli was pro-

86 /. Gavazzi et al. vided by courtesy of Dr R. L. Erikson and Dr S. Kellie and were also present (Fig. 1), although they were usually used at 1:50, 1:100 and 1:150 dilutions. Anti-azobenzyl less prominent than in other fibroblastic cell lines (Nicol phosphonate (ABP) antibodies were raised in rabbits, affinity- et al. 1987); the stress fibre bundles often splayed apart purified and characterised as previously reported (Comoglio et into single filaments at the termini to associate with the al. 1984). These antibodies were found to cross react specifi- membrane. cally with phosphotyrosine by radioimmunoassay, and to pre- cipitate selectively phosphotyrosine proteins. ABP antibodies In replicas of RSV/B4-BHK cells, large sheets of were used at 40, 26 or 20ng ml"'. ventral membrane were seldom seen, in agreement with Goat anti-rabbit IgG-lOnm or 15 nm gold conjugates were the IRM observation. Associated with the remaining purchased from Janssen (Beerse, Belgium) or Bio Clin (Cardiff, portions of the ventral membrane were actin filaments, UK). usually in random orientation, as well as clathrin sheets and pits, coated vesicles and clusters of small particles Light microscopy (LM) (Fig. 2 and 3A). Isolated small patches of clathrin-coated Cells and ventral membrane preparations were examined and membranes (Nicol & Nermut, 1987) were also frequently recorded (Kodak Technical Pan 2415 35 mm film) using a Leitz observed (Fig. 3B). Correlation with IRM showed that Ortholux II equipped with X50/1-0 and X 100/1-32 PHACO they corresponded to point contacts. Other components RK objectives for IRM. such as ribosomes and intermediate filaments were only Correlated LM and electron microscopy (EM) was per- occasionally observed on the membrane or the back- formed as described by Nicol and Nermut (1987). Briefly, a ground. reference mark (square) was scratched onto the coverslip prior to plating cells onto it. Cells within the labelled area were 'Ridged surface' vesicles (Nicol & Nermut, 1987) were photographed in IRM both before and after squirting. The observed in BHK cells, but not on the membranes of coverslip was then processed for EM and replicas of the marked RSV/B4-BHK cells. This may be due, in part, to the loss area were collected onto Formvar-coated G100 hexagonal grids. of larger and more loosely attached sheets of membrane in The regions of interest were then identified by direct compari- RSV/B4-BHK cells. son with the interference reflection micrographs. The rosette of podosomes Electron microscopy A correlated EM/lRM study was made to identify in Replicas and whole mounts of ventral membranes were viewed replicas of RSV/B4-BHK cells the area corresponding to using a Philips EM 300 or a JEOL 1200EX electron microscope the rosette of podosomes as seen by IRM. This involved operating at 60, 80 or 100 kV. Stereo pairs of electron micro- taking light micrographs of coverslip-attached cells be- graphs were taken with a 12° or 14° tilt angle, depending on fore and after squirting and preparing a replica of the magnification. Height measurements were carried out from stereo pairs of electron micrographs using a mirror stereoscope same coverslip (Nermut et al. 1986; Nicol & Nermut, (Cartographic Engineering Ltd, UK). 1987). Thus, the same rosette was observed in the living cell, i.e. before squirting, and also after squirting, both in IRM and in the replica by EM (Fig. 4A,B). Results The rosettes of podosomes vary in shape and size, but the most commonly observed are ring- or crescent-shaped Effect of lysis-squirting structures, 5-15 [im in diameter (both from IRM and in The effect of lysis-squirting on both cell lines in our EM). experimental conditions was assessed by IRM before and The basic structural components of the rosette (Figs 5 after squirting. It appeared that squirting removed not and 6) are small conical bodies (the individual podo- only the upper portion of the cell but also some of the somes) raised above the background and consisting of loosely attached ventral membrane. Large sheets of densely packed thin filaments. The podosomes (about membrane were seen in BHK cells, but much less in 0-4 nm in diameter) are often joined together so tightly RSV/B4-BHK cells. However, after squirting, both the that they form a continuous ridge (Fig. 5). Measure- area comprising the rosette of podosomes and small point ments from stereo micrographs showed that the height contacts were usually preserved. above the substratum in this region was 0-1-0-4 fun. The localisation of actin, vinculin and gelsolin in both Crater-like structures with well defined edges were also intact and squirted cells was also studied using immuno- seen both at the periphery and at other sites of the fluorescence microscopy. In squirted cells there was no network. obvious change in the pattern of labelling (results not Clathrin sheets and clusters of small particles were shown), suggesting that the organisation and overall observed on the plasma membrane, both at the periphery distribution of these cytoskeletal components were and in the central area of the rosette. In some cases the retained. central portion of the plasma membrane was removed by squirting (Fig. 5), and in others it was still present Ultrastructure of RSV/B4-BHK ventral membranes (Fig. 6A), slightly raised above the support and often Replicas of untransformed BHK cells were prepared first studded with small particles and clathrin sheets. Though to allow comparison of the morphological changes the general features of the rosette remain constant, details brought about by RSV-induced transformation. After can differ from case to case. Sometimes the individual lysis-squirting, rather large ventral membrane fragments podosomes were clearly distinguishable (Fig. 6A); in were found, covered with groups of small particles, other cases they merged into a tightly packed ring-like clathrin sheets and pits and thin filaments. Stress fibres structure, where individual podosomes were not easily

Podosomes in RSV transformed BHK cells 87 ?. yfc ••&••.:•&

Fig. 1. Platinum-carbon replica of a BHK ventral membrane, showing a field densely covered with microfilament bundles (stress fibres). Bar, 1 \im. (Photographically reversed to show shadows in black.) Fig. 2. Platinum-carbon replica of a RSV/B4-BHK ventral membrane. Note absence of stress fibres. The membrane is covered with single filaments, clathrin sheets (arrow), coated pits and groups of small particles. Bar, 1 ^tfn. (Photographically reversed to show shadows in black.) discerned (Fig. 5). These different appearances may nisation of the filamentous system in whole-mount prep- correspond to different stages in the development of the arations where the entire structure, and not just the rosette. The dense packing of actin in the rosette made it surface, can be visualized in stereo micrographs. Cells difficult to discern individual microfilaments. grown on grids produced many ventral membranes upon Since replicas reveal only the surface of podosomes, it squirting, with either individual podosomes or one large appeared necessary to study the three-dimensional orga- rosette of podosomes. Single podosomes appeared as

88 /. Gavazzi et al. Fig. 3. A. Groups of small membrane-associated particles on protoplasmic surface of ventral membranes of RSV/B4-BHK cells at higher magnification. Replica of freeze-dried specimen. Bar, 100nm. B. Patches of ventral membrane left behind after heavy squirting of RSV/B4-BHK cells. They are coated with clathrin sheets. Bar, 100 nm. (Photographically reversed to show shadows in black.) Fig. 4. A. IRM showing a rosette of podosomes (arrow) in RSV/B4-BHK ventral membrane after squirting. Bar, lOjim. B. Replica of the same rosette critical point-dried and shadowed. A stereo pair of micrographs revealed slight elevation of the central area (white in 'A') and a high network of coiled filaments at the periphery (dark ring in 'A'). Periphery of the rosette is marked with arrowheads, and the central elevation with a star. Bar, 1 /im. (Photographically reversed to show shadows in black.) dense bodies surrounded by filaments, which were well Gold-immunolabelling of proteins associated with visualized in positively stained preparations (Fig. 6B). podosomes The electron-dense cores contained small 'granules' Some of the proteins that were shown by immunofluor- (about 10-20nm). The thickness of the filaments was escence microscopy to be associated with cell-substratum 5-10 nm, suggesting that they are actin microfilaments. attachment sites were studied at the ultrastructural level This is also supported by immunofluorescence mi- using mono- or polyclonal antibodies and colloidal gold- croscopy, which showed the presence of actin in podo- conjugated secondary antibodies. somes (Marchisio et al. 19846; Tarone et al. 1985). This study was carried out using both replicas and

Podosomes in RSV transformed BHK cells 89 Fig. 5. Platinum-carbon replica of a rosette of podosomes. Individual podosomes are not distinguishable in this tightly packed rosette. Freeze-dried and shadowed. Note some of the filaments protruding high above the ridge (arrowheads). Below: height profile of the rosette along the line, as obtained from stereo micrographs. Zero point at right lower end of the line. Bar, 1 ^im. (Photographically reversed to show shadows in black.)

90 /. Gavazzi et al. Fig. 6. Whole mount preparation of RSV/B4-BHK ventral membrane. Critical point-dried and shadowed (A) or unshadowed (B). (A) shows the whole rosette with many dark podosomes densely packed in the upper part of the rosette (arrowheads), but still isolated in the lower part (arrows). Central area is free of filaments or podosomes, but unlike the view in Fig. 5 the membrane has not been removed. The filamentous nature of dense bodies is better shown in the positively stained (unshadowed) preparation (B). Note the filaments connecting individual podosomes (arrows). Bars, 500nm.

Podosomes in RSV transfonned BHK cells 91 whole mounts of ventral membranes. The latter, particu- filaments and within the electron-dense cores, as shown larly when combined with stereo observation, provided by stereo-imaging (not shown). information about the localisation of proteins within the pp60src. There is evidence from cell fractionation and structural complex, and not just at its surface. ultrathin sections that pprjCr"1^, in its phosphorylated Vinculin. A monoclonal vinculin antibody was used to form, is associated with the inner face of the plasma study the ultrastructural localisation of vinculin in membrane (Courtneidge et al. 1980; Kmeger et al. RSV/B4-BHK ventral membrane. Because commer- 1980a,6; Willingham et al. 1979). It has also been cially available gold-conjugated goat anti-mouse IgG reported that ppoO1"7 is associated with microfilaments at proved less satisfactory, a bridge technique was employed cell-substratum adhesion sites (Henderson & Rohr- using rabbit anti-mouse IgG followed by gold-conjugated schneider, 1987; Rohrschneider, 1980; Shriver & Rohr- goat anti-rabbit IgG. schneider, 1981). In replicas of RSV/B4-BHK ventral In both the replicas and whole mount preparations, the membranes labelled with polyclonal ppoO1"7 antibody, label was associated with aggregates of small particles or only a low level of gold labelling was found on top of the with the interconnecting filaments (Fig. 7A). Dense rosette; most of the gold particles were on clusters of 'cores' were only occasionally labelled. However, the membrane-associated particles, found on the membrane presence of gold particles within the 'cores' cannot be surrounding the rosette (Fig. 9B). The three-dimen- ruled out because of the high electron density of the sional network of actin filaments was very sparsely 'cores'. It is important to note that immunofluorescence labelled. labelling for vinculin revealed either a diffuse staining In whole mounts, gold labelling was usually confined correlating with the podosome in IRM (Tarone et al. to the area of the rosette, but with no preferential 1985, and our own observation) or a ring-like pattern, association with the filaments. In particular, it appeared suggesting the absence of vinculin from the central part of to be excluded from the electron-dense cores in the the podosome (Marchisio et al. 1987, 1988a). network of actin filaments. Clathrin patches were also A similar staining pattern was described by Nigg et al. occasionally labelled (not shown). (1986) but no correlation with IRM was shown in their paper. On ventral membranes prepared from untransformed Discussion BHK cells, vinculin antibody bound to small clusters of membrane-associated particles located specifically In the process of lysis-squirting, the cells swell or lyse and towards the periphery of the membrane, and at the the apical and lateral membranes, together with the termini of the stress fibres in focal adhesion (Fig. 7B). nuclei and cytoplasmic organelles, are swept away by a Gelsolin. Gelsolin localisation was studied using a stream of buffer. The protoplasmic surface of sub- monoclonal antibody, followed by a bridge (rabbit anti- stratum-adherent ventral membrane is thus exposed for mouse IgG) and then by goat anti-rabbit gold conjugate. both ultrastructural and immunocytochemical studies. In replicas of RSV/B4-BHK ventral membranes, the Structures that are not tightly associated with the mem- label was concentrated mainly in the areas of the rosettes brane are removed. Usually, cell-substratum adhesions (Fig. 8A). Residual stress fibres were also labelled, but with surrounding portions of the membrane are left without any specific pattern (Fig. 8B). Gold label was behind; after vigorous squirting only focal or point also observed on particles attached to clathrin-coated contacts are preserved. We tried to standardise the degree patches of membrane (not shown). of squirting using a special nozzle which allowed us to Whole-mount preparations (not shown) confirmed the squirt one coverslip in one or two runs evenly. results obtained with replicas. Gold particles were pres- We have not observed signs of major distortion or ent throughout the actin filament network and on the dislocation of membrane-associated structures, specifi- membrane surrounding the podosomes. They were cally actin stress fibres which are in the focus of our sometimes observed close to the electron-dense cores but interest. This applies to this study and to our work with did not seem to penetrate them. This, however, would be other cell lines as well. We find lysis-squirting better difficult to decide because of the high electron density of suited to the purpose than, for example, dry cleaving, the 'cores'. deep-etching or thin sectioning. The main advantage of Phosphotyivsine. In replicas of RSV/B4-BHK ventral lysis-squirting is that only membrane-associated struc- membranes, the level of labelling obtained with anti-ABP tures are retained. The other techniques do not discrimi- antibodies was rather high. Gold labelling could be seen nate between fortuitous and specific associations. The associated with groups of particles on the surface of the presence of complete cytoskeleton and cytomatrix pre- membrane, but most of them were seen on the rosette vents the visualisation of the membrane surface and (Fig. 9A). In cells retaining some residual stress fibres or impedes the penetration of antibodies or colloidal gold focal adhesions, gold particles were found both along the conjugates. fibres and in the contact area. Gold labelling was never The structural preservation of ventral membranes and observed on smooth areas of membrane. the definition in shadowed replicas is usually very good The spatial distribution of phosphotyrosine-containing (Nicol and Nermut, 1987; Nermut, 1989), but it suffers proteins in whole mounts of RSV/B4-BHK ventral during immunolabelling because of the mild prefixation membranes was similar to the distribution seen in repli- and exhaustive washing, which reduce the concentration cas. Most of the label was concentrated on the network of of aldehydes in the specimen.

92 /. Gavazzi et al. Fig. 7. Whole mount preparation of RSV/B4-BHK ventral membrane labelled with monoclonal antibody to vinculin and 15 nm colloidal gold. A. Gold particles are associated mainly with filaments, occasionally with dense cores (arrowed). B. Replica of a BHK ventral membrane labelled for vinculin as above. Gold particles are associated mainly with stress fibres termini. Bars, 100 nm.

Podosomes in RSV transfonned BHK cells 93 w

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Fig. 8. Part of a rosette labelled for gelsolin in an RSV/B4-BHK cell. A. Gold particles (10 nm) are associated with filaments within the rosette but are absent from the smooth regions of plasma membrane (pm). c, crater-like structures. B. Residual stress fibres are also labelled (as in untransformed cells). Freeze-dried replicas. Bars, 500 nm.

Transmission electron-microscopic observation of pla- been found associated with membranes after squirting: tinum-carbon replicas of ventral membranes thus ob- clathrin sheets and pits, coated vesicles, microfilaments, tained enables the viewing of the inner face of the plasma intermediate filaments, and many small membrane- membrane and of the membrane-bound portion of the associated particles. In the transformed cells, rosettes of cytoskeleton. The following structural elements have podosomes were invariably present. This suggests that

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Fig. 9. Ventral membranes of RSV/B4-BHK cells labelled for phosphotyrosine with an ABP antibody in A and a pp60"r antibody in B. Gold particles (10nm) are associated mainly with podosomes (arrows) or aggregates of 'particles' in A, but only with groups of membrane-bound particles in B, labelled with arrows. Freeze-dried replicas. Bars, 500nm.

Podosomes in RSV transformed BHK cells 95 the weakened adhesiveness of cells upon viral transform- adhesions in chondrocytes and osteoclasts have a bright ation cannot be due to reduced strength of the adhesion centre surrounded by a dark (adhesive) ring (Marchisio structures, but possibly to the reduced number of ad- et al. 1984a). It is not ruled out that both forms can exist hesion sites, and their different character. in one cell line, but it is necessary to correlate the The preparative techniques used in our work (freeze- distribution of vinculin and other proteins with the IRM drying, critical point-drying, replicas and whole mounts) image in every single case. allowed us to use stereo EM in our studies of the three- Using the gold-immunolabelling methods we have dimensional structure of podosomes. Sections published obtained more precise information about the spatial by others (Henderson & Rohrschneider, 1987; Marchisio distribution of some of the proteins known (from fluor- et al. 1984a; Tarone et al. 1985) showed a conical escence microscopy) to be associated with podosomes. network above the adhesion, but no correlation with the Gelsolin has been localised in platinum-carbon replicas IRM image was reported. Three-dimensional reconstruc- of RSV/B4-BHK ventral membranes mainly on podo- tion from serial sections is much more laborious and not somes, in agreement with LM observations (Marchisio et always rewarding. al. 1987; Wang et al. 1984), usually associated with Though height measurements from stereo microfilaments, and also on residual stress fibres. Gelsolin can graphs are less accurate than from sections, we did not reversibly disassemble actin filaments (this function be- z+ find it justifiable to prepare vertical sections because the ing regulated by physiological changes in Ca concen- range of values found was rather big, ruling out any need tration; Yin et al. 1981) and it is probably involved in the for high accuracy. Nonetheless, our dimensions are close rapid formation and dissolution of adhesion structures, to those obtained from micrographs published by Mar- and more generally in the reorganisation of actin fila- chisio et al. (1984a) and Henderson and Rohrschneider ments upon viral transformation. (1987). The location of vinculin in podosomes differs from that In platinum-carbon replicas the podosome appears to in normal cells, where vinculin is present predominantly be a dense agglomeration of actin oligomers and other in areas of the focal contacts. In podosomes, vinculin has proteins. Single podosomes are interconnected in the been found associated with microfilaments and, in many rosette by actin filaments. The contraction of these cases, far above the adhesions, as shown by stereo filaments may be responsible for the 'fusion' of single electron microscopy. This argues in favour of the conjec- podosomes into the rosette. In TEM of whole-mount ture that vinculin does not interact immediately with the preparations, the filaments were seen to emanate from plasma membrane but more probably with actin or a- electron-dense cores, 100-200 nm in diameter (presum- actinin on one side and possibly with talin on the other ably equivalent to the conical network observed in side. This is also supported by recent studies in our sections). These cores appear to be composed mainly of laboratory on colocalisation of vinculin and talin in NRK tightly packed short actin filaments, though the presence cells (Nicol and Nermut, in preparation). of small particles suggests that other components might It could be argued that the absence of vinculin from the be present, possibly actin bundling proteins. A structure 'cores' is a consequence of reduced penetration of anti- morphologically very similar to the rosette of podosomes, body-gold conjugates into the centre of the podosome, or i.e. an interlocking network of filaments radiating from their lower visibility in the preparative conditions used. electron-dense bodies, has been previously described in However, as shown in Fig. 7A, vinculin is associated with polymorphonuclear leukocytes (PMNs) by Boyles and the microfilaments high above the ventral membrane, Bainton (1979, 1981): they found filaments associated which is in contrast to the location in untransformed cells with the plasma membrane at sites of adherence to (Fig. 7B). In fluorescence microscopy, vinculin was phagocytosable or nonphagocytosable surfaces, appear- found either in spots correlating with punctate adhesions ing almost immediately after the cell attachment to a (Shriver and Rohrschneider, 1981; Tarone et al. 1985) or substratum. To our knowledge no data have been pre- at the periphery of these adhesions (Nigg et al. 1982, sented on the composition of the filament complex in 1986; Marchisio et al. 19846, 1988fl). However, it should PMNs, and this makes any further comparison with be noted that the latter adhesions were of the 'bright podosomes difficult. Amato et al. (1983) have described centre' type, as mentioned above. In our correlative punctate structures in substratum-adherent membranes experiments vinculin was found as a diffuse spot, not a of macrophages that were stained with rhodamine- ring. However, this is not proof that vinculin is associated labelled phalloidin, and that coincided with electron- with the ventral membrane because the observations can dense foci interconnected by radiating filaments and be explained by the low vertical resolution of light filament bundles. These, as well as other observations microscopy. (Caligaris-Cappio et al. 1986; Marchisio et al. 1984a, The src-oncogene product pp60 has been localised by 1987, 1988a), support the idea that podosomes are fluorescence light microscopy in punctate adhesions or adhesion structures which are not exclusively present in podosomes (as defined by IRM) in different RSV- transformed cells. transformed cell lines (Rohrschneider, 1979, 1980; However, it should be pointed out that two different David-Pfeuty and Singer, 1980; Shriver and Rohr- IRM images have been described: in RSV-transformed schneider, 1981; Nigg et al. 1982, 1986; Kellie et al. cells the punctate adhesions have a dark centre sur- 1986). It has also been found in residual focal contacts in rounded by a bright ring (Shriver and Rohrschneider, the early stages of transformation. 1981; Tarone et al. 1985), whereas the podosome-like The absence of pp6O"r from the membrane as shown

96 /. Gavazzi et al. ExM

% cs Fig. 10. Diagram of a podosome summarising data from electron microscopy and light microscopy data. CS, coverslip; ExM, extracellular matrix; PM, plasma membrane; SRC, pp60*rc; T, talin; V, vinculin; a, a--actinin; F, fimbrin; triangles, gelsolin; black dots, phosphotyrosine. Actin filaments are organised in a criss-cross manner forming a conical body. They probably interact with extracellular matrix receptors in the adhesion area. here is not surprising, but some uncertainty remains tron microscopy revealed an abundance of phosphotyro- about its presence on the membrane below the podo- sine on top of as well as within the podosomes. some, which could be expected on the basis of immuno- However, neither vinculin nor ppoO"^ itself can fully fluorescence. account for the labelling obtained with the anti-ABP Henderson and Rohrschneider (1987) reported the antibody. Phosphotyrosine-containing proteins are association of ppoO^ with microfilaments and focal clearly more abundant in the transformed cell, in particu- adhesions after detergent extractions and ferritin-immu- lar within the electron-dense cores, where both vinculin nolabelling. In our case, the antibody to pp60S7r labelled and ppoO"1^ are practically absent. This may indicate that mainly groups of small particles on the ventral membrane other hitherto unknown tyrosine phosphorylated mol- adjacent to the podosomes. The discrepancy between ecules (Comoglio et al. 1984), including potential sub- Henderson and Rohrschneider's and our data could be strates like talin (Pasquale et al. 1986) and the fibronectin explained by two circumstances: (1) Triton X-100 solu- receptor (Hirst et al. 1986), may be present in the bilizes the plasma membrane and may cause artifactual podosomes. association of cytosolic ppoO*"7 with the skeleton, and (2) Finally, what are the implications of our observations any soluble pp60"r is removed by our squirting method, for understanding cell-substratum adhesions in trans- which preserves the integrity of the plasma membrane formed cells? We shall answer this question best by and provides a high lateral and spatial resolution. Besides summarising our present knowledge of the spatial distri- ppoO"1^, other membrane-associated proteins can consti- bution of the protein components in podosomes. A tute the particles, which seem to be a general feature on tentative diagram in Fig. 10 (far from complete) should the protoplasmic surface of ventral membranes in cul- help in our attempt to demonstrate the most striking tured cells (Aggeler et al. 1983; Nermut ei a/. 1986; Nicol features of this peculiar structure. & Nermut, 1987) and they are not washed away under The first major difference from normal focal adhesions physiological conditions. Since ppoO^ is myristylated is the dense criss-cross packing of actin filaments. Their (Schultz et al. 1985), it is plausible to conclude that it is filamentous nature has been clearly demonstrated in our firmly associated with the plasma membrane and is study. Though many of the actin-bundling proteins seem transported to its destination by the diffusion or direct to be present in podosomes (see Burridge, 1986 and lateral flow of lipid molecules. This conclusion is sup- Marchisio et al. 1987) the stress fibres are missing. The ported by the report of Nermut and Eason (1987), who other striking difference from normal focal adhesions is found pp60src associated with the protoplasmic surface of the presence of vinculin on top of podosomes, mainly in ventral membranes in RSV-transformed rat fibroblasts association with actin filaments. Though there is no firm (LA-29) using the gold immunoreplica technique. evidence for the absence of vinculin from the area of By light microscopy, tyrosine-phosphorylated proteins actin—ventral membrane interaction, this finding sup- have been localised to podosomes in a diffuse pattern ports the idea that vinculin is not the link between actin (Comoglioef al. 1984; Taronee/a/. 1985). Immunoelec- and the extracellular matrix receptor (Burridge and Fath,

Podosomes in RSV transformed BHK cells 97 1989). As to the location of talin, we have to rely on light transformed fibroblasts: organization and regulation of cytoskeletal, microscopy evidence showing that talin is present in the membrane and extracellular matrix component at focal contacts. Cancer Rev. 4, 18-78. area of podosomes (Marchisio, 1988a). BURRIDGE, K. & CONNELL, L. (1983a). A new protein of adhesion There is a very reasonable correlation between light plaques and ruffling membranes. J. Cell Biol. 97, 359-367. microscopy and electron-microscopy findings as far as BURRIDGE, K. & CONNELL, L. (19836). Talin: a cytoskeletal gelsolin and phosphotyrosine are concerned. However, component concentrated in adhesion plaques and other sites of there is some discrepancy between our immunolocalisa- actin-membrane interaction. Cell Motil. 3, 405-417. BURRIDGE, K. & FATH, K. (1989). Focal contacts: transmembrane tion of ppoO^ and other people's observations, as already links between the extracellular matrix and the cytoskeleton. discussed. We have to stress again that we labelled only Bioessays 10, 104-108. membrane-associated protein and that its presence close CAUGARIS-CAPPIO, F., BERGUI, L., TESIO, L., CORBASCTO, G., to the adhesion structure is in line with its supposed Tousco, F. & MARCHISIO, P. C. (1986). Cytoskeleton function. reorganization is aberrantly rearranged in the cells of B chronic lymphocytic leukemia and hairy cell leukemia. Blood 67, 233-239. It is therefore tempting to speculate that the difference CAKLEY, W. W., BARAK, S. L. & WEBB, W. W. (1981). F-actin between podosomes and normal focal adhesions lies in aggregates in transformed cell9.7- Cell Biol. 90, 797-802. the absence of some of the bundling proteins, or of a CARLEY, W. W., BRETSCHER, A. & WEBB, W. W. (1985). F-actin signal for the formation of stress fibres. One could also aggregates in transformed cells contain ff-actinin and fimbrin but conclude that the stress fibres are actually not needed for apparently lack tropomyosin. Eur.J. Cell Biol. 39, 313-320. COMOGUO, P. M., Di RENZO, M. F., TARONE, G., GIANCOTTI, F. the formation of cell-substratum adhesions, coming into G., NALDINI, L. & MARCHISIO, P. C. (1984). Detection of play only when cells start spreading. Finally, the example phosphotyrosine-containing proteins in the detergent-insoluble of podosomes indicates that the interaction of actin with fraction of RSV-transformed fibroblasts by azobenzene the putative extracellular matrix receptors could be phosphonate antibodies. EMBOJ. 3, 483-489. mediated by yet another protein(s), perhaps specific to COURTNEIDGE, S. A., LEVINSON, A. D. & BISHOP, J. M. (1980). The protein encoded by the transforming gene of avian sarcoma virus the transformed state. (pp60"r) and a homologous protein in normal cells (pp60'ra'°"*rr) It should be added that besides the podosomes another are associated with the plasma membrane. Proc. natn. Acad. Sci. adhesion structure was observed in BHK-B4 cells: small U.SA. 77, 3783-3787. DAVID-PFEUTY, T. & SINGER, S. J. (1980). Altered distribution of patches of membranes coated with clathrin, as described the cytoskeletal proteins vincuhn and cr-actinin in cultured in normal fibroblasts by Nicol and Nermut (1987) and in fibroblasts transformed by Rous sarcoma virus. Proc. natn Acad. RSV-transformed fibroblasts by Nermut et al. (1988). Sci. U.SA. 77,6687-6691. In view of the observations by Amato et al. (1983), GEIGER, B. (1979). A 130K protein from chicken gizzard: its Grinnell (1984) and Marchisio et al. (1987) it seems that localization at the termini of microfilament bundles in cultured chicken cells. Cell 18, 193-205. neither of these two types of adhesion is specific to GRINNELL, F. (1984). 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Podosomes in RSV transformed BHK cells 99