Exp. Path. Bd. 14, S. 311-320 (1977)

Institute of Pathology (Head: Prof. Dr. F. BOLCK),Friedrich-Schiller University Jenaand Academy of Sciences of the GDR, Central Institute for Microbiology and Experimental Therapy(Head: Prof. Dr. U. TAUBENECK), Jena

Redistribution and internalisation of anti-ARel (anti-AHP )­

and Concanavalin A-binding sites

An electron microscopic double labeling study

By J. ROTH and M. WAGNER

With 4 figures

(Received May 30, 1977)

Key words: lectins; Concanavalin A; anti-AHel; anti-AHP electron microscopy; double labelingtechnique; plasma membrane; receptor redistribution; membrane internalisation

Summary

The redistribution and internalisation of two different lectin-binding sites on the same cell wasinvestigated electron microscopically on unfixed rat liver cell cultures. For these purposes an elec­tron microscopic double labeling technique was used for visualisation of the anti-AHel-binding sitesby the gold-labeled lectin and for the demonstration of the Concanavalin A-binding sites by theConcanavalin A-peroxidase technique. From the experiments it was evident that the ligand-inducedredistribution of the anti-AHel-binding sites effects a rearrangement of the Concanavalin A-bindingsites. Both markers were found in a clustered distribution on the cell surface. Following the redistribu­tion an internalisation of both lectin-binding sites could be observed on central and peripheral cellparts. In the lateral cell parts an accumulation of the both markers occurred on the plasma membrane.

The individual macromolecular components of the plasma membrane are diffusely andrandomly distributed in the unpertubed fluid plasma membrane. Evidence from a numberof laboratories indicates that the different cell surface components such as surface immuno­globulins or lectin-binding sites can be segregated from other components when cross-linkedby a multivalent ligand (TAYLOR et al. 1971, COMOGLIO and GUGLIELMONE 1972, DAVIS 1972,DE PETRIS and RAFF 1972, EDIDIN and WEISS 1972, KARNOVSKY et al. 1972, KOURILSKYet al. 1972, LOOR et al. 1972, SUNDQUIST 1972, UNANUE et al. 1972). It was first observedby TAYLOR et al. (1971) that after binding of anti-Ig to B lymphocytes the surface Ig re­ceptors were aggregated into clusters and patches and subsequently into a cap at one polof the cell. This ligand-induced redistribution apparently can occur because of the fluiditywithin the plasma membrane (SINGER and NICOLSON 1972), allowing extensive lateralmovements of membrane constituents (FRYE and EDDIN 1970). However, in contrast tothe original proposal that membrane proteins and lipids can diffuse freely over the cell sur­face at rates limited mainly by the viscosity of the lipid bilayer membrane matrix (SINGERand NICOLSON 1972), recent results indicate the operation of additional constraints on themobility of the membrane constituents (BERLIN and UKENA 1972, YAHARA and EDELMAN1972, 1973, YIN et al. 1972, EDELMAN et al. 1973, BERLIN et al. 1973, POSTE et al. 1975,JACOBSON et al. 1976, SCHLESSINGER et al. 1976, 1977, ZAGYANSKY and EDIDIN 1976). Theoccurrence of ligand-induced redistribution of cell surface components provides a usefultool for studying the mutual topological relationship of different components. In particular,when one specific component is suitably crosslinked and redistributed by a multivalent li­gand it is possible to invesitgate whether or not another specific component has redistributedtogether with the first.

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Recently, we have developed lectin double labeling techniques using colloidal gold, fer­ritin and horseradish peroxidase as markers (ROTH and WAGNER 1977, ROTH and BINDER1977, WAGNER and WAGNER 1977). The availability of morphological distinct markers offersa challenge to visualise different lectin-binding sites on the surface of the same cell and,furthermore, allows to study the influence of the redistribution and internalisation of onespecific lectin-binding site on the arrangement of another specific lectin-binding site.

In this communication we follow the redistribution and internalisation of two differentlectin-binding sites by cell surface double labeling with gold-labeled anti-ABel (the lectinfrom the edible snail Helix pomatia) and Concanavalin A-peroxidase complex-. We providesome direct evidence that the redistribution and internalisation of the anti-ABel-bindingsites effects also a rearrangement of the Concanavalin A-binding sites.

Materials and methods

CellsThe labeling experiments were performed on monolayer cultures of a non-tumorigenic epitheloid

cell line from rat liver established by KARSTEN et aI. (1976). All the labeling steps were done in situin the culture flasks on the non-prefixed cells at 37°C.

MaterialsThe Concanavalin A (Con A) was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden).

The lectin from the albumen gland of Helix pomatia was isolated and purified according to themethod of KUHNEMUND and KOHLER (1969) in a slight modification (WAGNER and WAGNER 1973).Horseradish peroxidase (po) (type I, RZ 3) was purchased from Boehringer (Mannheim, FRG), di­aminobenzidine tetrahydrochloride from Serva (Heidelberg, FRG), chloroauric acid from Merck(Darmstadt, FRG), .x-methyl-D-mannopyranosid (A grade) from Cal-biochem (San Diego, USA) andN-acetyl-D-galactosamine from Chemapol (CSSR).

Cytochemical techniquesThe cell surface binding sites for Con A were demonstrated by the method of BERNHARD and

AVRAMEAS (1971) using po as affinity marker for the membrane-bound Con A. The anti-ARel waslabeled with colloidal gold prepared according to FAULK and TAYLOR (1971) as reported earlier(WAGNER and WAGNER 1976).

Cell surface labeling conditionsIn a first set of experiments single labeling by one lectin was done. For visualisation of the

Con A-binding sites the cell cultures were incubated with Con A and po for 20 min· each step, fixedwith 1.6 % glutaraldehyde in PBS for 15 min and processed for the demonstration of the po activitywith the diaminobenzidine reaction (GRAHAM and KARNOVSKY 1966).

The anti-A Bel-bindiI1g sites were demonstrated by incubation of the cell cultures with the gold­labeled anti A-ReI for 30 min.

The cell surface double labeling was performed in the following manner. First the cell cul­tures were incubated with gold-labeled anti-ARel for 30 min at 37°C followed by three rinsing stepsto remove the unbound lectin-gold complex. Afterwards the Con A-po technique was carried out asdescribed. All the cell cultures were fixed with 1.6 %glutaraldehyde in PBS for 15 min after the cellsurface labeling. Postfixation was done with 2 % OS04 in 0.1 M cacodylate buffer (pH 7.2) for 2 hours.The cell cultures were embedded in the culture flasks with Vestopal W. Afterwards the culture flaskswere removed from the Vestopal which was subsequently sectioned perpendicular to the plane ofthe monolayer. The sections were examined in a JEM 100 B electron microscope.

Cytochemical controlsFor control of specificity of the labeling procedures the lectin-gold complex or the Con A and po

solutions, respectively, were preincubated with 0.2 M of the appropriate inhibitory sugar for 1 hour.Additional controls included the incubation with po without a prior Con A step for exclusion of anunspecific po binding, and the diaminobenzidine reaction alone for exclusion of demonstration ofendogenous enzyme activity.

ResultsThe incubation of the cell cultures in Con A and po for visualisation of the Con A-binding

sites only resulted in a dense and continously arranged reaction product on the cell surface(fig. 1 a). The microvilli were also covered by the reaction product indicating the presence

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Fig. 1. Cell surface single labeling. a,) The Con A-po technique results in a continous cell surfacestaining. b) Demonstr'ation of the anti-AHel-binding sites witb the anti-AHel-gold complex led toa dense cell surface labeling and c) under the same experimental conditions in some of the cells tothe redistribution of the binding.. sites (arrowheads'limit the membrane areas free from label) withsimultaenous internalisation (asteriks). X 52,000 (a), X 49,000 (b, c). The bars represent 0.25 pm.

of Con A-binding sites. Internalisationprocesses oflabeled membrane areas via membraneinvaginations and vesicle formation could not be ob~erved under the incubation conditionsused. The labeling of the cells with the anti-A,ii~I-g!3i4 complex for 30 min likewise resultedin a dense cell surface staining (fig. 1b). HOWllve~;"in the same preparation, cells could bedetected which exhibited a discontinous plasmahiembrane labeling by the gold particles(fig. 1c). Simultaneously, internalisation of labeled membrane areas with the formation ofvarious cytoplasmic vesicles densely studded with gold particles occurred in these regions.

In the double labeling experiments the incubation of the cells with the anti-A-Hei"gold complexes followed by Con A and po led to a continous cell surface staining as can be

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seen in figure 2a. However, similar to the single labeling with the anti-AHe1-gold complexalone, in some of the cells a discontinous labeling on the cell surface occurred (fig. 2 b, c, d).The discontinous labeling was related to both markers. That means, the gold particles (in­dicating the anti-AHe1-binding sites) as well as the diaminobenzidine reaction product (in­dicating the Con A-binding sites) were cluster-like arranged in the same membrane areas.

>.

Fig. 3. Cell surface double labeling. On the plasma membrane in the free margin of a cell both mar­kers are discontinously arranged (arrowheads). In some membrane areas the double label is aceumu­lated (arrows) and internalisation is visible (asterik). X 83,000. The bar represents 0.2 ,urn.

Fig. 2. Cell surface double labeling. a) Incubation of the cells with gold-labeled anti-AHe! followedby the Con A-po technique results in a continous cell surface double labeling. b) Ligand-inducedredistribution of both lectin-binding sites in a cluster-like arrangement on the plasma membraneand on a microvillus (arrowheads limit the membrane areas free from double label). c, d) Simul­taneously to redistribution in the plane of membrane (as marked out with arrowheads) interna,lis1ttionprocesses take place (arrows) which led to vesicle formation studded with gold particles only(asteriks) or with both markers. x 90,000 (a), X 99,000 (b), X 93,000 (c), X 74,000 (d). The barsrepresent 0.2 ,urn.

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The redistribution led to membrane areas and microvilli free from the double label (fig. 2b).Furthermore, internalisation of both labels together takes place via membrane invaginations(fig. 2 c) and vesicle formation (fig. 2d). In some cases intracytoplasmic vesicles studdedonly with gold particles could be observed (fig. 2c).

In the lateral cell parts the redistribution led to an accumulation of both labels on theplasma membrane with subsequent internalisation (fig. 3). Such an accumulation was alsoobserved in the lateral parts of two overlapping cells (fig. 4a). It is of interest, that in someareas of such accumulation regions of both labels the gold particles and the diamino-

Fig. 4. Cell surface double labeling. a) In the overlapping lateral parts of two cells accumulationand internalisation (arrows) of the cell surface double label. Note the predominance of the diamino­benzidine reaction product (arrowheads) whereas in neighbouring surface areas both labels are pre­sent in an equal ratio (asteriks), b) Detail from a). X 20,000 (a, the bar represents 1.0/-lm), X 54,000(b, the bar represents 0.25/-lm).

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benzidine reaction product are present in an equal ratio whereas in other cell surface regionsthe diaminobenzidine reaction product predominates. At sites of cell contact an internalisa­tion of both markers occurred (fig. 4 b).

In the cytochemical controls the cells remained free from surface label.

Discussion

In the present study we have used a lectin double labeling technique for demonstrationof two different lectin-binding sites on the cell surface: the anti-Hel for visualisation of N­acetyl-D-galactosamine-like residues and the Con A for non-reducing terminal and internal2-o-g1ycosidic bound mannopyranoside, glucopyranoside and fructofuranoside-like residues.In all of the double labeling studies with lectins, the demonstration of absence of a direct inter­action between the lectins used is of great importance. As we have already shown, the anti­Hel and the Con A do not interact with each other (ROTH and WAGNER 1977). Furthermore,we have obtained results speaking in favour of a topological distinct localisation of theboth lectin-binding sites on the cell surface (ROTH and WAGNER 1977). In further methodicalinvestigations on cell surface double labeling by use of lectins we have made the observationthat a sterical hindrance by one of the labeled lectins can occur which hinders the bindingof the second one (ROTH and BINDER 1977). Consequently, no evaluation of the numberof binding sites can be made in such experiments. In our opinion it is only the qualitativeaspect that has to be considered and, for example, can be used for investigations on cellsurface dynamics. The liver cell cultures used in the present experiments represent a usefulmodel system in this respect. The labeling of the Con A-binding sites only does not resultin a ligand-induced redistribution in these cells during the incubation periods and until areincubation time of 45 min (ROTH 1975). In contrast to the continous arrangement of theCon A-binding sites on the unfixed cells under these experimental conditions, the anti­AHe1-binding sites were redistributed upon the binding of the anti-AHel-gold complex al­ready during the period of incubation of 30 min. Basing on these observations in the singlelabeling experiments we have performed a double labeling with the gold-labeled anti-A Helas first incubation step. In this way we could observe that the ligand-induced redistributionof the anti-A Her-binding sites in a cluster-like arrangement also effects a redistribution ofthe Con A-binding sites. From this we can conclude that the movement of the anti-A He1­binding sites influences the localisation of the Con A-binding sites in such a manner thatthey move together laterally in the plane of the membrane. However, as already mentionedearlier (ROTH and WAGNER 1977), the question is still open whether the anti-A Hel and ConA-binding sites are saccharide residues located on one cell surface macromolecule or whetherthey are components of two different macromolecules. Similar results as reported by uswere presented by SHIII1IZU and YAII1ADA (1976) on rat ascites hepatoma cells by means ofCon A, Ricinus communis agglutinin and wheat germ agglutinin. In contrast to our experi­mental conditions they treated the unfixed cells in the first step with a native unlabeledlectin and, after fixation of the cells with glutaraldehyde, with another and ferritin-labeledlectin. In this type of double treatment of cells with lectins also a changed arrangement ofthe ferritin-labeled lectin could be observed. It was induced by the redistribution followingthe binding of the unlabeled lectin.

Beside of lectin-binding sites other cell surface constituents were object of such investi­gations. The lymphocyte surface Ig of different classes and the H-2 and HL-A histocom­patbility antigens in mouse and man behave as independent molecules (TAYLOR etal. 1971,BERNOCO et al. 1972, LAII1I11 et al. 1972, NEAUPORT-SAUTES et al. 1973, PREUD"HOMME et al.1972, STACKPOLE et al. 1974) whereas human HL-A and .B2-microglobulin are associated intheir movement (POULIK et al. 1973, SOLHEIM and THORSBY 1974). YAHARA and EDELMAN

(1972, 1973a, b) have reported the inhibition of cap formation of surface Ig of lympho-cytes by Con A. Similar findings were reported by LOOR et al. (1972, 1974) and by UNANUEand KARNOVSKY(1974). The results of DE PETRIS (1975) are consistent with these observationsbut indicate that surface Ig and probably 0 antigen are directly cross-linked by Con A

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because of their carbohydrate content. In consequence, the behaviour of the surface Igreflects essentially the behaviour of the Con A-binding sites.

Simultaneously to the ligand-induced redistribution in the plane of the plasma membranean internalisation of labeled membrane areas could be observed in the present study as alsoreported by others (BARAT and AVRAMEAS 1973, HUET and BERNHARD 1974, HUET et a1.1974, NICOLSON 1974, NICOLSON et a1. 1976, ROTH 1974, 1975). This internalisation wasrelated to both lectin-binding sites as indicated by the double labeling of the cytoplasmicvesicles. Such internalisation already occurred in the first incubation step in the doublelabeling experiments which led to the formation of vesicles studded with gold particles only.The internalisation also could be observed in the lateral margin of single cells and of over­lapping cells. In the lateral cell parts an accumulation of both labels occurred on the cellsurface. This accumulation may be due not only by the redistribution process. In additioncell surface movements related to cell locomotion (HARRIS 1973) could have led to the accu­mulation of the cell surface labels at the cell margin.

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Author's address:Dr. sc. med. J, ROTH, Friedrich Schiller University, Institute of Pathology, Ziegelmiihlenweg 1,

DDR - 69 Jena.

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