Planta 9 by Springer-Verlag 1977
Planta 136, 253-259 (1977)
Membrane Mobility and the Concanavalin A Binding System of the Plasmalemma of Higher Plant Protoplasts J. Burgess and P.J. Linstead John Innes Institute, ColneyLane, Norwich NR4 7UH, U.K.
Abstract. The binding of a colloidal gold-Concanavalin A (ConA) complex to the plasmalemma of tobacco leaf protoplasts has been investigated using scanning electron microscopy. At 5~ C the particles of goldConA appear to be randomly distributed over the surface of the protoplast. If the temperature is raised, the particles associate into clusters. Saturation of the membrane with particles can only occur when the weight of ConA in solution exceeds 1 lag/104 protoplasts in suspension, and when its concentration exceeds 15 ~tg/ml. These results are discussed in terms of the properties of the ConA binding site and the mobility of such sites within the membrane surface. Key words: Concanavalin A - Plasmalemma - Protoplasts - Scanning Microscopy.
Introduction
The lectin Concanavalin A (ConA) has been shown to bind to the plasmalemma of protoplasts from several plant sources (Burgess and Linstead, 1976a; Williamson et al., 1976). This binding may be accompanied by aggregation of the protoplasts (Glimelius et al., 1974; Burgess and Linstead, 1976a; Williamson et al., 1976). The binding of ConA to protoplasts has been studied by electron microscopy using either a colloidal gold ConA complex (AuConA) (Burgess and Linstead, 1976a), or a secondary labelling technique with haemocyanin (Smith and Revel, 1972; Williamson et al., 1976). The pattern of ConA labelling has been shown to depend on the source of the protoplast (Burgess and Linstead, 1976a) and on the conditions used for labelling (Williamson et al., 1976). ConA=Concanavalin A; AuConA=Colloidal gold-ConcanavalinA complex Abbreviations:
ConA coupled to an electron dense marker is a useful label for membranes in the electron microscope. It may also yield an insight into some of the properties of the membrane to which it is bound. In animal cells ConA is thought to bind to receptor sites which are mobile in the surface of the membrane (Nicolson, 1974; Rutishauser and Sachs, 1975). This mobility manifests itself in the clustering of bound ConA molecules due to their self-association. Clustering is enhanced by raised temperature (Comoglio and Filogamo, 1973; Nicolson, 1973) and is prevented by fixation of the membrane with glutaraldehyde prior to labelling the receptor sites (Comoglio and Guglielmone, 1972; Inbar et al., 1973). These observations accord with the fluid mosaic model for membrane structure proposed by Singer and Nicolson (1972). Thin sectioning techniques are unsuitable for examining the distribution of ConA on membrane surfaces, and the conclusions which can be drawn from such work are limited (Burgess and Linstead, 1976a). By the use of replica techniques, Williamson et al. (1976)have demonstrated clustering of haemocyanin as a secondary label to ConA on the surface of soybean protoplasts. The results presented here are an analysis of the behaviour of a colloidal goldConA complex at the surface of tobacco protoplasts by scanning microscopy. An attempt has been made to clarify the concentration dependence of ConA binding, and to demonstrate membrane mobility directly by temperature shift experiments.
Materials and Methods
Protoplasts were prepared as previously described (Burgess and Linstead, 1977). AuConAwas prepared as described by Burgess and Linstead (1976a). Methods for scanningelectron microscopy have been detailed elsewhere (Burgess et al., 1977). In order to examineprotoplasts prepared for scanningmicroscopyin thin sec-
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tions, the following procedure was adopted. Critical-point dried protoplasts were dusted on to the surface of a clean glass slide. The slide was then placed in a vacuum coating unit and coated with carbon and gold palladium (Burgess et al., 1977).The coated protoplasts were then washed from the slide with acetone, and embedded in araldite using standard procedure (Burgess and Fleming, 1974). For our initial labellingexperiments,batches of 1 5 x 106 protoplasts were washed in mannitol and resuspended in 8-10 ml of the AuConA solution. This procedure resulted in inconsistent labelling patterns due to exhaustion of the AuConA in dilute solutions (see Results, section (b)). In later experiments protoplasts were counted in a haemocytometerand the number of protoplasts added to a given volume of the AuConA solution was adjusted so as to obtain either saturation labelling or incomplete labelling, as desired.
conditions were large spherical projections which are a feature of many freshly prepared protoplasts (Fig. 2). Reference to thin sections of AuConA labelled protoplasts suggests that these projections overlay small vacuoles which protrude from the cytoplasm immediately beneath the plasmalemma (Fig. 3). Under the same conditions of labelling (see Methods) concentrations of ConA of 30 lag/ml or below did not always give rise to saturation labelling. Instead the A u C o n A particles appeared as clusters with more or less blank areas of membrane between them (Figs. 4, 5). This pattern was repeated when concentrations down to 10 gg/ml of ConA were used under our initial conditions. To test whether this effect was indeed one of concentration, protoplasts were exposed to a labelling solution of 30 ~tg/ml ConA in separate batches. The volume of the solution into which each identical batch of protoplasts from the same leaf was introduced was adjusted to give a range of ratios of weight of solute ConA/protoplast number. Saturation was achieved when this ratio exceeded 100 lag ConA/106 protoplasts. At levels of ConA below this saturation could not be achieved. Figures 4 and 5 are taken from experiments at 30 lag/ml where the ratio of ConA/protoplasts was 0.5 lag/104 protoplasts. If such protoplasts were subsequently incubated in further fresh labelling solutions at 30 lag/ml ConA, additional labelling occurred, and saturation could be obtained. In an attempt to show the lower concentration limit at which ConA in excess would lead to saturation cover of the membrane, protoplasts were incubated for 2 h at 30~ in solutions of AuConA of 15, 10 and 5 lag/ml so that the solute ConA was present at 2 lag/104 protoplasts. Saturation occurred at 15 lag/ml, but only about half the membrane area was covered with particles from the 10 lag/ml solution. 5 gg/ml gave more limited cover still.
Results
a) Appearance of AuConA in the Scanning Microscope ConA on its own did not give rise to any visible change at the surface of treated protoplasts when they were examined in the scanning microscope. When coupled to colloidal gold (particle size 16-20 nm), the complex appeared as an irregularly shaped particle (Figs. 5-11). The angular outline of A u C o n A serves to distinguish it from the more regularly spherical shapes of small protrusions which are a feature of freshly isolated and cultured protoplasts (Burgess and Linstead, 1976b). The identity of A u C o n A is inferred from the absence of such angular particles from the surface of non-labelled protoplasts. The presence of AuConA on the outer surface membrane of protoplasts prepared for scanning microscopy has been confirmed by embedding part of the population after metal coating. Thin sections of such preparations showed the layers of coating material overlying the colloidal gold particles (Fig. 1). This technique suggests that several particles of colloidal gold may give rise to a single coated particle as seen in the scanning mode (Fig. 1). This may explain both the variable size of AuConA particles seen under scanning conditions, and also the irregular outline of such particles. It emphasises the need for a cautious approach in the interpretation of the scanning microscope image.
b) Concentration Effects of ConA Binding Labelling of the membrane with AuConA at 125 lag/ ml resulted in saturation cover judged by thin sectioning techniques (Burgess and Linstead, 1976a). This has been confirmed for lower concentrations by scanning microscopy (Fig. 2). The only parts of the membrane which were not labelled under these saturating
c) Temperature Dependence of BOnding Protoplasts were examined after incubation for 30 rain in 30 lag/ml AuConA (insufficient to cause saturation, see above) at 5 ~ 25 ~ or 35 ~ C. At 5~ scattered random labelling was found with most of the particles in the form of units rather than clusters (Fig. 6). At 25~ under otherwise identical conditions, the label was present mainly in the form of clusters, with only a few units in between (Fig. 5). This pattern was repeated at 35 ~ C. Tighter clustering could be induced by prolonged incubation at room temperature or above. This is exemplified by Figure 7 where almost no flee units remain after 60 rain labelling at 35 ~ C.
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All Figures relate to tobacco leaf protoplasts, which have been washed twice with mannitol prior to the treatment described in the figure legend. Fig. 1. Thin section through a protoplast labelled in excess AuConA at 50 gg/ml, and embedded after fixation, drying and metal coating. The coating appears likely to simplify the image obtained by reflection, since in some cases several gold particles are seen to comprise a single coated particle (arrows) Fig. 2. Part of a similar protoplast to that shown in Figure 1, viewed by reflection scanning. The AuConA particles over the entire surface of the protoplast with the exception of large spherical projections (arrows) Fig. 3. Thin section through a protoplast treated as in Figure 1, but embedded conventionally. The only part of the surface membrane which is not covered with colloidal gold particles is a large projection Fig. 4. Scanning micrograph of a tobacco protoplast incubated for 30 min at 25 ~ C in a solution of AuConA at 30 pg/ml, insufficient to saturate the membrane. The particles of AuConA appear in clusters on the surface Fig. S. As Figure 4. Single particles are visibIe between the clusters (arrows) of AuConA
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Fig. 6. Scanning micrograph of a tobacco protoplast incubated for 30 min at 5 ~ C in a solution of A u C o n A at 30 gg/ml insufficient to saturate the membrane. The particles of A u C o n A are randomly distributed on the surface and form only very small clusters in a few places Fig. 7. Scanning micrograph of a tobacco protoplast incubated for 60 min at 3 5 ~ in solution of A u C o n A at 30 gg/ml insufficient to saturate the membrane. Large clusters of particles are visible. The m e m b r a n e between the clusters after prolonged incubation is almost devoid of A u C o n A particles Fig. 8. Scanning micrograph of a tobacco protoplast incubated for 30 min at 5 ~ C in a solution of 60 Itg/ml A u C o n A , insufficient to saturate the membrane. The A u C o n A is distributed randomly over the surface, predominantly as single particles Fig. 9. Scanning micrograph of a protoplast from the same population as Figure 8, but which was washed free of A u C o n A in solution and incubated at 35 ~ C for 30 min before fixation. The A u C o n A particles are predominantly clustered
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Fig. 10. Scanning micrograph of a tobacco protoplast incubated for 30 s at 35~ in a solution of AuConA at 30 gg/ml, sufficient to saturate the membrane. The surface is not completely covered at this time, and no clustering of AuConA particles is visible Fig. 11. Scanning micrograph of a tobacco protoplast incubated for 21/2min at 35~ C in a solution of AuConA at 30 gg/ml, insufficient to saturate the membrane. Some clustering of particles is evident even after this short labelling period
In a second series, protoplasts were prelabelled at 5 ~ C in a non-saturating a m o u n t of ConA at 60 gg/ ml for 30 rain. They were then washed free of the label in solution and incubated in mannitol at 25 ~ C or 35 ~ C. Protoplasts fixed immediately after the low temperature labelling showed a scattered distribution of single particles (Fig. 8, cf. Fig. 6). The same protoplast population after incubation for 30 min at 35 ~ C showed cluster formation (Fig. 9). This clustering was not observed on protoplasts which were fixed in glutaraldehyde prior to incubation at raised temperatures. Finally protoplasts were incubated at 5 ~ for 30 min with an excess of C o n A at 30 gg/ml. Complete saturation of the surface occurred suggesting that low temperatures do not impair the efficiency of binding or reduce the n u m b e r of available sites.
In the presence of an excess of ConA in solution at 30 ~tg/ml saturation of the surface occurred within 5 min at 35 ~ C. After only 30 seconds labelling under these conditions, a r a n d o m non-saturating label pattern was found (Fig. 10). At 5 ~ under the same conditions, saturation was not achieved until 30 min. Again the intermediate pattern was one of r a n d o m single particles. I f C o n A were not present in excess, the final pattern at 35 ~ C was one of cluster formation (Figs. 4, 5, cf. Fig. 7). The degree of clustering was time dependent. Even after 21/2 min of labelling, small clusters were visible under non-saturating conditions (Fig. 11). At 5 ~ C under these conditions, the pattern obtained was one of randomly spaced single particles whose density on the m e m b r a n e increased up to 30 min of incubation (cf. Figs. 6, 8).
d) Time Dependence of Binding Discussion Protoplasts were placed in A u C o n A solutions, then samples were withdrawn at various times for fixation. Patterns of labelling which were observed depended upon the ratio of ConA/protoplasts in solution, and the temperature at which the experiment was conducted.
The binding of the gold-ConA complex to the membrane ofprotoplasts is not easily interpreted in molecular terms. It is uncertain how the colloid and the protein associate, and whether this association is uniform within a given batch of AuConA, or between
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batches. Examination of sectioned material which has been otherwise prepared as for scanning microscopy suggests that a "single" particle of AuConA viewed in the scanning mode may embrace several particles of colloidal gold. This clearly implies that a single scanned particle may contain several molecules of ConA. In.addition to this difficulty of interpretation, it is known that ConA itself undergoes temperature and pH-dependent association (Huet et al., 1974). Furthermore the possibility exists of temperature dependent masking of binding sites (Inoue, 1974). For these reasons this discussion is limited to general patterns of behaviour of the AuConA complex. In particular, the terms " u n i t " , "cluster" and "saturation" are used to denote a microscopical appearance of the AuConA complex, and do not carry any direct molecular connotation. The first clear result which emerges is that it is possible to saturate the membrane with AuConA. This has been suggested previously (Burgess and Linstead, 1976a) using thin sectioning techniques and a high concentration of ConA at room temperature. We have confirmed this effect down to 15 pg/ml of ConA, and at temperatures of 5~ C. Masking of binding sites at low temperature therefore does not seem to occur in tobacco protoplasts (Inoue, 1974). Saturation labelling requires an excess of AuConA in solution. Attempts to quantify this effect have suggested that approximately 1 gg of ConA is needed to produce saturation o n 10 4 protoplasts. If this ratio is exceeded saturation will be obtained whether by means of a low volume of high concentration solution, or a large volume of a dilute solution, down to 15 pg/ml. This weight of ConA corresponds to the order of 108 ConA molecules per protoplast. It is very doubtful whether the number of binding sites can be deduced from such a figure however, due to uncertainties concerning the nature of the association between the colloid and the ConA, the size distribution of the protoplasts themselves in different populations, and the amount of ConA remaining in solution at the end of the labelling period. A figure of 3x 10v binding sites per cell has been given for fibroblasts (Collard and Temminck, 1975). The failure of the label at 30 pg/ml to produce saturation in an experiment such as that illustrated by Figure 4 is therefore due to the removal of ConA from solution, and the resultant reduction of its concentration to a low level. This has been demonstrated by reincubating such partially labelled protoplasts in further aliquots of AuConA solution at 30 pg/ml. Saturation results when the protoplasts have been exposed to a sufficient weight of ConA in solution. In order to obtain saturation the number of bind-
ing sites must be very large, or some non-specific mechanism of binding must be occurring. Non-specific binding is unlikely, since e-methyl mannoside is known to inhibit ConA binding to protoplasts (Burgess and Linstead, 1976a; Williamson et al., 1976). The nature of the binding site is unknown. Under conditions when the amount of ConA in solution is limiting, saturation of the surface cannot be obtained. The pattern of distributed particles which is found depends on the temperature and time of the experiment. Repeatedly it has been found that protoplasts labelled at 5~ C show a random distribution of predominantly single particles, whereas on those labelled at 25 ~ or 35~ the particles are clustered together. That this is a result of the redistribution of bound AuConA has been demonstrated directly by labelling protoplasts at 5~ C, then incubating them in a label-free environment at raised temperatures. Clustering of the particles can in this way be followed in a single protoplast population. Glutaraldehyde fixation after low temperature labelling prevents cluster formation. This protocol is preferable to labelling after glutaraldehyde fixation has been used to immobilise the receptor sites (Van Blitterswijk et al., 1976; Williamson et al., 1976), since it avoids the possibility of a glutaraldehyde-induced change in the binding process. This ability to label the membrane at low temperature illustrates the usefulness of AuConA as a marker; the ConA-haemocyanin double labelling method is not usable at 5~ C (Temminck et al., 1975) The mobility of the AuConA complex-in the membrane is not high. Periods of 30-60 min at 35~ C are necessary for the formation of tight clusters of particles. This is considerably longer than the time required for the clustering of Ferritin-ConA on turnout cell membranes (Nicolson, 1973). It explains why a clustered pattern of labelling is never observed in time course experiments leading to saturation of the surface. Presumably at least part of the comparative immobility of the AuConA complex could result from the size and density .of the gold particles themselves. The mobility of the AuConA complex which has been directly demonstrated in this study is consistent with the concept of the tobacco protoplast plasmalemma being a "fluid mosaic" (Singer and Nicolson, 1972). These results suggest that the tobacco protoplast plasmalemma contains a large number of binding sites which have a high affinity for ConA. These sites show increased mobility with raised temperature, and they are immobilised by fixation in glutaraldehyde. AuConA is clearly a useful probe for the investigation of membrane mobility. We have suggested elsewhere that polyethylene glycol fluidises the membrane on the basis of the behaviour of AuConA in the vicinity
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of the junction of adhering protoplasts (Burgess et al., 1977). It is hoped that other determinants of membrane mobility may now be identified.
Huet, C., Lonchampt, M., Huet, M., Bernadac, A. : Temperature effects on the Concanavalin A molecule and on Concanavalin A binding. Biochim. Biophys. Acta 365, 28 39 (1974) Inbar, M., Huet, C., Oseroff, A.R., Ben-Bassat, H., Sachs, L.: Inhibition of lectin agglutinability by fixation of the cell surface membrane. Biochim. Biophys. Acta 311, 594-599 (1973) Inoue, M.: Cell agglutination mediated by Concanavalin A and the dynamic state of the cell surface. J. Cell Sci. 14, 197-202 (1974) Nicolson, G.L. : Temperature-dependent mobility of Concanavalin A sites on tumour cell surfaces. Nature New Biol. 243, 218 220 (1973) Nicolson, G.L. : The interaction of lectins with animal cell surfaces. Int. Rev. Cytol. 39, 89 190 (1974) Rutishauser, U., Sachs, L.: Receptor mobility and the binding of cells to lectin coated fibres. J. Cell Biol. 66, 76 85 (1975) Singer, S.J., Nicolson, G.L. : The fluid mosaic model of the structure of cell membranes. Science 175, 720-731 (1972) Smith, S.B., Revel, J-P.: Mapping of Concanavalin A binding sites on the surface of several cell types. Dev. Biol. 27, 434 441 (i972) Temminck, J.H.M., Collard, J.G., Spits, H., Roos, E. : A comparative study of four cytochemical detection methods of Concanavalin A binding sites on the cell membrane. Exp. Cell Res. 92, 307-322 (1975) Van Blitterswijk, W.J., Walborg, E.F., Feltkamp, C.A., Hilkmann, H.A.M., Emmelot, P.: Effect of glutaraldehyde fixation on lectin-mediated agglutination of mouse leukaemia cells. J. Cell Sci. 21, 579-594 (1976) Williamson, F.A., Fowke, L.C., Constabel, F.C., Gamborg, O.L. : Labelling of Concanavalin A sites on the plasma membrane of soybean protoplasts. Protoplasma 89, 305 316 (1976)
References Burgess, J., Fleming, E.N. : Ultrastructural studies of the aggregation and fusion of plant protoplasts. Planta (Berl.) 118, 183-193 (1974) Burgess, J., Linstead, P.J.: Ultrastructural studies of the binding of Concanavalin A to the plasmalemma of higher plant protoplasts. Planta (Berl.) 130, 73 79 (1976a) Burgess, J., Linstead, P.J.: Scanning electron microscopy of cell wall formation around isolated plant protoplasts. Planta (Berl.) 131, 173-178 (1976b) Burgess, J., Linstead, P.J. : Coumarin inhibition of microfibril formation at the surface of cultured protoplasts. Planta 133, 267 273 (1977) Burgess, J., Linstead, P.J., Fisher, V.E.L.: Scanning microscopy of higher plant protoplasts. Micron (in press) (1977) Collard, J.G., Temminck, J.H.M. : Differences in density of Concanavalin A binding sites due to surface morphology of suspended normal and transformed 3T3 fibroblasts. J. Cell Sci. 19, 21 32 (1975) Comoglio, P.M., Filogamo, G.: Plasma membrane fluidity and surface motility of mouse C-1300 neuroblastoma cells. J. Cell Sci. 13, 415M20 (1973) Comoglio, P.M., Guglielmone, R.: Two-dimensional distribution of Concanavalin A receptor molecules on fibroblasts and lymphocyte plasma membranes. FEBS Letters 27, 256 258 (1972) Glimelius, K., Wallin, A., Eriksson, T.: Agglutinating effects of Concanavalin A on isolated protoplasts of Daucus carota. Physiol. Plant. 31,225-230 (1974)
Received 3 May; accepted 3 June 1977
Planta 136, 253-259 (1977)
Membrane Mobility and the Concanavalin A Binding System of the Plasmalemma of Higher Plant Protoplasts J. Burgess and P.J. Linstead John Innes Institute, ColneyLane, Norwich NR4 7UH, U.K.
Abstract. The binding of a colloidal gold-Concanavalin A (ConA) complex to the plasmalemma of tobacco leaf protoplasts has been investigated using scanning electron microscopy. At 5~ C the particles of goldConA appear to be randomly distributed over the surface of the protoplast. If the temperature is raised, the particles associate into clusters. Saturation of the membrane with particles can only occur when the weight of ConA in solution exceeds 1 lag/104 protoplasts in suspension, and when its concentration exceeds 15 ~tg/ml. These results are discussed in terms of the properties of the ConA binding site and the mobility of such sites within the membrane surface. Key words: Concanavalin A - Plasmalemma - Protoplasts - Scanning Microscopy.
Introduction
The lectin Concanavalin A (ConA) has been shown to bind to the plasmalemma of protoplasts from several plant sources (Burgess and Linstead, 1976a; Williamson et al., 1976). This binding may be accompanied by aggregation of the protoplasts (Glimelius et al., 1974; Burgess and Linstead, 1976a; Williamson et al., 1976). The binding of ConA to protoplasts has been studied by electron microscopy using either a colloidal gold ConA complex (AuConA) (Burgess and Linstead, 1976a), or a secondary labelling technique with haemocyanin (Smith and Revel, 1972; Williamson et al., 1976). The pattern of ConA labelling has been shown to depend on the source of the protoplast (Burgess and Linstead, 1976a) and on the conditions used for labelling (Williamson et al., 1976). ConA=Concanavalin A; AuConA=Colloidal gold-ConcanavalinA complex Abbreviations:
ConA coupled to an electron dense marker is a useful label for membranes in the electron microscope. It may also yield an insight into some of the properties of the membrane to which it is bound. In animal cells ConA is thought to bind to receptor sites which are mobile in the surface of the membrane (Nicolson, 1974; Rutishauser and Sachs, 1975). This mobility manifests itself in the clustering of bound ConA molecules due to their self-association. Clustering is enhanced by raised temperature (Comoglio and Filogamo, 1973; Nicolson, 1973) and is prevented by fixation of the membrane with glutaraldehyde prior to labelling the receptor sites (Comoglio and Guglielmone, 1972; Inbar et al., 1973). These observations accord with the fluid mosaic model for membrane structure proposed by Singer and Nicolson (1972). Thin sectioning techniques are unsuitable for examining the distribution of ConA on membrane surfaces, and the conclusions which can be drawn from such work are limited (Burgess and Linstead, 1976a). By the use of replica techniques, Williamson et al. (1976)have demonstrated clustering of haemocyanin as a secondary label to ConA on the surface of soybean protoplasts. The results presented here are an analysis of the behaviour of a colloidal goldConA complex at the surface of tobacco protoplasts by scanning microscopy. An attempt has been made to clarify the concentration dependence of ConA binding, and to demonstrate membrane mobility directly by temperature shift experiments.
Materials and Methods
Protoplasts were prepared as previously described (Burgess and Linstead, 1977). AuConAwas prepared as described by Burgess and Linstead (1976a). Methods for scanningelectron microscopy have been detailed elsewhere (Burgess et al., 1977). In order to examineprotoplasts prepared for scanningmicroscopyin thin sec-
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tions, the following procedure was adopted. Critical-point dried protoplasts were dusted on to the surface of a clean glass slide. The slide was then placed in a vacuum coating unit and coated with carbon and gold palladium (Burgess et al., 1977).The coated protoplasts were then washed from the slide with acetone, and embedded in araldite using standard procedure (Burgess and Fleming, 1974). For our initial labellingexperiments,batches of 1 5 x 106 protoplasts were washed in mannitol and resuspended in 8-10 ml of the AuConA solution. This procedure resulted in inconsistent labelling patterns due to exhaustion of the AuConA in dilute solutions (see Results, section (b)). In later experiments protoplasts were counted in a haemocytometerand the number of protoplasts added to a given volume of the AuConA solution was adjusted so as to obtain either saturation labelling or incomplete labelling, as desired.
conditions were large spherical projections which are a feature of many freshly prepared protoplasts (Fig. 2). Reference to thin sections of AuConA labelled protoplasts suggests that these projections overlay small vacuoles which protrude from the cytoplasm immediately beneath the plasmalemma (Fig. 3). Under the same conditions of labelling (see Methods) concentrations of ConA of 30 lag/ml or below did not always give rise to saturation labelling. Instead the A u C o n A particles appeared as clusters with more or less blank areas of membrane between them (Figs. 4, 5). This pattern was repeated when concentrations down to 10 gg/ml of ConA were used under our initial conditions. To test whether this effect was indeed one of concentration, protoplasts were exposed to a labelling solution of 30 ~tg/ml ConA in separate batches. The volume of the solution into which each identical batch of protoplasts from the same leaf was introduced was adjusted to give a range of ratios of weight of solute ConA/protoplast number. Saturation was achieved when this ratio exceeded 100 lag ConA/106 protoplasts. At levels of ConA below this saturation could not be achieved. Figures 4 and 5 are taken from experiments at 30 lag/ml where the ratio of ConA/protoplasts was 0.5 lag/104 protoplasts. If such protoplasts were subsequently incubated in further fresh labelling solutions at 30 lag/ml ConA, additional labelling occurred, and saturation could be obtained. In an attempt to show the lower concentration limit at which ConA in excess would lead to saturation cover of the membrane, protoplasts were incubated for 2 h at 30~ in solutions of AuConA of 15, 10 and 5 lag/ml so that the solute ConA was present at 2 lag/104 protoplasts. Saturation occurred at 15 lag/ml, but only about half the membrane area was covered with particles from the 10 lag/ml solution. 5 gg/ml gave more limited cover still.
Results
a) Appearance of AuConA in the Scanning Microscope ConA on its own did not give rise to any visible change at the surface of treated protoplasts when they were examined in the scanning microscope. When coupled to colloidal gold (particle size 16-20 nm), the complex appeared as an irregularly shaped particle (Figs. 5-11). The angular outline of A u C o n A serves to distinguish it from the more regularly spherical shapes of small protrusions which are a feature of freshly isolated and cultured protoplasts (Burgess and Linstead, 1976b). The identity of A u C o n A is inferred from the absence of such angular particles from the surface of non-labelled protoplasts. The presence of AuConA on the outer surface membrane of protoplasts prepared for scanning microscopy has been confirmed by embedding part of the population after metal coating. Thin sections of such preparations showed the layers of coating material overlying the colloidal gold particles (Fig. 1). This technique suggests that several particles of colloidal gold may give rise to a single coated particle as seen in the scanning mode (Fig. 1). This may explain both the variable size of AuConA particles seen under scanning conditions, and also the irregular outline of such particles. It emphasises the need for a cautious approach in the interpretation of the scanning microscope image.
b) Concentration Effects of ConA Binding Labelling of the membrane with AuConA at 125 lag/ ml resulted in saturation cover judged by thin sectioning techniques (Burgess and Linstead, 1976a). This has been confirmed for lower concentrations by scanning microscopy (Fig. 2). The only parts of the membrane which were not labelled under these saturating
c) Temperature Dependence of BOnding Protoplasts were examined after incubation for 30 rain in 30 lag/ml AuConA (insufficient to cause saturation, see above) at 5 ~ 25 ~ or 35 ~ C. At 5~ scattered random labelling was found with most of the particles in the form of units rather than clusters (Fig. 6). At 25~ under otherwise identical conditions, the label was present mainly in the form of clusters, with only a few units in between (Fig. 5). This pattern was repeated at 35 ~ C. Tighter clustering could be induced by prolonged incubation at room temperature or above. This is exemplified by Figure 7 where almost no flee units remain after 60 rain labelling at 35 ~ C.
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All Figures relate to tobacco leaf protoplasts, which have been washed twice with mannitol prior to the treatment described in the figure legend. Fig. 1. Thin section through a protoplast labelled in excess AuConA at 50 gg/ml, and embedded after fixation, drying and metal coating. The coating appears likely to simplify the image obtained by reflection, since in some cases several gold particles are seen to comprise a single coated particle (arrows) Fig. 2. Part of a similar protoplast to that shown in Figure 1, viewed by reflection scanning. The AuConA particles over the entire surface of the protoplast with the exception of large spherical projections (arrows) Fig. 3. Thin section through a protoplast treated as in Figure 1, but embedded conventionally. The only part of the surface membrane which is not covered with colloidal gold particles is a large projection Fig. 4. Scanning micrograph of a tobacco protoplast incubated for 30 min at 25 ~ C in a solution of AuConA at 30 pg/ml, insufficient to saturate the membrane. The particles of AuConA appear in clusters on the surface Fig. S. As Figure 4. Single particles are visibIe between the clusters (arrows) of AuConA
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Fig. 6. Scanning micrograph of a tobacco protoplast incubated for 30 min at 5 ~ C in a solution of A u C o n A at 30 gg/ml insufficient to saturate the membrane. The particles of A u C o n A are randomly distributed on the surface and form only very small clusters in a few places Fig. 7. Scanning micrograph of a tobacco protoplast incubated for 60 min at 3 5 ~ in solution of A u C o n A at 30 gg/ml insufficient to saturate the membrane. Large clusters of particles are visible. The m e m b r a n e between the clusters after prolonged incubation is almost devoid of A u C o n A particles Fig. 8. Scanning micrograph of a tobacco protoplast incubated for 30 min at 5 ~ C in a solution of 60 Itg/ml A u C o n A , insufficient to saturate the membrane. The A u C o n A is distributed randomly over the surface, predominantly as single particles Fig. 9. Scanning micrograph of a protoplast from the same population as Figure 8, but which was washed free of A u C o n A in solution and incubated at 35 ~ C for 30 min before fixation. The A u C o n A particles are predominantly clustered
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Fig. 10. Scanning micrograph of a tobacco protoplast incubated for 30 s at 35~ in a solution of AuConA at 30 gg/ml, sufficient to saturate the membrane. The surface is not completely covered at this time, and no clustering of AuConA particles is visible Fig. 11. Scanning micrograph of a tobacco protoplast incubated for 21/2min at 35~ C in a solution of AuConA at 30 gg/ml, insufficient to saturate the membrane. Some clustering of particles is evident even after this short labelling period
In a second series, protoplasts were prelabelled at 5 ~ C in a non-saturating a m o u n t of ConA at 60 gg/ ml for 30 rain. They were then washed free of the label in solution and incubated in mannitol at 25 ~ C or 35 ~ C. Protoplasts fixed immediately after the low temperature labelling showed a scattered distribution of single particles (Fig. 8, cf. Fig. 6). The same protoplast population after incubation for 30 min at 35 ~ C showed cluster formation (Fig. 9). This clustering was not observed on protoplasts which were fixed in glutaraldehyde prior to incubation at raised temperatures. Finally protoplasts were incubated at 5 ~ for 30 min with an excess of C o n A at 30 gg/ml. Complete saturation of the surface occurred suggesting that low temperatures do not impair the efficiency of binding or reduce the n u m b e r of available sites.
In the presence of an excess of ConA in solution at 30 ~tg/ml saturation of the surface occurred within 5 min at 35 ~ C. After only 30 seconds labelling under these conditions, a r a n d o m non-saturating label pattern was found (Fig. 10). At 5 ~ under the same conditions, saturation was not achieved until 30 min. Again the intermediate pattern was one of r a n d o m single particles. I f C o n A were not present in excess, the final pattern at 35 ~ C was one of cluster formation (Figs. 4, 5, cf. Fig. 7). The degree of clustering was time dependent. Even after 21/2 min of labelling, small clusters were visible under non-saturating conditions (Fig. 11). At 5 ~ C under these conditions, the pattern obtained was one of randomly spaced single particles whose density on the m e m b r a n e increased up to 30 min of incubation (cf. Figs. 6, 8).
d) Time Dependence of Binding Discussion Protoplasts were placed in A u C o n A solutions, then samples were withdrawn at various times for fixation. Patterns of labelling which were observed depended upon the ratio of ConA/protoplasts in solution, and the temperature at which the experiment was conducted.
The binding of the gold-ConA complex to the membrane ofprotoplasts is not easily interpreted in molecular terms. It is uncertain how the colloid and the protein associate, and whether this association is uniform within a given batch of AuConA, or between
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batches. Examination of sectioned material which has been otherwise prepared as for scanning microscopy suggests that a "single" particle of AuConA viewed in the scanning mode may embrace several particles of colloidal gold. This clearly implies that a single scanned particle may contain several molecules of ConA. In.addition to this difficulty of interpretation, it is known that ConA itself undergoes temperature and pH-dependent association (Huet et al., 1974). Furthermore the possibility exists of temperature dependent masking of binding sites (Inoue, 1974). For these reasons this discussion is limited to general patterns of behaviour of the AuConA complex. In particular, the terms " u n i t " , "cluster" and "saturation" are used to denote a microscopical appearance of the AuConA complex, and do not carry any direct molecular connotation. The first clear result which emerges is that it is possible to saturate the membrane with AuConA. This has been suggested previously (Burgess and Linstead, 1976a) using thin sectioning techniques and a high concentration of ConA at room temperature. We have confirmed this effect down to 15 pg/ml of ConA, and at temperatures of 5~ C. Masking of binding sites at low temperature therefore does not seem to occur in tobacco protoplasts (Inoue, 1974). Saturation labelling requires an excess of AuConA in solution. Attempts to quantify this effect have suggested that approximately 1 gg of ConA is needed to produce saturation o n 10 4 protoplasts. If this ratio is exceeded saturation will be obtained whether by means of a low volume of high concentration solution, or a large volume of a dilute solution, down to 15 pg/ml. This weight of ConA corresponds to the order of 108 ConA molecules per protoplast. It is very doubtful whether the number of binding sites can be deduced from such a figure however, due to uncertainties concerning the nature of the association between the colloid and the ConA, the size distribution of the protoplasts themselves in different populations, and the amount of ConA remaining in solution at the end of the labelling period. A figure of 3x 10v binding sites per cell has been given for fibroblasts (Collard and Temminck, 1975). The failure of the label at 30 pg/ml to produce saturation in an experiment such as that illustrated by Figure 4 is therefore due to the removal of ConA from solution, and the resultant reduction of its concentration to a low level. This has been demonstrated by reincubating such partially labelled protoplasts in further aliquots of AuConA solution at 30 pg/ml. Saturation results when the protoplasts have been exposed to a sufficient weight of ConA in solution. In order to obtain saturation the number of bind-
ing sites must be very large, or some non-specific mechanism of binding must be occurring. Non-specific binding is unlikely, since e-methyl mannoside is known to inhibit ConA binding to protoplasts (Burgess and Linstead, 1976a; Williamson et al., 1976). The nature of the binding site is unknown. Under conditions when the amount of ConA in solution is limiting, saturation of the surface cannot be obtained. The pattern of distributed particles which is found depends on the temperature and time of the experiment. Repeatedly it has been found that protoplasts labelled at 5~ C show a random distribution of predominantly single particles, whereas on those labelled at 25 ~ or 35~ the particles are clustered together. That this is a result of the redistribution of bound AuConA has been demonstrated directly by labelling protoplasts at 5~ C, then incubating them in a label-free environment at raised temperatures. Clustering of the particles can in this way be followed in a single protoplast population. Glutaraldehyde fixation after low temperature labelling prevents cluster formation. This protocol is preferable to labelling after glutaraldehyde fixation has been used to immobilise the receptor sites (Van Blitterswijk et al., 1976; Williamson et al., 1976), since it avoids the possibility of a glutaraldehyde-induced change in the binding process. This ability to label the membrane at low temperature illustrates the usefulness of AuConA as a marker; the ConA-haemocyanin double labelling method is not usable at 5~ C (Temminck et al., 1975) The mobility of the AuConA complex-in the membrane is not high. Periods of 30-60 min at 35~ C are necessary for the formation of tight clusters of particles. This is considerably longer than the time required for the clustering of Ferritin-ConA on turnout cell membranes (Nicolson, 1973). It explains why a clustered pattern of labelling is never observed in time course experiments leading to saturation of the surface. Presumably at least part of the comparative immobility of the AuConA complex could result from the size and density .of the gold particles themselves. The mobility of the AuConA complex which has been directly demonstrated in this study is consistent with the concept of the tobacco protoplast plasmalemma being a "fluid mosaic" (Singer and Nicolson, 1972). These results suggest that the tobacco protoplast plasmalemma contains a large number of binding sites which have a high affinity for ConA. These sites show increased mobility with raised temperature, and they are immobilised by fixation in glutaraldehyde. AuConA is clearly a useful probe for the investigation of membrane mobility. We have suggested elsewhere that polyethylene glycol fluidises the membrane on the basis of the behaviour of AuConA in the vicinity
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of the junction of adhering protoplasts (Burgess et al., 1977). It is hoped that other determinants of membrane mobility may now be identified.
Huet, C., Lonchampt, M., Huet, M., Bernadac, A. : Temperature effects on the Concanavalin A molecule and on Concanavalin A binding. Biochim. Biophys. Acta 365, 28 39 (1974) Inbar, M., Huet, C., Oseroff, A.R., Ben-Bassat, H., Sachs, L.: Inhibition of lectin agglutinability by fixation of the cell surface membrane. Biochim. Biophys. Acta 311, 594-599 (1973) Inoue, M.: Cell agglutination mediated by Concanavalin A and the dynamic state of the cell surface. J. Cell Sci. 14, 197-202 (1974) Nicolson, G.L. : Temperature-dependent mobility of Concanavalin A sites on tumour cell surfaces. Nature New Biol. 243, 218 220 (1973) Nicolson, G.L. : The interaction of lectins with animal cell surfaces. Int. Rev. Cytol. 39, 89 190 (1974) Rutishauser, U., Sachs, L.: Receptor mobility and the binding of cells to lectin coated fibres. J. Cell Biol. 66, 76 85 (1975) Singer, S.J., Nicolson, G.L. : The fluid mosaic model of the structure of cell membranes. Science 175, 720-731 (1972) Smith, S.B., Revel, J-P.: Mapping of Concanavalin A binding sites on the surface of several cell types. Dev. Biol. 27, 434 441 (i972) Temminck, J.H.M., Collard, J.G., Spits, H., Roos, E. : A comparative study of four cytochemical detection methods of Concanavalin A binding sites on the cell membrane. Exp. Cell Res. 92, 307-322 (1975) Van Blitterswijk, W.J., Walborg, E.F., Feltkamp, C.A., Hilkmann, H.A.M., Emmelot, P.: Effect of glutaraldehyde fixation on lectin-mediated agglutination of mouse leukaemia cells. J. Cell Sci. 21, 579-594 (1976) Williamson, F.A., Fowke, L.C., Constabel, F.C., Gamborg, O.L. : Labelling of Concanavalin A sites on the plasma membrane of soybean protoplasts. Protoplasma 89, 305 316 (1976)
References Burgess, J., Fleming, E.N. : Ultrastructural studies of the aggregation and fusion of plant protoplasts. Planta (Berl.) 118, 183-193 (1974) Burgess, J., Linstead, P.J.: Ultrastructural studies of the binding of Concanavalin A to the plasmalemma of higher plant protoplasts. Planta (Berl.) 130, 73 79 (1976a) Burgess, J., Linstead, P.J.: Scanning electron microscopy of cell wall formation around isolated plant protoplasts. Planta (Berl.) 131, 173-178 (1976b) Burgess, J., Linstead, P.J. : Coumarin inhibition of microfibril formation at the surface of cultured protoplasts. Planta 133, 267 273 (1977) Burgess, J., Linstead, P.J., Fisher, V.E.L.: Scanning microscopy of higher plant protoplasts. Micron (in press) (1977) Collard, J.G., Temminck, J.H.M. : Differences in density of Concanavalin A binding sites due to surface morphology of suspended normal and transformed 3T3 fibroblasts. J. Cell Sci. 19, 21 32 (1975) Comoglio, P.M., Filogamo, G.: Plasma membrane fluidity and surface motility of mouse C-1300 neuroblastoma cells. J. Cell Sci. 13, 415M20 (1973) Comoglio, P.M., Guglielmone, R.: Two-dimensional distribution of Concanavalin A receptor molecules on fibroblasts and lymphocyte plasma membranes. FEBS Letters 27, 256 258 (1972) Glimelius, K., Wallin, A., Eriksson, T.: Agglutinating effects of Concanavalin A on isolated protoplasts of Daucus carota. Physiol. Plant. 31,225-230 (1974)
Received 3 May; accepted 3 June 1977
