The appearance and internalization of transferrin receptors at the margins of spreading human tumor cells

Cell, Vol . 40, 1 99-208, January 1985, Copyright © 1985 by MIT

0092-8674/85/010199-10 $02 .00/0

The Appearance and Internalization of Transferrin Receptors at the Margins of Spreading Human Tumor Cells Colin R. Hopkins Department of Medical Cell Biology The Medical School University of Liverpool Liverpool L69 3BX, England Summary Using gold complexes stabilized with a monoclonal antibody specific for the human transferrin receptor, the distribution of transferrin receptors on the surfaces of human epidermoid carcinoma A431 cells has been mapped at high resolution . On prefixed cells and cells incubated at 5°C, the receptors are predominantly within and around clathrin-coated microdomains near the free cell margin . By preincubating the cells with saturating concentrations of free antibody at 5°C and warming them to 37°C in the presence of the gold complexes, the appearance of new receptors in the membrane has been followed . The majority first appear near the free cell margin and then move centripetally . At first, they are monodisperse, but as they move toward the site of internalization they form loose aggregates . Within the immediate vicinity of the clathrin-coated microdomains the migrating receptors form closely packed, ordered aggregates . These observations indicate recycling transferrin receptors move to their site of internalization without cross-linking . Introduction Studies of receptor-mediated endocytosis have identified several populations of plasma membrane receptor proteins that are continuously internalized and then recycled back to the cell surface (Goldstein et al ., 1979 ; Anderson et al ., 1982 ; Steinman et al ., 1983) . By carrying bound ligand on the inward leg of their journey these proteins are able to provide the cell with a continuous supply of metabolites . Since the original descriptions of the low density lipoprotein (LDL) uptake system (Anderson et al ., 1977a, 1977b ; Goldstein et al ., 1979 ; Anderson et al ., 1982) it has become clear that most recycling receptors are internalized by specialized microdomains of the cell surface : the clathrin-coated pits . It is also clear that some receptors have the capacity to aggregate within these microdomains regardless of whether or not they have bound ligand (Anderson et al ., 1982) . The site(s) at which newly inserted receptors first appear on the cell surface has not been identified, and although a genetic deficiency in which the receptor lacks the ability to transfer to coated pits has been described for the LDL receptor (Anderson et al ., 1977b), nothing is known about the mechanisms responsible for transferring recycling receptors to their sites of internalization . From morphological studies on receptors for signaling ligands such as those for epidermal growth factor, there is good evidence to suggest that ligand binding causes some kinds of receptor to become cross-linked and aggre-

gate on the cell surface (Maxfield et al ., 1978 ; Schlessinger et al ., 1978 ; Hopkins et al ., 1981) . From studies using monovalent ligands with these receptors there is accumulating evidence that this ligand-induced aggregation is the major requirement for the transfer of these receptors to the sites at which they are internalized (Kahn et al ., 1978; Schechter et al ., 1979 ; Hopkins et al ., 1981 ; Zidovetzki et al ., 1981 ; Gregory et al ., 1982 ; Yarden et al ., 1982 ; Schreiber et al ., 1983) . Whether or not receptors that internalize without binding ligand also need to aggregate in order to transfer to their site of internalization needs to be determined . Most published studies of receptor-mediated endocytosis in cultured cells have used cells with a fibroblast phenotype, and in the majority of these studies the sites at which receptor-mediated ligand internalization occurs have been shown, like their coated pits, to be distributed rather evenly over the upper cell surface (Anderson et al ., 1982 ; Schlessinger et al ., 1978) . Recently, however, there have been reports that the sites at which recycling ligands internalize may not always be distributed randomly throughout the coated pit population and that, in motile or spreading cells in particular, these sites may be distributed preferentially toward the leading, free cell margin (Bretscher, 1983 ; Bretscher and Thomson 1983 ; Ekblom et al ., 1983) . Earlier studies on newly synthesized plasma membrane components also indicated that these components may appear preferentially at the free margin of the cell (Marcus, 1962) . Together these observations suggest that in motile cells (and spreading epithelial cells) recycling receptors may be identifying domains at the cell margin in which membrane extension is being brought about by the insertion and subsequent rearward movement of "new" membrane components. The recycling receptors may thus be behaving like untethered "marker buoys ; identifying the direction of a generalized membrane flow . This idea, first suggested by the work of Bretscher (Bretscher et al ., 1980 ; Bretscher, 1982), has wide implications because it bears upon the fundamental question of whether or not there is a general, directed flow of membrane lipids and proteins within cell membrane boundaries (Abercrombie et al ., 1972 ; Bretscher, 1976) . We have used gold complexes carrying receptor-specific monoclonal antibodies to map, by electron microscopy, the distribution of transferrin receptors at the leading edge of human epidermoid carcinoma A431 cells . We have identified receptors appearing on preparations in which the resident receptors have been blocked with free antibody, and we have shown that the majority of the newly appearing receptors arise near the free cell margin . Following their appearance on the upper cell surface these receptors form loose aggregates and move centripetally to internalize within an extensive system of coated membrane domains in the vicinity of the most marginal microvilli and fillipodia . Our studies suggest that, unlike receptors for signal molecules such as epidermal growth factor, transferrin receptors do not become cross-linked before being transferred to their site of internalization . These studies also provide direct sup-

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Figure

1. Cells

Prefixed

in 2% Formaldehyde

before

incubation

with 8 nm Gold-B3/25

Complexes

Whole cell mount. The flattened margin of the cells gives off long spike-like fillipodia and, some distance from the free cell margin, shorter finger-like microvilli arise. Near the most peripheral microvilli there are numerous clusters of gold particles. There are few gold particles either at the free margin or over the main body of the cell. Ear: 50 pm. Inset: fluorescence micrograph of cells incubated with B3/25 antibody 15 min at 37X, fixed, then incubated with FITC rabbit anti-mouse antibody. Strong fluorescence at the periphery of the spreading cells is evident. Bar: 2 pm.

port for the suggestion that, in spreading cells, the leading edge is a site at which the insertion and removal of recycling receptors continuously occurs. Results In sparse culture on a plastic surface, A431 cells grow as small islands of cells. With cell division these islands grow larger, and as growth proceeds, their free margins extend over the substratum. The free margins of these enlarging cell islands vary in morphology. Some cells have broad flat pseudopodia and an elaborate array of narrow spike-like fillipodia (Figure 1) while others have thicker, blunt margins essentially free of fillipodia. Cells with flat pseudopodia and numerous fillipodia extend out from the main cell mass and form the leading edge of the spreading monolayer. In very sparse culture where there are many single cells, most of the cells have the more elaborate morphology. Away from the free margin of fillipodia-bearing cells the upper cell surface is elaborated into short, finger-like microvilli and low ridges that often bend over to form shallow folds. In prefixed preparations incubated with f33/25-gold complexes the majority of the gold particles occur on the

fillipodia-bearing cells. As shown in Figure 1 these gold complexes are distributed in irregular groups of up to 20 particles, usually at the inner margin of the flattened pseudopodia and often close to the base of the most peripheral microvilli. Over the rest of the cell surface and over the other cells in the aggregate, gold particles are sparser and in groups of only two or three. Only in dense culture where cells extensively overlap their neighbors and there are occasional free lateral margins does the density of gold particle labeling sometimes approach that at the free margin of fillipodia-bearing cells. The distribution of gold particles on unfixed cells incubated with B3/25-gold at 5% for 30 min is essentially the same (Figure 2). On cells incubated with 1 pglml free antibody at PC, rinsed at 5% and then incubated with B3/25-gold at 5% the levels of gold particles are negligible (less than 10 particles/10 pm2). The free antibody thus effectively blocks subsequent B3/25-gold binding. On cells incubated with free antibody at 5OC, rinsed at 5°C then transferred to medium containing B3/25-gold at 37% a significant number of bound gold particles is seen within 1 min. With longer incubations the number of gold particles increases (Table l), so that by 10 min it equals that in cells unincubated with

Transferrin 201

Figure

Receptors

2. Ceils

Incubated

in Spreading

A431 Cells

30 Min with 8 nm Gold-E3/25

at 5°C

(a-f) Particles clustered in shallow pits of varying depth. (c) Deeper pocket-like note increased granularity of background within pit. Bars: 100 nm.

free antibody. By 15 min it is more than 20% greater than that at 10 min. If cells are incubated with free antibody at 5%, rinsed at EPC, and then incubated at 37% for 1-15 min before being incubated at 5% with B3/25-gold complexes, the number of gold particles is about 30% fewer than in experiments where the gold complexes are present during the 37% incubation. However, the patterns of labeling are essentially the same (see below). We conclude from these observations that when cells are incubated with B3/25 at 5% and then warmed to 37% for 1-15 min most of the antibody remains bound. A similar result was obtained in an earlier study where the dissociation of B3/25 was followed

cavity

at base of microvillus

(arrow).

(f) Figure

printed

as a negative;

with 1251-B3/25 (Hopkins and Trowbridge, 1983). We believe that the majority of B3/25-gold particles which bind during a 37°C incubation that follows a 5% incubation in free antibody are bound to receptors which have appeared during the 37% incubation. Moreover, by incubating with B3/25-gold at 5% after, rather than during, the 37% incubation we can show that the distributions of particles we observe are not caused by B3/25-gold binding; nor do they simply reflect an increased binding of particles with time. By rotary shadowing cells at low angle we have been able to obtain some indication of the number of gold particles distributed on the exposed surfaces of the cell. As we



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Table 1 . Receptors Appearing after Cells Are Warmed to 37°C Min . at 37°C with Gold-83/25 following 30 min at 5°C with Free Antibody

Particles per µm2 within 2 .0 Nm of Free Cell Margin (± SEM)

% Particles with Halo

0 1 5 10 15

0 .15 0 .42 2 .40 3 .86 4 .04

34 84 84 53 26

± ± ± ± ±

0 .007 0 .01 0 .48 0 .46 1 .02

,

The number of particles per cell within 2 .0 µm of the free margin was counted on 300 cells for each time point (see Experimental Procedures) . The density of particles at the periphery of cells incubated at 5°C with 8 nm gold-83/25 without prior incubation with free antibody was 1 .89 ± 0 .46/µm 2 .

Table 2 . Quantitative Estimate of Receptor Aggregation Min . at 37°C with Gold-B3/25 following 30 min at 5°C with Free Antibody

Monodisperse

Loose Packed Aggregates Aggregates

0 1 5 10

0 .3 ± 0 .02 0 .2 ± 0 .05 0 .3±0 .04 0 .5±0 .10

10 .6 ± 1 .6 25 .3±1 .7 23 .4±3 .2

No . of Particles ± SEM

20 .6± 1 .6 38 .3±2 .1

The formation of aggregates was quantitated by enlarging micrographs to 50,000 x and using a 5 x 5 cm grid to score the number of particles with a neighboring particle within 2 nm (packed aggregates), the number of particles with neighboring particles within 5 nm but not 2 nm (loose aggregates), and the number of particles without a neighboring particle within 5 nm (monodisperse) .

showed in an earlier study of gold complexes mounted on a glass substrate (Tolson et al ., 1981), this kind of shadowing accentuates the edge of the gold-antibody complex and results in a 1-2 nm diameter halo around particles exposed to the shadowing beam . On a glass surface all of the complexes bear a halo. As shown in Table 1 the proportion of particles identified in this way is maximal during the first 5 min of the 37°C incubation with B3/25-gold . Thereafter, as gold complexes are internalized the proportion of particles with halos declines . As the B3/25-gold particles increase in number during the 37°C incubation they become redistributed (Table 2) . At 1 min the gold particles are monodisperse and randomly distributed but confined to an area within 1 µm of the free cell margin . At 2 min they remain within this area but are distributed as loose aggregates of 20 to 250 particles . At 5 min the loose aggregates of particles are still present but they have moved toward the body of the cell so that they are grouped around the bases of the short finger-like microvilli (Figure 3) . At this time gold particles are also distributed into more closely packed aggregates, in which they

often show some indication of an ordered array . These more closely packed particles lack a halo, rarely overlap, and when regularly ordered have a center-to-center spacing of approximately 20 rim . The aggregates formed by closely packed particles occur most often at the bases of the microvilli, but they are also present at the bases of fillipodia and folds . Occasionally, closely packed particles lacking halos occur at the edge of the loose particle aggregates, unrelated to any obvious topographical feature . To demonstrate that the changing distribution pattern observed during the 1-5 min incubation represented a sequence of steps rather than separate events, a pulse chase exercise was carried out . In this exercise cells were incubated for 10 min at 5°C with B3/25-coated 8 nm gold particles, rinsed, incubated with free antibody for 30 min at 5°C, then incubated for 5 min with B3/25-coated 1-3 nm gold particles at 37°C . The preparations displayed closely packed aggregates that contained predominantly 8 nm complexes at the base of the microvilli ; elsewhere the particles were predominantly 1-3 nm (Figure 4) . We conclude that the packed aggregates of particles at the bases of the microvilli arise by the loose aggregates of receptors moving into, and packing within, these areas . After 15 min at 37°C gold particles are still found in closely packed aggregates at the bases of the microvilli, but in addition there are short linear arrays of particles extending away from them (Figure 5) . Like the packed aggregates, these particles are without a halo and they often have a regular, linear spacing . Occasionally these linear arrays of particles form a branching reticulum and are similar, therefore, to the particle distributions described in earlier studies (Hopkins and Trowbridge, 1983 ; Hopkins, 1983) in which they were shown to be located within a system of tubular cisternae lying in the peripheral cytoplasm . Distribution of HLA Class I Antigens The distribution of class 1 HLA antigens on A431 whole mounts was demonstrated using a two-step procedure in which cells were incubated at 5°C with a specific monoclonal antibody (W6.32 .1) fixed in 3% formaldehyde and then incubated with protein A-8 nm gold complexes . Following this treatment the gold particles bound to sites distributed randomly over the entire cell surface . Local concentrations at the periphery of the cell in the vicinity of the microvilli and fillipodia similar to those observed with B3/25-gold complexes were not observed (Figure 6) . However, in these preparations, pit-like cavities (see below) became more obvious because HLA antigens were excluded from them (Figure 6) . In cells incubated with the W6 .32 .1 antibody at 37°C the distribution of HLA antigens was essentially the same. These experiments suggest that the appearance and removal of binding sites identified at the cell periphery with B3/25-gold complexes are related to the

Figure 3 . Cells Preincubated with Free Antibody at 5°C Then Incubated 5 Min at 37°C with 8 nm Gold-B3/25 Complexes Whole cell mount . The area is situated at the inner margin of the lateral pseudopod, where short finger-like microvilli and low folds first arise . Away from these surface protruberances the gold particles are often surrounded by a halo emphasized by the rotary shadowing with platinum . These particles are therefore exposed on the open cell surface where they form loose aggregates clearly distinguishable from the more closely packed particles found at the bases of the microvilli . Within the packed aggregates the particles lack halos, are often in linear array, and sometimes show a parallel or concentric arrangement . Bar: 200 nm .

Transferrin 203

‘

Receptors

in Spreading

-m . --4

.

A431

Cells

Cell 204

Figure

4. Gold-Antibody

Complexes

Associated

with Coated

Pits

(a) Cells preincubated with free antibody for 30 min at 5% then incubated 15 min at 37°C with 8 nm gold-B3/25 complexes. Whole cell mount. Most of the particles are grouped within clearly defined aggregates. Within these aggregates the particles are often distributed in parallel, linear arrays (small arrows). The relationship between the surface topography and the aggregates of granules is complicated by extensive overlapping and infolding of the membrane. The large arrow indicates an instance where the relationship between a particle aggregate and microvillus is clearly displayed. Compared with Figure 2 there are relatively fewer particles with halos and the closely packed aggregates are more extensive. (b) Cells incubated with free antibody for 30 min at 5% then incubated 10 min at 37% with 8 nm gold-B3/25 complexes. Thin section cut normal to ceil surface. Gold particles within shallow depression at base of microvillus and within more deeply invaginated pits (arrow). (c) Preparation as in(b), section cut parallel to cell surface in preparation fixed to preserve coated membranes. Coated pit at base of a microvillus not included in section plane is displayed. (d) Preparation as in (c) section cut normal to cell surface. (e) Preparation as in (c) displaying tangential section through gold particle containing pit. Arrows indicate particles in ordered linear array. (f) Cells incubated with 8 nm gold-B3/25 at VC, free antibody at 5%. and then incubated with

Transferrin 205

Figure

Receptors

in Spreading

5. Cells Preincubated

A431 Cells

with Free Antibody

30 Min at 5°C

Then

Incubated

15 Min at 37°C with 8 nm Gold-B3/25

Complexes

The edge of the cell is beyond the upper margins of this micrograph; the inner margin of the broad flat pseudopod runs along a line indicated by two large arrows. At the bases of the fillipodia and microvilli, packed aggregates of particles (without halos), often in an ordered linear array, are distributed. There are also some packed aggregates in areas lacking any obvious topographical feature (*). Near the lower margin of the micrograph the gold particles often form short straight lines (small arrows). These particles are probably within the tubular cisternae lying below the plasma membrane in the peripheral cytoplasm. Inset: lines of particles, probably within cytoplasmic cisternae, extending away from particle aggregates located at the bases of two microvilli. Bars: 0.5 urn.

transfer of a selected population of membrane components rather than the generalized movement of all membrane proteins. The Relationship between Aggregates of Gold Particles and Clathrin-Coated Membranes Rotary shadowing of whole mounts displays the surface topography of the cell surface in detail. At high resolution the surface microstructure displays the background granularity of the platinum/carbon coat, and in some areas it is also studded with larger rounded particles. It is very similar to the surface microstructure of other cell types that have been examined en face at high resolution (Heuser, 1980; Aggeler and Werb, 1982; Harding et al., 1983). On

cells incubated at 5% with B3/25-gold complexes pit-like cavities, usually containing several gold complexes, can be identified (Figure 2). They vary from shallow, usually spherical hollows (in the order of 100 nm diameter) to deep, narrow pits (in the order of 70 nm diameter). The cell surface within the shallow hollows consistently displays a coarser microstructure (Figure 2e), but the resolution of the technique prevents a more detailed analysis of its constituents. The pits occur on the open flat cell surface at the bases of microvilli and fillipodia, where they may extend as shallow pockets running laterally beneath the cell surface. In some instances groups of gold particles occur where the surface microstructure is typical of that found in pits but where the surface appears to be flat, without apparent

l-3 nm gold-B3/25 at 37% for 5 min. Whole cell mount. At the base of the microvillus (M) there is a packed aggregate of 8 nm particles essentially free of 1-3 nm particles whereas on the free cell surface there is an aggregate of 8 nm particles that includes 1-3 nm particles. Elsewhere on the free cell surface are distributed aggregates of l-3 nm particles. Bars: 200 nm.

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Figure

6. Histocompatibility

Antigen

Distribution

(a) Cells incubated with W6.32.1 antibody 30 min at V’C, fixed, and incubated with 8 nm gold-protein A complexes. Whole cell mount. Particles distributed evenly over the cell surface. (b) Cells incubated with W6.32.1 antibody 30 min at 37% before fixation and incubation with gold-protein A complexes. Particles excluded from shallow surface pits. Bars: 0.5 pm.

indentation. In preparations incubated at 37% and in which gold particles are packed into dense aggregates the distribution of the aggregates is similar to the distribution of the shallow indented areas and pits in cells incubated at 5%. However, in these more heavily labeled preparations, it is not possible to see the surface underlying the packed particle aggregates. In conventional thin sections of labeled preparations packed aggregates of particles are observed both in shallow depressions adjacent to microvilli and in deeper, flaskshaped coated pits (Figure 4). The distribution of coated membranes is not, however, clearly displayed, and for their optimal preservation we have used a procedure involving glutaraldehyde, tannic acid, and saponin (Maupin and Pollard, 1981). These preparations show clearly that closely packed gold particles are located over coated regions of membrane and in tangential sections; where gold particles and coated membrane can be seen in the same section plane, an ordered, linear distribution of the particles is sometimes displayed (Figure 4). Discussion Previous studies on transferrin receptors have shown that in steady state conditions between a quarter and a third of the total receptor population is on the cell surface (Bleil and Bretscher, 1982; Ciechanover et al., 1983; Hopkins and Trowbridge, 1983; Lamb et al., 1983). In several different systems these surface receptors have been shown to internalize rapidly, and after a brief processing within an intracellular compartment (the endosome) the majority of them are recycled back to the cell surface (Van Renswoude et al., 1982; Enns et al., 1983; Hopkins, 1983; lacopetta and Morgan, 1983; Klausner et al., 1983; Willingham et al., 1984). In A431 cells, where there is evidence that some recycling receptor populations contain a large proportion of receptors that remain on the cell surface (Anderson et al., 1981) we have shown that the majority of transferrin recep-

tors recycle with an intracellular processing time of approximately 7.5 min (Hopkins and Trowbridge, 1983). As in the LDL system (Anderson et al., 1982) the binding of transferrin is not thought to be a prerequisite for the internalization and recycling of the receptor (Hopkins and Trowbridge, 1983). Our studies show that the distribution of transferrin receptors on the surface of A431 cells is nonrandom, and under steady state conditions the majority of them are located within coated domains at the periphery of cells with free margins. Other surface antigens such as HLA and EGF receptors (unpublished observations) have a random distribution on these cells; and since we have found transferrin receptors to have a similar peripheral distribution on other epitheloid cell types (e.g. WISH cells, unpublished observations), we believe that the predominantly peripheral distribution of transferrin receptors we have observed on A431 cells in this study probably typifies this kind of recycling receptor. Although coated microdomains are most common at the periphery of A431 cells, pits containing transferrin receptors do occur over the entire cell surface. It seems probable, therefore, that the high density of transferrin receptors in the pits at the cell periphery arises because the majority of this rapidly recycling receptor population is being inserted and withdrawn in this vicinity. Our studies on prefixed cells show that on cells which have not been precooled the majority of transferrin receptors are already in coated pits. This is presumably because transit through these microdomains is the slowest step in the recycling pathway. In our experiments in which cells warmed to 37% were first cooled to 5°C we have been able to identify receptors on the cell surface en route to their site of internalization; presumably, this is because in these experiments movement in the plane of the surface membrane is also much reduced. The experiments on cells preincubated with free antibody clearly indicate that transferrin receptors recycling back to the plasma membrane are inserted into the peripheral pseudopodia close to the free

Transferrin Receptors in Spreading A431 Cells 2 07

cell margin . In these experiments the patterns of distribution at early time points (after transfer to 37°C) indicate that the newly inserted receptors probably appear as monodisperse singletons ; there is no indication in our studies of receptors making their first appearance as tightly packed clusters . The newly inserted receptors then move to form loose aggregates. Within these loose aggregates there is no indication of an ordered array, and although the density of their packing varies, it is rarely as close at 20 nm (center to center) . It is probable that these loose aggregates form passively and do not depend upon their constituent receptors being held together by direct intermolecular linkages . Close, ordered packing of gold particles is apparent when receptors reach the areas around the bases of the peripheral microvilli and fillipodia . In these regions a regular (center-to-center 20 nm) spacing is often seen, and it is worth noting that this array is compatible with the dimensions of the components within clathrin networks seen on the cytoplasmic surface of the membrane (30 nm diameter hexagons, bounded by 7-8.5 nm struts) . However, it will be necessary to obtain superimposed images of particles and baskets in order to pursue this question . In thin sections of A431 cells very few profiles suggest that coated microdomains give rise to free coated vesicles, and there is no other indication of how the internalized gold complexes might be transferred to an intracellular boundary. As reported in an earlier study (Hopkins and Trowbridge, 1983), the first intracellular compartment in the endocytic pathway of A431 cells appears to be a branching network of tubular cisternae that can be identified in whole mounts by its content of gold particles distributed in a characteristic linear array. In the preparations used in the present study there were (after longer, 10-15 min incubations) frequent images suggesting that elements of the tubular network containing the linearly arranged particles were close to the membrane carrying the packed aggregates . However, no profiles demonstrating continuity between these two membrane boundaries were observed in thin sections . Taken together the observations obtained in this study show that in A431 cells the majority of recycling transferrin receptors are inserted into the plasma membrane at the free cell margin . These newly inserted components then move centripetally. As they move they form loose aggregates, but it is only within the vicinity of coated membrane domains that the receptors become closely packed in ordered array. No clear insight into the mechanisms responsible for these movements in the plane of the plasma membrane, which transfer the receptors to the coated microdomains, is available, but the patterns of gold complexes observed suggest that these movements are not entirely random . The lines of particles extending from the edge of the cell to the inner margin of the peripheral pseudopodia and the buildup of loose aggregates of gold particles around the closely packed aggregates suggest there may be both general and localized flows of membrane . Thus, in addition to a stream of membrane flowing centripetally in from the edge, there may be local currents draining toward coated microdomains distributed around the peripheral microvilli and fillipodia .

Experimental Procedures Antisera, Reagents, Biochemicals The B3/25 monoclonal antibody was raised against the human hemopoietic cell K562 and is specific for the transferrin receptor (Trowbridge and Omary, 1981) . It was a gift from Dr . I . S. Trowbridge, Salk Institute, Cal . W6 .32 .1 is a monomorphic monoclonal antibody that reacts with native HLA-A, -B, and -C molecules . It was a gift from Dr . A . Williams, Dunn School of Pathology, Oxford, England . Manufacture and Labeling with Antibody-Gold and Protein A-Gold Complexes B3/25-gold and protein A-gold complexes were made as described previously (Tolson et al ., 1981 ; Hopkins and Trowbridge, 1983) . Before use they were washed by centrifugation in PBS containing 0 .1% carbowax on a Beckman airfuge (Beckman Instruments Inc ., Fullerton, Cal .) for 150,000 x g/min . By electron microscopy the suspensions of particles appeared entirely monodisperse except for 5 nm particles that sometimes showed slight aggregation . For incubating with cells, gold complexes were suspended in PBS containing 0 .1% BSAl0.1% carbowax . Processing for Electron Microscopy For whole mount microscopy, cells were fixed in 2% glutaraldehyde in 0 .1 M cacodylate buffer (pH 7.4) for 30 min . They were postfixed in 2% osmium tetroxide in 0.1 M phosphate buffer, dehydrated, and criticalpoint dried from 100% ethanol . The dried cells were rotary shadowed with platinum at an angle of 25° and coated with carbon in a Balzer freeze-drying unit (Balzer Ltd ., High Wycombe, England) . They were then floated off the coverslips with hydrofluoric acid, rinsed in distilled water, and mounted directly on copper grids . For preparing ultrathin sections, cell monolayers were fixed routinely in dilute Karnovsky fixative (Karnovsky, 1965), postosmicated, scraped from the coverslip, dehydrated in ethanol, and embedded in Epon as described previously (Hopkins and Trowbridge 1983) . To preserve coated membranes optimally, cells were fixed in glutaraldehyde, saponin, and tannic acid as described by Maupin and Pollard (1983) . Quantitation To quantitate the distribution of B3/25-gold particles on cell surfaces of whole mounts the number of particles per µm 2 were counted at the free margins of 300 cells for each time point . These counts were made on whole cell mounts magnified 9100x in the electron microscope by placing a 3 x 5 cm grid over the image and counting the particles that were visible within 2 µm of the free cell margin . Each particle was scored for the presence or absence of a halo. Cell Maintenance and Incubation Procedures A431 cells (provided by Dr . P Goodfellow, ICRF, London) were grown in Dulbecco's modified minimum essential medium with 5% fetal calf serum and usually plated out at 5 x 10 5 cells per 3.5 cm petri dish 24 hr before use . Before each experiment cells were rinsed free of serum . In experiments using free antibody, cells were incubated at 5°C with a saturating concentration of B3/25 monoclonal (1 µg/ml ; Hopkins and Trowbridge, 1983) for 30 min and then rinsed five times at 5°C before being incubated in 37°C medium with or without gold complexes for up to 15 min . In experiments in which the 37°C incubation was carried out in PBS alone, this incubation was followed by an incubation at 5°C with gold complexes . In double labeling experiments cells were incubated 5 min at 5°C with 8 nm gold-B3/25 then 30 min with 1 pglml free antibody before being incubated at 37°C with 3 nm gold-B3/25 . After final rinsing, cells were fixed in 2% glutaraldehyde and processed as described above. When prefixed preparations were used, cells were fixed 15 min in 2% formaldehyde. To localize HLA antigens, cells were incubated with hybridoma supernatant at 5°C or 37°C for 30 min, rinsed, fixed in 2% formaldehyde, rinsed thoroughly with 1% BSA, and then incubated for 60 min at 20°C with 8 nm gold-protein A (Tolson et al ., 1981) before being rinsed and fixed with 2% glutaraldehyde and prepared as whole mounts for electron microscopy . Acknowledgments For this work, Carole Thomas, Jenny Willcock, Angela Brennan, and Adrian Walsh provided excellent technical support . Their enthusiasm and willing help were essential to the success of the project .

Cell 208

Financial support was provided by project grants from the North West Cancer Research Fund and the Medical Research Council . I am also grateful to Dr. I . S . Trowbridge of the Salk Institute, San Diego, and Dr. Alan Williams of the Dunn School of Pathology, Oxford, for providing the monoclonal antibodies and to Dr. P. Goodfellow, of the ICRF, London, for providing the A431 cells . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U .S .C . Section 1734 solely to indicate this fact .

receptors in epidermoid carcinoma A431 cells . Cell 35, 321-330.

Received May 2, 1984 ; revised October 19, 1984

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