Ultrastructural localization of lectin binding sites on the surface of retinal photoreceptors and pigment epithelium

Erp. Eye Res. (1979) 29,181-194

Ultrastructural Localization of Lectin Binding Sites on the Surface of Retinal Photoreceptors and Pigment Epithelium IZHAK

NIB

AND

MICHAEL

0. HALL

Unit of Electron Microscopy, Tech&on-Faculty Isrcrel, and The Jules Stein Eye Irbstitute,

of Me&&e P.O.B. 9649. VCLA Rchool of Meclicitle. Los Angeles, C&j. 90024, 1r.A.A.

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(Received 9 October 1978 and in revised .form 27 Februar,q 1979, NW York) The ferritin-lectin complexes of Ricinus communis agglutinin (RCA-l 20). wheat germ agglutinin (WGA), concanavalin A (con A), soybean agglutinin (HBA) and fucose binding protein (FBP) have been used to study the distribution of sugars along the plasma membranes of frog photoreceptor and pigment epithelial cells. The tissues were fixed prior to labeling in order to prevent the lectin-induced rearrangement of binding sites. The plasma membrane of different regions of the rod and cone photoreceptor cells bound ea& lwtin to a different extent. In all cases. the outer segment plasma membrane bound more of the ferritin-lectin complex than did the inner segment plasma membrane. This might reflect the specific differentiat’ion of the outer segment plasma membranes. related to t’heir role in the visual process. The intensity of binding of each lect.in to the pigment epithelium plasma membrane was similar to its binding to t’he rod outer segment plasma membrane. In cone outer segments. where one side of t’he disk membrane is accessible to the incubation medium, direct binding of lectin to the disk edges, as well as to the plasma membrane. was seen. Cone outer segments bound more of the a-D-N-acetylgalactjosamine and Dgnlact’ose specific lectin, SBA. than did rod outer segments. Ko differenres were observed in t,he binding of ferritin-lectin complexes to rod outer segments from dark or light, adapted frogs. Kp!/ ~orrls; lect’ins; ferrit,in; plasma membranes; photoreceptors; pigment, epithelium.

1. Introduction Phagocytosis of photoreceptor tips by retinal pigment epithelium (PE) is essential for the survival of the photoreceptor cell (Young, 1976). The internalization of shed packets of disks presumably proceeds by a recognition and attachment step: which involves both the photoreceptor and the PE cell plasma membranes. Such recognition and attachment has been postulated to be a requisite step for phagocytosis t’o occur (Stossel, 1976). Cell surface saccharides are important to the maintenance of iutcrcellular interactions and are thought to be involved in the process of recognit,ion md attachment (Hughes, 1975). In the present study we have investigated the distribution of various saccha~rith 1In photoreceptor and pigment epithelial cell plasma membrane. Cytochemical procedures which employ ferritin labeled lectins were used for localization and visualization of membrane bound oligosaccharide chains (Nicolson and Singer, 1974; Hall and Nir, 1976). In order to elucidate the inherent topography of the surface saccharides, prefixation with glutaraldehyde was used to prevent lectin-induced rearrangement, of binding sites (Brown and Revel, 1976). Frog retina was selected so that both rods and cones could be studied. In view of recent reports of light-dependent shedding of rod outer segments (Basinger, Hoffman Reprint reyuest,s: Dr Michael Angeles, (‘alif. 90024, U.S.A. 0011-4835/79/080181+

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The suga~rs most commonly found in the oligosaccharicle side chills of ccl1 nl(au1brane glycoproteins are galactose, n1annose. S-acet~~lglucosatiii1i~~ .~-ucct’ylga,lactusamine, fucose and sialie acid (Hughes, 1975). Lectins specific for all of t,hese sugar,q have been isolated and purified from a number of sourceti (Sharon a.ntl Lis, 1972). WC have studied the binding of the lectins Riczkus comwwr& -120 agglutittitl, wtmt gtmt agglutinin. concanavalin A: soybean agglutinin and fucosc t~inding protein, specific (P-1.4-D-rl’-aCetylglUCOSatltiUe),; c/.-u-mannose; u.-rl-:\‘-acetylgalfor P-Y-galactose; actosalnine ad D-gak&Ose; atld x-L-fUCOse respectively.

2. Methods Preparation

of fewitin-lectin

complexes

Concanavalin A (Con A) and fucose binding protein (FBP) were purchased from Miles Laboratories, Inc., and used without further purification. Rici~a~s cornmun:is ngglut,iuin (RCA-120) was isolated from wild castor beans according to the procedure of Kicolsou aud Blaustein (1972) ; soybean agglutinin (SBA) was prepared according to Lietler (1953) and wheat ger1n agglutinin (WGA) was prepared according to Privat, Delmotte, Mialonier, Bouchard and Monsigny (1974). The purified plant agglutinins were conjugated to ferritin (Fer) (Miles Laboratories, Inc., 6X cryst)allized) using the method of Nicolson and Singer (1974). The ferritillconjugated lectins were separaLed from unconjugat)ed proteins by chromatography OII 1.5 >(:91)cm columns of either Agarose A 1.5 m (Con A, SBA, WGA aud FBP) or Hephadex G-200 (RCA-12(,). Ferritin-Con ii was &ted with 0.5 &I-NaCI in 0.G)5 u-sodium phosphate buffer at pH 6.5 while all of the other ferritin-lectin complexes were eluted with 0.2 ht NaCl in 0.~15I\I sodium phosphate buffer, pH 7.2. Fractions were rnonit~ored for absorbauc~e at 280 11111 and for agglutinability of sheep erythrocytes. Those fractious showing maximut1l agglutinat,ing activity were concentrated to 2-5 mg protein/ml before use. Fewitin-plant

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Frogs (Kana pilJie%S) were sacrificed after overnight dark adaptation, or after exposure to normal room light for 45 min. All operations with dark adapted frogs were the under dim red light. The retina and PE were removed from the eyecup and placed iu ice cold 0.1 til-phosphate buffer, pH 7.4. The buffer was replaced with cold 1+“/0 glutaraldehyde in 0.1 nf-phosphate buffer (pH 7.4) for 45 min. After fixation the t,issue was rillsed fog 10 min with buffer and cut into 1 mm squares. In order to block any remaining aldehytle groups which might otherwise cause nonspecific labeling, the tissue was treated with (j.1 m-NH&l in 0.1 M-phosphate buffer for 10 min as described by Brown and Revel (1976). Subseyuently the tissue was rinsed with buffer for 10 min, t,hen incubated for 30 rnin at room temperature with 2 mg/ml of ferritin-lectin complex. At the end of t,he incubation period, the tissue was rinsed for 30 min with phosphate buffer. In control experiments, the sugar inhibitor specific for the particular lectin used was added both to the incubation and rinsing solutions at a concentration of 0.1 M. The sugars used were: galactose for RCA-120, a-methyl mannoside for Con A. N-acetylglucosamine for WGA, N-acetylgalactosamine for SBA and fucose for FBP. Postfixation was carried out in 1% osmium tetroxide in 0.1 &I-phosphate buffer pH 7.0 for 60 min. The tissue was rinsed in buffer, dehydrated and embedded in Araldite 502. Thin sections were stained with lead citrate and uranyl acetate prior to exa1nination in :l JEOL 1OOB electron microscope.

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3. Results A diagrammatic representation of the structure of the outer retina is shown in Fig. 1. Binding sites for RCA-120, WGA, Con A and SBA were visualized on both photoreceptor and pigment epithelial cell plasma membranes. Marked differences were observed in the extent of binding of the lectins to outer and inner segment plasma membranes, and to cones as compared with rods. These results were obtained from a large number of different cells and representative micrographs are included in the following sections.

FIN:. 1. Diagrammatic representation of the outer retina. ROS, rod outer segmrnt: COS, cone outer segment: PE, pigment epithelium, MC, Miiller cell: X-, nucleus; PH, phagosome: PG. pigments granule: V. pigment epithelium microvilli: CC, connecting cilium: MCF. Altiller cell fibers: M. mitochontlria; 6. (+olgi apparatus: SB, synaptic body: OD, oil droplet. In t,he following micrographs similar abbrrviat’ions will be used in order to identify the various cellular structures.

Binding sites for FBP were not visualized on either the photoreceptors or pigment epithelial cell plasma membranes. It should be noted. however. that with the same preparation of Per-FBP, binding to the surface of erythrocytes in the choroidal capillaries was observed. Thus the Fer-FBP preparation used possessed the ability to bind to exposed surface receptors. Binding to outer segnwnt vs. inner segment Substantial binding was observed of RCA-120, WGA and Con A to rod outer segment (ROS) plasma membrane as compared with sparse binding to the rod inner segment. In Figs 2 and 3: the differential binding intensity of RCA-120 to the rod

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outer segment and to the rod inner segment plasma membrane is clearly visualized. Numerous ferritin granules can be seen on the outer segment plasma membrane (Fig. 2) while relatively few are present on the inner segment plasma membrane at the level of the ellipsoid and myoid zones (Fig. 3). The lower level of ferritin-lectin binding to the inner segment was not a result of limited availability of lectin due to restricted diffusion of the complex between the photoreceptor cells. This was evident from the binding of the lectins to the Miiller cell fibers which are adjacent to the inner segment at the level of the myoid zone (Fig. 1). In Fig. 4, intensive binding of RCA-120 to Miiller cell fibers is seen, while at. close proximity the inner segment plasma membrane shows a lower level of binding of the ferritin-lectin complex (Pig. 4, arrow). It is thus evident that these complexes are able to diffuse to the level of the photoreceptor cell inner segments.

FIG. 4. Binding of ferritin-RCA-120 to Miiller cell fibers. Note the intensive binding to the fibers as compared with sparse binding to the adjacent rod inner segment plasma membrane (arrow). Y 35 WI).

Differential binding was seen also in cones. In Fig. 12, binding of Per-SBA to the cone outer segment (COS) plasma membrane is clearly visualized, while the adjacent inner segment plasma membrane is almost completely devoid of ferritin granules.

Of the five lectins tested, binding sites for RCA-120 (Fig. 2): WGA (Fig. 5) and Con A (Fig. 6) were clearly visualized on the rod outer segment plasma membrane. Binding of SBA, in comparison, was very limited and only a few ferritin granules

m the FIG. 8. Inhibition of ferritin-RCA-120 binding to the rod outer segment plasma membranr presence of 0.1 iw-gala&w?. Very few ferrit,in gral lales are observed (compare with Fig. 2). Mote: This granules, if presrnt. would br clearly section was not stained with uranyl and lead Saks i so that ferritin seen. ,x 50 000.

LEC’TIN

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could be seen on the ROS plasma membrane (Fig. 7). This reduced binding of PerMBA was not the result of specific effects of the fixative or of pre-treatment wit’h KH&!l on SBX binding sites. The deletion of either t,reatment did not produce a. noticeable change in the binding of 8BL4 to the ROS plasma membrane. The binding CJE lectins was highly specific. It was greatly reduced when the appropriatca sugar inhiljitor was present in the incubation nledium. This was particularl! evident with t)he binding of RCA-120 which was almost c:mlpletelp inhibited in th(> presence of O-1 >I-gala&&e (Fig. 8).

In cones, where on one side the disk membranes are freely accessible to the ineuI)ation medium. direct binding of lectins to the exposed disk edges was ohservetl. This is most clearly shown in Fig. 9, where binding of Fer-WGA to the plasma membrane. which evelopes one side of the cone outer segment, as well as to the disk edges is seen. In addition, binding to the free surface of the most basal disk and to ljartly exposed surfaces of other disks is also visualized (Fig. 9, arrows). By following the I)ath of infolding of the plasma membrane which forms the basal disks, it is apparent that the ferritin-lectin complex is bound to the intradiscal facr of the membrane. Direct binding to the cone outer segment plasma membrane was also seen with the ferritin conjugates of RCA-120, Con A and SBA, but not with FKP.

FJG. 9. Binding of ferritin-WGA to a cone outer segment. Numerous ferritin granules are seen on the cone outer segment plasma membrane. Binding to disk edges and to exposed surfaces of disk membranes (arrows) can be also observed. ‘, 50 000.

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Most interesting however. was the otmxvation t,hat mow Fcr-SUA \va,s I the C’OS plasma menlhrane than t,o t’he R08 plasma Imn~lmne. ItI Figs 10 12, numerous Fer-SBA granules are seeu on the (‘OS plasn~ mwllm.ne. comparison fewer are observed on the RW plasma nmnhral?e (Pigs 7 and 11). of Per-SBA to freely uxessihle cone disk edges and to exl~owl disk meml also ohserved (Figs 10 and 11). In contrast to the cone outer segment plasma rnenllxane, very little hi]n&n R Fer-SBA to the adjacent cone inner segment plasma menll~rane is seen (‘Fig. arrow).

FIG. 10. Binding of ferribin-SBA to a cone outer segment. Ferritin granules are seen on the cone segment plasma membrane. Labeling of disk edges and of the exposed surface of a disk mem (arrow) can be also observed. x 52 000.

uter rane

LECTIN

of ferritin-SBA PI 1,:. 1. Binding bind ,in$ to the plasma membrane Fl ph31

bind occa

BINDING

TO

PHOTORECEPTORS

to cone outer segment disk edges. Sate that of an adjacent rod out,er segment. ,132 WO.

Binding of ferritin-SBA to a cone. There is nembrane (arrow), while the adjacent cone outer of Fcr-SBA (arrowhead). (The electron dense rim al accumulation of uranyl and lead ions in this

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almost no binding to the cone innel segment plasma membrane exhibits in the periphery of the oil droplet is c Gl 00lI. region.)

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Binding

to pigment

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epithelium

The intensity of binding of Per-RCA-120 (Fig. 13), FerWGA (Fig. 14) and FerCon A to the plasma membranes of the PE cell microvilli is comparable to the binding of these lectins to the ROS plasma membrane. Fer-SBA binding sites were even more sparse than on the ROS plasma membrane and were difficult to ohserve. The FerRCA-120 bound t]o the microvilli in relatively discrete clusters separated by areas of plasma membrane which showed sparse binding of the lectin (Fig. 13). The Fer-WGA binding &es were somewhat more regularly distributed (Fig. 14). effect of illun&atiol~

The localization of lectin binding sites was studied after 45 min of illumination following overnight dark adaptation and compared with results obtained from dark adapted animals. Particular attention was focused on the ROS tips which are reported to undergo extensive shedding during an illuminat)ion period which follows dark xdapt8ation. However, no significant differences were observed in t,he binding pattern of t’hrt various lectins, either to the ROS or to t’he PE in light and dark adaptetl atiinials. 4. Discussion Prefixation with glutaraldehyde inhibits h&n-induced rearrangement of binding sites (Brown and Revel, 1976): but does not affect the specificity or intensity of lectin binding to membranes (Nicolson and Singer, 1974; Oliver, Ukena and Berlin. 1974). Thus, it can be assumed that in cells fixed before treatment with lectins, the distribut,ion of ferritin-lectin particles represents the inherent topography of the membrane binding sites on the retinal phot,oreceptor and pigment epithelial cell l)lasma membranes. The plasma membrane of different regions of both the rod and cone phot’oreceptor cells bound each lectin to a different extent. Thus in all cases. the outer segment 1Jastua membrane bound more ferritin-lectin complex than did the inner segment plasma membrane. This suggests that the composition of surface saccharides on the l)hot’oreceptor cell plasma membrane differs at different areas along the same cell. A similar regional distribution of lectin binding sites has been found on sperm cells (Nicrjlson. Noriko, Yanagimachi, Yanagimachi and Smith, 1977). The higher le.vels of saccharides on the ROS and PE plasma membranes, in cotiil)arison to the plasma membrane of the inner segment, might reflect the specific tlifferentiation of these membranes, related t’o their role in the visual process. The oligosaccharide side chains might also play a role in the physical and lnetdJ(J~ic interactions which occur between the PE and the ROS, e.g. phagocytosis of &et1 KOS tips and the exchange of vitamin d and ot*her metabolites. The presence of rhotlopsin in the ROS plasma membrane (Dewey. Davis, Blasie and Barr, 1969; Jan and Revel. 1974; Basinger, Bok and Hall, 19’76) could account in part for the bincling sites for WGL4 and Con A, since the oligosaccharide sicle chain of rhodopsin coma ins mannose and S-acetylglucosamine (Heller aud Lawrence, 1970; Plantner and &an. 1976). The RCA-120 specific -3 ’ sugar, galactose, is not usually found in rhodopsin, anti the heavy binding of this ferritinlectin complex indicates that the ROS plasula membrane contains major glycoproteins other than rhodopsin. SBA binding sit,es were very fen;: thus suggestmg a limit,ed availability of SBA-specific sugars on the

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ROS plasma membrane. It is of int,erest t,o il0t.e that substantial differences iir t,hc: binding of SBA as compared wit,h RCA have been report~etl in other cell t,ypes (S;IC~Ibar, Oppenheim and Null, 1976). The intensive binding of lectins to the Miiller fibers indicates the presence of ii rich carbohydrate coat on the surface of the Miiller cells. This might, l)e associa,tetl with the possible participation of these cells in the transport of nutrients to the inner retinal layers. The substantial binding of RCA-120, Con A, WGA and SBA to the Miiller cell fibers, which are located adjacent to the base of the photoreceptors (Fig. 1). clearI> indicates that the ferritin-lectin complexes were able to diffuse freely bet,ween t,he photoreceptor cells. Thus, differences in binding to outer and inner segment plasma membranes are not a result of limited diffusion of the ferritin-lectin complexes to the inner regions of the photoreceptor cell layer. In this regard it should also be mentioned that the photoreceptors and pigment epithelial cell plasma membranes are impermeable to the lectin-ferritin complex (Hall and Nir. 1976): so that. only membrane surface binding sites are visualized. The intracellular granular densities sometimes seen in the outer segments (Figs 2. 3 and 7) are not, due to ferritin4ect’in penetration into this organelle, since they also appear in sections which have not lieen exposed to the lectin complex. The origin of these deposits is unknown, i)ut, tht>y are probably due to the staining of cellular components with either 0~0, or lest1 autl uranyl salts. The cones are only partially enveloped by a plasma membrane (Cohen, 1963). so that on one side, disk membranes are freely accessible to the incubation medium. This structural arrangement allows for direct binding of the ferritin-lectin complexes to the disk membranes. However, the ferritin labeled lectins were unable to tlifftlse between the disks, and binding was limited to the free surface of the most, basa,l disk, and to the disk edges. No significant differences were observed when the ljindirlg of RCA-120. WGA and Con A to C!OS and ROS was compared, except that, direct, binding of the various lectins to the inner surface of COS disk membranes was revealed. It should be noted that binding of Con A to the intradiscal surface of the ROM disk membrane has been demonstrated by Riihlich (1976). Of particular interest is the substantial binding of Per-SBA to COS. The binding of this lectin to the disks and plasma membrane of C’OS is much heavier than its binding to the ROS plasma membrane, on which very few binding sites were visualized. This suggests that the chemical composition of the CO8 plasma membra~ne differs from that of the ROS plasma membrane in containing an elevated proportion of one or both of the SBA-specific sugar:: cc-D-X-acetylgalactosamine or D-galactose. This observation complements and extends the recent autoradiographic work (Jf hnt, (1978), showing that rabbit cones incorporate “[H-lfucose while rods do not. In general, the binding of all the lectins tested was similar in dark adapted and in illuminatecl retinas. No changes were observed in the binding of lectins to ROS which were reported to undergo extensive shedclinp under similar illumination conditions (Basinger, Hoffman and Matthes, 1976; Hollyfield et al., 1976; LaVail, 1976). Our observations are in agreement with a recent report in which exposure of re.tinas to light, prior to fixation and labeling with (!on A conjugated microsphereu. had no effect on the distribution of the marker (Molday, 1976). Recently, O’Brien (1976) has proposed that galactose or fucose might be added to the tip of the ROS as a signal to induce shedding. We were unable to detect a cha,nge in the amount of Fer-RCA-120 bouncl to ROS tips after light exposure, nor did w-e

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observe any binding of fucose specific Fer-FBP to either dark or light adapted ROS. It should be noted, however, that the addition or removal of a few sugar molecules could take place without being detected by the present experimental procedures. In addition, separation of the retina from the PE might result in a change in the endogenous distribution of lectin binding sites on the PE and photoreceptor cell plasma membranes. In the present study the effect of light was studied 45 minutes after the onset of illumination. The possibility that transient changes occur following short periods of illumination is currently being investigated.

ACKNOWLEDGMENTS

We wish to thank Mrs E. Geresh and Mr D. Hamill for expert technical assistance, and Mrs H. Hillel for typing the manuscript. This work was supported in part by a Sammy Davis, Jr. Award of Fight For Sight, Inc., New York City, and by grants EY 00046 and EY 00331 from the National Institutes of Health (to M.O.H.). Dr I. Nir was a Research To Prevent Blindness, Inc. International Research Scholar. REFERENCES

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Sicolson, G. L., Soriko, U., Yanagimachi, It., Yanigimachi, H. and Smith. .J. K. (1977). Lectin binding sites on the plasma membranes of rabbit spermatozoa. J. Cell. Biol. 74, 950-62. O’Brien, P. J. (1976). Rhodopsin as a glycoprotein: a possible role for the oligosaccharidc in phagocytosis. Eqn. Eye Res. 23, 127-37. O’Day, W. T. and Young, R. W. (1978). Rhythmic daily shedding of outer segment membranes by visual cells in the goldfish. J. Cell Biol. 76, 593-604. Oliver, J. M., Ukena, T. E. and Berlin, R. D. (1974). Effects of phagocytosis and colrhicine on the distribution of lectin binding sites on cell surfaces. Proc. Nnt. Acnd. Sci. 7’J.A. 71, 394-8. Planter, J. J. and Kean, E. L. (1976). The carbohydrate composition of bovine rhodopsin. J. Hiol. Chem. 251,1548-52. Privat, H. P., Delmot,te, F., Mialonier, G., Bouchard, I’. and Monsigny, M. (1974). Fluorescence studies of saccharide binding to wheat-germ agglutinin (lect.in). Eur. J. Biocochenl.47,5-14. Riihlich, P. (1976). Photoreceptor membrane carbohydrate on the intradiscal surface of retinal rod disks. Nature (Lo&on) 263, 789-91. Sharon, N. and Lis, H. (1972). Lectins: Cell-agglutinating and sugar specific proteins. Science

177,949-59. Stossel, T. P. (1976). The mechanism of phagocytosis. J. ReticuZoendotheE. Sot. 19, 23745. Young, R. W. (1976). Visual cells and the concept of renewal. Invest. Ophthdmol. 10, 70G25.