organisation of glycoproteins into plasma membrane domain...

Journal of Cell Science 101, 437-448 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

437

Organisation of glycoproteins into plasma membrane domains on Fucus

serratus eggs

C. J. STAFFORD, J. R. GREEN* and J. A. CALLOW*

School of Biological Sciences, University of Birmingham, PO Box 363, Birmingham B15 2TT, UK

*To whom correspondence should be addressed

Summary

Fertilisation in the brown alga Fucus involves theinteraction of motile, blflagellate sperm with sphericaleggs. The eggs differ from those of animals in not havingthe equivalent of a vitelline layer, jelly coat or zonapellucida outside the plasma membrane, and in additionthey are not surrounded by a cell wall. Previous studieson Fucus eggs have shown that the lectin ConcanavalinA (ConA) binds in patches on the egg surface, suggestingthat there is a non-uniform distribution of plasmamembrane glycoproteins. In this paper we have furtherinvestigated the occurrence of domains on the Fucus eggplasma membrane using monoclonal antibodies (mAbs)and the lectins Con A and Fucose Binding Protein (FBP).Confocal laser scanning microscopy (CLSM) has beenused to observe the binding of probes to the Fucus eggcell surface. Four mAbs (FS2, FS4, FS5 and FS9) raisedto Fucus serratus sperm have previously been shown tocross-react with crude egg membrane vesicles. Three ofthese mAbs (FS2, FS4 and FS5) have now been shown tobind to the egg cell surface and they recogniseglycoproteins which are organised into domains. mAbFS4 labels large areas of the egg surface, whereas mAbsFS2 and FS5 bind to smaller patches. The lectins Con Aand FBP also bind to smaller, discrete domains. Westernblotting results and competition binding assays haveshown that mAbs FS2 and FS5 compete for binding to

the same set of glycoproteins, and FS5 is used insubsequent experiments; FS4 also binds to severalglycoproteins but produces a different pattern oflabelling on Western blots compared to FS5, thoughthere may be some common components. ConA labels asubset of the glycoproteins recognised by mAb FS5, andFBP recognises one major glycoprotein which is alsorecognised by ConA and FS5. Double labelling exper-iments using the CLSM, with FITC- and Au-labelledprobes, have shown that the regions on the egg surfacelabelled by FS4 and FS5 are mainly exclusive, with smallareas of overlap. There are also areas which are notlabelled by either of these antibodies. The domainsrecognised by mAb FS5 contain smaller areas which arelabelled by ConA. Overall the results show that theFucus egg surface is heterogeneous with different sets ofglycoproteins being organised into domains. With theprobes used it is possible to distinguish between FS4+

FS5", FS4~ FS5+, FS4+ FS5+ and FS4~ FS5~ regions.Within the FS5+ domains smaller sets of glycoproteinsare recognised by ConA and within these latter regionsthere are glycoproteins recognised by FBP.

Key words: confocal microscopy, domains, eggs, Fucus,glycoprotein, monoclonal antibody, plasma membrane.

Introduction

Fertilisation in the brown alga Fucus involves theinteraction of motile, biflagellate sperm with spherical,unicellular eggs (Evans et al. 1982). Unlike most plantcells these gametes are not surrounded by cell walls,and the eggs differ from those of animals (e.g. seaurchin, mouse) in not having the equivalent of avitelline layer, jelly coat or zona pellucida outside theplasma membrane (Trimmer and Vacquier, 1986;Wasserman, 1988). However, after species-specificfertilisation, the egg releases polysaccharides to form asurrounding wall of alginic acid, cellulose and fucoidan(Evans et al. 1982).

Monoclonal antibodies (mAbs) raised to Fucusserratus sperm have been used to show that somesurface antigens are highly concentrated in particularregions or domains, including the anterior flagellumplasma membrane, the mastigonemes on the anteriorflagellum and the sperm body (Jones et al. 1988). Thesefindings are in line with those for mammalian sperm,where antigens are restricted to particular domains ofthe plasma membrane (e.g. head, tail) with specificfunctions (Primakoff and Myles, 1983; Saling, 1986). Incontrast to the situation for sperm, there is littleinformation about egg plasma membranes and there isno clear evidence for the occurrence of domains on eggsurfaces. However, previous studies on Fucus eggs have

438 C. J. Stafford and others

shown that the lectin Concanavalin A (ConA) binds inpatches on the egg surface, suggesting that there is anon-uniform distribution of plasma membrane glyco-proteins (Catt et al. 1983).

In this paper we have further investigated theoccurrence of domains on the Fucus egg plasmamembrane using mAbs and lectins. The four mAbs(FS2, FS4, FS5 and FS9) used in this study were raisedto Fucus serratus sperm and have been previouslyshown to cross-react with crude egg membrane vesicles(Jones et al. 1988, 1990). FS2 and FS5 bind tomastigonemes on the anterior flagellum of the spermand recognise a series of proteins of 90-250 kDa andthese antibodies compete with each other for binding.FS4 and FS9 bind to the sperm body and both flagella.They bind to carbohydrate epitopes and FS4 recognisesan immunodominant 205 kDa glycoprotein, whereasFS9 does not Western blot. The lectins ConA andFucose Binding Protein (FBP) used in this report havepreviously been shown to inhibit fertilisation in Fucusby binding to eggs (Catt et al. 1983).

Fucus eggs are difficult to study using conventionalfluorescence microscopy due to their size (up to 80 umin diameter), shape and optically dense contents (Cattet al. 1983). In the present study, confocal laserscanning microscopy (CLSM) was used, allowing clearobservation of fluorescent- and Au-labelling on the eggcell surface. The results observed using this technique,along with blotting experiments to identify the proteins/glycoproteins recognised by the mAb and lectin probes,have shown that glycoproteins are organised intodomains of differing size and composition on the Fucusegg cell surface.

Materials and methods

Plant materialSexually mature plants of Fucus serratus L. were collectedfrom Watchet, Somerset, UK (Crothers, 1976), sexed bymicroscopic examination of hand sections of the receptaclesand stored at 4°C in ventilated plastic trays for up to 18 days.Eggs were released from excised receptacles and washed toremove any adhering mucilage (Callow et al. 1978).

Monoclonal antibodiesThe mAbs used in this study (FS2 [IgGl], FS4 [IgM], FS5[IgM] and FS9 [IgG2b]), were raised against Fucus sperm asdescribed by Jones et al. (1988, 1990). A mAb raised againstrat bone cells (UBIM 22) was used as a negative control(provided by Dr E. Green, University of Birmingham). ThemAbs were all used in the form of tissue culture supernatant.

Indirect immunofluorescence (IIF)Cells were labelled in screw-necked 2 cm3 white soda vials(FBG Trident, Temple Cloud, Bristol, Avon, UK). To asuspension of eggs (104 cm"3) an equal volume of fixative (3%(w/v) glutaraldehyde, 2% (w/v) paraformaldehyde, in 50 mMsodium cacodylate-HCl, pH 7.3, with 1.4 mM CaCl2, isotonicto seawater) was added and the mixture incubated at 4°C for20 min. After washing cells with 5 x 2 cm3 seawater, the non-specific binding sites were blocked by treatment with 5 mgcm"3 bovine serum albumin (BSA, Sigma) in seawater for 30

min at 4°C. The tubes were aspirated, leaving a minimalvolume (c. 200 /il) to which 200 JJ\ of the mAb was added.Control tubes received a mAb which had been raised againstrat bone cells. Following a 1 h incubation at 20°C withfrequent agitation, eggs were washed twice with 2 cm3

seawater, before receiving 0.5 cm3 (10 ng cm"3) of fluoresceinisothiocyanate-conjugated rabbit anti-mouse immunoglobu-lins (FITC-RAM1G; Dakopatts) diluted with 0.5 mg cm"3

BSA in seawater. The eggs were treated for 1 h at 20°C beforewashing thoroughly with seawater, and they were observed onMultiwell microscope slides (Flow, UK). Treatments whichrequired the addition of two primary antibodies (e.g. FS4 andFS5) were labelled concurrently with 100 /.i\ each of therelevant mAb, and incubated for 90 min at 20°C. These werethen treated as for single labelled cells.

Direct fluorescenceEgg cells were fixed and washed as above before directlabelling with FITC-conjugated lectins Concanavalin A(FITC-ConA; Sigma) or Fucose Binding Protein (FITC-FBP,isolated from Lotus tetragonolobus; Sigma). Stock solutionsof both lectins were made at 1 mg cm"3 (FITC-ConA in 10mMTris-HCl, pH 7.2, containing 150 mM NaCl, 1 mM CaCl2,1 mM MnCl2 and FITC-FBP in PBS). Lectins were used at afinal concentration of 200 fig cm"3 with the egg suspensions,and incubated for 45 min at 20°C. Cells were subsequentlywashed three times with seawater before viewing. Controlsincluded incubations with 100 mM o^methyl D-mannose (forthe FITC-ConA) and 100 mM a-L-fucose (for the FITC-FBP).

Double labellingEgg cells were fixed and treated as for single-labelled cells,unless otherwise stated. Sodium azide (0.02%) was alsoincluded throughout in all incubations. Eggs labelled withFITC-conjugated lectin were incubated with 5% goat serum(GS) for 30 min (SeraLab, Sussex, UK) in seawater after 5 x2 cm3 washes. Cells were subsequently labelled with mAb in1% GS, for 1 h at 20°C before 5 x 2 cm3 washes with 10 mMphosphate buffer, pH 7.4, comprising 0.45 M NaCl, 1% BSA.They were then treated with goat anti-mouse immunoglobu-lins (GAMIG) conjugated to 30 nm gold particles (AuroprobeEM G30, Amersham, UK). The second antibody was diluted1:10 with 0.1% GSin the above buffer, and incubated with thecells at 4°C for 16 h. Eggs were then washed using 10 mMphosphate buffer, before examination. Cells pre-labelled withmAb were incubated with FTTC-RAMIG in 0.5 mg cm"3

BSA in PBS for 16 h at 4°C, to saturate all the binding sites.Cells were then treated with a second primary mAb after 5 X2 cm3seawater washes. Eggs were then incubated withGAMIG G30, as above, for 10 h or 16 h at 4°C.

Confocal laser scanning microscopyThis was carried out using the MRC-600 confocal imagingsystem (Bio-Rad, Microscience Division, Watford, Herts.),attached to the photo-port of a conventional optical micro-scope (Zeiss Axioplan, Oberkochen, FRG). Images werecollected using a water immersion lens (Zeiss, 20x, NA 0.5).A mountant containing 0.1% p-phenylenediamine in 10%phosphate buffered saline (PBS), 90% glycerol was used toprevent fluorochrome bleaching. An argon ion laser (IonLaser Technology Inc., Salt Lake City, Utah) operating withthe 488 nm line was used as the excitation source and thescanner was used in the dual label mode with a scan time of 4 sper frame and rastor size of 512 lines. Kalman filtering wasused to integrate the signal collected over ten frames in orderto reduce noise. By using the CLSM in its dual label mode it

Domains on Fucus egg plasma membranes 439

was possible to eliminate the autofluorescence of chloroplastswhich otherwise hampered observations. Three optical sec-tions were obtained for each specimen, by moving the samplestage. The first two sections were taken through the centreand topmost regions of the egg cell, (these were normallyseparated by approximately 30 to 35 j«m). The third image wastaken between these two (approximately 15 to 18 ^m abovethe equatorial plane). A focal series was also collected foreach specimen. The focus step between these sections wasgenerally 3 pm, an egg cell typically requiring about tenoptical sections. Focal series were then processed to producesingle composite images, combining high spatial resolutionand high depth of field. Pixel intensity profiles were obtainedby drawing transects across the composite images at the pointsof maximum diameter.

Double labelled cells, i.e. those incubated with FITC- andAu-conjugated probes, were also mounted in anti-fadesolution, but were gently covered with a glass coverslip. Focalseries were collected for each specimen, from the topmostregion of the egg, using a 40x/0.75NA water-immersionobjective (Zeiss). The CLSM was operated in dualfluorescence/reflection mode, enabling the two probes to beanalysed simultaneously. The coverslip was used to minimiseegg surface reflection, but typically resulted in a more oval-shaped cell. For this reason, 8 images of 0.1 ;<m step werefound to be adequate; each z-series was collated, and the twoimages, one from each channel, merged. Pseudo-colouringthe image with green (to represent FITC-labelling) and purple(for Au-labelling) then highlights any overlap between thetwo probes with a resultant white (after an idea by Dr. G.Johnson, Dept of Immunology, University of Birmingham).

EM-immunogold labellingTo a suspension of whole eggs (3 x 106 cm"3) in seawater anequal volume of fixative (3% (w/v) formaldehyde, 2.5% (v/v)glutaraldehyde in 100 mM Pipes, pH 7.2, with 5 mM CaCl2)was added and the mixture incubated at 4°C for 3 h. Cellswere washed with five changes of seawater. Preparations werethen dehydrated and embedded in LR White (LRW).Polymerisation of LRW was carried out at -18°C using 360nm (longwave) u.v. light, in the presence of a resin initiator.Cut sections were placed onto uncoated nickel grids (200mesh size) and treated with each of the following solutions insequence: (i) 25 [A of blocking buffer (2% goat serum, 10 mMTris-HCl, pH 7.5, 0.9% (w/v) NaCl, 0.5 mg cm"3 PEGcompound (20K) with 2,2'-[(l-methyl-ethylidene) bis (4,1-phenyleneoxymethylene)] bisoxirane, 0.02% (w/v) azide) for1 h at 20°C; (ii) 25 /A of wash buffer A (blocking buffer minusgoat serum) 4 x 5 min; (iii) 25 /il of primary mAb diluted 1:1with incubation buffer (0.1% gelatin, 10 mM Tris-HCl, pH7.5, 0.9% (w/v) NaCl, 0.1% goat serum) for 16 h at 4°C in ahumid chamber, (iv) 2 x 5 min washes with wash buffer A(above); (v) 4 x 5 min washes with buffer B (wash buffer Aplus 0.2% goat serum); (vi) 25 fA of gold-conjugated goat anti-mouse immunoglobulin (15 nm, Au-RAMIG; Janssen LifeSciences Products, Wantage, UK), which had been diluted1:20 with incubation buffer, for 6 h at 4°C in a humidchamber; (vii) 4 x 5 min washes with incubation buffer.Sections were finally washed for 2 x 10 min in distilled waterbefore drying with velin tissue. Controls received either (i) amAb which had been raised against rat bone cells, or (ii) noprimary antibody (incubation buffer only). Sections werefinally stained with 1% uranyl acetate for 1 h, followed by 1min with lead citrate, before observing with a Philips(Eindhoven, The Netherlands) 301 or Joel Electron Micro-scope, at 80 keV.

Preparation of egg membrane vesiclesEgg membrane vesicles were prepared as described byBolwell et al. (1980). The plasma membrane-enrichedfraction (C. J. Stafford et al. in preparation) derived from 1cm3 packed volume of eggs was resuspended in either 1 cm3

PBS, pH 7.2, for preparative work or reducing sample bufferfor SDS-polyacrylamide gel electrophoresis (SDS-PAGE).The protease inhibitor, p-phenylmethyl sulphonyl fluoride(100 mM in isopropanol; Sigma), was added to a finalconcentration of 0.5 mM, and iodoacetamide to 5 mM, to allegg preparations, before SDS-PAGE or storage at —70°C.Protein was determined using the Bio-Rad Bradford proteinassay performed according to the manufacturer's instructions(BioRad, UK).

ELISACompetition assays

These were performed on Immulon I microtitre plates(Gibco, Uxbridge, UK) coated with 100 [A per well of 10 ngcm"3 poly-L-lysine for 2 h followed by 100 jA per well of eggmembrane vesicles (at 25 /ig protein cm"3) for 2 h at 4°C. Theadsorbed egg vesicles were washed with PBS prior to blockingthe plate with 200 [A per well of blocking solution containing10 mg cm"3 BSA, 100 mM glycine and 0.02% (w/v) azide inPBS, for at least 12 h at 4°C. For the competition assaysinvolving pairs of mAbs, 50 /A of each was added to the wellsin the appropriate combinations for 90 min at 20°C (seeResults). Controls involved using individual mAbs undilutedand diluted 1:1 with PBS. After washing with 4 x 200 ̂ PBS,binding of mAb was detected after incubating each well with100 /A of alkaline phosphatase-conjugated rabbit anti-mouseimmunoglobulin (AP-RAMIG) diluted 1:400 and mixed withunlabelled RAMIG (Dakopatts) diluted 1:20 in PBS contain-ing 0.5 mg ml"1 BSA. After incubation for 3 h at 20°C, wellswere washed with 4 x 200 fA PBS, after which 100 /A of 1 mgcm"3 p-nitrophenylphosphate freshly dissolved in 9.7% (v/v)diethanolamine buffer, pH 9.8, was added to each well for 1 hat 20°C. 50 [A per well of 3 M NaOH was added to stop thereaction. Optical density was read at 405 nm.

Competition experiments using ConA and mAbs involvedthe following: immobilised egg vesicles were washed oncewith TBSCT (20 mM Tris, pH 7.4, 0.5 M NaCl, 1 mM CaCl2,1 mM MnCl2 and 0.5% (v/v) Tween 20). Wells were incubatedwith 50 fA ConA (500 jug cm"3) for 90 min at 20 °C. The wellswere washed with 4 x 200 /il TBSCT, after which mAbs wereadded (50 jA per well) and incubated as described above.Incubations with AP-RAMIG, washing procedures anddetection of antibody binding were as described above.Control experiments without ConA or with ConA plus 0.2 Ma^methyl D-mannose were treated with mAbs as above.

Periodate oxidationImmobilised egg vesicles were washed once with 50 mMsodium acetate buffer (pH 4.5). Buffer containing 20 mMNaIO4 was added to treated wells, while control wellsreceived buffer alone. Following incubation for 6 h indarkness at 4°C, wells were washed three times with acetatebuffer and once with PBS before blocking as described above.Reduction of the periodate-generated sites with sodiumborohydride (7 mg cm"3) for 2 h was found to be withouteffect on mAb binding. Incubations with mAbs, AP-RAMIGand washing procedures were as described above except thatmAbs were used undiluted and the AP-RAMIG was diluted1:600 with 0.5 mg cm"3 BSA in PBS. Antibody binding wasdetected as described above.

440 C. /. Stafford and others

Pronase digestionImmobilised egg vesicles were washed once with PBS,Pronase (Protease XTV, Sigma) was added at 0.1 unit per j/gof egg vesicles and the samples were incubated at 37°C for 6 h.The plate was washed four times with PBS and blocked asabove. Controls received PBS without Pronase. Detection ofantibody binding involved the same procedures described forthe periodate oxidation treatments.

SDS-PAGE and Western blottingmAb binding

This was carried out using the Bio-Rad minigel/transblotapparatus in accordance with the manufacturer's instructionsand modifications of the methods of Birk and Koepsell (1987)and Laemmli (1970). Egg membrane vesicles, prepared asdescribed above, were resuspended in reducing sample buffer(0.125 M Tris-HCl, pH 6.8, 20% (v/v) glycerol, 5% (w/v)SDS, 5% (v/v) 2-/5-mercaptoethanol, 0.0005% (w/v) bromo-phenol blue) and incubated at 95°C for 5 min. Aggregatedmaterial was removed by a final centrifugation step (11 600 g,30 s). Approximately 20 jig protein (20 fA) was loaded on toeach well of a 10% acrylamide gel on the minigel apparatuswhich was run at 100 V for 60 min at 20°C. Proteins on the gelwere then electrophoretically transferred to nitrocellulose(Hybond C; Amersham International) with the transblotapparatus (Towbin et al. 1979) at 4°C for 90 min at 75 V. Theblotted membrane was then incubated in PBS containing0.1% (w/v) sodium azide for 16 h at 30°C for renaturation ofthe blotted proteins. To check for protein transfer to thenitrocellulose, the edges of the membrane were removed andstained for 5 min at 20°C in 10% (v/v) amido black (Sigma),45% (v/v) methanol and 7% (v/v) acetic acid in water, andsubsequently destained in 90% (v/v) methanol, 2% (v/v)acetic acid in water. The remaining area of the blot was cutinto strips for probing with mAbs. The strips were incubatedwith constant agitation first in PBS containing 10% (v/v)newborn calf serum (NCS; Gibco) and 0.05% (v/v) Tween 20(Sigma), for 3 h at 20°C, and then in mAb tissue culturesupernatant diluted 1:1 with PBS containing 10% (v/v) NCS,0.5% (v/v) Tween 20, 2 M D-glucose (BDH) and 20% (v/v)glycerol, for 16 h at 4°C. Following three washes, each of 10min, with PBS containing 0.5% (v/v) Tween 20, the stripswere incubated in AP-RAMIG (Dakopatts) diluted 1:200with PBS containing 10% (v/v) NCS and 0.5% (v/v) Tween 20for 3 h at 20°C. After a further six washes (2 x 0.5% (v/v)Tween 20 in PBS; 2 x 0.05% (v/v) Tween 20 in PBS; 2 xPBS), strips were incubated, in the dark, with substratesolution [0.1 mg cm"3 of Nitroblue tetrazolium (Sigma), 0.05mg cm of 5-bromo-4-chloro indolyl phosphate (Sigma), 4^M MgCl2 in 0.15 M sodium barbitone pH 9.6 (Sigma)]. Thestrips were subsequently washed three times with water andstored dry in darkness.

Lectin bindingBlotted glycoproteins which bound to the lectin ConA wereidentified. Two strips of nitrocellulose were washed in TBSCT(20 mM Tris, pH 7.4, 0.5 M NaCl, 1 mM CaCl2,1 mM MnCl2and 0.5% (v/v) Tween 20). One strip was treated with 50 /jgcm"3 ConA conjugated to horseradish peroxidase (HRP-ConA; Sigma) in TBSCT, the other with HRP-ConA inTBSCT plus 0.2 M ar-methyl D-mannose, and they wereincubated for 90 min at 20°C on a shaker. The strips were thenwashed 2 x 10 min with PBS containing 0.05% Tween 20,followed by PBS, before incubating in HRP developer [1volume of methanol containing 3 mg cm"3 4-chloro-l-naphthol (Sigma), 5 volumes of PBS, 0.01% (v/v) hydrogen

peroxide (Sigma)] for 30 min at 20°C in darkness. Blots weresubsequently washed three times with water and stored indarkness. Blotted glycoproteins were also probed withperoxidase-labelled FBP (HRP-FBP, Sigma). This was pre-pared in PBS containing 0.05% Tween 20, and used as forHRP-ConA, except for control incubations which contained0.2 M a^L-fucose.

Competition with ConAEgg membrane vesicles, subjected to SDS-PAGE andWestern blotting, were probed with unconjugated ConAbefore incubation with mAbs, to see whether this affected theblotting pattern of the mAbs in any way. Blotted nitrocellu-lose membranes were cut into strips, half of which weretreated with mAbs as described above, using GS in place ofNCS. The remaining membrane was washed in TBSCT for 60min, before treating with 500 [i% cm"3 ConA for 90 min, on ashaker, at 20°C. The strips were then washed 3 x 5 min withTBSCT before blocking with 10% GS, 0.05% Tween 20 inPBS, for 2 h. Strips were then incubated with mAbs as perprotocol. Controls included (i) probing ConA-treated stripswith ConA-HRP (to check for saturation with the lectin) and(ii) probing untreated nitrocellulose strips with ConA-HRP inthe presence of 0.2 M a'-methylmannose.

Detection of FBP binding proteinsExcept where stated, all procedures were performed at 4°C.Egg membrane vesicles (500 ng protein) were microfuged at11 600 g for 10 min. The pellet was gently solubilised in 0.25cm3 10 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 1%Nonidet P-40 (Sigma, UK), 5 mM iodoacetamide and 0.5 mMPMSF. Following a 30 min incubation, with constant rotation,the preparation was microfuged at 11 600 g for 10 min toremove debris. 0.25 cm3 of a packed volume of FBP-agarose(Sigma) was added to the supernatant, after washing with 1 MNaCl in Tris buffer to alleviate the problem of free lectinrelease. The mixture was rotated for 3 h, the beads removedby centrifugation, and then resuspended in Tris buffer, andloaded on a discontinuous sucrose gradient in the same buffer(0.5 cm3 20% sucrose: 0.5 cm3 10% sucrose). Aftermicrofuging for 5 min, the beads were treated with threeconsecutive washes of 1 cm3 each of 10 mM Tris-HCl (asabove). The fraction was finally eluted from the beads in a 30min incubation in 0.2 cm3 10 mM Tris-HCl, pH 8.0,containing 150 mM NaCl, 1% NP-40, 0.4 M oL-fucose, 5 mMiodoacetamide and 0.5 mM PMSF. The supernatant (approxi-mately 15 /ig protein) was then prepared for SDS-PAGE, asfor the FBP-depleted fraction. Control preparations receivedthe above treatments, but were solubilised in buffer contain-ing 0.4 M a^L-fucose.

Results

Localisation of mAb binding to Fucus eggsEggs were incubated with mAbs FS2, FS4, FS5 andFS9, followed by FITC-RAMIG, and surface labellingwas observed using the confocal laser scanning micro-scope (CLSM). For mAbs FS4, FS5 and FS2, obser-vations at three different focal planes in the egg areshown (Fig. 1). The fourth image in each series is acomposite image made up from ten sections, approxi-mately 3 [an apart, of one hemisphere of the egg;similar composite images were also obtained wheneighteen individual sections were summed (not shown).


Fig. 1. (A-V) Fluorescence confocal images of Fucus serratus eggs, acquired using a CLSM. mAb binding was followed byFITC-RAM1G in each case. A-D, FS4; E-H, FS5; I-L, FS2; M-P, FITC-ConA; Q, FITC-FBP; R, FTTC-ConA with o--methyl D-mannose; S, FS9; T, FITC-FBP with o^L-fucose; U, mAb control (UBIM 22); V, FS2 with FS4. A-C, E-G, I-Kand M-O: serial sections through individual eggs. D, H, L, P-V: composite images of an average of ten optical sections, 3/itn apart. Cells in each case were approximately 65 pm in diameter.

442 C. /. Stafford and others

» 100 •

10 20 30 40 50 60 70

DISTANCE ACROSS THE EGG (urn)

B 100

z tr

UJx

2 0 -

DISTANCE ACROSS THE EGG (jim)

Fig. 2. Pixel intensity plots of egg cells labelled with eitherFS4 (A) or FS2 (B) and FITC-RAMIG. Transects weredrawn across the equator of the cell in each case to obtainthe profiles. The results shown are representative of a setof transects obtained.

mAb FS4 clearly labels the periphery (cell surface) ofthe egg when observing a section through the equatorialregion of the egg (Fig. 1A). When observations aremade between this region and the top of the egg thelabelling observed is in large diffuse patches on the cellsurface (Fig. 1B,C). This is also shown in the compositeimage (Fig. ID). FS5 and FS2 also label the egg cellsurface (Fig. IE,I), but in contrast to FS4, these mAbslabel smaller patches (FS5: Fig. 1 E-H; FS2: Fig. 11-L).A typical comparison of the intensity of labelling of FS2with FS4 across the egg is shown in Fig. 2 and this re-inforces the observations that in general FS4 labelsmuch larger areas on the egg surface, though somesmaller patches are also observed. Similar results were

obtained when immunolabelling either fixed or unfixedcells, suggesting that the labelling patterns observedafter antibody binding were not caused by cross-linkingof receptors. A fourth mAb used in this study, FS9, didnot bind to the egg cell surface as assessed by IIF (Fig.IS).

In an experiment in which eggs were labelled withboth FS2 and FS4 followed by FITC-RAMIG, the eggswere similar in appearance to those labelled by FS4alone (Fig. IV). Thus there were still regions on the eggsurface which were unlabelled by either of theseantibodies. However it was not possible using thismethod to determine whether the mAbs FS2 and FS4labelled mutually exclusive regions on the egg surface.This was further investigated using two different secondantibody probes (see below).

When eggs were labelled with the lectins FITC-ConA(Fig. 1 M-P) or FITC-FBP (Fig. 1Q), both labelledsmall patches on the egg surface. FITC-FBP labellingproduced the most discrete patches of all the probesused. Controls with the relevant haptens for theselectins showed that they were exhibiting specific binding(Fig. 1R,T).

EM-immunogold labelling of egg sections with FS2showed that this mAb bound to the plasma membrane(Fig. 3). This also confirmed that FS2 bound in patcheswhich seemed to form small protuberances on the eggcell surface and there was negligible internal labelling ofthe egg. Although FS4 and FS5 labelled eggs by IIF,significant levels of immunogold labelling of the cellsurface were not observed and FS4 showed someinternal binding (not shown). FS9 bound to osmiophilicbodies containing polyphenols lying just beneath theplasma membrane (not shown).

Competition binding assaysBefore double labelling experiments were attempted aseries of competition assays were performed, using thevarious mAb and lectin probes. In the first of these, anELISA was used to see whether the mAbs FS2, FS4 andFS5 showed any competition with each other forbinding to egg vesicles. The results showed (Table 1A)that FS2 and FS5 clearly compete with each other forbinding, whereas FS4 does not compete with either FS2or FS5: in the latter instances the binding to egg vesiclesis additive. As a result of these tests, FS5 was used inmost of the subsequent experiments. In the secondassay, egg vesicles attached to a microtitre plate wereincubated with ConA. After washing away excesslectin, the egg vesicles were incubated with mAbs,followed by AP-RAMIG. The results of this bindingassay (Table IB) showed that binding of mAbs FS2 andFS5 to egg vesicles was partially blocked by the pre-incubation with ConA, suggesting that the lectin iseither binding to a subset of the components recognisedby FS2/5 or is causing a more general steric hindrance.In contrast, mAb FS4 binding was increased after pre-incubation of the egg vesicles with ConA, suggestingthat this mAb actually binds to the lectin. Similarexperiments to the above, but substituting FBP for

Fig. 4. Confocal images of Fucus serratus eggs, double labelled with FTTC- and Au-conjugated probes. Composite imagesfrom eight sections 0.1 /an apart. Image is pseudo-coloured with green representing FTTC-labelling and purple representingAu-labelling. Any overlap is shown in white. (A,B) FS4/FTTC-RAMIG followed by FS5/Au-RAMIG. (C,D) FS4/Au-RAMIG followed by FS5/FITC-RAMIG. (E,F) FTTC-ConA followed by FS5/Au-RAMIG. Bar, 10 yon.


m v

\V

B

.#• .

^ <

Fig. 3. Localisation of FS2 binding by EM-immunogoldlabelling of egg sections. (A,B) FS2 labelled egg sections.(C) Control. Egg plasma membrane is indicated byarrowheads. Mitochondria (m), peripheral vesicle (v),polyphenol body (p). Bar, 200 nm.

Con A, showed no effects of this lectin on binding of anyof the mAbs (not shown).

Double labelling of Fucus eggsDouble labelling experiments were performed usingFITC-conjugated probes in conjunction with 30 nm Au-labelled probes. It was not possible to use Texas Red-

Table 1. Binding of mAbs to egg vesicles in ELISA:competition assays and effects of ConA pre-incubation

(A) Competition between mAbs

mAbELISA

(absorbance at 405 nm)

FS2FS4FS5FS2+FS5FS2+FS4FS4+FS5

0.35±0.020.45±0.030.30±0.020.37±0.020.83±0.020.80±0.04

(B) Effect of ConA on mAb binding

ELISA (absorbance at 405 nm)

mAb Without preincubation With preincubation

FS2FS5FS4

0.37±0.020.45±0.010.40±0.03

0.15±0.010.18±0.010.75±0.03

ELISA plates were coated with Fucus egg membrane vesicles.(A) mAbs were either added to wells individually or in pairs

and binding was detected as described in the Methods, afterincubating with AP-RAMIG followed by its substrate.

(B) Wells were pre-incubated with ConA (500 ^g ml"1), washedand then incubated with mAbs, whose binding was detected as for(A). Controls involved pre-incubation with TBSCT.

Results are means of four replicates with s.d.

labelled reagents due to the intense autofluorescence ofchloroplasts in the red channel of the CLSM in its duallabel mode. Using a combination of fluorescence(FITC) and reflection (Au) filters on the CLSM it waspossible to overcome the above problem, and to furtherinvestigate the distribution of surface components. Theresults of the competition assays imposed certainrestrictions on combinations of probes which could beused for double labelling experiments on intact eggs.The results from the combinations which yielded usefulinformation are shown in Fig. 4. These experimentsinvolved observations on double labelling with mAbsFS4 and FS5, and with ConA and FS5. In theexperiments with the mAbs, reciprocal combinations ofmAb and second antibody were used. Thus, Fig. 4A,Bshows two representative pseudocolour images ob-tained after eggs were labelled with FS4, which werevisualised using FITC-RAMIG (shown in green), andFS5, which was visualised with Au-RAMIG (shown inpurple). Both mAbs were used at saturating levels. Theresults show that in general FS4 and FS5 label differentregions on the egg cell surface. However there is someoverlap (seen in white), which can be seen in particularin Fig. 4A, showing that the labelling is not absolutelymutually exclusive. There are also areas of the eggsurface which are unlabelled with either mAb (inparticular seen in Fig. 4B). In the reciprocal exper-iment, mAb FS4 binding was visualised with Au-RAMIG (purple) and FS5 labeling was observed usingFITC-RAMIG (green; Fig. 4C,D). As in Fig. 4A,B,mAb FS4 labels larger areas than those labelled by FS5.The two mAbs in general label different areas, thoughthere are some areas of overlap, in agreement with the


Con A FBPAg FS4 FS5 C ConA FBP aMM af

205 -

11697.4

66

45

29 -

B

2 0 5 -

11697.4

6 6 -

45 -

2 9 -

FS9

Fig. 5. Detection of proteinsrecognised by mAbs and lectinson blots. A Fucus serratus eggmembrane vesicle preparationwas subjected to SDS-PAGE(reducing conditions, 10% gel)with subsequent blotting tonitrocellulose and was probedwith mAbs (FS4, FS5 and FS9),binding of which was detectedusing AP-RAMIG, or lectinsHRP-ConA and HRP-FBP. Theappropriate substrates were thenused. Lectins were also incubatedin the prescence of theappropriate sugar haptens: ConAwith a^methyl-D-mannose(aMM) and FBP with o<-L-fucose(aF). Controls involved using amAb raised to rat bone cells (C).A silver stained gel (Ag) is alsoshown for comparison.

results shown in Fig. 4A,B- In a further experiment,eggs were incubated with FITC-ConA (green) and thenmAb FS5, which was visualised using Au-RAMIG(purple). Two representative images are shown in Fig.4E,F. Apart from small areas of overlap (shown inwhite) ConA and FS5 seem to label different regions onthe egg surface. However, since ConA partially blocksFS5 binding (see competitive binding assay above)these results have to be interpreted with caution (seeDiscussion).

Overall, the localisation results show that there arelarge domains on the Fucus egg surface recognised bymAb FS4, and smaller domains recognised by FS2 andFS5 and the lectins ConA and FBP. The regionslabelled by FS4 and FS5 are mainly exclusive, thoughthere are small areas of overlap.There are also regionswhich are not labelled by FS4 or FS2/5.

Antibody and lectin labelling of blotted eggmembranesProteins from egg vesicle preparations enriched inplasma membranes were separated by SDS-PAGE,blotted onto nitrocellulose paper and probed witheither mAbs or lectins (Fig. 5). As the silver stained gelshows, there is a complex ladder of proteins over theentire molecular mass range. mAb FS4 (Fig. 5) labelledmajor bands of apparent molecular mass 43, 48, 57, 62,70, 85, 96, 105, 115, 125 and 170 kDa. mAbs FS2 andFS5 gave similar labelling patterns in Western blots,binding to proteins mainly between 47 and 185 kDa(only FS5 result shown). They recognised majorcomponents of apparent molecular mass 47, 52, 58, 62,72, 78, 85, 110-113, 150, 185 kDa and also labelled aseries of finer bands. The pattern of labelling wasdifferent to that obtained for FS4, though there weresome common bands at 62, 70, 85 and 105 kDa. FS9(Fig. 5B) recognised components of 34-37, 42-48, 54, 77and 138 kDa.

Incubation of blotted egg glycoproteins with HRP-ConA and HRP-FBP revealed a number of labelled

bands (Fig. 5). The bands identified by HRP-ConAincluded those at 52, 62, 72, 76, 88, 102, 110, 128 and150 kDa. HRP-FBP labelled one major band at 62 kDa,and minor bands 72, 88 and 128 kDa. (Fig. 5). Thelabelling of all the bands by ConA and FBP wasabolished in the presence of the relevant hapten sugars.

Several different procedures were used in order toobtain information about the relationship between theproteins/glycoproteins identified by the mAbs FS4 andFS5 and the two lectins. In one experiment, egg vesicleproteins which were bound to FBP-agarose beads wereeluted with <*-L-fucose, separated by SDS-PAGE andprobed with mAbs and lectins on blots. The results(Fig. 6) show that FBP labels a major band at 62 kDa aswould be expected. The minor bands observed pre-viously in the blot of untreated egg vesicles were notdetected. This 62 kDa protein which had been elutedfrom the FBP-agarose beads was also recognised byConA and by FS5; these probes also bound tocomponents at 66 kDa which were not labelled by FBP.In contrast FS4 did not bind to the proteins which werebound to the FBP-agarose. The results suggest that thelectins FBP and ConA and the mAb FS5 all bind to acommon 62 kDa protein. The protein(s) labelled atapproximately 62 kDa by FS4 using untreated eggvesicles (see Fig. 5) does not bind to FBP and must bedifferent to the FBP-binding component at 62 kDa.

In a second experiment, which was a follow-up to thecompetition ELISA result shown in Table IB, blottedegg vesicle proteins which had been separated by SDS-PAGE, were incubated with ConA, and after washing,with FS5 followed by AP-RAMIG. ConA bindingproteins were visualised by incubating some strips withHRP-ConA. The results (Fig. 7) showed that severalbands recognised by FS5 were not labelled after theConA pre-incubation; these were at 47, 52, 62, 66, 102and 150 kDa. Thus this set of glycoproteins must berecognised by both probes. There are other glyco-proteins recognised by FS5 which are not labelled byConA, but there do not seem to be any glycoproteins


labelled by ConA which are not labelled by FS5.However the important result is that the glycoproteinsrecognised by FS5 contain a subset recognised byConA. The equivalent experiment using FS4 instead ofFS5 was uninterpretable since this mAb bound to the

FBP ConA FS5 FS4

Mr

2 0 5 -

U-J6 6 -

4 5 -

2 9 -

Fig. 6. Binding of mAbs and lectins to proteins which bindto FBP-agarose beads. An egg vesicle detergent lysate wasincubated with FBP-agarose. Bound material was eluted,separated by SDS-PAGE, blotted onto nitrocellulose andsamples were incubated with lectins HRP-FBP (FBP infigure) or HRP-ConA (ConA) and mAbs FS5 and FS4followed by AP-RAMIG. Appropriate substrates were thenused. A mAb raised to rat bone cells was used as a control(C).

Con A

C T

FS5

C T

(xiO

205 H

Mr- 3 , 116

'97.4

66

45 -

29 -

Fig. 7. Effect of ConA on FS5 binding to egg vesicles inWestern blots. Egg membrane vesicles were subjected toSDS-PAGE and blotted onto nitrocellulose. After treatingappropriate strips with ConA (T) and controls with TBSCT(C) and washing, strips were incubated with either HRP-ConA (ConA in figure) or FS5. mAb binding was detectedafter incubation with AP-RAMIG. Arrowheads indicatebands which are present on the strip incubated with FS5(control) compared with the ConA pre-treated strip(FS5/T).

Table 2. Effects of periodate and Pronase treatmentson binding of mAbs to egg membrane vesicles in

ELISA

mAb

FS2FS5

FS4FS9

Relativeimmunoreactivity (%)

Periodate

140.9162.2

64.7115.9

Pronase

14.1

50.0115.6

ELISA plates were coated with Fucus egg membrane vesicles,which were then either incubated with periodate or Pronase for 6h. mAb binding was determined by ELISA. Mean optical densitieswere calculated for four replicate wells in each case, comparedwith that for the control wells, and expressed as a percentage ofthe control mean (relative immunoreactivity).

ConA itself, in agreement with the previous ELISAdata (result not shown).

A third protocol involved an attempt to depletedetergent-solubilised egg vesicle proteins of com-ponents recognised by either mAb FS4 or FS5. Westernblots of the remaining proteins were probed with mAbsin order to investigate the possibility of commoncomponents identified by these two antibodies. Unfor-tunately these experiments were unsuccessful, since themAbs did not immunoprecipitate their respectiveantigens efficiently.

mAb binding to egg membrane vesicles, which werepre-incubated with either periodate or Pronase, wastested using an ELISA (Table 2). Binding of FS2 andFS5 was increased by pre-treatment of the vesicles withperiodate, whereas binding of FS4 was reduced by up to35% of control levels. Pronase treatment of immobi-lised antigens resulted in a reduction in FS2, FS5 andFS4 binding. Trypsin treatment of antigens had similareffects to those produced by Pronase whilst Endo-Ftreatment of antigens had no effect on antibody binding(data not shown). FS9 binding was not affected by thesetreatments.

Discussion

In the present study we have used mAb and lectinprobes to show that sets of proteins/glycoproteins in theFucus egg plasma membrane are organised intodomains. The results extend previous observations withFITC- and Au-conjugated ConA which showed bindingof this lectin to patches on the Fucus egg surface (Cattet al. 1983). Using CLSM it was possible to observelabelling of the egg by a variety of probes at differentlevels of the cell. The eggs were labelled at theirperiphery by the lectins ConA and FBP and the mAbsFS2, FS4 and FS5 when observed at the equatorialregion, showing that labelling is at the cell surface. Thiswas confirmed for FS2 using EM-immunogold tech-niques which showed that this mAb specifically labelledplasma membrane components. Previous results withAu-labelled lectins used at the EM level have also


shown that these probes bind to the plasma membrane(Catt et al. 1983). A fourth mAb used in this study, FS9,did not show any labelling of eggs using IIF, and EMstudies showed that this mAb recognised polyphenolbodies in the cytoplasm beneath the plasma membrane.This shows that this antibody was not able to penetratethe plasma membrane of intact eggs during labelling forIIF in order to reach internal components, supportingthe view that the other probes are only labelling surfacecomponents of the egg in the UF procedures.

By using CLSM it was also possible to observe theorganisation of surface components on the egg labelledby the different probes, in particular by assemblingcomposite images comprising many individual sectionsof the egg. The results showed that ConA and FBPlabelled very discrete regions on the egg cell surface.The mAbs FS2, FS4 and FS5 also labelled proteins/gly-coproteins organised into domains of differing sizes.The patches labelled by FS4 tended to be larger thanthose labelled by FS2 and FS5, though smaller areaswere also labelled. The EM-immunogold labellingresults showed that FS2 bound to components of theegg surface arranged in clusters which formed protuber-ances on the egg surface, confirming the observationsmade by IIF. Previous results with Au-conjugatedConA and FBP suggested that they also bound toclusters on the egg surface (Catt et al. 1983). Theseresults, and the fact that the results using FITC-conjugated probes were obtained on both fixed andunfixed cells, suggested that the observations were notdue to patching or capping of surface proteins by themAbs or lectins.

Double labelling experiments on eggs were per-formed in order to study the relative distribution ofproteins/glycoproteins recognised by the lectins andmAbs. The most informative combination of probesinvolved using FS4 and FS5. Using the CLSM in thereflection mode (as used by White et al. 1989) allowedobservations to be made with Au-conjugated secondantibodies in combination with FITC-labelling. Theresults showed that there are clearly regions on the eggsurface which are labelled by either FS4 or FS5 and theytherefore label mainly mutually exclusive areas. Thereare some areas labelled by both probes and there arealso areas on the egg surface which are not labelled byeither probe. The results of double labelling exper-iments using ConA and FS5 suggested that there wereareas on the cell surface which were FS5+ ConA" andothers which were FS5~ ConA+; however these resultsneed to be interpreted with the molecular datadiscussed below.

Blotting of egg vesicle preparations enriched inplasma membranes with the mAbs FS2 and FS5 showedthat they bound to similar sets of proteins. Theseresults, taken together with similar effects of periodateand Pronase on mAb binding in ELISA, and the factthat they compete with each other in ELISA on eggvesicles, suggest that these mAbs are binding to thesame or overlapping epitopes. For this reason only oneof these mAbs, FS5, was chosen for further study. FS4gives a different pattern of labelling to that for FS5 on

Western blots, although there seem to be somecomponents of similar size recognised by both mAbs.FS4 binding to egg vesicles is inhibited by periodate andPronase treatment, suggesting that the mAb recognisescarbohydrate epitopes of glycoproteins. FS4 is clearlybinding to a different epitope than FS5, since binding ofthe latter is increased by periodate treatment ofantigens.

Binding of ConA to egg glycoproteins reduced thebinding of FS5 in ELISA, and the results of exper-iments in which blotted egg proteins were pre-incubated with ConA, showed that this lectin labels asubset of the proteins labelled by FS5. Double labellingexperiments on eggs using FITC-labelled ConA andAu-conjugated antibodies showed that there are someregions on the egg surface which are FS5+ ConA".Presumably the glycoproteins in these areas are thoseidentified as being FS5+ ConA" in the Western blottingexperiments (Fig. 6). There are also regions on the eggcell surface which appear to be FS5~ ConA+. HoweverConA blocks the binding of FS5 to some glycoproteins,and the Western blots show that there is a subset ofglycoproteins which are FS5+ ConA+; there is noevidence for a set of glycoproteins which are FS5~ConA+. Thus it is likely that the FS5" ConA+ regionsobserved on the eggs actually contain glycoproteinswhich are also normally recognised by FS5.

FBP labels very discrete areas on the Fucus egg cellsurface and blotting experiments show that it primarilylabels a glycoprotein at 62 kDa. This glycoprotein isalso recognised by FS5 and ConA, but not FS4. ThusFBP, ConA and FS5 recognise larger and largerfamilies of glycoproteins held within small domains onthe egg plasma membrane.

Overall the results of the localisation and molecularstudies suggest that there are regions on the egg surfacewhich contain different groups of glycoproteins. Theseare FS4+ FS5+ (small domains), FS4+ FS5~ (mainlylarge domains), FS4~ FS5+ (mainly small domains) andFS4~ FS5~ (variable size). Within the FS5+ domains,smaller sets of glycoproteins are recognised by ConA,so that there are FS5+ ConA+ and FS5+ ConA"regions. Also within the FS5+ ConA+ regions lie theglycoproteins recognised by FBP. There are clearlyareas on the egg which are not labelled by any of theseprobes. The results described show that the Fucus eggplasma membrane is very complex in its organisation.Information on egg plasma membranes from organismsother than Fucus is rather limited. Mouse eggs have amicrovillus-free area on their cell surface and thisregion labels to a lesser extent with ConA comparedwith the rest of the plasma membrane (Wolf andZiomek, 1983). The results suggest that there aredifferences in membrane protein diffusion rates in thetwo regions of the mouse egg; however the glyco-proteins recognised by the lectin have not beenidentified. Localisation results for the bindin receptoron the sea urchin egg show that it is uniformlydistributed over the vitelline layer (Ruiz-Bravo et al.1989). Organisation of surface antigens into particulardomains is not unusual for polarised or morphologically


highly differentiated cells e.g. epithelial cells or spermcells (Primakoff and Myles, 1986; Simons and Fuller,1985). In these instances specific proteins are main-tained within the domains by barriers to free diffusion(Cowan et al. 1987; Gumbiner and Louvard, 1985). Thisis unlikely to operate in a spherical, apolar cell like theFucus egg and it is more probable that the cytoskeletonhas an important part to play in the assembly andmaintenance of the domain structure. This aspect isnow under investigation.

The mAbs used in this study were selected from apanel of antibodies raised to Fucus serratus sperm andthey demonstrated two types of binding pattern to thesperm cell surface (Jones et al. 1990). FS4 and FS9 labelthe entire sperm, including the body and both flagella,whereas binding of mAbs FS2 and FS5 is restricted tothe mastigonemes on the anterior flagellum of thesperm. FS4 binding to eggs and sperm is periodate-sensitive. It recognises a 205 kDa glycoprotein on thesperm surface, while on eggs it recognises a set ofglycoproteins none of which is less than 160 kDa. Thusthe mAb recognises carbohydrate epitopes on differentglycoproteins on the two gametes. mAbs FS2 and FS5both label a broad band of proteins from 90-250 kDa onsperm and compete for binding (Jones et al. 1990). Adifferent set of proteins is labelled by these mAbs oneggs. Though binding of FS2/5 to eggs is increased byperiodate treatment of antigens, the identification ofmultiple bands on blots suggests that these mAbs areprobably recognising a carbohydrate epitope on glyco-proteins. FS9 binds to a carbohydrate epitope on thesperm surface but does not Western blot. However oneggs this mAb recognises a series of glycoproteinspresent in the osmiophilic, polyphenol bodies. Thus theglycoproteins recognised by each mAb on the sperm aredifferent to those recognised by the mAbs on the egg.

Since the glycoproteins in domains on sperm andother cell types have specific roles, the functionalsignificance of the above results is also of interest. Theprimary function of the Fucus egg is to interact withsperm in a species-specific fashion by direct contact oftheir plasma membranes. It is therefore highly likelythat the complex domain structure of the plasmamembrane on Fucus eggs has some direct relevance tosperm binding and fusion. ConA and FBP have beenshown to inhibit fertilisation by binding to eggs and thisand other evidence suggests that the egg-bound spermreceptor is a glycoprotein(s) containing mannose andfucose (Callow et al. 1985). The results in this paperhave shown that these lectins bind to glycoproteins heldwithin the small domains on the egg surface. Non-uniformity of the Fucus egg cell surface has also beensuggested by Brawley (1990), since sperm, uponreaching the egg cell, actively probe the surface beforefusion occurs, as shown in a cinemicrographic study byFriedmann (1961). The present study suggests that theFucus sperm may probe the egg surface in order toreach specific receptors held in discrete domains.

C.J.S. thanks the Science and Engineering ResearchCouncil for a research studentship during this work. Mr P.

Stanley is thanked for help with EM techniques, and Mr H.Munasinghe for help with the Western blotting procedures.

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organisation of glycoproteins into plasma membrane domain...

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