Colocalization of discoidin-binding ligands with discoidin in developing Dictyostelium discoideum

IIEVELOPMEKTAL

BIOLOGY

106,

59-70 (1984)

Colocalization of Discoidin-Binding Ligands with Discoidin in Developing Dictyostelium discoidem DOUGLASN. W. COOPERAND SAMUEL H. BARONDES' Departments

of Biology and Psychiatry, University of California, San Diego, La Jolla, California Veterans Administration Medical Center, San Diego, California 92161 Received Januaq

92093, and

27, 1984; accepted March 21, 1984

The Dictyostelium discoideum lectins, discoidin I and discoidin II, and the endogenous ligands to which they bind were immunohistochemically localized in sections of this organism at successive stages of development. For these studies, an axenic strain, AX3, was grown in a macromolecule-depleted medium rather than on bacteria, which themselves contain discoidin-binding ligands. Discoidin I-binding sites (endogenous ligands) in sections of D. discoideum were concentrated in the slime coat around aggregates, whereas discoidin II-binding sites were observed in a vesicle-like distribution in prespore cells and also in spore coats. In contrast, discoidin II did not bind to the slime coat and discoidin I bound relatively poorly to prespore cells and spore coats. The distributions of the endogenous lectins themselves were the same in axenically grown cells as previously reported for cells raised on bacteria. Discoidin I was concentrated in the slime coat and around stalk cells, and discoidin II was prominent in and around prespore cells. The congruent localization of each lectin with its endogenous ligand suggests that discoidin I normally functions in association with glycoconjugates in the slime around aggregates, and discoidin II with the galactose-rich spore coat polysaccharide. INTRODUCTION

The cellular slime mold Dictyostelium discoideum synthesizes two lectins, discoidin I (d1)’ and discoidin II (dII), during differentiation. d1, the more abundant lectin, is made as cells aggregate and may, at that time, comprise about 1% of the cell protein (Barondes et al., 1982). This lectin has been immunohistochemically localized in materials surrounding aggregates and slugs (Barondes et al, 1983). dI1 is also synthesized by aggregating cells, but immunohistochemical studies indicate that there is a second burst of synthesis later in development that correlates with the terminal differentiation of the two major cell types. It is concentrated in prespore cells and appears to be externalized as they mature (Barondes et al, 1983). Although the role of these lectins is still not clearly understood, we have assumed that they, like soluble vertebrate lectins (reviewed in Barondes, 1984), function by interacting with endogenous glycoconjugates in or around the cells. In the case of dI1, the relevant ligand ’ To whom reprint requests should be addressed at: Department of Psychiatry; M-003; University of California, San Diego, La Jolla, Calif. 92093. ’ Abbreviations used: d1, discoidin I; dI1, discoidin II; TBS. 50 mM Tris, 150 mM NaCl, pH 7.6; BSA, bovine serum albumin; lactosylBSA, lactose conjugated to BSA (37 mole/mole); SDS, sodium dodecyl sulfate; PBS, 75 mM Na2HPOa/KH2P04, 75 mM NaCl, pH 7.2; GS, goat serum; TBS-GS-BSA, TBS including 2.5% goat serum and 0.4% bovine serum albumin. 59

appears to be a polysaccharide composed largely of galactose and N-acetylgalactosamine, which is synthesized late in development as spore cells mature (Cooper et ah, 1983). This ligand, which appears to be identical to a previously identified spore coat polysaccharide, is synthesized at a time when dI1 is becoming increasingly prominent. Another discoidin ligand has also been detected in extracts of D. discoideum prepared in sodium dodecyl sulfate (Cooper et ak, 1983). This ligand interacts well with d1, is found at early stages of development, but has not yet been characterized. To evaluate the possible function of these endogenous ligands, we sought to localize them immunohistochemically by reacting sections of developing colonies with purified d1 or dI1 and detecting sites to which the lectins bound by immunohistochemical procedures. We also compared the localization of the discoidin-binding ligands with the localization of the endogenous lectins already present in the tissue sections. These studies were done with an axenic strain grown on a macromolecule-depleted liquid medium instead of on bacteria, which themselves contain discoidin-binding ligands (Cooper et aZ., 1983). The major finding of these studies is that discoidinbinding ligands, as visualized by binding of added purified lectin, are localized in a pattern very similar to that of the endogenous discoidins. Furthermore, these ligands show marked specificity in differentially binding d1 and dI1. d1 and its ligand are both prominently con0012-1606/84 $3.00 Copyright All rights

Q 1984 by Academic Press, Inc. of reproduction in any form reserved.

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DEVELOPMENTALBIOLOGY

centrated around the exterior of aggregates. Both are also found around maturing stalk cells. dI1 and its ligand are prominent in prespore cells in what appear to be vesicular structures, and then are found around maturing spore cells. MATERIALS

Growth and D-$wentiation

AND

METHODS

of D. discoideum

Cultures of D. discoideum, strain NC4, were raised on nutrient agar in association with Klebsiella aerogenes, harvested in growth phase by washing in cold water, and seeded for development on moist filters (Barondes et al, 1978) or, for histological studies, on nonnutrient agar (Barondes et al., 1983) as previously described. D. discoideum axenic strain AX3, obtained from Dr. W. Loomis (University of California, San Diego), was initially cultured from spores stored on silica gel by seeding on a lawn of K. aerogenes. Fruiting bodies were picked and inoculated in a dialysate of nutrient medium with streptomycin sulfate. This medium was prepared by dialyzing a four times normal concentration against 3 vol of distilled water and autoclaving, as previously described (Cooper et ah, 1983). The dialysate, which is thus freed of macromolecules that bind to discoidin, was used for all subculturing. Prior to experimental studies, cells were subcultured at least five times from a density of 2 X lo5 to a density of 5 X lo6 in order to minimize carryover of material from bacterial culture. For differentiation, AX3 cells were harvested from log growth phase, washed once in cold distilled water, and 2 X lo8 cells were placed on sterile nonnutrient agar in 100 X ZO-mm petri dishes for immunohistochemical studies or on moist filters for the biochemical studies, all as previously described (Barondes et al., 1983; Cooper et al., 1983). Antibody

Preparation

VOLUME105.1984

immunoglobulins that cross-react with dI1 was achieved by circulation overnight through an equivalent column of immobilized pure dI1. The unbound antibody was stored frozen in aliquots. The rabbit antiserum raised against dI1 was affinity purified and cross-adsorbed in the same manner. The mouse antiserum against dI1 was not affinity purified, but was similarly cross-adsorbed by circulation of 1 ml diluted with 9 ml of TBS containing 0.4% BSA through a l-ml column of 100 pg of immobilized d1. Rabbit antiserum to a spore coat protein, SP96, was generously provided by Dr. K. Devine and Dr. W. Loomis (University of California, San Diego). This serum is specific for a glycoprotein component of spore coats and specifically stains prespore vesicles (Devine et al., 1983). Evaluation

of Antibody

Specificity

The specificity of the antibody preparations was determined by immunoblotting, using procedures detailed previously (Springer and Barondes, 1983). Crude cell extracts from various developmental stages and purified discoidin were first solubilized by boiling under reducing conditions in SDS and electrophoresed with SDS in 15% polyacrylamide slab gels. Separated proteins were transferred to nitrocellulose sheets using an electrophoretic blotting chamber (CBS Scientific, Del Mar, Calif.) at 1 V/cm, 20 mA overnight followed by 3 hr at 4 V/cm, 50 mA. Sheets were either stained with amido black or incubated for 1 hr in TBS-GS-BSA prior to immunostaining. Such precoated sheets were incubated for 1 hr with appropriate concentrations of affinity-purified cross-adsorbed antibody and washed for 30 min in several changes of the above buffer. Sheets were then incubated for 30 min in a 1:lOOdilution of goat antibody to rabbit IgG (Cappel Laboratories, Inc., Cochranville, Pa.), washed, and incubated for 30 min in a 1:lOOdilution of rabbit antiperoxidase-peroxidase mixture (Cappel Laboratories, Inc.). After several washes in TBS, sheets were reacted with a fresh solution of TBS containing 0.3% HzOz and 0.16 mg/ml 4-chloronaphthol (Polysciences, Inc., Warrington, Pa.). After completion of the reaction, sheets were washed and stored in distilled water.

d1 and dI1 were prepared by affinity chromatography (Cooper et al, 1983) and used to raise specific antisera in rabbits and mice as previously reported (Barondes et al, 1983). To further increase the already considerable specificity (Barondes et al, 1983) of these reagents, affinity-purified antibodies were prepared. Six milliliters of rabbit antiserum against d1 was circulated overnight Immunoassay of dI and dII through a Z-ml column containing 200 /*g of pure d1 To determine the lectin content of crude samples, a (Cooper et al, 1983) immobilized on cyanogen bromideactivated Sepharose 4B (Pharmacia Fine Chemicals, solid-phase immunoassay was designed, in which anPiscataway, N. J.). Bound antibody was eluted with 0.2 tigen bound to immobilized antibody was quantitated M HCl (pH adjusted to 2.2 with 2 M glycine) and the by reaction with a second biotin-conjugated antibody pH immediately neutralized with 1 M K,HPO,. This (Wilchek, 1980). Polystyrene wells (Immunolon I Reaffinity-purified antibody was diluted to 6 ml with TBS movawells, Dynatech Laboratories, Alexandria, Va.) and adjusted to 0.4% BSA. Further purification to adsorb were saturated with IgG prepared from rabbit antisera

COOPER AND

BARONDES

against d1 or dI1 by chromatography on DEAE Affi-Gel Blue (Bio-Rad Laboratories, Richmond, Calif.). In preliminary studies, we found that 10 pg of either IgG preparation in 0.2 ml TBS saturated the surface of the well. After they were washed four times with TBS containing 0.2% BSA and 0.3 M galaetose, wells were incubated for 1 hr with a serial dilution of sonicated extract of D. discoideum in TBS containing 0.1% Emulphogen (Sigma Chemical Co., St. Louis, MO.), 0.2% BSA, and 0.3 1Mgalactose. Wells were again washed four times in this buffer. Biotin-conjugated rabbit IgG specific for d1 or for dI1, which had been prepared with the Nhydroxysuccinimide ester of biotin (Calbiochem-Behring Corp., La Jolla, Calif.) as described by Heggeness and Ash (1977), was then added for 1 hr at a concentration of 10 pg in 0.2 ml of the above buffer. After the wells were washed four times, they were incubated for 30 min with 150 ~1 of avidin-conjugated peroxidase (ABC Vectastain standard kit, Vector Laboratories, Burlingame, Calif.) prepared according to the manufacturer’s instructions and then diluted with 4 vol of TBS containing 0.2% BSA and 0.3 M galactose. Wells were then washed six times with this diluent before addition of 200 ~1 of o-phenylenediamine (Bionetics Laboratory Products, Kensington, Md.) in 0.1 M sodium citrate, pH 4.5, with 0.02% HeOz. The calorimetric reaction was allowed to proceed until standard wells containing high discoidin concentration reached saturation and was stopped by addition of 50 ~1 of saturated NaF. Results were recorded spectrophotometrically at 410 nm (Minireader, Dynatech Laboratories). Assay of Developmentally

Regulated Enzyme Activity

To help define the developmental stage of differentiating cultures, the developmentally regulated enzyme a-mannosidase (Loomis, 1970) was assayed. Protein concentrations were determined by a calorimetric method (Lowry et al., 1951). Assay of Discoidin-Binding

Ligands

To quantitate levels of discoidin-binding glycoconjugates we measured their competitive effect on lz51lactosyl-BSA binding to immobilized d1 or dI1 as previously described (Cooper et al, 1983). In this assay, pure d1 or dI1 is adsorbed to a polystyrene well and appropriate concentrations of the iodinated neoglycoprotein are reacted with the well in the presence or absence of varying concentrations of extract. The neoglycoprotein binds to the lectin’s active site and glycoconjugate ligands in the extract competitively inhibit this interaction. In the present experiments, SDS was used to solubilize crude extracts, in contrast with the treatments used previously (Cooper et al, 1983). Samples,

Discoidin-Binding

61

Ligands

suspended in water, were sonicated and boiled as described previously, then SDS was added to a final concentration of O.l%, and appropriate dilutions in this detergent were tested. The detergent itself did not significantly influence 1251-lactosyl-BSA binding to the immobilized lectins. Cell Preparation

for Immunohistochemistry

Colonies were fixed on agar either in Carnoy’s fixative as previously reported (Barondes et al., 1983) or in 3% paraformaldehyde, 1% glutaraldehyde in PBS. After 30 min in paraformaldehyde-glutaraldehyde at room temperature, colonies were detached from the agar by gentle agitation and sedimented by centrifugation for 1 min at 2009 in a Sorvall table top centrifuge. Cells were gently resuspended in PBS with 0.3% paraformaldehyde and 30% sucrose and stored overnight at 4°C. Further processing for immunohistochemical observation followed our previously described protocol (Barondes et ah, 1983), except that sections were picked up on glass slides precoated in 1% BSA instead of gelatin. Immunohistochemical Lectins and SP96

Localization

of Endogenws

Slides of colonies preserved in Carnoy’s fixative were prewashed for 1 hr in TBS-GS-BSA. This was used throughout for all washes and as an antibody diluent. Rabbit affinity-purified cross-adsorbed antibody to d1 and mouse cross-adsorbed antiserum to dI1 were mixed for primary staining at respective dilutions of 1:50 and 1:25. Slides were incubated with this mixture for 30 min and washed for 20 min with two changes of TBS-GSBSA. Control slides were similarly treated with preimmune serum. Alternatively, slides were incubated with rabbit antibody to SP96 (1:25 dilution) and cross-adsorbed mouse antibody to dII(1:25 dilution). Slides were then stained for 30 min with a mixture of rhodamineconjugated goat antibody to rabbit IgG and fluoresceinconjugated goat antibody to mouse IgG (Cappel Laboratories, Inc.), both at dilutions of 1:lOO. After washing for 20 min with two changes of TBS-GS-BSA, slides were drained and mounted with coverslips in 90% glycerol. Immunohistochemical Discoidin-Binding

Localization

of

Sites

To localize discoidin-binding sites, sections of colonies fixed in paraformaldehyde-glutaraldehyde were preincubated for 1 hr in TBS containing 0.5 M glycine and for another hour in 0.4% BSA. Slides were then incubated for 30 min in 50 pg/ml of d1 or dI1 in TBS with 0.4% BSA. Control slides were incubated in buffer alone or

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DEVELOPMENTALBIOLOGY

VOLUME105.1984

with 50 pg/ml lectin in buffer including 0.3 M N-acetylgalactosamine which reacts with the lectin or glucose which does not. After washing in the same medium for 30 min with three changes, slides were transferred to a 1:50 dilution of rabbit affinity-purified cross-adsorbed antibody to dI or to dI1 for 20 min. Fluorescent secondantibody staining was carried out as above with rhodamine-conjugated goat antibody to rabbit IgG. For double-labeling studies of endogenous dI1 and exogenous discoidin-binding sites, Carnoy’s fixed sections were prewashed and stained as above with a mixture containing 50 pg/ml d1 and a 1:25 dilution of crossadsorbed mouse antiserum to dI1. After washing, the slides were incubated for 30 min with affinity-purified cross-adsorbed rabbit antibody to dI(1:50 dilution) and again washed before staining for 30 min with fluoresceinconjugated goat antibody to mouse IgG and rhodamineconjugated goat antibody to rabbit IgG. Controls included deleting the exogenous d1 or deleting the antibody to d1.

ng /ml

RESULTS

Quantitation

of dI and dII in Cells Crown on

Bacteria OT Axenically As a prelude to the immunohistochemical studies, we quantitated the levels of d1 and dI1 at various stages of development with solid-phase immunoassays using antisera prepared against highly purified preparations of each lectin. These antisera discriminated well, but not perfectly between them (Fig. 1). Each assay was linear in the concentration range from 1 to 10 rig/ml, and cross-reaction was so minimal that it was not considered in computing the results. Using this assay with D. discoideum strain NC4 that had been grown on bacteria, we verified the early burst of d1 synthesis and the later secondary rise in dI1 levels (Fig. 2) that had been previously indicated qualitatively by immunohistochemical studies (Barondes et al, 1983). Synthesis of a-mannosidase, a developmentally regulated enzyme, was measured in the same samples to supplement morphological observations of the stage of differentiation. The pattern of its synthesis closely resembled that observed previously (Loomis, 1970). Since it was necessary to use axenic cells grown on macromolecule-depleted medium to study endogenous ligands for discoidin, we also determined levels of d1 and dI1 in such cells with differentiation. Axenically grown cells, which are known to prematurely synthesize a number of developmentally regulated proteins (Quance and Ashworth, 1972), had high levels of d1, dI1, and a-mannosidase while still in their growth phase (Fig. 2). However, the abnormalities in developmental regulation of the lectins did not have a major effect on the

rig/ml

FIG. 1. Quantitation of d1 and dI1 with a solid-phase immunoassay. Polystyrene wells were saturated with anti-d1 IgG (upper panel) or anti-d11 IgG (lower panel). After washing, the wells were incubated with serial dilutions of either purified d1 or dI1, as shown, or with extracts containing these lectins (not shown). Wells were washed and incubated with biotinylated anti-d1 (upper panel) or biotinylated antidI1 (lower panel). Binding of biotinylated antibody was quantitated with avidin-conjugated peroxidase, as described under Materials and Methods.

localization of the endogenous lectins at various stages in comparison with bacterially grown wild-type cells (Barondes et al., 1983), as will be indicated below. Nevertheless, the abnormality necessitated that we confirm the ligand distributions observed in such cells by also examining bacterially grown wild-type cells, as considered later. Quantitation

of Endogenous Ligands for dI and dII

In previous studies in which we solubilized axenically grown D. discoideum, either with a nonionic detergent or Pronase or by sonication, we detected a discoidinbinding ligand only late in development (Cooper et al, 1983). This polysaccharide inhibited ‘251-lactosyl-BSA binding to dI1 at significantly lower concentrations than

COOPER AND

BARONDES

Discoidin-Binding

63

Ligands

8

0

0

6

12

III

24

30

HOURS FIG. 2. Quantitation of d1 and dII during development of NC4 cells raised on bacteria (left panels) and AX3 cells raised axenically (right panels). Levels of each lectin were measured at intervals throughout development by immunoassay of crude extracts solubilized by sonication and addition of 0.1% Emulphogen. Sketches depict colony morphology at the times shown. o-Mannosidase activity was determined in the same samples.

it did for d1 (Cooper et ak, 1983). However, solubilization with SDS revealed potent inhibitory activity for d1 in early aggregates (Cooper et aZ., 1983). To evaluate the levels of this latter material, we grew cells axenically in media free of discoidin-binding ligands, differentiated them on filters and extracted samples at different stages with SDS. We then measured the effect of these extracts on binding of ‘251-lactosyl-BSA to either immobilized d1 or dII. Slime mold ligands that competed with d1 binding of ‘251-lactosyl-BSA were solubilized by SDS even in axenic growth-phase cells, and levels were relatively constant until late in development (Fig. 3). Endogenous ligands that competed with dI1 binding of this iodinated neoglycoprotein were, in contrast, undetectable until late in development (Fig. 3) as shown previously (Cooper et al., 1983). Apparently, the ligand for dI that appears early and requires SDS for solubilization is quite specific for that lectin, as also observed in the histochemical studies considered below. SpeciJcity

of Afinity-Purified

FIG. 3. Effect of SDS extracts of axenic cells on ‘=I-lactosyl-BSA binding to immobilized d1 or dI1. Strain AX3, grown axenically in macromolecule-depleted medium, was differentiated on filters and cells were collected at intervals in water, sonicated, boiled, and solubilized with 0.1% SDS. Concentrations of extract representing lo7 cells/ml of assay mixture were analyzed for inhibition of ‘251-lactosyl-BSA binding to d1 or dI1, expressed as percentage inhibition relative to control binding in 0.1% SDS. Under these solubilization conditions, 4 mM lactose inhibits binding to each lectin by 50%.

been separated by polyacrylamide gel electrophoresis in SDS. The antibodies showed a high degree of specificity when reacted with proteins from aggregating cells (Fig. 4) and other developmental stages (not shown). In no case did either antibody react with any protein other than the lectin. However, when very high concentrations of purified lectin were tested by this procedure, faint cross-reaction was observed (not shown). For doublelabel immunohistochemical studies, we also used a

I

-

lr

Antibodies

Although the rabbit antisera raised against dl and dI1 were highly specific (Barondes et al., 1983), we improved their specificity by affinity purification on columns conjugated with the lectin to which they were raised, followed by adsorption over a similar column of the other isolectin. The resulting affinity-purified and cross-adsorbed antibodies were reacted with nitrocellulose blots of whole D. discoideum proteins that had

FIG. 4. Identification of antigens reacting with affinity-purified and cross-adsorbed antibodies. Whole colonies at the tight aggregate stage of development were harvested in water, solubilized, and 8 pg protein was electrophoresed in each lane of a polyacrylamide slab gel. Separated proteins were then electrophoretically blotted onto nitrocellulose paper and reacted with affinity-purified cross-adsorbed antibody to d1 (left column) or dI1 (right column). Bound antibody was visualized as described under Materials and Methods.

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DEVELOPMENTAL BIOLOGY

mouse antiserum to dII which was absorbed over a column of immobilized d1. Immunoblotting revealed that this serum also was specific for dI1 with slight crossreaction for dI (not shown). Localization Axenically

of dI and dII in LIiIerentiating Grown Cells

Because it was necessary to use axenically grown cells to determine the localization of endogenous ligands for discoidin, we first determined the localization of endogenous d1 and dI1 in such cells during differentiation. It seemed especially important to compare this distribution with that of wild-type cells grown with bacteria, because the developmental expression of discoidin was so different in axenic cells (Fig. 2). As with wild-type cells, the distribution of both lectins was determined in the same sections by a double-label immunofluorescence technique using Carnoy’s fixative, which preserves immunologically recognized determinants of both dI and dI1. Despite the different patterns of lectin synthesis in axenically cultured amoebae, the distribution of both lectins in developing colonies closely resembled that previously described in bacterially grown cells (Barondes et al, 1983). As with bacterially grown cells, dI appears to be exteriorized soon after the initiation of development (Fig. 5A) and accumulates in extracellular material surrounding aggregates (Figs. 5C, E). However, d1 appeared more smoothly distributed without the focal intracellular and extracellular accumulations observed with bacterially raised cells (Barondes et ak, 1983). This suggests that the focal concentrations may have been due to association of this lectin with bacterial products. Also, in axenically raised cells, a layered distribution of d1 close to the aggregate surface was more apparent (Fig. 5C). Although much of the d1 had been lost from the late aggregate, a small amount remained around maturing spores (Fig. 5G), and the lectin was quite prominent in association with the stalk (Fig. 51). The dI1 distribution was also very similar to that found with bacterially grown cells (Barondes et al, 1983), although much more dII was found early in development in axenic cultures. This dI1 appeared to be exteriorized early in development (Fig. 5B). Later, it was concentrated in wavy layers parallel to the aggregate surface (Fig. 5D), a pattern also seen in bacterially grown cells (Barondes et al., 1983). As in that case, a new pattern was observed at the slug stage, when dII was found in a punctate, apparently vesicular, distribution in prespore cells (Fig. 5F). With maturation of the spore cells, dII appeared to have been secreted around them (Fig. 5H). In mature fruits, dI1 staining was no longer detectable (Fig. 55).

VOLUME 105, 1984

Localization

of Endogenous Ligands for d1 and dI1

In order to localize endogenous ligands that bind discoidin in developing colonies, we attempted to prepare various labeled lectin derivatives. Unfortunately, none retained sufficient carbohydrate-binding activity to make them useful for direct binding studies. As a result, we used an indirect procedure. We took advantage of the fact that fixation with 1% glutaraldehyde virtually eliminates immunological reactivity of both lectins in tissue sections, but would not be expected to influence the structure of the glycoconjugates that bind exogenous lectins. Therefore, by incubating such sections with purified lectin followed by antiserum to the lectin, it should be possible to identify discoidin-binding sites. Since, under these conditions, the discoidins intrinsically present in the section do not bind antibody, only sites that bound exogenously added lectin would be visualized. dI-binding sites were observed in growing axenic cells (Fig. 6A). As with d1 itself, these ligands were exteriorized with development and became concentrated at the periphery of differentiating aggregates (Fig. 6C). In slugs, some d1 binding was observed in a pattern resembling the pun&ate endogenous dI1 distribution (Fig. 6E), but much stronger staining was found around the colony (Fig. 6E). Upon culmination, most d1 binding was associated with stalk material (Fig. 6G), but there was also some binding around spores (Fig. 6G). At each of these stages, d1 binding was blocked by N-acetylgalactosamine, but not by comparable concentrations of glucose. This indicates that, under these fixation conditions, fluorescent antibody is labeling specifically bound exogenous and not endogenous antigen. dII-binding sites were localized in the same manner. No binding was observed until the formation of slugs, at which time dII-binding sites appeared in a pun&ate distribution similar to that observed for dI1 itself (Fig. 7A). Later in development, dI1 bound well to spore coats (Fig. 7C), but not to stalk (Fig. 7C). Inclusion of Nacetylgalactosamine with the lectin incubation and wash buffer blocked dI1 binding at these stages (not shown). Are dI1 and Complementary Prespm-e Vesicles?

Glycoconjugates

in

In previous double-label studies with an antiserum raised to whole spores and antiserum to dI1 (Barondes et al., 1983), we found staining patterns which indicated that dI1 might be in prespore vesicles. To further assess whether the punctate staining of dII in slugs represented prespore vesicles, we carried out double-label experiments with a rabbit antiserum specific for SP96, which has been shown to specifically label prespore vesicles and spore coats (Devine et al., 1983), and a mouse an-

anti-d1

FIG. 5. Immunohistochemical localization of endogenous d1 and dI1 in D. disctideum. Strain AX3, grown axenically in macromoleculedepleted medium, was differentiated on nonnutrient agar and fixed at intervals in Carnoy’s fixative. Frozen sections were reacted with rabbit affinity-purified cross-adsorbed antibody to d1 (anti-d1) and mouse cross-adsorbed antiserum to dI1 (anti-dI1). Bound antibody was visualized with rhodamine-conjugated goat anti-rabbit IgG (left column) and Ruorescein-conjugated goat anti-mouse IgG (right column). (A and B) 0.5 hr development. (C and D) 8 hr development (loose aggregate). (E and F) 18 hr development (standing slug). (G and H) 22 hr development (culminating slug). (I and J) 24 hr development (mature fruits). sp, Spore; st, stalk. Bar, 2 pm; x2500. 65

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DEVELOPMENTALBIOLOGY

VOLIJME105,19&i

II:

IB

I

I

FIG. 6. Immunohistochemical localization of binding sites for exogenous d1 on sections of D. discoideum. Strain AX3, grown axenically in macromolecule-depleted medium, was differentiated on nonnutrient agar and fixed at intervals in 1% glutaraldehyde, which virtually abolishes immunological reaction of endogenous discoidin in the tissue. Frozen sections were incubated with 0.5 M glycine and 0.4% BSA to block reactive aldehyde. After incubation with 50 pg/ml d1, the slides were washed either with (B, D, F, H) or without (A, C, E, G) 0.2 M Nacetylgalactosamine, followed by affinity-purified cross-adsorbed antibody to d1. Antibody binding was visualized with rhodamine-conjugated goat anti-rabbit IgG. (A and B) 0 hr development. (C and D) 4 hr development (streaming). (E and F) 18 hr development (standing slug). (G and H) 24 hr development (mature fruit). sp, Spore; st, stalk. Bar, 2 Frn; X2500.

FIG. 7. Immunohistochemical localization of binding sites for exogenous dI1 on sections of D. discoideuwz. Frozen sections of axenic strain AX3 were prepared as for Fig. 6 with glutaraldehyde fixation to block immunological reactivity of endogenous discoidin in the tissue. After incubation with 50 @g/ml of dI1, the slides were reacted with affinity-purified cross-adsorbed antibody to dI1, followed by rhodamine-conjugated goat anti-rabbit IgG, and visualized by fluorescence (left) or phase-contrast microscopy (right). (A and B) 20 hr development (standing slug). (C and D) 24 hr development (mature fruit). Bar, 2 pm; X2500.

COOPER AND

BARONDES

tiserum specific for dI1. When SP96 first appeared just prior to slug formation, it was found in a pun&ate distribution (Fig. 8A), but dII was not (Fig. 8B). Later in development, the distributions of these antigens showed some overlap, but were never completely congruent (Figs. 8C, D). Whereas it was clear that both antigens were in the same cells, it was not possible to determine at this resolution whether or not they were ever in the same vesicles. Since SP96 antigen then appeared on the spore surface at a time when dI1 still appeared largely intracellular (Figs. 8E, F), these antigens did not appear to be secreted together. Later in development, dI1 also appeared to be externalized (Fig. 8H), although its distribution was not identical with SP96 (Fig. 8G). The association of dI1 with the spore coat was only evanescent (Fig. 8J), whereas SP96 remained (Fig. 81). We could not use similar colocalization methods to determine whether ligands which bind dI1 colocalize with endogenous dI1, since we rely on destroying the reactivity of endogenous lectin with glutaraldehyde to examine exogenous lectin binding. However, dI binds somewhat to the polysaccharide which is probably the endogenous ligand for dI1 (Cooper et aZ., 1983), but d1 is not found in prespore cells. We, therefore, compared the distribution in prespore cells of bound exogenous d1 added to the sections with that of endogenous dI1. The localization of d1 binding sites appeared to be similar to the localization of endogenous dI1, within the limits of resolution of fluorescence microscopy (Fig. 9). This is consistent with the possibility that dI1 and complementary glycoconjugates are in the same vesicles. Binding of Exogenous dI and dII to Sections of Bacterially Grown Cells

To be sure that the distribution of endogenous discoidin ligands in axenically grown cells is not abnormal compared to wild-type cells, we repeated some of these studies with bacterially grown cells of strain NC4. The only difference we found was early in development. In aggregating cells grown on bacteria, there were focal accumulations of dI binding material in and around cells. This was not observed in comparable sections of axenically grown cells. The clumps probably represent bacterial products that bind d1 (Cooper et ah, 1983) and have been found in association with endogenous d1 by immunohistochemistry with the electron microscope (P. L. Haywood-Reid, D. N. W. Cooper, and S. H. Barondes, unpublished observations). Later in development, the discoidin-binding patterns were the same in cells grown axenically or on bacteria. DISCUSSION

These results extend previous findings (Cooper et al,, 1983) suggesting that D. discoideum cells contain an

Discoidin-Binding

Ligands

67

endogenous ligand specific for d1 early in development, and another with relative specificity for dI1 late in development. They also confirm, with an axenically grown cell line, the distinct distribution of dI and dI1 during development previously reported for bacterially grown cells (Barondes et al, 1983). The major new finding is that distribution of the endogenous receptors, as determined histochemically is, in general, very similar to the distribution of the endogenous lectins. This provides further strong evidence that the lectins and their specific complementary glycoconjugate ligands interact in vivo in D. discoideum. Since the endogenous lectins have specificity for their complementary ligands and show distinct patterns of synthesis and cellular distribution, the functions of these two lectins must be distinct. After its synthesis, d1 tends to become localized extracellularly. Its most prominent localization in aggregates and slugs is in the amorphous extracellular material surrounding these colonies, which we refer to as slime coat to distinguish it from the much thinner sheath observed with electron microscopy (Farnsworth and Loomis, 1975) which is immediately adjacent to the cellular component of the aggregate. Like the lectin, the endogenous d1 ligand is also concentrated in this slime coat that is secreted around developing aggregates. It is notable that little dI1 is found at this site and that added dI1 does not bind to this material. d1 and its ligands are also colocalized in association with the stalk of mature fruits where a material believed to be analogous to the slime coat has previously been noted (Gezelius, 1959; Shaffer, 1965). These specific localization studies suggest that d1 and its complementary ligand participate in some manner in the formation and/or function of the slime coat. The slime coat, which is difficult to purify and, therefore, largely uncharacterized (Freeze and Loomis, 1977; Smith and Williams, 1979) is believed to function as a protective sheath and to preserve colony integrity during slug migration (Smith and Williams, 1979; Garrod, 1969). Interaction of d1 with its complementary glycoconjugate ligand could influence the viscoelastic properties of the slime to give rise to a gel-like structure which not only assures hydration of the surface of the aggregate but also a proper local medium for aggregate migration. dI1 and its complementary ligand also are found in association with each other, both in and around prespore cells. Although dII is synthesized earlier in development, no binding sites for this lectin are observed until the slug stage. The ligand, which is then synthesized late in development, binds selectively but not exclusively to dII in an in vitro assay. In immunohistochemical studies, it is localized in and around prespore cells as shown by selective binding of dI1. Our studies are consistent with the possibility that dI1 and its ligand are contained in identical vesicles

anti-sp96

anti-dlI

FIG. 8. Immunohistochemical localization of spore coat protein 96 and dI1 in D. diswideum. Strain AX3, grown axenically in macromoleculedepleted medium and differentiated on nonnutrient agar for varying periods, was fixed in Carnoy’s fixative. Frozen sections were reacted with affinity-purified rabbit antibody to SP96 (antiSP96) and mouse antiserum to dI1 that had been cross-adsorbed with d1 (anti-dI1). Bound antibody was visualized with fluorescein-conjugated goat anti-rabbit IgG (left column) and rhodamine-conjugated goat anti-mouse IgG (right column). (A and B) 14 hr development (Mexican hat). (C and D) 20 hr development (standing slug). (E and F) 22 hr development (culminating slug). (G and H) 24 hr development (maturing fruit). (I and J) 72 hr development (mature fruit). Bar, 2 pm; X2500. 68

COOPER AND BARONDES

anti-dIl

FIG. 9. Immunohistochemical localization of dI1 and discoidin-binding sites in sections of slug-stage D. discoideum. Frozen sections prepared as in Fig. 8 were reacted with a mixture of cross-adsorbed mouse antiserum to dI1 (anti-dI1) and 50 pg/ml of d1, followed by affinitypurified cross-adsorbed rabbit antibody to d1 (anti-d1). Bound antibody was visualized with rhodamine-conjugated goat anti-rabbit IgG (A) and fluorescein-conjugated goat anti-mouse IgG (B). Bar, 2 pm; X2500.

within prespore cells and may be secreted together. However, as the spore coat matures, dI1 is no longer found associated with it. This might be due to masking of the antigenic determinants of the lectin with physicochemical changes in the spore coat. Another possibility is that dII does not remain in stable association with its ligand after secretion, in contrast with the dIligand complex. The finding that added d1 and dI1 each bind so selectively to sections of D. discnideum was initially surprising since it seemed possible that there would be many widely distributed glycoconjugates containing galactose or N-acetylgalactosamine that might bind these lectins. Indeed, previous studies in which a chicken lectin was reacted with chicken tissues sometimes showed extensive binding to sites where the endogenous lectin was not concentrated (Beyer and Barondes, 1980). However, the highly specific staining that we observed in the present study is consistent with the finding that galactose and N-acetylgalactosamine containing glycoproteins are not common in D. discoideum cell extracts (Ivatt et ah, 1981) or plasma membranes (Gilkes et al., 1979, Gilkes and Weeks, 1977). They are, however, prominent constituents of extracellular polysaccharides, including a spore coat polysaccharide (White and Sussman, 1963, Cooper et al, 1983), especially during development (Ivatt et aL, 1981, Yamada et al., 1974a,b). Apparently, these highly specialized glycoconjugates have evolved along with the endogenous lectins to serve specific cellular functions. The finding that endogenous slime mold lectins are externalized in association with complementary glycoconjugates during differentiation is reminiscent of

Discoidin-Binding

69

Ligand.s

similar findings with endogenous vertebrate lectins. For example, chicken lactose-lectin-II is prominent in the secretory vesicles of intestinal goblet cells along with mucin, which has been shown to bind to the lectin. Both the lectin and its glycoconjugate ligand are apparently secreted together onto the mucosal surface (Beyer and Barondes, 1982). The results we report here for dI1 and its ligand suggest analogous compartmentalization. Other endogenous lectins, including chicken lactose-lectin-1 (CLL-I) and a related rat P-galactoside-binding lectin, have been localized in extracellular matrix. For example, CLL-I is secreted around developing chicken myotubes (Barondes and Haywood-Reid, 1981) and also appears to form a stable association around pancreatic acini (Beyer et ab, 1979). The related lectin from rat has been localized extracellularly in elastic fibers of the lung (Cerra et al., 1983). Another chicken lectin, which binds to heparin-like glycosaminoglycans, is also secreted with development of muscle cells (Ceri et ak, 1979). An endogenous lectin in Xenops laevis oocytes is secreted following fertilization and is believed to precipitate a component of the jelly coat to form part of the fertilization envelope (Schmell et ah, 1983). A similar frog lectin is found in the glycoconjugate-rich cleavage furrows of developing embryos (Roberson and Barondes, 1983). These findings, coupled with those reported here, strongly support the general conclusion that soluble lectins and their complementary glycoconjugates may often function in the formation of extracellular matrices (Barondes, 1984). This work was supported by grants from the United States Public Health Service (HD-13542) and the McKnight Foundation, and by the Veterans Administration Medical Center. Douglas Cooper is a doctoral candidate in the Department of Biology. REFERENCES BARONDES, S. H. (1984). Soluble iectins: A new class of extraceiiuiar proteins. Science 223, 1259-1264. BARONDES, S. H., COOPER, D. N., and HAYWOOD-REID, P. L. (1983). Discoidin I and discoidin II are localized differently in developing Dictyostelium discoideum. J. Cell Biol. 96, 291-296. BARONDES, S. H., and HAYWOOD-REID, P. L. (1981). Externalization of an endogenous chicken muscle leetin with in viva development. J. Cell Biol. 91, 568-572. BARONDES, S. H., ROSEN, S. D., FRAZIER, W. A., SIMPSON, D. L., and HAYWOOD, P. L. (1978). D. discoideum agglutinins (discoidin I and II). In “Methods in Enzymology” (V. Ginsburg, ed.), Vol. 50, pp. 306-312. Academic Press, New York. BARONDES, S. H., SPRINGER, W. R., and COOPER, D. N. (1982). Cell Adhesion. 1n “The Development of Dictyostelium disccrideum” (W. F. Loomis, ed.), pp. 195-231. Academic Press, New York. BEYER, E. C., and BARONDES, S. H. (1982). Secretion of endogenous lectin by chicken intestinal goblet cells. .J. Cell Biol 92, 28-33. BEYER, E. C., and BARONDES, S. H. (1980). Chicken tissue binding sites for a purified chicken lectin. J. Supramol. Struct. 13, 219-227. BEYER, E. C., TOKUYASU, K., and BARONDES, S. H. (1979). Localization

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of an endogenous lectin in chicken liver, intestine and pancreas. J. Cell Biol 82, 565-571. CERI, H., SHADLE, P. J., KOBILER, D., and BARONDES, S. H. (1979). Extracellular lectin and its glycosaminoglycan inhibitor in chick muscle cultures. J. Supramol. Struct. 11, 61-67. CERRA, R. F., HAYWOOD-REID, P. L., and BARONDES, S. H. (1984). Endogenous mammalian lectin localized extracellularly in lung elastic fibers. J. Cell Biol. S&1580-1589. COOPER, D. N., LEE, S.-C., and BARONDES, S. H. (1983). Diseoidinbinding polysaccharide from Dictyostelium dticddeum J. Biol Chem 258, 8745-8750. DEVINE, K. M., BERGMANN, J., and LOOMIS, W. F. (1983). Spore coat proteins of Dictyostelium dticoideum are packaged in prespore vesicles. Dev. BioL 99, 437-446. FARNSWORTH, P., and LOOMIS, W. F. (1975). A gradient in the thickness of the surface sheath in pseudoplasmodia of Dictyostelium &cm’o!eum Dev. Biol 46, 349-357. FREEZE, H., and LOOMIS, W. F. (1977). Isolation and characterization of a component of the surface sheath of Dictyostelium discoideum. J. Biol. Chem 252, 820-824. GARROD, D. R. (1969). The cellular basis of movement of the migrating grex of the slime mould Dictyostelium discvideum. J. Cell Sci. 4,781798. GEZELIUS, K. (1959). The ultrastructure of cells and cellulose membranes in acrasiae. Exp. Cell Res. 18, 425-453. GILKES, N. R., LAROY, K., and WEEKS, G. (1979). An analysis of the protein, glycoprotein and monosaccharide composition of Dictye stelium discordearn plasma membranes during development. B&him Biophys. Acta 551, 349-362. GILKES, N. R., and WEEKS, G. (1977). The purification and characterization of Dictyostelium discoideum plasma membranes. B&him. Biophys. Acta 464, 142-156. HEGGENESS, M. H., and ASH, J. F. (1977). Use of the avidin-biotin complex for the localization of actin and myosin with fluorescence microscopy. J. Cell Biol 73, 783-788. IVATT, R. J., PREM DAS, O., HENDERSON, E. J., and ROBBINS, P. W. (1981). Developmental regulation of glycoprotein biosynthesis in Dictyostelium. J. Supramol Struct. Cell B&hem. 17, 359-368.

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LOOMIS, W. F. (1970). Developmental regulation of a-mannosidase in Dictyostelium discoideum. J. Bacterial 103, 375-381. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol Chem. 193, 265-275. QUANCE, J., and ASHWORTH, J. M. (1972). Enzyme synthesis in the cellular slime mould Dictyostelium discoideum during the differentiation of myxamoebae grown axenically. Biochem J 126, 609615. ROBERSON, M. M., and BARONDES, S. H. (1983). Xenqpus luevis lectin is localized at several sites in Xen0pu.s oocytes, eggs and embryos. J. Cell Biol. 97, 1875-1881. SCHMELL, E. D., GULYAS, B. J., and HEDRICK, J. L. (1983). Egg surface changes during fertilization and the molecular mechanism of the block to polyspermy. In “Mechanism and Control of Animal Fertilization” (J. F. Hartmann, ed.), pp. 365-413. Academic Press, New York. SHAFFER, B. M. (1965). Cell movement within aggregates of the slime mould Dictyostelium discaideum revealed by surface markers. J. Embryo1 Exp. Mcrrphol 13, 97-117. SMITH, E., and WILLIAMS, K. L. (1979). Preparation of slime sheath from Dictyostelium discoideum FEMS Microbial. L&t. 6, 119-122. SPRINGER, W. R., and BARONDES, S. H. (1983). Monoclonal antibodies block cell-cell adhesion in Dictyostelium disco-ideum. J. Biol. Chem. 258,4698-4701. WHITE, G. J., and SUSSMAN, M. (1963). Polysaccharides involved in slime-mold development II. Water-soluble acid mucopolysaccharides. B&him. Biophys. Acta 74, 179-187. WILCHEK, M. (1980). The avidin-biotin complex in solid phase radioimmunoassays. J. Solid-Phase B&hem. 3, 193-195. YAMADA, H., YADOMAE, T., and MIYAZAKI, T. (1974a). Polysaccharides of the cellular slime mold I. Extracellular polysaccharides in growth phase of Dictyostelium discoideum. Biochim. Biophys. Acta 343,371381. YAMADA, H., YADOMAE, T., and MIYAZAKI, T. (1974h). Polysaccharides of the cellular slime mold II. Change of intra- and extracellular polysaccharides during growth phase of Dictyostelium discoideum NC-4. B&him Biophys. Acta 362, 167-174.