DEVELOPMENTAL GENETICS 11:49%506 (1990)

Formation of the Dictyostelium Spore Coat CHRISTOPHER M. WEST AND GREGORY W. ERDOS Department of Anatomy and Cell Biology, College of Medicine, and Department of Microbiology and Cell Science, IFAS, University of Florida, Gainesville

ABSTRACT The spore coat forms as a rigid extracellular wall around each spore cell during culmination. Coats purified from germinated spores contain multiple protein species and an approximately equal mass of polysaccharide, consisting mostly of cellulose and a galactose/N-acetylgalactosamine polysaccharide (GPS). All but the cellulose are prepackaged during prespore cell differentiation in a regulated secretory compartment, the prespore vesicle. The morphology of this compartment resembles an anastomosing, tubular network rather than a spherical vesicle. The molecules of the prespore vesicle are not uniformly mixed but are segregated into partially overlapping domains. Although lysosomal enzymes have been found in the prespore vesicle, this compartment does not function as a lysosome because it is not acidic, and a common antigen associated with acid hydrolases is found in another, acidic vesicle population. All the prespore vesicle profiles disappear at the time of appearance of their contents outside of the cell; this constitutes an early stage in spore coat formation, which can be detected both by microscopy and flow cytometry. As an electrondense layer, the future outer layer of the coat, condenses, cellulose can be found and is located immediately beneath this outer layer. Certain proteins and the GPS become associated with either the outer or inner layers surrounding this middle cellulose layer. Assembly of the inner and outer layers occurs in part from a pool of glycoproteins that is shared between spores, and unincorporated molecules loosely reside in the interspore matrix, a location from which they can be easily washed away. When the glycosylation of several major protein species is disrupted by mutation, the coat is assembled, but differences are found in its porosity and the extractibility of certain proteins. In addition, the retention or loss of proteolytic fragments in the mutants indicates regions of spore coat proteins that ore required for association with the coat. Comparative examination of the macrocyst demonstrates that patterns of molecular distributions are not conserved between the macrocyst and spore coats. Thus spore coat assembly is choracterized by highly specific intermolecular interactions, leading to saturable associations of individ-

0 1990 WILEY-LISS, INC.

ual glycoproteins with specific layers and the exclusion of excess copies to the interspore space.

Key words: Prespore vesicle, cellulose, polysaccharide, acid hydrolase, spore coat protein, secretion, flow cytometry, confocal microscopy, macrocyst, cellular slime mold

INTRODUCTION The commonly studied developmental cycle of Dictyostelium results in the formation of a pseudoplasmodium, or slug, which later culminates into a fruiting body. There are four obvious extracellular matrix types formed in these structures: the slime sheath, which envelopes the entire mass of the slug: the stalk tube, which is formed during culmination and surrounds the stalk cells; the stalk cell wall, which is interposed between stalk cells; and the spore coat, which surrounds each spore. A less commonly studied developmental cycle can be induced in which the cell aggregate develops into a macrocyst, which is surrounded by a macrocyst coat. Each of these five matrix types consists of cellulose, probably other polysaccharides, and glycoproteins. At least some of the glycoproteins are distinct between matrix types. Because the spore coat is jettisoned after germination of the enclosed amoeba, this matrix has been purified to homogeneity (GonzalezYanes et al., 1989; Lam and Siu, 1981; Orlowski and Loomis, 1979) and has been most extensively studied. We will examine the spore coat and its assembly in this papers. Because the macrocyst coat serves a homologous function, some comparisons will be made with this matrix type. The Dictyostelium spore coat appears to offer a special opportunity for understanding assembly of a n extracellular matrix. For example, assembly is developmentally regulated and can be resolved into multiple steps. Several proteins found in the coat have been

Received for publication July 31, 1990. Address reprint requests to Christopher M. West, Dept. of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610.

SPORE COAT FORMATION IN DICTYOSTELIUM cloned and can now be subjected to genetic analysis. Dictyostelium extracts are capable of synthesizing a pl4 glucan (Blanton and Northcote, 1990), which might be related to the cellulose observed in the coat. Finally, as we discuss in this report, evidence is accumulating that the molecular contacts between coat molecules are specific and saturable. Our intent here is to summarize, in temporal order, the current understanding of the events of spore coat genesis, drawing on both published findings and our own observations.

RESULTS AND DISCUSSION The Prespore Vesicle (PSV), a Storage Organelle Composition. The composition of the PSV has been examined both microscopically and biochemically. Stored spore coat proteins are the primary residents (see Table 1). Of these glycoproteins, SP70 (Gomer et al., 1986), SP75, SP80, SP85, and SP96 (Devine et al., 1983; Erdos and West, 1989; West and Erdos 1988; West et al., 1986)have been localized excusively to the PSV using fluorescence and electron microscopic (EM) immunolocalization methods. The galactose/N-acetylgalactosamine-containing spore coat polysaccharide (GPS) (Cooper et al., 1983; White and Sussman, 1963) has been similarly localized to this compartment (Erdos and West, 1989; Ikeda and Takeuchi, 1971). SP60 has been inferred to be located in the PSV based on coimmunoprecipitation studies (Devine et al., 1983). The possible localization of numerous other more minor spore coat species has not been examined. The EB4 protein (Nellen, personal communication) and a group of lysosomal enzymes have also been reported to be located within PSVs. The enzymes, which include amannosidase and acid phosphatase, sediment as two discrete density classes when isolated from gently lysed slug cells. One of these cosediments with PSVs (Lenhard et al., 1989a). The acid phosphatase found in this denser class appears to be the vegetative form retained from before development, whereas the lighter class is believed to be associated with lysosomes from prestalk cells or prespore cells. Immunoglod localization confirms t h a t a-mannosidase resides in the PSV (Lenhard et al., 198913). Freeze et al. (1990) suggests that lysosomal enzymes gradually enter PSVs during the life of the prespore cell, with complete transfer being achieved before sporulation begins. It is important to note that PSVs do not function as lysosomes, because they do not share a n acidic pH with other organelles found in these cells (Fig. 1A). Furthermore, common antigen-1, a glycoantigen recognized by mAb mLE2 (5G7) (Freeze et al., 19901, which is associated primarily with lysosomal enzymes earlier in development, is found in these acidic organelles but not in PSVs (Fig. 1B). Origin. The PSV is assumed to derive from a Golgi apparatus. Takeuchi and colleagues have described a morphological Golgi stack in forming prespore cells

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(Takemoto et al., 1985). Since this morphology is not observed a t other stages of development, it is a s if the stack arrangement is assumed only in times of high throughput (or back-up), which has a n analogy in yeast (Schekman, 1982). The posttranslational modifications of SP60, SP70, and SP75 are consistent with prior transit through the rough endoplasmic reticulum (RER) and Golgi. SP60, SP70 (Fosnaugh and Loomis, 1989b), and SP96 (Fosnaugh and Loomis, 1989a) have typical signal peptides, and the signal peptides of SP60 and SP70 are known to be cleaved. SP75 is known to be N-glycosylated because its apparent molecular weight (MW) is increased in a modA mutant (M31) (unpublished data), which lacks the trimming enzyme a-glucosidase-I1 (Freeze et al., 1990). SP96, SP70, and SP75 are extensively phosphorylated (Devine et al., 1982) (unpublished data) on serine residues (Akalehiywot and Siu, 1983). Phosphorylation of secretory proteins has not been closely examined in any organism but is presumed to occur in the Golgi apparatus. The Golgi is also presumed to be the location of UDP-galactose polysaccharide transferase (Ikeda, 1981), a n enzyme involved in the assembly of the GPS. Finally, some of the spore coat proteins are 0-glycosylated, presumably in a Golgi compartment or earlier in the secretory pathway. SP96 and SP75 are modified with small 0linked glycans, which are smaller than six glucose units as judged by @-eliminationand P4 gel filtration (Riley, West, and Henderson, unpublished data). This 0-linked glycan(s) contains fucose and probably expresses the fucose epitope recognized by monoclonal antibody (mAb) 83.5. SP80 and SP85 are modified by the modB-dependent pathway (Aparicio et al., 1990), which also appears to be associated with a n 0-linked glycan (K. Williams, personal communication). The assignments of known modifications are summarized in Table 1. The molecular contents of the PSV do not appear to be homogeneously intermixed. Double-label immunogold localization reveals that specific epitopes tend to be clustered in discrete domains in separate parts of the vesicle. Different epitopes typically occupy partially overlapping domains. These epitopes include two glycoantigens (GAS-X and -XI), a-mannosidase, and the GPS (detected with the lectin RCA-I) (Fig. 2). These distributions are not consistent with preassembly of the spore coat, because the associations and separations do not appear to mirror what is known about associations in the finished spore coat (see below). It is possible that the clustering of epitopes in the PSV is a reflection of quanta1 deposits from small delivery vesicles from the Golgi.

Secretion Delivery of Golgi products to the PSV and delivery of the contents of the PSV to the cell surface presumably involve fusion events between the PSV membrane and the membrane of a delivery vesicle and the plasma

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Fig. 1. Identification of acidic and common antigen-1 containing vesicles. For identification of acidic compartments, NC-4 cells (either slug or early culmination) were partially dissociated by shearing between coverslips, incubated for 15-30 min in DAMP, fixed, embedded (Erdos and West, 1989), and probed with anti-DNP antibody and goat antimouse IgGkolloidal gold as described (Erdos and West, in preparation; Anderson, 1989). Prespore vesicles (PSVs) failed to accumulate DAMP, and hence their luminal pH must be similar to, or greater

than, that of the cytoplasm (A) x 78,000. A vesicle population with a n electron-dense luminal content but of unknown identity accumulated this probe and can be concluded to have a n acidic pH relative to the cytoplasm. B Vesicle profiles with a similar morphology were found to contain common antigen-l when probed with mAb mLE2 (5G7) and goat antimouse IgGicolloidal gold as previously described (Erdos and West, 1989). PSVs also do not contain this antigen, which is primarily associated with acid hydrolases. x 74,000.

membrane, respectively. Future work to substantiate these ideas must take into account the morphology of the PSV, which is a n anastomosing tubular network with bulges, rather than a simple spherical vesicle. This has been determined both by reconstruction of serial thin sections viewed by transmission EM (TEM), and by serial optical sections taken after FITC-RCA-I labeling in a scanning confocal microscope (Fig. 3). There are probably only one or a few PSVs per cell, as has been found for mitochondria in certain yeast, peroxisomes in sebaceous glands (Gorgas, 1984), and lysosomes in macrophages (Swanson et al., 1987). This tubular morphology also raises the possibility that secretion does not necessarily transfer all the PSV membrane to the cell surface. The existence of a tubular channel could allow delivery of PSV contents analogous to squeezing gel from a tube of toothpaste. The first ultrastructural indication of spore coat for-

mation is given by the absence of PSVs. If a section is probed for spore coat epitopes, these epitopes are now found a t the cell surface, but otherwise there is no morphological evidence for their presence (Fig. 4A). The cellular profile remains more or less amoeboid, with a scalloped border, rather than having the smooth, rounded border possessed by spore cells. Flow cytometric analyses of papain-treated culminating cells has revealed a subpopulation of cells that express both SP29 (a prepsore cell surface glycoprotein) and SP96 (Browne et al., 1989). These cells presumably correspond to the cells that express cell surface spore coat epitopes in sections. Forward light scattering angle measurements indicated that these cells are smaller than normal prespore cells. Further analysis revealed two additional subpopulations of SP96-positive cells. The second subpopulation, which consisted of cells smaller than the first SP29-, SP96-positive cells, no

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TABLE 1. Contents of the Prespore Vesicle (PSV), Spore Coat, and Interspore Matrix Mo1ecu 1e SP96 SP85 SP80 SP75 SP70 SP60 a-Mannosidase, acid phosphatase, and other lys. enz. EB4 protein GPS cellulose

PSV +arb +h +b +b +c

Inner -

+h -

Spore coat Middle Outer

Interspore matrix

-

+b -

-

+h

-e

? ?

+a

? ?

+h

+h -

?

fa

? ?

+d

+d

-

-

+m

?

?

?

?

+h -h

-

-

-

+’

+h

-h

+

+’

+b

Molecule defined by Seq.‘, mAbs A6.2g, MUD3h mAb 16.1’ mAbs 5.1, 83.53 mAb 83.5g Ab 2H3C*g,seq.k Seq.k

Known modifications PO,”.q, GA-X” GA-XI‘ GAS-X, -XIJ PO,”, GA-X“, N“ FUC~

mAb 2H9, enz. act.d Seq.’ Lectins RCA-I, SBAh Cellulaseb,morphology

CA-1P

aDevine et al., 1983. bErdos and West, 1989. ‘Gomer et al., 1986. dLenhard et al., 1989. ‘N-Glycosylation, this paper. ‘Fosnaugh and Loomis, 1989a. gGonzalez-Yanes et al., 1989. hBrowne et al., 1989. ‘West et al., 1986. JAparicioet al., 1990. kFosnaugh and Loomis, 198913. ’Orlowski and Loomis, 1979. “Seshadri et al., 1986. “Akalehiywot and Siu, 1983. “West and Loomis, 1985. PFreezeet al., 1990. qDevine et al., 1982. ‘W. Nellen, personal communication. longer expressed SP29 but instead expressed another cell surface glycoprotein (gpl17) previously identified on aggregation-stage cells. The third subpopulation, consisting of spores, expressed only SP96. Brown et al. (1989) have thus proposed that this initial phase of spore coat formation includes two intermediate stages, pdspl and pdsp2, referring to partially differentiated spores 1 and 2. Both these stages presumably precede deposition of cellulose (see below). It remains to be determined whether the pdspl and pdsp2 states indeed represent successive steps in differentiation or were artificially induced by pretreatment of cells with papain. In view of its synchronous disappearance from the cell, the PSV has been assumed to be a n example of a “regulated” secretory compartment. Since the original definition of regulated vs. constitutive in reference to secretion was based on a direct measurement of the rate of secretion (Burgess and Kelly, 19871, this classification of the PSV must be regarded a s tentative. For example, some epitopes found “uniquely” in the PSV are also found in the slime sheath (West and Erdos, 1988), suggesting constitutive secretion of this PSVassociated epitope, and the nature of the secretagogue is unknown. Exocytosis of the PSV coincides with the disappearance of UDP-galactose polysaccharide galactosyltans-

ferase (GPS-GT) from the cell (Sussman and Lovgren, 19651, presumably from the Golgi apparatus (Ikeda, 1981). Discoidin I1 also appears to be excreted from the cell from a n unidentified vesicle population at this time (Barondes et al., 1983). The appearance of the majority of cellular acid hydrolases in the interspore matrix (see below) (Seshadri et al., 1986) is consistent with the earlier entry of these enzymes into the PSV as discussed above. It is not known whether the absence of two acid hydrolases (including N-acetyl-f3-D-glucosaminidase) from the interspore matrix is because they are not secreted or because they are incorporated into the spore coat. In addition, two GAS (-XXX and -XXXII), which are each associated with multiple secretory glycoproteins, accumulate in the interspore matrix but not the spore coat (West and Erdos, 1988); a similar observation has also been made for a protease using a different method (North et al., 1990). These GAS do not normally accumulate in the PSV. The coincidental secretion of markers from at least three different organelles suggests that Golgi- and other vesicle-associated proteins might terminally collect in the PSV, a s a n intermediate compartment in a pathway designed to flush cells of developmental isozymes that are not to be retained after germination. Alternatively, there are multiple secretory pathways that potentially contribute to the spore coat.

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Fig. 2. Localization of spore coat protein epitopes in prespore vesicles (PSVs). Sections similar to those shown in Figure 1 were probed simultaneously with RCA-Iigold 15 nm), to localize the galactoseiNacetyl-galactosamine containing polysaccharide (GPS) (A,B), and either monoclonal antibody (mAb) 83.5 and goat antimouse IgGigold (5 nm), to localize the fucose epitope, or mAb 16.1 and goat antimouse

IgGigold (5 nm), to localize SP85. Although all three probes localize to the PSV, they are nonuniformly and noncoextensively distributed within this compartment. Contents of the PSV are not homogeneously intermixed. A, x 66,000, B, x 91,000 (Reproduced from Erdos and West, 1989, with permission of the publisher.)

Assembly of the Spore Coat

and spore coat formation would be cell autonomous. This question was tested by allowing spores of two strains [one whose spore coat proteins SP75, and SP80, and SP96 were marked with the fucose epitope (F') and one whose proteins were not marked (F-) (because of a genetic defect in their ability to fucosylate protein (Gonzalez-Yanes et al., 1989)l to form together in comixtures. Spores were treated with 8 M urea, which should strip adventitiously associated proteins, but is gentle enough to activate germination (Cotter and O'Connell, 1976). Spores were labeled with a fluorescent antibody, which recognizes the fucose-epitope, and their fluorescence was analyzed using flow cytometry. Normal ( F + )spores were, a s expected, highly fluorescent, and mutant (F-) spores were nonfluorescent (Fig. 5). When F + and F- strains were allowed to sporulate together, their repective spore fluorescences were not conserved. Instead, F- cells became more fluorescent, wheras F + cells became less fluorescent. A 5050 mixture of input cell types yielded a n almost homogeneous, monodisperse, fluorescence distribu-

Cellulose is detected in the forming coat only in conjunction with the appearance of a superficial electrondense layer (Fig. 4B).During this period, the plasma membrane of the cell assumes a more gradual contour. Since this electron-dense layer contains the fucose epitope (GA-X), it is the future outer layer of the mature spore coat. Cellulose is detected immediately deep to this layer, and above the plasma membrane. The earlier amorphous distribution of cell surface spore coat molecules indicated by immunogold labeling suggests that spore coat assembly is a postsecretion event coincident with cellulose deposition. This interpretation is consistent with the finding that spore coat proteins secreted from cells developed in suspension are not sedimentable (West and Erdos, 1988). If this interpretation is correct, it would be expected that spore coat molecules are diffusive and that spore coat assembly is nonce11 autonomous. On the other hand, if some degree of preassembly occurred prior to secretion, this would be expected to inhibit diffusive exchange,

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Fig. 3. Morphology of the prespore vesicle (PSV) compartment. Slugs were partially dissociated by shearing, fixed for immunofluorescence, and labeled with FITC-RCA-I as described (Gonzalez-Yanes et al., 1989). The GPS distribution was visualized using scanning confocal microscopy. Fifteen optical sections were scanned and superimposed, and the resulting representation was photographed for stereo viewing. A stereo viewer is required to visualize the three-dimensional image. The distribution of the GPS indicates that the PSV compartment is not vesicular but rather a tubular anastomosing net-

work. To verify the morphology suggested by the RCA-I fluorescence, serial thin sections were cut from embedded material, and tracings of PSV membrane profiles from 30 successive sections were digitized and reconstructed using a Jandel PC-3D program. In addition, tracings were manually converted to a Styrofoam model and visualized. The results of EM serial sectioning confirmed the observations made by scanning confocal microscopy (Erdos and West, in preparation). x 10.000.

tion. These results indicate that, during spore coat formation, F- and F + molecules are shared between neighboring spores and incorporated into both coats. This model is consistent with the initial amorphous distribution of PSV contents after secretion rather than preassembly. As is discussed below, quantitative analysis of a complete series of strain proportions indicates that a n antibody accessibility (porosity) phenotype of the F- cells is also shared between neighboring spores at higher proportions of mutant cells (West and Erdos, in preparation). The GPS may help localize the assembly process by increasing the viscosity a t the cell surface, since the spore coat can assemble in single sporogenous mutant cells in the absence of neighboring cells. There is no obvious evidence for biochemical changes in the spore coat proteins as they assemble to form the spore coat, a s far as can be determined by one- and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) and epitope analysis. More subtle changes have not been ruled out that might serve to regulate the assembly process. pH does not seem to regulate

assembly since the PSV luminal pH, cytoplasmic pH, and extracellular pH all seem to be similar (see Fig. 1).

Spore Coat Structure Composition. The composition of the spore coat has been determined from coats shed upon germination and purified to homogeneity on density gradients. Its dry weight is about equally divided between protein and cellulose (Aparicio et al., 1990). The major glycoprotein species (Aparicio et al., 1990; Gonzalez-Yanes et al., 1989; Lam and Siu, 1981; Orlowski and Loomis, 1979) have already been listed in Table 1 as constituents of the PSV. Numerous more minor species can be identified on gels and, because of the purity of spore coat preparations, can be confidently ascribed to spore coats. It is unknown, however, whether they are distinct protein species or are isoforms or degradation products of the major species. Direct extraction of intact spores with sodium dodecyl sulfate (SDS) and/or urea and reducing agent selectively releases spore coat proteins, and similar major species are identified (Wilkinson and Hames, 1983) (unpublished data). It is

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Fig. 4. Initial stage of spore coat formation. A developmental time course was sampled hourly be sectioning and probing with mAb 83.5 and goat-antimouse IgGigold for localizing the expression of the fucose epitope (GA-X) as described (West and Erdos, 1988). The first evidence of spore coat formation is given by the example in A. PSVs are no longer found in the cell, and the fucose epitope is entirely extracellular, near the cell surface, but not organized into a layer. A

later stage of assembly was detected in sections that had been labeled with cellulaseigold (B). Cellulose was found only around cells surrounded by a condensed layer of electron-dense material, which is to be the future outer layer ofthe spore coat. A, x 36,000.B, x 37,000. (B is reproduced from Erdos and West, 1989, with permission of the publisher.)

questionable, however, whether some of the lower MW species extracted from intact spores actually are spore coat proteins, since they do not correlate with proteins from purified spore coats and they have the electrophoretic behavior of cytoplasmic proteins (unpublished data). Substructure. The mature spore coat is organized as a trilaminar structure (Fig. 6) (Erdos and West, 1989; West and Erdos, 1988). As determined by immunogold localization studies on sections of washed spores, the outer electron-dense layer contains the spore coat proteins SP75, SP80, and SP96 (see Table 1). The fucose epitope associated with these proteins precisely straddles either side of the electron-dense plane in a double track. According to protease-sensitivity studies, SP60 and SP70 are found at the surface of the spore coat, whereas SP96 is inaccessible (Orlowski and Loomis, 1979). Since SP96 is proteolyzed in coats isolated from a glycosylation mutant only after germination (Gonzalez-Yanes et al., 1989), this glycoprotein may require germination to be solvent exposed or is protease accessible from the inside out. The inner elec-

tron-dense layer, near the plasma membrane, contains SP85, a-mannosidase (Lenhard et al., 1989131, and the GPS (Erdos and West, 1989). Cellulose intervenes as a sheet between these two layers. The inner layer components tend to blend gradually with the intermediate layer with respect to gold labeling density. Freeze-fracture analysis has resolved the cellulose into two layers, with the predominant fibrillar orientations a t a different angle (Hemmes et al., 1972). Since no microscopically visible residue remains after alkaline hydrolysis followed by acetolysis, the spore coat of Dictyostelium discoideum does not seem to contain significant quantities of nonhydrolyzable polymers (Maeda, 1984). The inner layer of the spore coat appears to have some physical association with the plasma membrane. This is inferred from the finding that plasma membrane remains attached to fragments of the spore coat after mechanical disruption of the spore and centrifugation (Cotter et al., 1969), although this association is apparently weak because fragments of spore coats often do not have associated membrane after purification by density gradient centrifugation (data not shown).

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499

4

FL2

FL2

Fig. 5. Exchange of protein between forming spore coats. Ax3 (F + ) and HL250 (F-1 amoebae (axenically grown) were allowed to develop together. Sari were picked, extracted with urea, and immunolabeled with mAb 83.5, which recognizes the fucose (F)epitope synthesized on Ax3 glycoproteins only. Labeled spores were subjected to dual-parameter flow cytometry to quantitate fluorescence intensity (amount and accessibility of the fucose epitope) and forward light scattering (size). Results are shown in the left column, and histograms of fluorescence intensities are shown in the right column. The top and bot-

tom panels show that mutant and normal spores have low and high fluorescence levels, respectively. When mutant and normal spores (1:l ratio) form adjacent to one another, however, all spores have a similar level of fluorescence between that of mutant and normal spores formed alone. This implies that each spore, regardless of its genotype, expresses a similar, accessible quantity of normal (F ) glycoproteins synthesized by normal spores. Thus the spore coat appears to assemble from a pool of glycoproteins shared between neighboring spores.

The weakness of this association can also be inferred from the ease with which the cell shrinks away from the coat in hyperosmotic media (plasmolysis) (Cotter, 1981)and the observation that the plane of separation

as a result of shrinkage during fixation and embedding usually occurs between the membrane and the inner layer (unpublished observations). The separateness of the spore coat and the plasma membrane is consistent

+

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INTERSPORE IlRTRlX (USUALLY EXTRRCTED)

GI ycoprotei ns (GAS-X, -XX), GPS G1 ycoprotei ns (GAS-X, - X X )

OUTER LAYER O F S.C.

Cellulose

IllDDLE LRYER OF S.C G1 ycoproteins ( G A - X I ) , GPS

INNER LFIVER OF S.C. PLRSUR UEUBRANE

Fig. 6. Trilaminar organization of the spore coat. The results of EM gold localization studies of the distribution of various molecules of the spore coat are tabulated in Table 1 and pictorially represented here. The three layers of the coat are surrounded by the plasma membrane (bottom)and the interspore matrix (top). (Reproduced from Erdos and West, 1989, with permission of the publisher.)

with the absence of obvious hydrophobic domains in the spore coat proteins that have been sequenced thus far, including SP60, SP70, and SP96 (Fosnaugh and Loomis, 1989a,b), and the finding that secretory SP85 can be recovered from suspension-developed cells in soluble form (West and Erdos, 1988). In conclusion, there are probably weak associations between the plasma membrane and the spore coat; these may be sufficient to provide polarity to the coat during its morphogenesis. As might be expected from the molecular distributions, there is evidence of a functional distinction between spore coat layers. According to Cotter et al. (1969), the two outer layers split and can retract during the swelling phase of germination, exposing the outer surface of the inner layer, which remains continuous over the surface of the cell. Completion of germination disrupts the inner layer resulting in its shedding. Interspore matrix. Additional information about the spore coat is obtained if unwashed spores are examined (Erdos and West, 1989; West and Erdos, 1988). When intact sori are fixed in the presence of cetylpyridinium chloride, a polycationic precipitant of polyanions, elements of the interspore matrix are preserved (Fig. 6, Table 1).This region contains GPS and glycoproteins carrying the fucose epitope. In contrast, SP85 is not detected i n this region. The presence of the fucose epitope in the interspore matrix provides a n explanation for the functional sharing of this epitope between spore coats during spore coat assembly (West and Erdos, in preparation): The interspore matrix appears to be the location of a functional pool of spore coat precursors. The interspore matrix is probably also the location of secreted GPS-GT, of the pool of lysosomal enzymes that do not become incorporated into the inner layer of the spore coat (Seshadri et al., 1986), and of a group of glycoproteins carrying GAS-XXX and -XXXII

(West and Erdos, 1988). The presence of a pool of proteins in the interspore matrix is consistent with the observation that spore coat proteins are secreted in soluble form from cells that are developed in suspension (West and Erdos, 1988). The quantity of SP96 and SP75 present in the interspore matrix was estimated by washing spores with water and 8 M urea (a treatment that activates germination). Aliquots of each fraction, proportioned to a n equivalent per spore basis, were compared by SDSPAGE and Western blotting (Fig. 7). SP75 was most prevalent in the spore fraction, with lesser amounts found in the water and urea washes. In contrast, SP96 was most prevalent in the water wash, with lesser amounts found in the urea wash and the spore. As was anticipated from the immunogold localization studies, individual proteins thus have characteristic associations with the spore coat proper and the interspore matrix. These associations do not seem to involve disulfide bonds, because hot urea extraction in the presence or absence of 1%2-mercaptoethanol was equally effective in eluting SP75 and SP96 (Aparicio et al., 1990). In addition, high-MW species seen in SDS gels, which have previously been shown to be oligomers of lower MW proteins, including SP75 and SP96, as determined by reelectrophoresis (Gonzalez-Yanes et al., 1989), tend to be excluded from the spore coat proper and instead appear in the washes. It is interesting to consider the partitioning of SP75 and SP96 relative to intermolecular binding between these two proteins. SP96 can be separated from most other spore coat proteins by DEAE-ion exchange highperformance liquid chromatography (HPLC), taking advantage of its highly acidic PI. Surprisingly, SP75, which has a more neutral PI (on 2D gels), uniquely copurifies with SP96 even after boiling in 8 M urea with 1%2-mercaptoethanol (Fig. 8). Taken together,

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Since SP96 is phosphorylated (Devine et al., 1982) and has a very acidic PI (Devine et al., 1983; West and Loomis, 1985), this protein may also be highly phosphorylated. Extensive phosphorylation of cytoskeletal proteins can influence their length and stiffness (see, e.g., Hagestedt et al., 1989). The potential role of oligosaccharide substituents on spore coat proteins (summarized above and in Table 1)is discussed below in the section on mutants. The amino acid sequences of SP60, SP70, and SP96 have been deduced from their cDNAs and from partial sequences of the proteins (Fosnaugh and Loomis, 1989a,b). Although the structural basis for the association of these proteins with the coat has not been elucidated, the specificities of intermolecular associations suggested by examination of SP96 (see above) and SP80 (see mutant section below) indicate that this may be experimentally approachable. In contrast to most of the other major spore coat proteins, SP96 and SP80 are not apparently linked to the spore coat by disulfide bonds (Aparicio et al., 1990; Wilkinson and Hames, 1983). Each of the three sequenced proteins possesses several repeat sequences, some of which are shared with the others, and all three proteins seem (see above) to reside in the outer layer of the coat. The presence of repeat sequences within the same polypeptide chain suggests the possibility of a repetitive structural motif, which might be significant for the shape of the moleFig. 7. Partitioning of spore coat glycoproteins between the coat cule. Such a motif may be involved in intermolecular and the interspore matrix. One-day-old Ax3 (axenically grown) spores were picked with a loop, suspended in water, and sedimented. The recognition, a s has been indicated for repeated motifs water wash was reserved and the spores were resuspended in 8 M in proteins associated with intracellular filaments, urea in 10 mM KINaPO,, pH 6.8. After 0.5 hr, spores were again such as MAP2, T protein (see e.g., Hagestedt et al., sedimented. The urea wash was reserved, and the spores were ex1989), and cytokeratins. The occurrence of extensive tracted with hot SDS-sample buffer containing 2-mercaptoethanol for 3 min. The three extracts were resolved by SDS-PAGE, western blot- phosphorylation may inf h e n c e the shape and stiffness of individual protein molecules (Hagestedt et al., 1989). ted onto nitrocellulose, and probed for the fucose epitope (GA-X) using

96%

75”

mAb 83.5

A6.2

mAb 83.5 or for SP96 using mAb A6.2 (as in Gonzalez-Yanes et al., 1989).SP96 (denoted 96 in margin) and higher MW species (which are aggregates of lower MW species) were enriched in the wash fractions (representing the interspore matrix), and SP75 (denoted 75 in margin) was enriched in the spore coat.

the results suggest that SP75, with which SP96 tightly associates, is limiting for SP96 association with the spore coat. Rather than becoming trapped in the inner or intermediate layers of the spore coat, excess SP96 overflows into the interspore matrix. Two levels of specificity are thus indicated: 1)SP96 may bind to the coat via SP75, and 2) excess SP96 is specifically excluded from other layers of the coat. Properties of glycoproteins. SP96 has been suggested to be of low abundance in the spore coat based on a weak signal after Coomassie blue staining (Lam and Siu, 1981), but, since Coomassie blue staining of this glycoprotein is labile (unpublished data), this interpretation is probably incorrect. In contrast, SP96 readily stains with Stains-All (unpublished data). The highly phosphorylated protein phosvitin also stains poorly with Coomassie blue but intensely with Stains-All.

Analysis of Mutants Three glycosylation mutations are known to affect spore coat glycoproteins. The modA mutation, which affects initial glucose trimming of N-linked glycans, affects SP75, as noted above, but the effects of this mutation have not been examined further. In the second mutant (HL250), GDP-fucose cannot be formed from GDP-mannose at any stage of development; thus all fucosylation in the cell is blocked (unless exogenous fucose is supplied by bypass this block) (GonzalezYanes et al., 1989). The fucose epitope (GA-X), unique to the PSV and spore coat and recognized by mAb 83.5, is not formed. This epitope is found on SP75, SP80, and SP96 (summarized in Table 1)and is located in association with the outer electron-dense layer of the spore coat. The third kind of mutation (modB) appears to block the assembly of 0-linked glycans on threonines in SP29 (K.L. Williams, personal communication), and thus may also inhibit 0-glycosylation of the spore coat proteins SP80 and SP85 (Aparicio et al., 1990). SP85 is located in the inner electron-dense layer of the spore coat, and SP80 is probably located in the outer elec-

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-96 -75

f.t.

0.3

0.5M NaAc

Fig. 8. HPLC fractionation of spore coat proteins. Intact spores formed from 32P0,-labeled Ax3 slugs were treated with hot urea/ 2-mercaptoethanol and the extract was passed over a DEAE (TSK DEAE-5PW 8 x 75 mm) anion exchange HPLC column as described (Gonzalez-Yanes et al., 1989).Every fifth fraction, in addition to fractions containing high DPM (this allowed identification of fractions with spore coat proteins), was resolved by SDS-PAGE and stained with Coomassie blue. The left lane contains flow-through (f.t.) material, which did not adsorb to the column. The next lane contains material that eluted a t the beginning of the salt gradient (--1 M NaAc). The higher MW proteins are spore coat proteins and, as predicted

from their apparent pls according to 2-D gels (Gonzalez-Yanes et al., 1989), were eluted later in the gradient. However, SP75, which has an apparent pl of 5.5-6.0, coeluted at 0.5 M NaAc with SP96, with an apparent pl of ca. 3.5. This anomolous chromatographic behavior suggests that SP75 and SP96 strongly interact, in a manner that is not affected by urea or 2-mercaptoehtanol but is dissociated by SDS. All copies of SP96 and SP75 participate in this interaction because there are no fractions that contain either of these proteins individually. As discussed in the text, excess copies of SP96 accumulate in the interspore matrix. SP85 and SP70 elute earlier (0.3 M NaAc) and do not participate in this interaction.

tron-dense layer (Erdos and West, 1989; West and Erdos, 1988). Both mutants display a germination-defective phenotype relative to their parental strains. HL250 spores germinate poorly after 7 days despite normal germination after 2 days (Gonzalez-Yanes et al., 1989). Young mod3 mutant spores germinate normally in response to heat but not to urea (Aparicio et al., 1990). Both mutant types also display a spore coat phenotype. This was evaluated by testing the ability of a lectin, FITCRCA-I, to label GPS in urea-extracted spores. Both mutants labeled more readily with the lectin, implying

that the mutant coats have a more porous architecture, leading to greater accessibility of the lectin. The molecular composition of the mutant coats appeared to be normal with respect to polysaccharide and protein species, and the trilaminar organization of these components was not affected as far a s could be determined with the probes available. A change in the spore coat was corroborated by findings that glycoproteins that were affected by the respective mutations tended to be proteolytically cleaved during germination. In each case, however, proteolysis followed emergence of the amoeba, and thus did not appear to play a causative role

SPORE COAT FORMATION IN DICTYOSTELIUM in the germination defects. Finally, proteins of the modl3 mutant coat were found to be differentially sensitive to extraction by low and high pH compared with normal coats. Taken together, the results indicate that protein-linked oligosaccharides contribute to intermolecular associations in the spore coat. Although the mutated oligosaccharides do not provide all of the specificity of the spore coat protein interactions, their disruption may alter the strength of protein associations with the coat, rendering the coat more porous to macromolecules and increasing exposure to secreted proteases after germination.

Comparison of Spore Coat With Other Extracellular Matrices Slime sheath and stalk tube. As discussed in the Introduction, three separate matrices are formed in addition to the spore coat during the fruiting body cycle. In addition, a fifth matrix type, the macrocyst coat, forms during the macrocyst cycle. Each of these matrix types possesses cellulose organized in fibrillar arrays. The stalk tube and slime sheath have been biochemically purified, although purification of these structures has required extraction with detergents and/or protein denaturants, which, as we know from studying the spore coat, probably strips glycoproteins that might normally be associated with them. One study attempted to assay for glycoproteins biochemically using slime trails, which are slime sheaths through which slugs have passed (Grant and Williams, 1983), but it is difficult to determine whether the proteins found are residents of the sheath or are derived from contaminating cells that tend to break away from the slug. There is better evidence from the study of denatured sheaths for the presence of small proteins (ca. 10 kD) (Freeze and Loomis, 1977, 1978), which have no obvious counterparts in the spore coat. No evidence for abundant polysaccharides other than cellulose or pl-4 glucans has been found in slime sheaths or stalk tubes. However, a fibrillar residue remains at the site of the stalk tube following alkaline hydrolysis and acetolysis, which Maeda (1984) has inferred to indicate the presence of a carotenoid polymer called sporopollenin. It has been possible to address the question of protein and polysaccharide composition of these matrices by assaying for the presence of protein and oligosaccharide epitopes using immunogold and lectinlgold localization methods. Briefly, the slime sheath shares some GAS with the spore coat (andlor interspore matrix) and possesses some GAS not found in the spore coat (West and Erdos, 1988). For example, the fucose epitope (GAX recognized by mAb 83.5) is found in both the spore coat and the slime sheath, although its expression in the slime sheath is only within that part of the sheath that lies over prespore cells, the cells of origin of GA-X. Three GAS, GA-XXX, GA-XXXI, and GA-XXXII, are found in the slime sheath and not in the spore coat, although two of them are trapped in fixed interspore

503

matrix (GA-XXXII and GA-XXX). I n contrast, none of these GAS, with the exception of GA-X, is found in the stalk tube, in that part of the stalk tube associated with the prespore region. It is noteworthy that two GAS, GA-XX and GA-XI (recognized by mAbs 54.2 and 16.1, respectively), are not found in either the slime sheath or the stalk tube. Finally, two proteins, ST310 and ST430, are synthesized only by prestalk cells and are found in both the slime sheath and the stalk tube but not the spore coat (McRobbie et al., 1988). Several conclusions can be drawn from these findings. 1)Proteins are a general feature of cellulose-based Dictyostelium matrices, and different proteins are associated with different matrix types. There is evidence that the specificity of protein associations with different matrices is governed both by the location of synthesis and at the level of incorporation. 2) There must be a secretory pathway not involving the PSV to deposit some of the proteins (that carry GAS-XXX, -XXXI, and -XXXII) extracellularly. Since these GAS can be detected intracellularly, i t is not clear whether this pathway(s) is constitutive or regulated. 3) There must be some pathway that can secrete GA-X without secreting GA-XI, despite the fact that the only intracellular accumulation of these two GAS is in the PSV. This strongly implies that there is a constitutive secretory pathway for GA-X in addition to the regulated pathway involving the PSV. Macrocyst coat. The macrocyst coat is more complex in that i t consists of three walls, which are deposited a t three separate times. Their names, given in order of time of deposition, are primary (deposited by the aggregate), secondary, and tertiary (deposited by the zygote after phagocytosis of the surrounding haploid amoebae), the primary wall thus being the outermost layer. Each of these walls is separated by a “space” microscopically, which may be fluid. All three walls contain cellulose. Each of the spore coat epitopes, GA-X, GA-XI, and GA-XX, and the GPS are found in the macrocyst coat, although some are specific to one wall or another (Fig. 9). Western blotting reveals that these epitopes are associated with glycoproteins whose apparent MWs in some cases differ from, and in other cases are similar to, those of spore coat proteins (unpublished data). An unexpected finding is that these GAS and the GPS tend to be distributed uniformly throughout the cellulose-containing layers, because in the spore coat these GAS and the GPS are concentrated away from the zone of cellulose. In addition, GA-X and GA-XX, which are distributed together in the spore coat, are localized to separate walls in the macrocyst coat. As in the spore coat, GA-XI and the GPS are found together (in the tertiary wall) in the macrocyst coat. If the principles of GA assembly are the same in the macrocyst and spore coats, then these findings imply that 1)cellulose is not essential for incorporation of GA-containing proteins, 2) cellulose does not interfere with incorporation of these proteins, and 3) the incor-

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Ab156 - - - - - mmAb (Ga-XI)- 16.1

-

-

-

-

I

-

-

-

-

-

A

Fig. 9. Distribution of polysaccharides and glycoproteins in the macrocyst coat. Macrocyst (NC-4iV12)thin sections were probed with cellulaseigold (B), mAb 5.1 (which recognizes the modB-dependent GA-XX) followed by goat-antimouse IgGigold (0, RCA-Iigold (which recognizes the GPSi (D), or mAb 83.5 [which recognizes the “fucose epitope” (GA-Xi](E), followed by goat-antimouse IgGigold. Methods are a s in Erdos and West (1989).The full thickness of the coat, with the exception of the primary wall, which continues out of view to the right, is shown in E. Since the thicknesses of layers and colloidal

regions between layers varies, portions of the interlayer colloid have been cropped in ELD to permit alignment of the cellulose-containing layers. Data from these and other sections labeled with other antibodies are summarized in A. The macrocyst coat is formed during three intervals. The primary (first) layer, formed by the cell aggregate, becomes the outer layer (to the right). The secondary and tertiary layers are deposited later by the zygote. The protoplast itself is to the left of the field of view. x 29,000.

SPORE COAT FORMATION IN DICTYOSTELIUM porations of proteins bearing GA-X and -XX are not interdependent. The pattern of deposition of GAS and GPS in the macrocyst coat layers also indicates that the timing of protein synthesis and the activity of the regulated secretory pathwayk) that supplies these proteins extracellularly undergo interesting modifications over the time course of macrocyst maturation. EM views of thin sections do not reveal PSV-like structures (not shown), and it has been difficult to identify secretory vesicles because of poor fixation of the macrocyst protoplast.

SUMMARY AND SPECULATION Perhaps the most significant question to ask about spore coat formation is whether it is in fact a n assembly process characterized by ordered, dependent associations of molecules or a nonspecific congelation of whatever proteins and polysaccharides happen to be present a t the time of cellulose deposition. A second question is whether proteins are really important for the structure and function of the coat. Data are now available that address these questions. If assembly is a n ordered process, then it would be expected that individual molecules would have characteristic locations in the coat, and these locations would be mediated by specific intermolecular interactions. Most spore coat proteins are restricted to either the outer or the inner layer of the coat, suggesting that their localizations are specific. SP85, from the inner layer, and SP75, from the outer layer, are unusual in that they are not also present in the interspore matrix. In contrast, all other proteins examined and the GPS are also found in the interspore matrix. Our model for assembly is t h a t SP85 and SP75 in some way define the extent of the inner and outer layers, respectively. We propose that the incorporation of other molecules into these layers is limited by the number of copies of these “core” molecules. It is noteworthy that excess copies of these other molecules are excluded from the rest of the coat, and instead appear in the interspore matrix, which is easily washed away. These characteristics are not consistent with a nonspecific congelation mode of assembly. Two additional observations support the model of specific, ordered assembly. First, SP96 forms a molecular pair with SP75 that resists treatment with strong denaturing and reducing agents. Furthermore, there are no free copies of SP75 or SP96 extracted from the coat. The only free copies of SP96 are found in the interspore matrix. This implies that SP75 is limiting for association of SP96 with the coat, and this limitation is imposed by the binding event between SP96 and SP75. The second supporting observation derives from analysis of germinating spores from modB mutants (Aparicio et al., 1990). SP80, which appears to reside in the outer layer, is proteolytically cleaved after germination. Coincident with this cleavage event, the larger fragment becomes dissociated from the coat. No other

505

proteins are known to be released. This means that SP80 is anchored to the coat by a telopeptide of ca. 12 kD. This mode of association is consistent with the existence of a specific binding site for SPBO in the outer layer. Finally, the phenotypes of the two glycosylation mutants provide evidence that proteins of the coat contribute to its structure and function. The fucosylation and modB mutants affect the glycosylation of separate but partially overlapping groups of glycoproteins in the inner and outer layers of the coat. Both mutant coats have an increased porosity to a macromolecular probe, and one of them has been shown to have different chemical sensitivity to extraction of proteins. Both mutants have a germination phenotype, which might be related to changes in the coat. In conclusion, the assembly of the coat appears to involve three steps. First, the noncellulosic components are secreted from the PSV. Organization of the outer and inner proteinaceous layers begins. At least some copies of these proteins are assembled from a soluble extracellular pool. During this time, cellulose deposition begins. It is not known whether these latter two steps are separate or interdependent. The assembly of proteins into the coat appears to be a specific process important for the structure and function of the coat. These characteristics suggest that the assembly process can be productively studied by genetic manipulation of structureifunction relationships using cloned genes that have been isolated for some of the resident proteins.

ACKNOWLEDGMENTS The research from our laboratories was supported by NIH grants R 0 1 GM-33015 and GO1 GM-37539. Flow cytometry was supported by NIH grants S10 RR-02666 and S10 RR-03454 and funds from the University of Florida Division of Sponsored Research and Interdisciplinary Center for Biotechnology Research. We are grateful to Neal Benson for his assistance with flow cytometry and Dr. W.A. Dunn for his advice on the DAMP method. The help of B.J. OBrien, 0. Aparicio, R. Davis, B. Gonzalez-Yanes. and A. Choate is also gratefully acknowledged. REFERENCES Akalehiywot T, Siu C-H (1983): Phosphorylation of spore coat proteins of Dictyostelium discoideum. Can J Biochem Cell Biol61:9961001. Anderson RGW (1989): Postembedding detection of acidic compartments. Methods Cell Biol 31:463-472. Aparicio JG, Erdos GW, West CM (1990):The spore coat is altered in modB glycosylation mutants of Dictyostelium discoideum. J Cell Biochem 42:255-266. Barondes S, Cooper D, Haywood-Reid P (1983): Discoidin I and discoidin I1 are localized differently in developing Dictyostelium discoideum. J Cell Biol 96:291-296. Blanton RL, Northcote DH (1990):A 1,4-P-D-glucan-synthase system from Dictyostelium discoideum. Planta 180:324-332. Browne LH, Sadeghi H, Blumberg D, Williams KL, Klein C (1989): Re-expression of 117 antigen, a cell surface glycoprotein of aggre-

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gating cells, during terminal differentiation of Dictyostelium discoideum prespore cells. Development 105657-664. Burgess TL, Kelly RB (1987): Constitutive and regulated secretion of proteins. Annu Rev Cell Biol 3:243-293. Cooper D, Lee S, Barondes S (1983):Discoidin-bindingpolysaccharide from Dictyostelium discoideum. J Biol Chem 258:8745-8750. Cotter D, Miura-Santo L, Hohl H (1969): Ultrastructural changes during germination of Dictyostelium discoideum. J Bacteriol 100: 1020-1026. Cotter D, OConnell R (1976):Activation and killing of Dictyostelium discoideum spores with urea. Can J Microbiol 22:1751-1755. Cotter DA (1981): Spore activation. In Turian G, Hohl HR (eds): “The Fungal Spore: Morphogenetic Controls.” New York Academic Press, pp 385-411. Devine K, Bergmann J , Loomis W (1983): Spore coat proteins of Dictyostelium discoideum are packaged in prespore vesicles. Dev Biol 99:437-446. Devine K, Morrissey J , Loomis W (1982): Differential synthesis of spore coat proteins prespore and prestalk cells of Dictyostelium. Proc Natl Acad Sci USA 79:7361-7365. Erdos GW, West CM (1989): Formation and organization of the spore coat of Dictyostelium discoideum. Exp Mycol 13:169-182. Fosnaugh KL, Loomis WF (1989a): Sequence of the Dictyostelium discoideum spore coat gene SP96. Nucleic Acids Res 17:9489. Fosnaugh KL, Loomis WF (1989b): Spore coat genes SP60 and SP70 of Dictyostelium dzscoideum. Mol Cell Biol 95215-5218. Freeze H, Loomis W (1977): Isolation and characterization of a component of the surface sheath of Dictyostelium discozdeum. J Biol Chem 252:820-824. Freeze H, Loomis W (1978):Chemical analysis of stalk components of Dictyostelium discoideum. Biochim Biophys Acta 539529-537. Freeze HH, Bush JM, Cardelli J (1990):Biochemical and genetic analysis of a n antigenic determinant found on N-linked oligosaccharides in Dictyostelium. Dev Genet 11:463-472. Gomer R, Datta S, Firtel R (1986): Cellular and subcellular distribution of a CAMP regulated prestalk protein and prespore protein in Dictyostelium discoideum: A study on the ontogeny of prestalk and prespore cells. J Cell Biol 103:1999-2015. Gonzalez-Yanes B, Mandell RB, Girard M, Henry S, Aparicio 0, Gritzali M, Brown RD, Erdos GW, West CM (1989): The spore coat of a fucosylation mutant in Dictyostelium discoideum. Dev Biol 133: 576-587. Gorgas K (1984): Peroxisomes in sebaceous glands. Anat Embryo1 169:261-270. Grant W, Williams K (1983):Monoclonal antibody characterization of slime sheath: the extracellular matrix of Dictyostelium discoideum. EMBO J 2:935-940. Hagestedt T, Lichtenberg B, Wille H, Mandelkow E-M, Mandelkow E (1989): Tau protein becomes long and stiff upon phosphorylation: Correlation between paracrystalline structure and degree of phosphorylation. J Cell Biol 109:1643-1651. Hemmes DE, Kojima T, Buddenhagen ES, Hohl HR (1972):Structural and enzymic analysis of the spore well layers in Dictyostelzum discoideum. J Ultrastruct Res 41:406-417. Ikeda T (1981): Subcellular distributions of UDP-ga1actose:polysaccharide transferase and UDP-glucose pyrophosphorylase involved in biosynthesis of prespore-specific acid mucopolysaccharide in Dictyostelium discoideum. Biochim Biophys Acta 675:69-76.

Ikeda T, Takeuchi I (1971): Isolation and characterisation of a prespore specific structure of the cellular slime mold, Dictyostelium discoideum. Dev Growth Differ 13:221-229. Lam T, Siu C-H (1981): Synthesis of stage-specific glycoproteins in Dictyostelzum discoideum during development. Dev Biol 83:127137. Lenhard JM, Kasperek E, Moore BR, Free SJ (1989a): Developing Dictyostelium discoideum cells contain two distinct acid hydrolasecontaining vesicles. Exp Cell Res 182:242-255. Lenhard JM, Siege1 A, Free SJ (1989b): Developing Dictyostelzum cells contain the lysosomal enzyme a-mannosidase in a secretory granule. J Cell Biol 109:2761-2769. Maeda Y (1984): The presence and location of sporopollenin in fruiting bodies of the cellular slime molds. J Cell Sci 66:297-308. McRobbie SJ, Jermyn KA, Duffy K, Blight K, Williams J G (1988): Two DIF-inducible, prestalk-specific mRNAs of Dictyostelium encode extracellular matrix proteins of the slug. Development 104: 275-284. North MJ, Cotter DA, Franek KJ (1990): Dictyostelium discoideum spore germination: Increases in proteinase activity are not directly coupled to emergence of myxamoebae. J Gen Microbiol (in press). Orlowski M, Loomis W (1979): Plasma membrane proteins of Dictyostelzumt The spore coat proteins. Dev Biol 71:297-307. Schekman R (1982):The secretory pathway in yeast. TIBS 7:243-246. Seshadri J , Cotter D, Dimond R (1986): The characterization and secretion pattern of the lysosomal trehalases of Dictyostelium discoideum. Exp Mycol 10:131-143. Sussman M, Lovgren N (1965): Preferential release of the enzyme UDP-galactose polysaccharide transferase during cellular differentiation in the slime mold, Dictyostelium discoideum. Exp Cell Res 38:97-105. Swanson J , Burke E, Silverstein SC (1987):Tubular lysosomes accompany stimulated pinocytosis in macrophages. J Cell Biol 104:12171222. Takemoto K, Yamamoto A, Takeuchi I (1985): The origin of prespore vacuoles in Dictyostelium discoideum cells as analysed by electronmicroscopic immunocytochemistry and radioautography. J Cell Sci 77:93-108. West CM, Erdos GW (1988): The expression of glycoproteins in the extracellular matrix of the cellular slime mold Dictyostelium discoideum. Cell Differ 23:l-16. West CM, Erdos GW, Davis R (1986): Glycoantigen expression is regulated both temporally and spatially during development in the cellular slime molds Dzctyostelium discoideum and Dictyostelium mucoroides. Mol Cell Biochem 72:121-140. West CM, Loomis WF (1985): Absence of a carbohydrate modification does not affect the level or subcellular localization of three membrane glycoproteins in modB mutants of Dictyostelium discoideum. J Biol Chem 260:13803-13809. White G, Sussman M (1963): Polysaccharides involved in slime mold development 11. Water soluble acid mucopolysaccharide(s).Biochim Biophys Acta 74:179-187. Wilkinson DG, Hames BD (1983): Characterisation of the spore coat proteins of Dictyostelium dzscoideum. Eur J Biochem 129:637-643.