1 Dept of Anatomy and Cell Biology, ICBR, University of Florida, Gainesville, FL 32610-0235, USA
2 College of Medicine and Electron Microscopy Core Laboratory, ICBR, University of Florida, Gainesville, FL 32610-0235, USA
Correspondence
Christopher M. West
westcm{at}ufl.edu
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ABSTRACT |
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INTRODUCTION |
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The spore coat of Dictyostelium is a cell wall specialized for long-term protection of the enclosed spore cell from environmental extremes. The spore coat consists of a thick middle layer composed primarily of cellulose fibrils sandwiched between an electron-dense inner layer next to the plasma membrane and an electron-dense outer layer that is exposed to the environment (West, 2002). Cell walls from many organisms, including yeast, fungi and epidermal cells of vascular plants, exhibit a similar trilaminar organization. The mechanism by which multiple layers are formed is poorly understood, but a recent study in Dictyostelium implicates the spore coat protein SP85 in a feedback loop that regulates cellulose synthesis (West et al., 2002
). Absence of SP85 appeared to lead to precocious cellulose synthesis, whereas expression of a fragment of SP85 containing two of its domains inhibited cellulose synthesis. In addition, cellulose deposition appears to be required for subsequent condensation of the outer electron-dense layer (Zhang et al., 2001
).
Wild-type spores show low permeability to a fluorescent lectin probe that binds to a polysaccharide rich in galactose and N-acetylgalactosamine associated with the inner layer of the coat (Erdos & West, 1989). The location and intense electron density of the outer layer suggests that it composes the permeability barrier to fluorescent lectins, which was supported by increased permeability of a glycosylation mutant that affects the fucosylation of coat proteins associated with the outer layer (Gonzalez-Yanes et al., 1989
). Multiple proteins, including SP96, SP87, SP80, SP70 and SP60, are associated with the outer layer based on immunocytochemical localization (West & Erdos, 1988
; Erdos & West, 1989
) and protease accessibility studies (Orlowski & Loomis, 1979
). Mutant spores lacking SP96, SP70 or SP60 as a result of gene targeting show increased labelling by a fluorescent lectin (Fosnaugh et al., 1995
) and an antibody against the coat protein SP85, also associated with the inner layer (Srinivasan et al., 2000
). Labelling is even greater in strains lacking two or three of the proteins, suggesting that they each contribute independently and additively to outer-layer function. These findings implicate the outer layer as the permeability barrier but morphological evidence to support this model has been lacking.
Spores lacking SP85, associated with the inner layer, also exhibit increased lectin labelling compared to normal spores, but the effect is more subtle as it requires pre-extraction with hot urea and 2-mercaptoethanol (Zhang et al., 1999). A similar effect is observed in a mutant targeting the O-glycosylation of mucin-like regions of SP85 (Aparacio et al., 1990
). These studies suggested that SP85 contributes to outer-layer functionality by cross-bridging other molecules in the coat based on the similar effects of expressing fragments of SP85 hypothesized to have independent binding functions. For example, the C-terminal (C1C2) domain of SP85 binds cellulose and another coat protein, SP65, in vitro (Zhang et al., 1998
, 1999
), and other studies suggest that SP85 can be isolated as a multiprotein complex (McGuire & Alexander, 1996
). Based on these binding activities, SP85 is hypothesized to contribute to the organization of protein-cellulose interfaces between the coat layers. New studies reported here document that deletion of SP85 or expression of SP85 fragments renders ultrastructural effects that are, for some constructs, similar to the deletion of outer-layer proteins. Biochemical studies show that SP85 is important for the incorporation of the outer-layer coat protein SP96, and mutational studies suggest that the cellulose-binding activity of SP85, which maps to its Cys-rich C1 domain, contributes to SP85 action. In addition, the appearance of novel, regularly spaced, punctate electron densities and curvilinear thin filaments in the spore coats of specific mutant strains suggests previously unrecognized units of organization of the outer layer of the coat.
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METHODS |
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Spore coats were purified from intact spores as described by Zhang et al. (1998).
Transmission electron microscopy.
Spores were fixed in glutaraldehyde or a combination of glutaraldehyde and formalin at pH 7·2 (McDowell & Trump, 1976), collected on poly-L-lysine-treated Millipore membrane filters and covered with 3 % agarose. Agarose-embedded spores were peeled away from the filters, post-fixed in 1 % buffered osmium tetroxide, stained en bloc and dehydrated in a graded ethanol series and 100 % acetone. Spores were embedded in EmBed 812 epoxy resin and polymerized at 60 °C for 2 d. Thin sections of approximately 70 nm were collected on Butvar-coated 100-mesh copper grids and post-stained with 2 % aqueous uranyl acetate, followed by Reynold's lead citrate. Grids were examined on a Hitachi H-7000 transmission electron microscope (Hitachi Scientific Instruments). Digital micrographs were taken on a BioScan/Digital Micrograph 2·5 (Gatan) at an exposure level optimized for viewing the outer layer and processed with MEGA View III/AnalySIS 3.1 (Soft Imaging System).
For immunolocalization studies, spores were fixed in 1 % (v/v) glutaraldehyde, 4 % (w/v) paraformaldehyde in 15 mM sodium phosphate, 135 mM NaCl, pH 7·2 (PBS), for 1 h at 4 °C. Spores were recovered and dehydrated as above and embedded in Unicryl acrylic resin (British Biocell International) under UV light at -10 °C for 2 d. Thin sections (70 nm) were collected on Butvar-coated 400-mesh nickel grids. Sections were treated with 1 % (w/v) non-fat dry milk in 0·1 % Tween-20, 0·5 M NaCl, 0·05 M Tris/HCl, pH 7·2 (HST), incubated overnight in a humid chamber at 4 °C in mAb 83.5 (1 : 1000 ascites) in HST, washed in HST and incubated with a secondary antibody (goat anti-mouse IgG, 1 : 30 diluted in PBS) conjugated to 18 nm colloidal gold (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Sections were post-stained and observed as above.
Mutant and normal strain Ax3 spores were prepared in parallel to ensure comparable fixation and staining. NC1- and mutant NC1-expressing spores were also processed in parallel. Micrographs shown are representative of images from at least two independent clones.
Site-directed mutagenesis.
pVSBNC1 (West et al., 2002) was modified by site-directed mutagenesis using the QuikChange kit (Stratagene) as described by Sassi et al. (2001)
. The nucleotide sequence for W352EN was changed from TGGGAAAAT to TCAAAACAT; the sequence for R390GK was changed from CGTGGTAAG to AGTGGTGAG; the sequence for K417GQ was changed from AAAGGTCAA to CAAGGTGAA; and the sequence for R445RGE was changed from AGACGTGGTGAA to AGTGCTGCTCAA. For some of the constructs it was necessary to design the oppositely oriented mutagenic primers in staggered (35 nt), rather than perfectly overlapping fashion to amplify plasmid DNA. Simultaneous introduction of these mutations in pVSBNC1 was performed as described by Wang & Malcolm (1999)
.
Anti-SP85 antibody.
A frozen spleen from a mouse immunized with the bacterially expressed, T7-tagged N domain of SP85 (NT7) that produced polyclonal antibody (pAb) NT7 used in a previous study (Zhang et al., 1999) was thawed and cells dissociated from the tissue were fused with plasmacytoma SP2/0 cells to create hybridomas as described by Simrell & Klein (1979)
. Hybridomas were grown in HAT selective media supplemented with 20 % horse serum. Hybridoma clones were chosen based on the ability of antibodies in their culture supernatants to immunoprecipitate soluble SP85 from the cA-ISM fraction (see above) and recognize SP85 on Western blots. Clone 5F5 from this screen is used in this report.
Cellulose-binding assay.
Avicel powder (type PH-101; FMC corporation) was stored as a 50 mg ml-1 suspension in H2O, 0·02 % NaN3 at 4 °C. For each assay, a volume containing 9 mg Avicel was transferred with a wide-bore pipette tip to a 1·5 ml polypropylene tube, centrifuged at 10 000 g for 1 min and the supernatant removed by aspiration. cA-ISM (200 µl) was pre-centrifuged at 13 000 g for 5 min to remove any insoluble material and used to resuspend the Avicel pellet. The suspension was agitated at 22 °C for 1316 h. Binding was incomplete at 2 h (data not shown). The bound and non-bound fractions were separated by centrifugation at 10 000 g for 1 min and the supernatant was transferred to a new tube (non-bound fraction). The pellet was resuspended in 0·5 ml 50 mM NH4Ac (pH 6·8), centrifuged again and the supernatant aspirated. In some experiments, 150 µl of the non-bound supernatant was reincubated with 9 mg Avicel and the process repeated. 1x, 2x times; or 4x times; Laemmli sample buffer containing 50 mM dithiothreitol was added to each sample, which was boiled, precleared by centrifugation and loaded onto individual lanes of a 720 % linear gradient SDS-PAGE gel. Gels were electroblotted onto 0·45 µm pore-size nitrocellulose film and probed with primary antibodies (Table 1). Primary antibody labelling was detected using an appropriate alkaline phosphatase-conjugated secondary antibody, which was developed colorimetrically with 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium (Zhang et al., 1999
).
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RESULTS |
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Expression of SP85 fragments disrupts the outer layer
The sequence of SP85 suggests that it consists of a series of globular domains (West, 2002), named N (for NH2-terminal), M (for middle), C1 (for COOH-terminal 1) and C2 (for COOH-terminal 2), as depicted in Fig. 3
(a). The M domain is flanked by mucin-like domains which consist of tetrapeptide repeats, named TR1 and TR2. Individual domains or domain combinations were previously expressed in prespore cells to investigate the role of SP85 in the regulation of coat formation and in coat permeability (Zhang et al., 1999
; West et al., 2002
). These included the N domain, the C1C2 domain and fusions of the N domain with either C1C2, C1 or C2. The latter constructs were designed to ensure delivery to the prespore vesicle as the N domain carries targeting information (Zhang et al., 1999
) and to promote folding of the short C1 and C2 domains (C. M. West and others, unpublished data). Each construct was incorporated efficiently into the coat (or precoat in the case of NC1), except for N which was recovered in the interspore matrix (Zhang et al., 1999
; West et al., 2002
). Further analysis of NC1 and NC2 expression using domain-specific antibodies (see Table 1
) showed partial cleavage of these constructs (Fig. 4
a), releasing the N domain into the interspore matrix, and retention of free C1 with the cell (C2 was not detected). Cleavage appeared to occur at the internal myc epitope, which separates the N and C1 domains, based on absence of mAb 9E10 binding and correspondence of apparent Mr values of the N and C1 domains with their expected values (data not shown). Thus, expression of the NC1-construct resulted in the accumulation of NC1, N and C1; expression of NC2 resulted in the accumulation of NC2 and N.
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A second kind of defect was seen in spores expressing the NC1 domain fusion. Only about 15 % of the prespore cells of this strain deposit cellulose and complete sporulation because of failure to execute an early checkpoint in coat formation (West et al., 2002). Images from the mature spores that formed revealed an unusual mat of novel, filamentous projections at their surfaces (Fig. 5
b, c). The filaments replaced the normally continuous electron density of the outer layer and extended outwardly approximately one thickness of the coat. They had apparent diameters on the order of 10 nm, similar to the apparent thickness of the outer layer. The filamentous mat covered most of the surface of the spore (Fig. 6
a). Occasional spores had an attenuated or missing middle layer but still exhibited the modified outer layer (data not shown), indicating that the filaments did not depend on the full deeper layers of the coat. The filamentous mat was also formed when NC1 was expressed in SP85-null HW70 cells (Fig. 5d
), showing that filaments do not require SP85 and are not the result of checkpoint delay which does not occur in this strain (West et al., 2002
). The filaments appear to contain coat proteins because they were immunogold-labelled with mAb 83.5 (Fig. 6c
), which recognizes outer-layer proteins (Fig. 6b
).
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Site-directed mutagenesis of the NC1 domain fusion
The C1 domain consists of four tandem repeats of the C4C motif (Fig. 3a), which is about 25 aa in length and predicted to fold with two disulfide bonds (Table 2
), based on sequence similarity to the N-terminal subdomain of the EGF motif (West, 2002
). In an attempt to dissect the contributions of each C4C motif to these functions, the third predicted loop of each motif was subjected to site-directed mutagenesis in which one to two residues on each side of the predicted
-turn in the middle of the loop were modified (Table 2
). These mutations were designed to alter the chemical character of the loops without disrupting global folding of the motif (Handford et al., 2000
). Each mutant construct, NC1(B), NC1(C), NC1(D) and NC1(E), and a quadruple mutant NC1(BE) in which all four motifs were mutagenized simultaneously, was expressed in strain Ax3 and clones expressing high levels of the NC1 mutant proteins were chosen for further analysis.
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Strains expressing mutant NC1s formed fruiting bodies with 3060 % of the normal number of cellulose-positive spores, compared to the 10 % produced by normal NC1-expressing cells in these experiments (Table 3). This indicated that the mutations partially inactivated the inhibitory effects of NC1 on checkpoint execution, showing that each of the four C4C motifs contributes to its previously described checkpoint blockade activity (West et al., 2002
).
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Cellulose-binding activities of normal and mutant NC1s
A recombinantly expressed version of the C region, consisting of the C1 and C2 domains, was previously shown to bind cellulose in vitro (Zhang et al., 1998). To determine whether binding maps to the C1 or C2 domain, the binding activities of the NC1 and NC2 constructs were compared. Cells expressing the domain fusions in a dcsA-null background (see Table 1
), to eliminate endogenous cellulose, were sporulated in suspension in the presence of 20 mM 8-Br-cAMP and the supernatants (cA-ISM) were used as a source of the NC1 and NC2 proteins. The cA-ISMs were incubated in the presence of Avicel cellulose as described in Methods and separated into bound and unbound fractions by centrifugation. As shown in Fig. 7
(a), the majority of NC1 bound to Avicel. In contrast, very little binding of NC2 was detected. Similar results were observed when the domain fusions were expressed in SP85-null cells (Fig. 7a
) or normal Ax3 cells (see below), showing that it was not necessary to express the protein in the absence of cellulose and that binding did not depend on any SP85 in these preparations.
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DISCUSSION |
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The coats from HW70, lacking SP85, also exhibit loss of electron density from the outer layer (Fig. 2b). The residual material does not show as clear a periodic pattern as TL56, but grazing sections suggest the existence of residual strands (Fig. 2d
). The similar spacing of these densities with those of TL56 coats suggests that they may be related structures. This is supported by the appearance of periodic dots when NC2 is expressed in SP85-null spores (see below), suggesting that in SP85- spores the dots may be obscured by additional material. SP85-null coats are missing SP65 (Zhang et al., 1999
) and the outer-layer protein SP96 (Figs 2e and 4b
), each of which is recovered in the interspore matrix. Failure of SP85-null spores to incorporate SP96 suggests that the same end point, in-fill of outer-layer proteins, is inhibited and implies that a hierarchy of proteinprotein interactions involving SP85 and SP65 is required for incorporation of SP96. The less severe defect seen in SP85- compared to TL56 outer layers correlates with the less severe permeability defect observed in SP85- spores, which required pre-treatment with urea and reducing agent to detect (Zhang et al., 1999
).
Role of SP85 in outer-layer formation
The outer layer is also altered when fragments and domain fusions of SP85 fragments are expressed. NC2-expressing spores had an outer-layer morphology similar to that of TL56 spores with the appearance of a regular array of similarly spaced punctate dots (Fig. 3). Consistent with this interpretation, cells expressing NC2 fail to incorporate the outer-layer protein SP96 into the coat and instead accumulate a truncated isoform of SP96 in the interspore matrix (West et al., 2002
), as now also observed for SP85-null spores (Fig. 4b
). Spores expressing the N domain are missing patches of the outer layer altogether. This correlates with other defects including increased coat permeability, decreased coat buoyant density and more rapid germination, as reported previously for this strain (Zhang et al., 1999
). The mechanism of action of N may involve binding to a critical distal component as N is not incorporated into the mature coat and is recovered in the interspore matrix (Zhang et al., 1999
). Coats of spores expressing C1C2 exhibit a mosaic character, with some regions resembling NC2. Thus the effect of NC2 does not seem to obligatorily depend on the presence of the N domain, or the accumulation of free N domains observed when NC2 was overexpressed. Spores expressing C1C2 also showed increased permeability, decreased buoyant density and more rapid germination (Zhang et al., 1999
). The NC1C2 domain fusion was least severe, consistent with the greatest similarity to full-length SP85 and this correlated with the least severe effect of this construct on coat functions (Zhang et al., 1999
). These results are consistent with a cross-bridging model for normal SP85 function in which discrete domains bind other components in the coat, thereby linking them together. Overexpression of individual domains would be expected to compete with endogenous SP85 for ligands, resulting in a failure to cross-bridge them into the inter-dot regions of the outer layer. The somewhat more severe effects of expressing the domains compared to deletion of SP85 suggest the existence of mechanisms that compensate for the absence of SP85, which are also effectively competed by the domain constructs.
In contrast to the other mutant constructs, NC1 expression led to the formation of a novel structure. This consists of a thick mat of filamentous material which replaces the normal continuous outer layer (Figs 5 and 6). The filaments may represent a displaced normal coat subunit. The only linear structures known in the coat are cellulose microfibrils. We have been unsuccessful in labelling these filaments using cellulase-gold probes (C. M. West and others, unpublished data), but the sensitivity was low and number of microfibrils per filament may be small. A coat protein epitope is associated with the filaments (Fig. 6c
) though it cannot be determined if this represents the core or a coating. Instead of cellulose, the filaments might be formed of protein or another polysaccharide. SP85 is not required for the filaments as the double mutant (expression of NC1 in SP85-null cells) also expresses them, albeit with altered appearance. NC1 might seed the ectopic formation of filamentous structures at the outer layer, as a result of improper localization due to the absence of a critical localizing domain present in full-length SP85. Elsewhere, fibres with radial orientation have been implicated in the formation of cell walls in Chlamydomonas and Cryptococcus (Woessner & Goodenough, 1994
; Pierini & Doering, 2001
).
Involvement of cellulose-binding activity in SP85 function
Previously, a recombinant version of the C1C2 domain fusion was found to bind cellulose in vitro (Zhang et al., 1998) and a comparison of the NC1 and NC2 domain fusions here shows that this activity maps to the C1 domain (Fig. 7
). The C1 domain contains four Cys-rich C4C motifs. Based on the hypothesized similarity to the N-terminal subdomain of the EGF-module (Table 2
), the sequence between the third and fourth conserved Cys-residues is predicted to form a loop with a centrally positioned Gly (or similar) residue forming a critical
-turn, possibly analogous to the conformation of the RGD motif of fibronectin (Tani et al., 2002
). Residues adjacent to the Gly residue were altered in each C4C motif individually and also simultaneously in a fifth construct in an attempt to disrupt cellulose-binding. Although mutagenesis of any of the C4C motifs interfered with the ability of NC1 to block the early checkpoint in cellulose deposition, only the quadruple mutation interfered with cellulose binding. This was also the only mutant that affected the filament-inducing activity of NC1. Although it is not known if cellulose binding is the only activity affected by these mutations, as C1C2 also binds SP65 via a site separate from cellulose binding (Zhang et al., 1999
), this correlation suggests that cellulose binding is important for NC1 action. If the above model that NC1 nucleates or anchors naturally occurring filaments in an ectopic location is correct, then the apparent dependence on cellulose binding could be explained if cellulose is the basis for NC1 attachment to the coat, or if the filaments contain cellulose to which NC1 binds. In plant walls, cellulose fibrils are thought to be anchored by hemicelluloses (Delmer, 1999
; Brett, 2000
), but additional mechanisms are likely to be involved especially in walls like the spore coat that lack this polysaccharide (West, 2002
). In any case, the result implicates cellulose binding in the contribution of SP85 to outer-layer formation.
Relationship of outer-layer formation to earlier events in coat formation
Secretion of coat protein precursors is thought to be the initiating event of the extracellular phase of coat assembly (West, 2002). An early function of SP85 is to prevent cellulose synthesis until coat proteins are secreted, hydrated and possibly reorganized to form a precoat (West et al., 2002
). The outer layer itself does not appear until cellulose deposition is initiated (Zhang et al., 2001
). Since SP85 contributes in an essential way to outer-layer formation and the dominant negative effect of NC1 depends on structural motifs also required for efficient cellulose binding, it is likely that the role of cellulose in outer-layer formation is transmitted at least in part through SP85. The contribution of SP85 to outer-layer formation is expected to be indirect because it is localized in the inner layer and is not long enough to stretch across the estimated 160 nm thickness of the middle layer of the coat. If the 108 aa of the TR regions fold as left-handed polyproline type II helices (Kelly et al., 2001
), they would have a combined length of 35 nm, and the remaining 405 residues, which are likely to fold as globular domains, are unlikely to contribute more than this length again. As summarized above, SP85 appears to contribute to the outer layer via a hierarchy of intermolecular contacts. Based on previous studies (Zhang et al., 1999
) and results shown here, a cellulose-SP85-SP65-SP96 linkage might compose the molecular backbone that links elements of the outer layer to the coat (West, 2002
). The C1 domain mediates cellulose binding (Fig. 7
), and appears to also bind SP65 (C. M. West and others, unpublished data) via a separate site, and the C2 domain may mediate SP96 binding based on the effects of NC2 on SP96 incorporation (Fig. 4b
). SP70 and SP60, previously suggested to interact with SP85 (McGuire & Alexander, 1996
), might also be anchored via this proposed module. The filaments seen in NC1-expressing spores might represent misplaced copies of this module or, alternatively, reflect an additional function for SP85 in the packaging of cellulose microfibrils, which are disorganized in these mutants based on abnormal sensitivity to acid hydrolysis (West et al., 2002
).
The previously described contribution of SP85 to early checkpoint regulation (West et al., 2002) appears to be separate from its outer-layer function. First, the early effect of NC1 on checkpoint function does not depend on cellulose or its cellulose-binding activity, whereas its ability to induce outer-layer filaments seems to depend on its cellulose-binding activity. Second, NC1 mutations that affect checkpoint execution do not affect filament induction (Table 3
). Third, the effect of NC1 on checkpoint execution depends on endogenous SP85, whereas induction of filaments does not (Fig. 5d
). Finally, some of the SP85 fragment constructs have other effects on the outer layer without altering the timing of checkpoint execution.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bayer, E. A., Shimon, L. J., Shoham, Y. & Lamed, R. (1998). Cellulosomes structure and ultrastructure. J Struct Biol 124, 221234.[CrossRef][Medline]
Blanton, R. L., Fuller, D., Iranfar, N., Grimson, M. J. & Loomis, W. F. (2000). The cellulose synthase gene of Dictyostelium. Proc Natl Acad Sci U S A 97, 23912396.
Boulianne, R. P., Liu, Y., Aebi, M., Lu, B. C. & Kues, U. (2000). Fruiting body development in Coprinus cinerus: regulated expression of two galectins secreted by a non-classical pathway. Microbiology 146, 18411853.
Brett, C. T. (2000). Cellulose microfibrils in plants: biosynthesis, deposition, and integration into the cell wall. Int Rev Cyt 199, 161199.
Delmer, D. P. (1999). Cellulose biosynthesis: exciting times for a difficult field of study. Annu Rev Plant Physiol Plant Mol Biol 50, 245276.[CrossRef]
Erdos, G. W. & West, C. M. (1989). Formation and organization of the spore coat of Dictyostelium discoideum. Exp Mycol 13, 169182.
Fosnaugh, K. L., Fuller, D. & Loomis, W. F. (1995). Structural roles of the spore coat proteins in Dictyostelium discoideum. Dev Biol 166, 823825.[CrossRef]
Frisardi, M., Ghosh, S. K., Field, J., VanDellen, K., Rogers, R., Robbins, P. & Samuelson, J. (2000). The most abundant glycoprotein of amebic cyst walls (Jacob) is a lectin with five Cys-rich, chitin-binding domains. Infect Immun 68, 42174224.
Gonzalez-Yanes, B., Mandell, R. B., Girard, M., Henry, S., Aparicio, O., Gritzali, M., Brown, R. D., Erdos, G. W. & West, C. M. (1989). The spore coat of a fucosylation mutant in Dictyostelium discoideum. Dev Biol 133, 576587.[Medline]
Handford, P. A., Downing, A. K., Reinhardt, D. P. & Sakai, L. Y. (2000). Fibrillin: from domain structure to supramolecular assembly. Matrix Biol 19, 457470.[CrossRef][Medline]
Harb, O. S., Gao, L.-Y. & Abu Kwaik, Y. (2000). From protozoa to mammalian cells: a new paradigm in the life cycle of intracellular bacterial pathogens. Environ Microbiol 2, 251265.[CrossRef][Medline]
Kelly, M. A., Chellgren, B. W., Rucker, A. L., Troutman, J. M., Fried, M. G., Miller, A.-F. & Creamer, T. P. (2001). Hostguest study of left-handed polyproline II helix formation. Biochemistry 40, 1437614383.[CrossRef][Medline]
Loomis, W. F. (1971). Sensitivity of Dictyostelium discoideum to nucleic acid analogues. Exp Cell Res 64, 484486.[Medline]
Maeda, M. (1992). Efficient induction of sporulation of Dictyostelium prespore cells by 8-bromocyclic AMP under both submerged- and shaken-culture conditions and involvement of protein kinase(s) in its action. Dev Growth Differ 34, 263275.
Mateos, F. V., Rickauer, M. & Esquerre-Tugaye, M.-T. (1997). Cloning and characterization of a cDNA encoding an elicitor of Phytophthora parasitica var. nicotianae that shows cellulose-binding and lectin-like activities. Mol Plant Microbe Interact 10, 10451053.[Medline]
McDowell, E. M. & Trump, B. F. (1976). Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100, 405414.[Medline]
McGuire, V. & Alexander, S. (1996). PsB multiprotein complex of Dictyostelium discoideum: demonstration of cellulose binding activity and order of protein subunit assembly. J Biol Chem 271, 1459614603.
Nakao, H., Yamamoto, A., Takeuchi, I. & Tasaka, M. (1994). Dictyostelium prespore-specific gene (DP87) encodes a sorus matrix protein. J Cell Sci 107, 397403.
Orlowski, M. & Loomis, W. F. (1979). Plasma membrane proteins of Dictyostelium: the spore coat proteins. Dev Biol 71, 297307.[Medline]
Pierini, L. M. & Doering, T. L. (2001). Spatial and temporal sequence of capsule construction in Cryptococcus neoformans. Mol Microbiol 41, 105115.[CrossRef][Medline]
Sassi, S., Sweetinburgh, M., Erogul, J., Zhang, P., Teng-umnuay, P. & West, C. M. (2001). Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium. Glycobiology 11, 283295.
Simrell, C. R. & Klein, P. A. (1979). Antibody responses of tumor-bearing mice to their own tumors captured and perpetuated as hybridomas. J Immunol 123, 23862394.[Medline]
Srinivasan, S., Griffiths, K. R., McGuire, V., Champion, A., Williams, K. L. & Alexander, S. (2000). The cellulose-binding activity of the PsB multiprotein complex is required for proper assembly of the spore coat and spore viability in Dictyostelium discoideum. Microbiology 146, 18291839.
Tani, P. H., Loftus, J. C. & Bowditch, R. D. (2002). In vitro selection of fibronectin gain-of-function mutations. Biochem J 365, 287294.[CrossRef][Medline]
Wang, W. & Malcolm, B. A. (1999). Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange site-directed mutagenesis. Biotechniques 26, 680682.[Medline]
Wang, Y., Slade, M. B., Gooley, A. A., Atwell, B. J. & Williams, K. L. (2001). Cellulose-binding modules from extracellular matrix proteins of Dictyostelium discoideum stalk and sheath. Eur J Biochem 268, 43344345.
West, C. M. (2002). Comparative analysis of spore coat formation, structure and function in Dictyostelium. Int Rev Cyt 222, 237293.
West, C. M. & Erdos, G. W. (1988). The expression of glycoproteins in the extracellular matrix of the cellular slime mold Dictyostelium discoideum. Cell Differ 23, 116.[CrossRef][Medline]
West, C. M., Mao, J., van der Wel, H., Erdos, G. W. & Zhang, Y. (1996). SP75 is encoded by the DP87 gene and belongs to a family of modular Dictyostelium discoideum outer layer spore coat proteins. Microbiology 142, 22272243.[Abstract]
West, C. M., Zhang, P., McGlynn, A. C. & Kaplan, L. (2002). Outsidein signaling of cellulose synthesis by a spore coat protein in Dictyostelium. Euk Cell 1, 281292.[CrossRef]
Woessner, J. P. & Goodenough, U. W. (1994). Volvocine cell walls and their constituent glycoproteins: an evolutionary perspective. Protoplasma 181, 245258.
Zhang, P., McGlynn, A. C., Loomis, W. F., Blanton, R. L. & West, C. M. (2001). Spore coat formation and timely sporulation depend on cellulose in Dictyostelium. Differentation 67, 7279.[CrossRef][Medline]
Zhang, Y., Brown, R. D. & West, C. M. (1998). Two proteins of the Dictyostelium spore coat bind to cellulose in vitro. Biochemistry 37, 1076610779.[CrossRef][Medline]
Zhang, Y., Zhang, P. & West, C. M. (1999). A linking function for the cellulose-binding protein SP85 in the spore coat of Dictyostelium discoideum. J Cell Sci 112, 43674377.
Received 10 September 2002;
revised 6 November 2002;
accepted 11 November 2002.