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3 ß1 in Sulfate-Starved Marine Sponge Cells: Functional Correlates
Marine Biological Laboratory, Woods Hole, Massachusetts 02543
Integrins are a large family of heterodimeric transmembrane glycoproteins that attach cells to fibronectin and collagen and other extracellular matrix proteins of the basement membrane. The attachment is by way of recognition sequences—RGD in the case of fibronectin. The transmembrane and cytoplasmic domains of integrins provide a conduit for outside-in as well as inside-out signaling (1). Integrins or their domains have been highly conserved over many millions of years as judged from their presence in sponges (2). Cell adhesion and motility are regulated by integrins, but the pathways that modulate this function are unclear.
Our previous studies have shown that the properties of adhesion and motility are lost when isolated sponge cells in rotation are deprived of inorganic sulfate (3). We hypothesized that this kind of stress would be likely to cause membrane alterations and would therefore be a useful model for studying integrins and integrin-ligand binding. Herein we describe the effects of sulfate starvation upon the expression of 
3 and ß1 integrins in Microciona prolifera, a marine sponge.
Microciona cells were prepared from intact sponge as previously described (3). Aliquots of the cell suspension at a concentration of 2 x 107/ml were placed in 50-ml centrifuge tubes and spun at low speed in a table model centrifuge at 16°C. The cells were resuspended in either sulfate-free artificial seawater (i.e., less than 10 nM SO42-), or in seawater with a normal sulfate concentration (26 mM; +SO42-). Each flask was rotated for 8 h; the cells were then centrifuged, the supernatant discarded, and the cell pellets resuspended in fresh ASW, either normal sulfate or sulfate-free. This cycle was repeated four more times.
SDS-PAGE analyses were carried out with lysates prepared by Triton X100 extraction of normal and sulfate-deprived sponge-cell pellets. The proteins separated by gel electrophoresis were probed with rabbit antibodies prepared against integrins 3, 5, and ß1 following their electro-transfer to nitrocellulose (NC). The NC was cut into seven lanes to account for reference standards (1 lane), integrin staining of (+SO42+) lysate (3 lanes), and integrin staining of (-SO42+) lysate (3 lanes). The primary antibodies (rabbit anti-integrins) were applied to their substrates for 1 h and removed, and the NC strips washed with PBS. The secondary reagent (goat anti-rabbit horseradish peroxidase (HRP) conjugate) was applied for 1 h and removed, and the NC was washed again with PBS. Color was developed with ECL reagent at a dilution of 1:1 applied to the NC strips, which were then autoradiographed.
For immunohistochemistry, chemically dissociated cells conditioned in normal or sulfate-free ASW were fixed in 10% formalin ASW and the centrifuged pellets embedded in paraffin as described (3). Tissues were sectioned at 6 µm and stained using mouse monoclonal antibodies (MAB) raised against integrins 3 and ß1; following a wash with PBS, the sections were treated with the secondary antibody (goat anti-mouse HRP conjugate), washed with PBS, and developed with 3', 3' diaminobenzidine. The sections were counterstained for 5 min with Harris’ hematoxylin.
The stained Western blots revealed marked differences between the integrins derived from Microciona cells prepared in normal ASW and those processed in sulfate-free ASW (Fig. 1a). The expression of 3 and ß1 integrins was considerably greater in sulfate-free ASW than in normal ASW. The distinction was particularly clear in the case of 3 integrin, which displayed a single prominent band at about 65 kDa in the (-) lane, whereas the (+) lane displayed a corresponding band at considerably lower intensity. The differences were maintained in the lanes stained with anti-integrin ß1, but the 65-kDa bands were much more intense. The molecular sizes are somewhat less than those reported in other sponge species (2,4). The ß1 integrin derived from the sulfate-free lysate also displayed a very dense broad band at 200–205 kDa. The multiplicity of bands could be accounted for, either by cross-reactions between the anti-integrins and non-integrin proteins, or by glycosylation variants of the primary bands. The anti-integrin 5 failed to react with either cell lysate.
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3 displayed many large darkly stained cells along with a considerable amount of stained matrix (Fig. 1b). In contrast, the normal counterpart showed cells and matrix with far less staining intensity, and a more prominent counterstain (Fig. 1c). Similar differences between cells preconditioned in sulfate-free ASW and normal ASW were found in sections stained with anti-integrin ß1, but the distinctions were less pronounced than those observed in cells stained with anti-integrin 3.
This study confirms those of other workers, and it indicates that sponges—the oldest animal phylum with a multicellular lineage—already had membrane structures that provide for controlled reactions between cells, and for cell matrix reactions (2,4). The inverse correlation between integrin up-regulation and sponge cell motility is of interest and has been described in systems other than the sponge (5). In some cells, the expression of integrins that bind fibronectin RGD is correlated with reduced cellular motility (6). This is important when considering the possible relationships between Microciona aggregation factor (MAF), Microciona cells, and the 3ß1 integrins. MAF proteins that are expressed from cDNA possess RGD binding sequences (see ref. 7, p. 29,548, MAF p3/p4 form C), which could potentially ligate MAF to the integrins to initiate a trans-cellular motility-reduction signal, a strategy distinct from the carbohydrate-carbohydrate binding thought to mediate aggregation. Under usual conditions, the high sugar content of MAF might preclude effective integrin-RGD peptide binding, but this situation might change when cells are exposed to low levels of environmental sulfate. The availability of pure MAF RGD peptide sequences and of integrin peptides may allow for binding correlations between these synthetic compounds and their natural counterparts in normal and sulfate-stressed sponge. The ability to manipulate sponges in vivo by sulfate reduction will provide a powerful tool toward a further understanding of signaling pathways and their relationships to adhesion and motility.
Footnotes
1 Hospital for Sick Children, 555 University Ave. Toronto, Ontario, Canada M5G 1X8. (Author) for correspondence. 
2 Freidrich Miescher Institute, Ch 4002, Basel, Switzerland.
3 Mount Holyoke College, Department of Biology, South Hadley, MA 01075.
4 Faculty of Pharmacy, University of Barcelona, Barcelona, Spain.
Literature Cited
This article has been cited by other articles:
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  C. Sabella, E. Faszewski, L. Himic, K. M. Colpitts, J. Kaltenbach, M. M. Burger, and X. Fernandez-Busquets Cyclosporin A Suspends Transplantation Reactions in the Marine Sponge Microciona prolifera J. Immunol., November 1, 2007; 179(9): 5927 - 5935. [Abstract] [Full Text] [PDF]
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