Biosilica formation in spicules of the sponge Suberites domuncula: synchronous expression of a gene cluster

The formation of spicules is a complicated morphogenetic process in sponges (phylum Porifera). The primmorph system was used to demonstrate that in the demosponge Suberites domuncula the synthesis of the siliceous spicules starts intracellularly and is dependent on the concentration of silicic acid. To understand spicule formation, a cluster of genes was isolated. In the center of this cluster is the silicatein gene, which codes for the enzyme that synthesizes spicules. This gene is flanked by an ankyrin repeat gene at one side and by a tumor necrosis factor receptor-associated factor and a protein kinase gene at the other side. All genes are strongly expressed in primmorphs and intact animals after exposure to silicic acid, and this expression is restricted to those areas where the spicule formation starts or where spicules are maintained in the animals. Our observations suggest that in S. domuncula a coordinated expression of physically linked genes is essential for the synthesis of the major skeletal elements.     

The complete set of ribosomal proteins from the marine sponge Suberites domuncula

The siliceous marine sponge Suberites domuncula is a member of the most ancient and simplest extant phylum of multicellular animals-Porifera, which have branched off first from the common ancestor of all Metazoa. We have determined primary structures of 79 ribosomal proteins (r-proteins) from S. domuncula: 32 proteins from the small ribosomal subunit and 47 proteins from the large ribosomal subunit. Only L39 and L41 polypeptides (51 and 25 residues long in rat, respectively) are missing. The sponge S. domuncula is, after nematode Caenorhabditis elegans and insect Drosophila melanogaster the third representative of invertebrates with known amino acid sequences of all r-proteins. The comparison of S. domuncula r-proteins with r-proteins from D. melanogaster, C. elegans, rat, Arabidopsis thaliana and Saccharomyces cerevisiae revealed very interesting findings. The majority of the sponge r-proteins are more similar to their homologues from rat, than to those either from invertebrates C. elegans and D. melanogaster, or yeast and plant. With few exceptions, the overall sequence conservation between sponge and rat r-proteins is 80% or higher. The phylogenetic tree of concatenated r-proteins from 6 eukaryotic species (rooted with archaeal r-proteins) has the shortest branches connecting sponge and rat. Both model invertebrate organisms experienced recently accelerated evolution and therefore sponge r-proteins very probably better reflect structures of proteins in the ancestral metazoan ribosome, which changed only little during metazoan evolution. Furthermore, r-proteins from the plant A. thaliana are significantly closer to metazoan r-proteins than are those from the yeast S. cerevisiae.     

Silicatein-mediated incorporation of titanium into spicules from the demosponge Suberites domuncula

Primmorphs (a three-dimensional sponge primary cell culture system) have been revealed to be a cell/tissue nano-factory for the production of tailor-made hybrid nanostructures. Growth of primmorphs is stimulated by the presence of a titanium alkoxide precursor tolerating titania (TiO₂) concentrations up to 250 μM. The presence and activity of silicatein in primmorphs has been analyzed by gel electrophoresis and Western blotting. Results of studies by scanning transmission electron microscopy with energy-dispersive X-ray spectroscopy have revealed silica and titania to be co-localized on nanosized spicules. Our findings suggest that the incorporation of titania into the nanosized spicule is enzymatically mediated via active silicatein in an orchestrated mechanism.     

Iron induces proliferation and morphogenesis in primmorphs from the marine sponge Suberites domuncula

Dissociated cells from marine demosponges retain their proliferation capacity if they are allowed to form special aggregates, the primmorphs. On the basis of incorporation studies and septin gene expression, we show that Fe3+ ions are required for the proliferation of cells in primmorphs from Suberites domuncula. In parallel, Fe3+ induced the expression of ferritin and strongly stimulated the synthesis of spicules. This result is supported by the finding that the enzymatic activity of silicatein, converting organosilicon to silicic acid, depends on Fe3+. Moreover, the expression of a scavenger receptor molecule, possibly involved in the morphology of spicules, depends on the presence of Fe3+. We conclude that iron is an essential factor in proliferative and morphogenetic processes in primmorphs.     

Cultivation of primmorphs from the marine sponge Suberites domuncula: morphogenetic potential of silicon and iron

Marine demosponges (phylum Porifera) are rich sources for potent bioactive compounds. With the establishment of the primmorph system from sponges, especially from Suberites domuncula, the technology to cultivate sponge cells in vitro improved considerably. This progress was possible after the elucidation that sponges are provided with characteristic metazoan cell adhesion receptors and extracellular matrix molecules which allow their cells a positioning in a complex organization pattern. This review summarizes recent data on the cultivation of sponges in aquaria and--with main emphasis--of primmorphs in vitro. It is outlined that silicon and Fe(+++) contribute substantially to the formation of larger primmorphs (size of 10 mm) as well as of a canal system in primmorphs; canals are probably required for an improved oxygen and food supply. We conclude that the primmorph system will facilitate a sustainable use of sponges in the production of bioactive compounds; it may furthermore allow new and hitherto not feasible insights into basic questions on the origin of Metazoa.     

Bruton tyrosine kinase-like protein, BtkSD, is present in the marine sponge Suberites domuncula

Sponges, the simplest and most ancient phylum of Metazoa, encode in their genome complex and highly sophisticated proteins that evolved together with multicellularity and are found only in metazoan animals. We report here the finding of a Bruton tyrosine kinase (BTK)-like protein in the marine sponge Suberites domuncula (Demospongiae). The nucleotide sequence of one sponge cDNA predicts a 700-aa-long protein, which contains all of the characteristic domains for the Tec family of protein tyrosine kinases (PTKs). The highest homology (38% identity, 55% overall similarity) was found with human BTK and TEC PTKs. Sponge PTK was therefore named BtkSD. Human BTK is involved in the maturation of B cells and mutations in the BTK gene cause X-linked agammaglobulinemia. Kinases from the Tec family are not present in Caenorhabditis elegans and, until now, they were found only in insects and higher animal taxa. Our finding implies that the BTK/TEC genes are of a very ancient origin.     

Apposition of silica lamellae during growth of spicules in the demosponge Suberites domuncula: biological/biochemical studies and chemical/biomimetical confirmation

Recently it has been discovered that the formation of the siliceous spicules of Demospongiae proceeds enzymatically (via silicatein) and occurs matrix guided (on galectin strings). In addition, it could be demonstrated that silicatein, if immobilized onto inorganic surfaces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailed studies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamellae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that the appositional growth of the spicules in radial and longitudinal direction proceeds in the extracellular space along hollow cylinders; their surfaces are formed by silicatein. The extracellularly located spicules are surrounded by sclerocytes which are filled with both electron-dense and electron-poor vesicles; energy dispersive X-ray analysis/scanning electron microscopical studies revealed that the electron-dense vesicles are filled of silicon/silica and therefore termed silicasomes. The release of the content of the silicasomes into the hollow cylinder suggests that the newly formed silica lamella originate there; in addition the data are compatible with the view that the silicatein molecules, attached at the centripetal and centrifugal surfaces, mediate biosilica formation. In a chemical/biomimetical approach silicatein is linked onto the organic material-free spicules after their functionalization with aminopropyltriethoxysilane [amino groups]-poly(acetoxime methacrylate) [reactive ester polymer]-N(epsilon)-benzyloxycarbonyl L-lysine tert-butyl ester-Ni(II); finally His-tagged silicatein is immobilized. The matrix-bound enzyme synthesized a new biosilica lamella. These bioinspired findings are considered as the basis for a technical use/application/utilization of hollow cylinders formed by matrix-guided silicatein molecules for the biocatalytic synthesis of nanostructured tubes.     

Ethylene modulates gene expression in cells of the marine sponge Suberites domuncula and reduces the degree of apoptosis.

Sponges (phylum Porifera) live in an aqueous milieu that contains dissolved organic carbon. This is degraded photochemically by ultraviolet radiation to alkenes, particularly to ethylene. This study demonstrates that sponge cells (here the demosponge Suberites domuncula has been used), which have assembled to primmorphs, react to 5 microM ethylene with a significant up-regulation of intracellular Ca(2+) concentration and with a reduction of starvation-induced apoptosis. In primmorphs from S. domuncula the expression of two genes is up-regulated after exposure to ethylene. The cDNA of the first gene (SDERR) isolated from S. domuncula encodes a potential ethylene-responsive protein, termed ERR_SUBDO; its putative M(r) is 32,704. Data bank search revealed that the sponge polypeptide shares high similarity (82% on amino acid level) with the corresponding plant molecule, the ethylene-inducible protein from Hevea brasiliensis. Until now no other metazoan ethylene-responsive proteins have been identified. The second gene, whose expression is up-regulated in response to ethylene is a Ca(2+)/calmodulin-dependent protein kinase II. Its cDNA, SDCCdPK, encodes a M(r) 54,863 putative kinase that shares 69% similarity with the corresponding enzyme from Drosophila melanogaster. The expression of both genes in primmorphs from S. domuncula is increased by approximately 5-fold after a 3-day incubation period with ethylene. It is concluded that also metazoan cells, with sponge cells as a model, may react to ethylene with an activation of cell metabolism including gene induction.     

Identification of highly conserved genes: SNZ and SNO in the marine sponge Suberites domuncula: their gene structure and promoter activity in mammalian cells(1)

Recently, we reported that cells from the sponge Suberites domuncula respond to ethylene with an increase in intracellular Ca(2+) level [Ca(2+)](i), and with an upregulation of the expression of (at least) two genes, a Ca(2+)/calmodulin-dependent protein kinase and the potential ethylene-responsive gene, termed SDSNZERR (A. Krasko, H.C. Schr?der, S. Perovic, R. Steffen, M. Kruse, W. Reichert, I.M. Müller, W.E.G. Müller, J. Biol. Chem. 274 (1999)). Here, we describe for the first time that also mammalian (3T3) cells respond to ethylene, generated by ethephon, with an immediate and transient, strong increase in [Ca(2+)](i). Next, the promoter for the sponge SDSNZERR gene was isolated from S. domuncula. It was found that the SDSNZERR gene is positioned adjacent to the SNZ-related gene (SNZ-proximal open reading frame) (SDSNO) and linked, as in Saccharomyces cerevisiae, in a head-to-head manner. Until now, neither homologues nor orthologues of these two genes have been identified in higher metazoan phyla. The full-length genes share a bidirectional promoter. 3T3 cells were transfected with this promoter; the activity of the SDSNZERR promoter was strong and twice as high as that of the SV40 promoter, while the SDSNO promoter was less active. Surprisingly, the activity of the SDSNZERR promoter could not be modulated by ethylene or salicylic acid while it is strongly upregulated, by 4-fold, under serum-starved conditions. It is concluded that the modulation of the level of [Ca(2+)](i) by ethylene in mammalian cells is not correlated with an upregulation of the ethylene-responsive gene SDSNZERR. The data indicate that in mammalian cells, the activity of the SDSNZERR promoter is associated with the repression of serum-mediated growth arrest.     

Stimulation of protein (collagen) synthesis in sponge cells by a cardiac myotrophin-related molecule from Suberites domuncula.

The body wall of sponges (Porifera), the lowest metazoan phylum, is formed by two epithelial cell layers of exopinacocytes and endopinacocytes, both of which are associated with collagen fibrils. Here we show that a myotrophin-like polypeptide from the sponge Suberites domuncula causes the expression of collagen in cells from the same sponge in vitro. The cDNA of the sponge myotrophin was isolated; the potential open reading frame of 360 nt encodes a 120 aa long protein (Mr of 12,837). The sequence SUBDOMYOL shares high similarity with the known metazoan myotrophin sequences. The expression of SUBDOMYOL is low in single cells but high after formation of primmorph aggregates as well as in intact animals. Recombinant myotrophin was found to stimulate protein synthesis by fivefold, as analyzed by incorporation studies using [3H] lysine. In addition, it is shown that after incubation of single cells with myotrophin, the primmorphs show an unusual elongated, oval-shaped appearance. It is demonstrated that in the presence of recombinant myotrophin, the cells up-regulate the expression of the collagen gene. The cDNA for S. domuncula collagen was isolated; the deduced aa sequence shows that the collagenous internal domain is rather short, with only 24 G-x-y collagen triplets. We conclude that the sponge myotrophin causes in homologous cells the same/similar effect as the cardiac myotrophin in mammalian cells, where it is involved in initiation of cardial ventricular hypertrophy. We assume that an understanding of sponge molecular cell biology will also contribute to a further elucidation of human diseases, here of the cardiovascular system.     

Silica spicules and axial filaments of the marine sponge Stelletta grubii (Porifera,Demospongiae)

Summary In all cases an organic axial filament within the silica spicules of Stelletta grubii forms the core of the major axes of the glass. In the small, star-shaped silica spicules (asters) the filament is shown for the first time to be radial with an enlarged center; in the large four-rayed spicules (triaenes) it is four-rayed; and in the large single-rayed spicules (oxeas) the filament is single-rayed. In situ, the filament is not dissolved by boiling nitric acid and thus is apparently protected by encasement within the glass which can also be stratified. The small silica asters are formed by single cells which resemble the so-called spherulous cells of other sponges. The very large size of triaenes and oxeas suggests that they may possibly be formed by more than one cell. The diameter of the filament in the much smaller asters is much narrower than the filament in the larger spicules, indicating a possible relationship between filament diameter and spicule diameter. While the axial filament in larger spicules frequently has a triangular cross-section it can also be hexaognal. Some aster filaments also retain a close to hexagonal cross-section. Filaments freed from large spicules by hydrofluoric acid display a complex morphology; possibly there is an internal silicified core. Some reported aspects of filament morphology are, however, probably artefacts of desilicification with hydrofluoric acid. Offprint requests to: T.L. Simpson, Department of Biology, University of Hartford, West Harford, Connecticut 06117, USA (Permanent affiliation)     

Increased gene expression of a cytokine-related molecule and profilin after activation of Suberites domuncula cells with xenogeneic sponge molecule(s)

Porifera (sponges) constitute the lowest metazoan phylum. Experiments examined whether sponges can recognize self/nonself molecules. Cells from the marine sponge Suberites domuncula were incubated with membranes from either S. domuncula or another marine sponge, Geodia cydonium, as well as with recombinant alpha-integrin from G. cydonium. The cells responded immediately with a rise of intracellular Ca2+ ([Ca2+i]) if they were treated with membranes from G. cydonium but not after treatment by those from S. domuncula. This change of [Ca2+i] was also recorded with G. cydonium alpha-integrin. In parallel, the expression of two genes was strongly upregulated; one codes for a cytokine-related molecule, pre-B-cell colony-enhancing factor, and the other for profilin. These genes have previously been found to be highly expressed in human or echinoderm cells in the presence of xenogeneic proteins. Our data support the hypothesis that a primordial immune response system is present in sponges.     

Origin of the integrin-mediated signal transduction. Functional studies with cell cultures from the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with beta integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The beta subunit of the integrin receptor was cloned from S. domuncula; the estimated 91-kDa molecule comprises the characteristic signatures. Evolutionary conservation of the beta integrin was assessed by comparison with corresponding beta integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1-2 min) increase in the intracellular Ca2+ concentration which is further augmented in the presence of Ca2+. Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.