Spicule formation in the calcareous sponge Sycon ciliatum

Summary The spicule primordium is formed in an intercellular cavity within a group of sclerocytes. This cavity contains organic material which ensheaths the growing spicule but does not appear to determine the nature of the mineral morph (magnesian calcite) or the crystallographic orientation of the spicule. The tip of each growing spicule ray is seated in a dense cup in the cytoplasm of the sclerocyte concerned. Both ends of monaxons are initially inserted each into a dense cup. As rays elongate the sclerocyte membrane around the tip becomes invaginated and forms a system of converging spaces that possibly indicate high secretory activity in that region. Spicule growth involves the displacement and expansion of the organic sheath by the enlarging spicule. Fully formed spicules which are exposed to the mesohyl become surrounded by collagen fibrils. However, these fibrils are in no way concerned with the process of mineral deposition and are never found within the spicule calcite.     

Hexagonal shaped ice spicules in frozen antifreeze protein solutions

In the presence of antifreeze proteins from both Antarctic and Arctic fishes, water freezes in the form of long c-axis spikes or spicular-like crystals. Transmission electron microscopy of the Pt/C replicas of the freeze fractured spicular ice in a small capillary revealed the presence of many hexagonally shaped structures whose cross-sectional dimensions were between 0.5 and 10 microm. Well-defined parallel faces were associated with most fractured and etched spicules. When fracture planes occurred near the tip of a spicule, well-defined pyramidal faces were apparent. Steps were sometimes associated with these pyramidal spicular crystal faces. On some of the replicas obvious roughening of certain crystal faces of the spicule was observed, suggesting that the antifreeze proteins may have adsorbed to those faces.     

Formation of giant spicules in the deep-sea hexactinellid <Emphasis Type="Italic">Monorhaphis chuni</Emphasis> (Schulze 1904): electron-microscopic and biochemical studies

The siliceous sponge Monorhaphis chuni (Hexactinellida) synthesizes the largest biosilica structures on earth (3 m). Scanning electron microscopy has shown that these spicules are regularly composed of concentrically arranged lamellae (width: 3–10 μm). Between 400 and 600 lamellae have been counted in one giant basal spicule. An axial canal (diameter: ~2 μm) is located in the center of the spicules; it harbors the axial filament and is surrounded by an axial cylinder (100–150 μm) of electron-dense homogeneous silica. During dissolution of the spicules with hydrofluoric acid, the axial filament is first released followed by the release of a proteinaceous tubule. Two major proteins (150 kDa and 35 kDa) have been visualized, together with a 24-kDa protein that cross-reacts with antibodies against silicatein. The spicules are surrounded by a collagen net, and the existence of a hexactinellidan collagen gene has been demonstrated by cloning it from Aphrocallistes vastus. During the axial growth of the spicules, silicatein or the silicatein-related protein is proposed to become associated with the surface of the spicules and to be finally internalized through the apical opening to associate with the axial filament. Based on the data gathered here, we suggest that, in the Hexactinellida, the growth of the spicules is mediated by silicatein or by a silicatein-related protein, with the orientation of biosilica deposition being controlled by lectin and collagen. Carsten Eckert was previously with the Museum für Naturkunde, Invalidenstrasse 43, 10115 Berlin, Germany. The collagen sequence from Aphrocallistes vastus reported here, viz., [COL_APHRO] APHVACOL (accession number AM411124), has been deposited in the EMBL/GenBank data base. This work was supported by grants from the European Commission, the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung Germany (project: Center of Excellence BIOTECmarin), the National Natural Science Foundation of China (grant no. 50402023), and the International Human Frontier Science Program.     

Expression of silicatein in spicules from the Baikalian sponge Lubomirskia baicalensis

Lake Baikal harbors the largest diversity of sponge species [phylum Porifera] among all freshwater biotopes. The abundantly occurring species Lubomirskia baicalensis was used to study the seasonal silicatein metabolism; the spicules of this species have an unusually thick axial filament, consisting of silicatein, which remains constant in diameter during their growth. In the course of maturation, the size of the silicic acid shell grows, until the final diameter of the spicules of about 8 microm is reached. The seasonal content of silicatein was assessed by use of antibodies raised against silicatein; they stained specifically the axial filaments. In addition we determined, by application of the enzyme-linked immunosorbent assay system, that the proteinaceous content of the spicules, the silicatein, increases from spring to late summer by 8-fold. As molecular markers to quantify the seasonal changes in expression levels of genes coding for proteins/enzymes, the genes for the calumenin-like protein and the kinesin-related protein, were selected. The expression of calumenin-like gene, involved in the intracellular signaling, is highest during September, whereas the expression of the kinesin-related protein does not change during the annual course. These results suggest that the highest metabolic activity of L. baicalensis occurs in late summer (September), in parallel with the highest accumulation of silicatein, a structural protein/enzyme of the spicules.     

Acquisition of structure-guiding and structure-forming properties during maturation from the pro-silicatein to the silicatein form

Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structure-guiding and structure-forming properties.     

Nanostructural features of demosponge biosilica

Recent interest in the optical and mechanical properties of silica structures made by living sponges, and the possibility of harnessing these mechanisms for the synthesis of advanced materials and devices, motivate our investigation of the nanoscale structure of these remarkable biomaterials. Scanning electron and atomic force microscopic (SEM and AFM) analyses of the annular substructure of demosponge biosilica spicules reveals that the deposited material is nanoparticulate, with a mean particle diameter of 74+/-13 nm. The nanoparticles are deposited in alternating layers with characteristic etchant reactivities. Further analyses of longitudinally fractured spicules indicate that each deposited layer is approximately monoparticulate in thickness and exhibits extensive long range ordering, revealing an unanticipated level of nanoscale structural complexity. NMR data obtained from differentially heated spicule samples suggest that the etch sensitivity exhibited by these annular domains may be related to variation in the degree of silica condensation, rather than variability in the inclusion of organics. In addition, AFM phase imaging in conjunction with results obtained from HF and alkaline etching experiments suggest that at various stages in spicule biosynthesis, regions of unusually low silica condensation are deposited, indicating a possible interruption in normal spicule formation. While this discovery of nanoparticulate silica aggregation in demosponge skeletal elements is likely to reflect the intrinsic kinetic tendency of silica to form such particles during polycondensation, the heirarchical organization of these nanoparticles is biologically unique.     

Intra-epithelial spicules in a homosclerophorid sponge

Attempts to understand the intricacies of biosilicification in sponges are hampered by difficulties in isolating and culturing their sclerocytes, which are specialized cells that wander at low density within the sponge body, and which are considered as being solely responsible for the secretion of siliceous skeletal structures (spicules). By investigating the homosclerophorid Corticium candelabrum, traditionally included in the class Demospongiae, we show that two abundant cell types of the epithelia (pinacocytes), in addition to sclerocytes, contain spicules intracellularly. The small size of these intracellular spicules, together with the ultrastructure of their silica layers, indicates that their silicification is unfinished and supports the idea that they are produced "in situ" by the epithelial cells rather than being incorporated from the intercellular mesohyl. The origin of small spicules that also occur (though rarely) within the nucleus of sclerocytes and the cytoplasm of choanocytes is more uncertain. Not only the location, but also the structure of spicules are unconventional in this sponge. Cross-sectioned spicules show a subcircular axial filament externally enveloped by a silica layer, followed by two concentric extra-axial organic layers, each being in turn surrounded by a silica ring. We interpret this structural pattern as the result of a distinctive three-step process, consisting of an initial (axial) silicification wave around the axial filament and two subsequent (extra-axial) silicification waves. These findings indicate that the cellular mechanisms of spicule production vary across sponges and reveal the need for a careful re-examination of the hitherto monophyletic state attributed to biosilicification within the phylum Porifera.     

深海六放海绵大骨针的结构与特性

在海绵动物(多孔动物)中,六放海绵和寻常海绵为硅质骨骼.生活在深海(1 000 m)中的六放海绵是最古老的海绵动物,其中间单根海绵和春氏单根海绵有长达3 m的骨针,是地球上最长的生物硅结构.利用电子显微技术观测, 这些直径达8 mm的巨大根须骨针具有同心层状结构,其横截面显示明显的构造分界:中间为含有轴丝的轴管,外围是一50-150 μm厚的轴筒,最外面为区状区(300-500层,每层厚度3-5 μm).生物化学研究显示其主要的蛋白质为35 kD大分子,另外,还检测到23-24 kD 多肽,可能是硅蛋白相关蛋白.依据现有的红血球凝聚活性,从骨针提取物中也检测到了凝集素.由电子探针获得其化学成分主要为Si,K和Na.此外,骨针的光传输实验表明,该巨大根须骨针用作光纤可传输600 nm至1 400 nm范围的光,而滤掉小于600 nm的光(类似高通滤波器)和大于1 400 nm 的红外光(类似低通滤波器).另外,从六放海绵的空囊泡沫海绵中分离出一个基因并确证了其推导的编码蛋白序列,该蛋白编码一个光裂合酶相关蛋白,蛋白相似性比较结果显示属于光裂合酶相关蛋白中多细胞动物隐色素一类.基于以上数据给出了六放海绵硅质骨针形成的示意图.另外,由单根海绵骨针可作为波导传输光/电和/或化学信号,推断在海绵动物中有类似神经系统的网络系统[动物学报 53(3):557-569,2007].     

Allocation of chemical and structural defenses in the sponge Melophlus sarasinorum

Sponges have evolved a variety of chemical and structural defense mechanisms to avoid predation. While chemical defense is well established in sponges, studies on structural defense are rare and with ambiguous results. We used field and laboratory experiments to investigate predation patterns and the anti-predatory defense mechanisms of the sponge Melophlus sarasinorum, a common inhabitant of Indo-pacific coral reefs. Specifically, we aimed to investigate whether M. sarasinorum is chemically or structurally defended against predation and if the defenses are expressed differently in the ectosomal and choanosomal tissue of the sponge. Chemical defense was measured as feeding deterrence, structural defense as feeding deterrence and toughness. Our results demonstrated that chemical defense is evenly distributed throughout the sponge and works in conjunction with a structurally defended ectosome to further reduce predation levels. The choanosome of the sponge contained higher protein levels, but revealed no structural defense. We conclude that the equal distribution of chemical defenses throughout M. sarasinorum is in accordance with Optimal Defense Theory (ODT) in regards to fish predation, while structural defense supports ODT by being restricted to the surface layer which experiences the highest predation risks from mesograzers.     

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.