Axial growth of hexactinellid spicules: Formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis

Journal of Structural Biology 164 (2008) 270–280

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Axial growth of hexactinellid spicules: Formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis Xiaohong Wang a, Alexandra Boreiko b, Ute Schloßmacher b, David Brandt b, Heinz C. Schröder b, Jinhe Li c, Jaap A. Kaandorp d, Hermann Götz e, Heinz Duschner e, Werner E.G. Müller b,* a

National Research Center for Geoanalysis, 26 Baiwanzhuang Dajie, CHN-100037 Beijing, PR China Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany c Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, CHN-266071 Qingdao, PR China d Section Computational Science, University of Amsterdam, Kruislaan 403, 1098 SJ Amsterdam, The Netherlands e Angewandte Struktur- und Mikroanalytik der Universität (Bau 911), Universität Mainz, Obere Zahlbacherstr. 63, D-55131 Mainz, Germany b

a r t i c l e

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Article history: Received 15 June 2008 Received in revised form 24 August 2008 Accepted 26 August 2008 Available online 6 September 2008 Keywords: Sponges Hexactinellida Monorhaphis chuni Spicules Silicatein Axial growth

a b s t r a c t The glass sponge Monorhaphis chuni (Porifera: Hexactinellida) forms the largest bio-silica structures on Earth; their giant basal spicules reach sizes of up to 3 m and diameters of 8.5 mm. Previously, it had been shown that the thickness growth proceeds by appositional layering of individual lamellae; however, the mechanism for the longitudinal growth remained unstudied. Now we show, that the surface of the spicules have towards the tip serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show a pronounced size tendency. The apical elongation of the spicule proceeds by piling up cone-like structural units formed from silica. As a support of the assumption that in the extracellular space silicatein(-like) molecules exist that associate with the external surface of the respective spicule immunogold electron microscopic analyses were performed. With the primmorph system from Suberites domuncula we show that silicatein(-like) molecules assemble as string- and net-like arrangements around the spicules. At their tips the silicatein(-like) molecules are initially stacked and at a later stay also organized into net-like structures. Silicatein(-like) molecules have been extracted from the giant basal spicule of Monorhaphis. Applying the SDS–PAGE technique it could be shown that silicatein molecules associate to dimers and trimers. Higher complexes (filaments) are formed from silicatein(-like) molecules, as can be visualized by electron microscopy (SEM). In the presence of ortho-silicate these filaments become covered with 30–60 nm long small rod-like/cuboid particles of silica. From these data we conclude that the apical elongation of the spicules of Monorhaphis proceeds by piling up cone-like silica structural units, whose synthesis is mediated by silicatein(-like) molecules. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction Sponges [phylum Porifera] are grouped into the phylogenetic oldest taxon the siliceous sponges, with the classes Hexactinellida and Demospongiae, and the younger calcareous sponges (class Calcarea); Bergquist (1978). Both sponge taxa are characterized by their skeletons, which are composed of elements, termed spicules. Like for most bio-mineral constituents in Metazoa, including the human skeletal system, we only recently began to understand the formation of sponge spicules on subcellular (biochemical and molecular biological) level; see: Bäuerlein (2007). A major obstacle is the lack of biochemical and biophysical techniques to follow and understand the processes that cause deposition of minerals, e.g. calcite, apatite or bio-silica. Furthermore, it is difficult to apprehend * Corresponding author. Fax: +49 6131 392 5243. E-mail address: [email protected] (W.E.G. Müller). 1047-8477/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2008.08.005

conceptually which morphogenetic mechanisms should be regarded as starting points to investigate the construction plan of skeletal structures. Using the demosponge Suberites domuncula as a model it was shown that the formation of the spicules starts intracellularly, while their sizes and shapes are completed extracellularly (Müller et al., 2005, 2006). The extracellular growth of the spicules follows the same pattern in both groups of siliceous sponges, namely appositional lamellar growth (Pisera, 2003; Schröder et al., 2006). While in demosponges the distinct lamellar organization of the spicules can be resolved only after etching with hydrofluoric acid, and then mainly in the early growth phases of the spicules (Schwab and Shore, 1971; Pisera, 2003), the lamellar apposition continues in Hexactinellida throughout all growth phases (Schulze, 1904; Pisera, 2003; Sandford, 2003). One distinguished model suitable to study the appositional, lamellar growth of siliceous spicules are the giant basal spicules of the hexactinellid sponge Monorhaphis chuni (Monorhaphis

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intermedia); Schulze (1904). These spicules represent the largest bio-silica structures on Earth; they may reach sizes of 3 m [length] and 8.5 mm [diameter]. Based on electron microscopic as well as on biochemical studies it could be shown that up to 500 concentric rings/lamellae of the giant basal spicules of M. chuni are stacked on top of each other without any interjacent proteinaceous layers (Wang et al., 2007; Müller et al., 2007b). One unique feature of siliceous sponges is that the formation of their bio-silica matrix is enzymatically mediated, as has first been demonstrated for the demosponges Tethya aurantium (Shimizu et al., 1998; Cha et al., 1999) and S. domuncula (Krasko et al., 2000). The responsible enzyme, silicatein, follows the conventional enzymatic parameters (Müller et al., 2008b). By immuno-biochemical techniques (Müller et al., 2008a) and later by cloning of the gene (Müller et al., 2008c), it became clear that the silicatein enzyme exists also in hexactinellids. Hence, silicatein represents an autapomorphic character of all siliceous sponges. In M. chuni silicatein exists within and not between the siliceous lamellae, thus forming a composite material with the inorganic bio-silica (Müller et al., 2007c; Miserez et al., 2008). Both in Demospongiae (Eckert et al., 2006) and in Hexactinellida (Müller et al., 2007b) collagen participates in spicule formation. However, in contrast to the report by Ehrlich et al. (2008a) neither in S. domuncula (Demospongiae) nor in M. chuni (Hexactinellida) was evidence found for collagen within individual lamellae or even within the lamellar arrangements of the spicules; collagen can be detected exclusively on the surface of the spicules as a covering net or mantle. Based on Fourier transform infrared (FT-IR) spectroscopic data some evidence has been presented that spicules from the hexactinellid glass sponge Rossella fibulata contain chitin (Ehrlich et al., 2008b). However, these studies await further biochemical and molecular biological support. It is the aim of our study to clarify and elucidate the pattern of lamella formation by application of sensitive and high-resolution imaging methods, focusing mainly on the tips of the spicules. Our studies base on the pioneering description of the giant basal spicules from M. chuni by Schulze (1904). Schulze assumed that the organic layers (‘‘Spiculinlamellen”) which he observed between individual siliceous lamellae (‘‘Siphon”) are not involved in the extension/elongation of the hexactinellid spicules. Furthermore, he postulated that the formation of new lamellae on top of existing ones is mediated and guided by organic material that is released from cells surrounding the respective spicules. Our experiments indicate that cone-like appositions (hoods) of new lamellae are formed at the tips of the spicules. In addition, experimental data suggest that silicatein that accumulates on the outside of the siliceous spicules, is incorporated into the axial filament of the giant basal spicules probably through channeling into the pre-existing axial filament. Finally, we give data to demonstrate that extracts from the lamellae of the giant basal spicule (comprising (only) one protein species most likely to be the 25 kDa large silicatein) possess hydrolytic activity, as it has already been demonstrated for the silicatein from demosponges (Shimizu et al., 1998; Cha et al., 1999; Müller et al., 2008b). Finally, it is shown that the silicatein from Monorhaphis forms larger filaments, with sizes over 1 lm, if this enzyme is incubated in the absence of glycerol. These filaments/aggregates facilitate the precipitation of silica on their surfaces.

2. Materials and methods 2.1. Chemicals, materials and enzymes The sources of most chemicals and enzymes used here, were given earlier (Krasko et al., 2000; Wang et al., 2007; Müller et al., 2007a).

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2.2. Sponges, giant basal spicule and primmorphs Specimens of the hexactinellid M. chuni [Monorhaphis intermedia (Li, 1987)] (Porifera: Hexactinellida: Amphidiscosida: Monorhaphididae) had been collected by dredging during expeditions organized by the Institute of Oceanography (Chinese Academy of Sciences, Qingdao) in the Okinawa Trough in a depth of 800 m. The giant basal spicules had been stored at 4 °C for eight years prior to their use in the present study. The spicules were freed from the (partial) organic coat first by mechanical cleaning (ultrasonics), and subsequently by treatment with 10% sodium dodecyl sulphate (overnight) for all experiments except for the microtomographic analysis. Then the spicules were washed in distilled water and finally were treated with acetone. For the determination of the cracking zones (cross-breaks) of spicules, 60 mm large comitalia had been used. They were bent to 90° prior to the measurement in the nanopositioning and nanomeasuring (NPM) machine. For another series of experiments, the demosponge S. domuncula (Porifera, Demospongiae, Hadromerida) was used. Specimens were collected in the Northern Adriatic near Rovinj (Croatia), and then kept in aquaria in Mainz (Germany) at a temperature of 17 °C for more than 5 months. A cell culture system growing in three dimensions, the primmorphs system (Krasko et al., 2000), was employed for the analyses. Five days after preparation of those three dimensional aggregates from single cells, sections were prepared to identify silicatein in cross-sections by immunohistological techniques, using polyclonal antibodies (PoAb-aSilic) raised in rabbits against the purified filaments from spicules (Müller et al., 2005). The specimens were analyzed by electron microscopy. 2.3. Microtomography (MicroCT) analysis The analysis was performed as described previously (Stock et al., 2003; Cooper et al., 2007) using a Desktop Cone-Beam MicroCT Scanner (lCT40, SCANCO Medical AG, Brüttisellen; Switzerland). The system was operated at 55 kV tube potential, 145 lA tube current, and a total integration time of 1.5 s for each projection (5  300 ms). The high-resolution mode with an angular increment of 0.18° (2000 projections/360°) ensured the minimal achievable isotropic pixel size of 6 lm. The four different locations, with a z-dimension of 3.11 mm each, were reconstructed to 518 slices with a 2048  2048 grid and a slice thickness of 6 lm. The total measuring time was 5.2 h for each location. 2.4. Distance measurement in nanometer scale The nanomeasurements were performed with the NPM machine, developed by SIOS Meßtechnik GmbH (Ilmenau; Germany). This apparatus allows the measuring in a free arrangement in all three axes within an area of 25  25  5 mm3 and a resolution of 0.1 nm, as described (Schmidt et al., 2007). For the recording of the outer lamellae separated from the cross cracking zones, the device had been optimized by use of several displacement interferometers. These were operated by He–Ne lasers, which had been connected and compared with an iodine-stabilized reference laser. To avoid (potential) angular misalignments of the linear guides, the data of the control system of the NPM had been set against the remaining first- and second-order errors. During the course of the measurements the respective specimen was moved by a corner mirror, which simultaneously represented the reference coordinate system. Voice coil actuators were used to achieve a high-resolution motion over the entire working range with constant velocity. For the determinations of the outer lamellae of the cracked comitalia, an NPM machine was used that had been advanced by several single-beam homodyne polarization-optical Michelson interferometers, fitted with planar reflectors. The determinations

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were conducted directly on the planar outer surfaces of the reference corner mirror. To suppress heat sources inside the system the light of the laser was coupled in by optic fibers. 2.5. Extract from spicules Outer lamellae, that had been pealed off from SDS (sodium dodecyl sulfate)-cleaned siliceous spicules, were used and treated with 2M HF/8M NH4F (pH 5.0) for 20 h. The suspension was dialyzed against a 500 mM Tris–glycerol buffer (pH 7.2; 100 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 2% glycerol). Temperature in all steps was 4 °C. 2.6. Self-association of mono-/oligomeric silicatein into fibrils For the self-association of silicatein into fibrils this procedure the Microcon Centrifugal Filter Devices was applied. The lamellar extract, containing 30 lg/ml of protein was incubated for 0 min up to 120 min at room temperature in a test tube. Then aliquots were taken and were used for further analyses. The samples were transferred either (i) into 500 mM Tris buffer (see above), supplemented with 1% glycerol, or (ii) into 500 mM Tris buffer without glycerol. In a final series of experiments, a sample was incubated in this glycerolfree Tris buffer in the presence of 60 lM ortho-silicate. For the analysis of oligo/polymerization of silicatein, the protein was analyzed by SDS–PAGE (see below) and with the high-resolution field emission scanning electron microscope [SEM] (Leo 1530; Gemini, Oberkochen; Germany). Prior to this analysis, the 1-ll aliquots were dropped onto a copper electron microscopy grid, coated with Formvar. After adsorption for 1 min the solvent was removed by Whatman filter paper. Subsequently, each grid was washed with distilled water and allowed to dry in air overnight. 2.7. SDS–PAGE, Western blot analysis Samples containing 1 to 3 lg of protein were dissolved in loading buffer (Roti-Load (Roth, Karlsruhe; Germany)) and subjected to 12% polyacrylamide gel electrophoresis (PAGE), containing 0.1% or (only) 0.01% sodium dodecyl sulphate (SDS–PAGE), as indicated. The samples had not been pre-heated. After separation, the gels were washed in 10% methanol (supplemented with 7% acetic acid) for 30 min and were then stained in Coomassie brilliant blue, as described (Wiens et al., 2006). For Western blot analysis, the polypeptides were transferred from the polyacrylamide gel to a nitrocellulose membrane and incubated with polyclonal anti-silicatein antibodies (PoAb-aSilic) from S. domuncula as described (Müller et al., 2005, 2008e). The immunocomplexes were visualized with biotinylated goat antirabbit IgG secondary antibody followed by the peroxidase reaction with TMB (SK-4400 [Linaris Biologische Produkte GmbH, Wertheim; Germany]). In a control PoAb-aSilic was adsorbed with recombinant silicatein (Müller et al., 2005). This serum did not stain any protein on the gel.

with a Leo 1530 microscope. For the analysis of cross-breaks, the surfaces of spicules remained either unpolished or were polished with emery paper (silicon carbide; Matador, Hoppenstedt, Darmstadt, Germany); during that polishing process the quality of the surface was permanently inspected under a stereomicroscope (enlargement: about 30). 2.9. Immunogold labeling of silicatein Electron immunogold labeling for TEM [transmission electron microscopy] analysis was performed with tissue samples that had been treated in glutaraldehyde/paraformaldehyde as described (Müller et al., 2005). After dehydration, slices (60 nm thick) were cut and blocked with bovine serum albumin in phosphatebuffered saline and then incubated with the primary antibody PoAb-aSilic (1:1000) for 12 h at 4 °C. For the detection of the antigen–antibody complexes, a secondary antibody (1.4-nm nanogold anti-rabbit IgG [Nanoprobes, Yapbank, NY; USA]) was applied. After washing, an enhancement of the immunocomplexes was achieved with silver as described (Danscher, 1981). The samples were examined using a Tecnai 12 microscope. In controls, pre-immune serum was used; in these sections no antigen–antibody could be detected (Müller et al., 2005). 2.10. Catalytic activity of Monorhaphis extract Extracts from Monorhaphis spicules (lamellae) were prepared, after dissolving the silica of the spicules with HF, using the 500 mM Tris–glycerol buffer. Hydrolytic activity was measured by determining the hydrolysis of the Z-Phe-Arg-AMC [AMC: 7Amino-4-methyl coumarin] substrate (Bachem, Bubendorf; Switzerland), as described (Müller et al., 2008a). Protein extracts were pre-incubated in 80 mM Na-acetate buffer (pH 5.5; 8 mM L-cysteine, 4 mM EDTA) for 10 min at room temperature. Five parallel assays each were conducted in 96-well plates, containing 20 ll of protein extract (10.5 lg protein), 10 ll of substrate (final concentrations 5, 10, 15, 20 lM) and the buffer to reach a final volume of 200 ll. Plates were incubated (22 °C; 40 min) and fluorescence (excitation: 355 nm, emission: 460 nm) was determined in an Anthos 2020 ELISA reader. The activity was calculated and is given in nmol AMC released mg 1 protein and min 1 (Dvorak et al., 2005). The kinetic parameters were calculated from five parallel experiments (Cleland, 1979; Sachs, 1984). As controls (Müller et al., 2008b), the protein extract was replaced by bovine serum albumin (10 lg) or the extract was pretreated with heat (5 min; 95 °C). The data are given in a double reciprocal plot of velocity versus substrate concentration (Lineweaver–Burk plot). 2.11. Analytical method For the quantification of protein the Bradford method (Compton and Jones, 1985; Roti-Quant solution—Roth) was used.

2.8. Scanning electron microscopic analysis

3. Results

Scanning electron microscopic analysis (SEM) was used to study the morphology of the spicules, as described (Wang et al., 2007; Müller et al., 2007b). Cleaned spicules were mounted on aluminum stubs (SEM-Stubs G031Z; Plano, Wetzlar; Germany) and inspected

3.1. Growth of Monorhaphis The Monorhaphis animals live in a depth range of 1600 m (Somalia basin [East Africa]; (Schulze, 1904)) to 1000 m (New

" Fig. 2. MicroCT analysis of a giant basal spicule. (A) Fixation of a giant basal spicule (sp) in a plastic holder (ho) which allows the scanning at an appropriate angle to analyze the spicule at a resolution of 10 lm. The regions within the spicule where the images (B–D) have been taken are marked (B, C and D). (B) 3D reconstruction of the tip of the spicule. The spicule (sp) is not surrounded by an organic envelope and displays a smooth surface with its lamellar architecture (la). (C) More distant to the tip, the spicule is surrounded by an organic envelope (env). It becomes obvious that protrusions are regularly arranged on the spicule (sp) that follow a helical structural (hs) arrangement. (D) The section shows the organic envelope (env) that tightly surrounds the inorganic spicule (sp). The outer surface of the spicule (><) shows a serrated relief structure.

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Caledonia; (see: Müller et al., 2007b)) or 800 m (Okinawa Trough (see: Müller et al., 2007b)). The soft body of each specimen with its large atrial openings, that are linearly arranged, is stabilized by 14 different types of smaller spicules of sizes between a few lm and 50 mm, and one giant basal spicule per specimen (Schulze, 1904). As all other hexactinellids, also Monorhaphis specimens remain sessile during their complete life cycle. The cells form one giant basal spicule that anchors the animal to the substratum. During growth the specimen elongates together with its giant basal spicule (Fig. 1A a–c). From the different fragments of Monorhaphis, collected during the Valdivia Expedition (Schulze, 1904), and during the expeditions organized by the Institute of Oceanography (Qingdao), the schematic growth scheme can be deduced. Older specimens apparently lose the basal portions of their soft body and expose the giant basal spicule (Fig. 1B). 3.2. MicroCT analysis

Fig. 1. Growth of Monorhaphis specimens. (A) Schematic representation of the growth phases. The soft body of the sessile animals is formed around one giant basal spicule (gbs) which anchors them to the substratum; a further characteristics of this sponge are their linearly arranged large atrial openings (at) of approximately 2 cm in diameter. During the growth cycle, the soft body dies off in the basal region; the bare giant basal spicule (a–c) is exposed. (B) A dried specimen of a ‘‘moderate” size of 120 cm showing the bare giant basal spicule (gbs) in the basal part; atrial openings (at).

MicroCT has become a standard technique for the visualization and quantification of the 3D structure of hard-structured materials below 10 lm. After applying this procedure described under Section 2, representative 2D cross-sections were prepared and used for 3D reconstructions. A 10 cm long part of a giant basal spicule with a diameter of 2 mm, was inserted into a holder and exposed to the scanner (Fig. 2A). Five hundred and eighteen individual 2D cross-sections were recorded as the dataset for 3D reconstructions. Several 3D renderings yielding different views of the giant basal spicule are shown in Fig. 2. A cross section displays an organic envelope, which surrounds the inorganic spicule (Fig. 2C and D). This rendering already discloses that the surface of the silica material is not even but has a serrated relief structure. A higher magnification discloses that the protrusions are arranged in an organized pattern; an almost regular helical succession of small projections (Fig. 2C). At the tip of the spicules, towards the apex, the organic

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envelope is missing; there a smooth surface is seen that displays their lamellar architecture (Fig. 2B).

If the sample remains in the glycerol-free Tris buffer for 30 min, monomers, dimers and trimers are visible after gel separation (Fig. 6C lane b).

3.3. Scanning electron microscopy analysis SEM analysis was performed to show the lamellar organization of the giant basal spicules. The cross section reveals the concentric arrangement of the lamellae around the central core of the spicule, the axial cylinder (Fig. 3A). This axial cylinder harbors the axial canal, which is solid and thus not distinguishable in older segments of the spicules. A longitudinal section discloses the regular organization of the lamellae (Fig. 3B). The surfaces of the spicules are covered by a collagen coat which is interspersed with holes of diameters of around 10 lm (Fig. 3C). The collagen fibrils have a diameter of approximately 25 nm and a periodicity of 65 nm (Müller et al., 2007b; Ehrlich et al., 2008a). Interestingly, the surface of the giant basal spicules has ‘‘hemi-spherical” protrusions (knobs) of the same dimensions as the holes of the collagen net (Fig. 3D). By application of microCT analysis a serrated relief structure is disclosed on the surface of the spicules (Fig. 2C). Analysis by SEM revealed that those protrusions are formed by the siliceous layers (Fig. 3D).

3.6. Kinetic parameters for the hydrolytic activity in the Monorhaphis extract Only one protein species (size 25 kDa) had been identified in the lamellar extracts from the giant basal spicules. We term this preparation ‘‘extract”. Following the previously described procedure (Müller et al., 2008a), and using Z-Phe-Arg-AMC as substrate, we determined now the different kinetic parameters by establishment of the double-reciprocal plot (Lineweaver–Burk plot); Fig. 7. The plot, 1/V versus 1/S, was constructed and the Km value (Michaelis–Menten constant) for this substrate was determined with 15.4 ± 3.8 lM and a corresponding Vmax (maximal velocity) of 961.5  10 4 (±158.3) lM/min. Two sets of controls were included to support the conclusion that the reaction is enzymatically controlled. First, the extract had been thermally denatured (5 min; 95 °C) and second, bovine serum albumin had been added at the same amount to the reaction mixture. In both assays the reaction velocity dropped to <20% of the values seen in the assays with native silicatein during a 10 min-incubation period.

3.4. Measurement in nanometer scale 3.7. Self-assembly of the monomeric silicatein: SEM analysis The determination of the thickness of the lamellae within one spicule was performed with an NPM machine, as described under Section 2. Giant basal spicules were cross fractured and the sizes of the lamellae were precisely determined in two different spicules (Fig. 4A and B). Taking the spicule shown in Fig. 4B, the thickness of 20 lamellae was determined in two series; a first series of lamellae that are adjacent to the central axial cylinder (Fig. 4C) and a second series from the surface inward (Fig. 4D). The values were plotted (Fig. 4E) and the graph shows that the first eight inner layers measure approximately 7 lm, while the remaining outer lamellae are about 4.5 lm thick. From previous force displacement studies it is known (Mayer and Sarikaya, 2002; Mayer, 2005; Müller et al., 2008d) that the large spicules from hexactinellids are characterized by high stability especially towards perpendicular force actions. This stability is due to a sequential and independent breaking of the lamellae. In turn, bending of the spicules results first in a cracking of their outer lamellae (Fig. 5A). For the studies, described here, 60 mm large comitalia had been used. After bending, the diameters at the cracking zones of the outer lamellae were determined in the NPM machine. They measure at the tip 194 lm (Fig. 5A) and the diameter increases to 250 lm. This fracture pattern suggests that during longitudinal growth of the spicules the individual lamellae are layered on top of each other as cone-like structural units (Fig. 5B a–e); further details are given under Section 4. 3.5. Self-assembly of the monomeric silicatein: PAGE analysis The silicatein(-related) monomeric protein from spicules of Monorhaphis has a size of approximately 25 kDa, if analyzed by SDS–PAGE in the presence of 0.1% SDS (Wang et al., 2007; Müller et al., 2007b; Fig. 6A lane a). This protein band cross-reacts with anti-silicatein antibodies as described under Section 2 (Fig. 6B lane a). If this antibody preparation had been pre-adsorbed with recombinant silicatein, no immunocomplexes can be visualized (Fig. 6B lane b). The process of self-assembly of monomeric silicatein can be followed by SDS–PAGE as well, if the sample exists in a Tris-buffer, lacking glycerol. If the protein sample, immediately after transfer into this buffer, is subjected to PAGE at low SDS concentration (0.01%) again the 25 kDa band can be identified (Fig. 6C lane a).

Silicateins from demosponges have the property to self-assemble (Murr and Morse, 2005; Müller et al., 2007a). Here we show that the Monorhaphis protein, isolated from lamellae of spicules, forms only small aggregates of sizes between 50 and 100 nm if the Tris buffer contains the viscogenic glycerol (1%); Fig. 8A–D. However, if the protein is transferred into the glycerol-free medium, it forms (almost) filamentous structures (Fig. 8E and F), already after 2 min. An extended incubation results in the formation of larger filaments of lengths of around 1 lm (Fig. 8G) or larger aggregates (Fig. 8H) with a smooth surface. Addition of ortho-silicate to the reaction assay during this filament formation does not considerably change the size of the protein aggregates but allows silicates to precipitate on their surface (Fig. 8J–L). The first clusters are seen after 7 min (Fig. 8J). The silica nature of these clusters had been confirmed by energy dispersive X-ray spectroscopy (not shown). These self-assembly studies were performed in triplicate, and all of them gave qualitatively the same results. At a shorter incubation period, 30 s, no filaments or silicate precipitations (>10 nm) are seen (Fig. 8I). 3.8. Axial growth of spicules in S. domuncula: Immunogold localization Using the in vitro cell culture system of the three dimensionally growing cells, the primmorphs, the development and growth of spicules had been analyzed in a straightforward manner (Müller et al., 2005). At present, the biochemical tools to investigate the arrangement of the silicatein molecules in the extra-spicular space of Monorhaphis, namely antibodies against the silicatein from this species, are not available. Hence, an analogous study has been performed with S. domuncula. Sections through primmorphs were prepared and reacted with anti-silicatein antibodies (PoAb-aSilic); subsequently, the slices were evaluated by TEM. In cross-sections through the spicules (Fig. 9A–C) it becomes evident that the spicules [the 150 lm long tylostyles] are surrounded by string- and net-like structures. In the extra-spicular space, the antibodies recognized the corresponding antigen that is organized in concentric rings around the growing spicules (Fig. 9A–C). In addition, the PoAb-aSilic antibodies recognized, as expected, also the organic material within the axial canal, the axial filament (Fig. 9 C). This axial filament of a diameter of 300–600 nm, is compact and reacts

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Fig. 3. SEM analysis of a giant basal spicule. (A) A cross section through a spicule shows the concentric, lamellar arrangement of the silica layers (la) which surround the axial cylinder (cy). (B) A longitudinal section shows the foliated arrangement of the lamellae (la). (C) Smaller spicules are surrounded by a collagen (col) net which is regularly punctured with holes (h) through which the underlying siliceous spicular material is visible. (D) Polished cross section through a spicule, discloses ‘‘hemi-spherical” protrusions of the same size as the holes in the collagen net seen in (C). The dimension of these protrusions fit into the space uncovered by the holes within the collagen sheet (>h<). Lamellae (la) are forming the protrusions and follow the curvings. (C and D) modified after Müller et al. (2008c).

strongly with the antibodies. Inspections of growing spicules in the axial direction, using the immunohistochemical approach, showed that silicatein molecules exist massively around their tips. There, strong antibody–antigen complexes can be visualized, indicating a high accumulation of this protein (Fig. 9D–F). It is interesting, that the high accumulation of the grains at the tip of each growing spicule is seen also between the string- and net-like rings surrounding the surface of each silica lamella, and on the silica surface. In controls, it had been established that the antibodies recognized specifically silicatein, since the anti-serum that had been pre-adsorbed with recombinant silicatein failed to recognize any molecule in the sections (not shown here). 4. Discussion The essentiality of silicon in biota has been well described and its crucial role during bone formation and connective tissue synthesis, as well as its protective role against heart disease, have been documented (reviewed in: Exley, 1998). It was the discovery of silicatein (Shimizu et al., 1998; Cha et al., 1999; Krasko et al., 2000) which attracted attention of biomaterial research to the siliceous sponges (Wang and Wang, 2006). These animals bio-fabricate their siliceous skeleton enzymatically (Cha et al., 1999; Müller et al., 2008b) raising the hope to copy this nature’s ability and to make complex silica structures under mild conditions (reviewed in: Schröder et al., 2008). By means of this enzyme and of ortho-silicate, in concert with the silicic acid pump (Schröder et al., 2004), the sponges can form their skeleton that is made of polymerized silicic acid [bio-silica]. While until recently these processes had exclusively been studied on biochemical level only in demosponges, efforts have now been started to understand the related pro-

cess in the second class of siliceous sponges, the Hexactinellida (Wang et al., 2007; Müller et al., 2007b). Triggered also by the impressive design of the large silica skeleton assembled from building blocks, in the hexactinellid Euplectella aspergillum (Aizenberg et al., 2005) and by its unusual mechanical stability (Mayer, 2005), studies with the largest hexactinellid sponge synthesizing also the largest bio-silica structure on Earth, Monorhaphis, had been intensified (Wang et al., 2007; Müller et al., 2007b). First a 24–26 kDa structural protein was identified in the giant basal spicules (Müller et al., 2007b), followed by a lectin that has been assumed to be involved in formation of an organic cylinder around the spicules (Wang et al., 2007). Finally a third component had been discovered, collagen, that functions as a stiffening for this cylinder and acts as one component involved in shaping the spicules to their filigree construction (Müller et al., 2008c). Recent studies, claiming that collagen is the key polypeptide forming bio-silica (Ehrlich et al., 2008a) await support by experimental results. The giant basal spicule of M. chuni is surrounded by a massive collagen net which is punctured with holes of approximately 10 lm. Based on the SEM images, shown here (Fig. 3), it can be accepted that the protrusions, originally described by Schulze (1904), existing on the surface of the bio-silica spicules, fit into those holes. This observation supports earlier assumptions (Wang et al., 2007; Müller et al., 2007b) that the collagen net acts as a tight-fitting bracing for the organic cylinder mediating bio-silica formation. The application of the technique of MicroCT revealed in the 3D reconstructions from the spicules that these protrusions exist in the initial phase of silica formation when the bracing tightly surrounds the spicules. At later stages, the protrusions seem to melt and the collagen bracing loses its contact to the siliceous spicules.

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Fig. 4. Measurement if the thickness of the lamellae in the giant basal spicules; SEM analysis. (A and B) Cross breaks of two giant basal spicules that have been used for the determination of the thickness of the lamellae. In the centers the axial cylinders (cy) exist which appear, under the conditions used here, as more solid centers. (C and D) The determination of individual lamellae was performed in two series; the more central lamellae (C) and the peripheral lamellae (D). (E) The width of the individual lamellae, from the center (surface of the axial cylinder) to the surface, has been plotted. The determination has been performed with an NPM machine, as described under Section 2.

Fig. 5. Axial growth of the spicules. (A) Determination of the cracking zones within a spicule (comitalia) after its bending by 90° (perpendicular force actions); determination was performed in the NPM machine. The diameter of the spicules at the cracking zones measures at the tip 194 lm. (B) Schematic outline of the longitudinal growth of the spicules by cone-like structural units (in green). The cone #3 is layered onto the underlying cones #2 and #1. The axial canal, which harbors the axial filament (af), that is composed of silicatein (sil), represents a continuous structure that traverses the individual cones. It is proposed that silicatein (sil) is deposited from the extra-spicular space onto the spicules, forms there the lamellae, and is then intrudes the axial filament.

The giant basal spicules, and also the comitalia, have a characteristic lamellar patterning. Up to 500 concentric rings form the giant basal spicules (Schulze, 1904), which surprisingly have an almost uniform widths of approximately 6 lm, as analyzed here by

the NPM machine. A continuous decrease of the thickness with layer number from 10 to 2 lm (Levi et al., 1989; Miserez et al., 2008) could not be supported by the measurement with the spicules used here. More detailed, our measurement showed two sets

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Fig. 6. Self-assembly of silicatein; PAGE analysis. (A) Identification of monomeric silicatein in extracts from lamellae of Monorhaphis, after SDS–PAGE in the presence of 0.1% SDS; one bright band of 25 kDa becomes prominent (lane a). M: protein size markers were run in parallel. (B) Western blot of a gel after separation in 0.1% SDS. The blot was reacted either with the non-treated antibodies (PoAb-aSILIC antibodies) [lane a], or with a preparation that had been pre-adsorbed (ad) with recombinant silicatein [lane b]. (C) Self-assembly of silicatein, analyzed in SDS– PAGE in the presence of the low SDS concentration (0.01%). Size separation was performed either immediately after obtaining the sample in glycerol-free Trisbuffer (lane a) or after allowing the sample to stand at room temperature for 30 min (lane b). After staining the gel with Coomassie brilliant blue monomers (mon), dimers (dim) and trimers (tri) were seen.

Fig. 7. Lineweaver–Burk plot for the determination of the hydrolytic activity of the protein extract from Monorhaphis lamellae. Various concentrations of substrate (ZPhe-Arg-AMC) were added to the standard assay. After incubation (22 °C; 40 min), the degree of fluorescence of the released AMC was determined and used for the calculation of the enzymatic parameters; the values are expressed in nM AMC released per min, or in M substrate applied. A double reciprocal plot of velocity versus substrate concentration (Lineweaver–Burk plot) was computed.

of layer thicknesses, the internal more enlarged lamellae (size range around 7 lm) and the slightly smaller stack of lamellae, which are surrounding the core lamellae towards their surface (size around 4–5 lm). The biological basis for the formation of lamellae of defined thickness is not known. Annual, seasonal changes of the environment can hardly account for this multi-layer structure, since Monorhaphis lives in the deep sea of 1000 m. It is more likely that biomechanical principles/forces guide the lamellae formation. As initially reported by Levi et al. (1989), and later outlined in detail (Mayer, 2005), Monorhaphis spicules comprise unique biomechanical properties, featured by a combination between toughness/energy dissipation, stiffness, and resilience. The toughness of the spicule was found to be an order of magnitude higher, than that of a solid silica rod (Mayer, 2005; Müller et al., 2008d). This high stability/flexibility of the spicules, that are fabri-

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cated of amorphous quartz-glass (Müller et al., 2008e), can only be reached by a lamellar organization of the spicules. Even more, the existence of protein within the silica lamellae amplifies this property (Müller et al., 2008d,e). Hence, we assume that during the long history of evolution of the hexactinellids, and especially of Monorhaphis, during their 500–800 million years (Kruse et al., 1998), they have acquired both the optimal composite composition and the ideal size dimensions for the highest possible degree of mechanical stability. After bending, the comitalia spicules from Monorhaphis show a distinct breaking pattern. Cracking zones of distinct, successive fractures are generated exhibiting a cone-like formation of the tips of the spicules (Fig. 5A). It is proposed here that during longitudinal growth of the spicules cone-like silica structures are formed via silicatein, that result in an basal-apical increase of the number of lamellae within the spicules (Fig. 5B a–e). This mode of spicule growth is perfectly suitable for the formation of an increasing number of lamellae along the apical-basal axis of the spicules, and follows a suggestion that had already been proposed by Schulze (1904). The basal-apical growth pattern, increasing number of lamellae towards the basis, is facilitated by the fact that spicules from hexactinellids show an open tip. This structure allows the elongation of the axial filament within the axial canal (Müller et al., 2007b). From studies using demosponges (S. domuncula) it is known that the first (few) silica lamella(e) of a spicule is/are formed intracellularly, while most of the silica material of the spicules is produced extracellularly by appositional layering of the lamellae (Müller et al., 2005; Schröder et al., 2006). In demosponges it remains unsolved by which mechanism an elongation of the axial filament can occur, since their tips are (always) closed. However, using the three-dimensional cell culture from S. domuncula and the antibodies against silicatein as a tool it can be demonstrated that in growing spicules the silicatein molecules are arranged around the outer surface of the silica rod as a cylinder. At the tips of the growing spicule a strong immunological reaction occurs, suggesting a high accumulation of silicatein molecules. At a later stage, the stack-like accumulation of the silicatein molecules is lost under formation of net-like rings formed from silicatein molecules. Building on the data on spicule formation in demosponges, the 25 kDa structural protein from the Monorhaphis giant basal spicules [prepared from their lamellae] was isolated and—now successfully—analyzed for enzymatic activity. By using increasing concentrations of the substrate Z-Phe-Arg-AMC a linear double-reciprocal (Lineweaver–Burk) plot had been computed. The Km value calculated was 15.4 lM, a value close to that determined for the recombinant silicatein from S. domuncula with 22.7 lM (Müller et al., 2008a). The kinetic parameters for the S. domuncula silicatein were identified with the substrate bis(p-aminophenoxy)-dimethylsilane. Furthermore, the Kcat value (turnover number) for the Monorhaphis protein was determined with 0.47 s 1, a value which is 10-fold lower than that determined for the S. domuncula silicatein. However, it should be stressed here that the two substrates used are synthetic ones and certainly do not represent the natural substrate (reviewed in: Müller et al., 2008a). Under in vivo conditions, the spicule formation (in demosponges) is a rapid process; e.g. the growth of 100–300 lm long spicules from Ephydatia fluviatilis needs about 40 h (Weissenfels, 1989). Using the protein (silicatein) preparation from Monorhaphis oligo- and polymerization studies were performed. Following the concept applied in T. aurantium (Murr and Morse, 2005) and in S. domuncula (Müller et al., 2007a) to understand the self-assembly property and the pattern formation of higher ordered structures, built of silicatein, the enzyme was isolated from the spicules under mild conditions. Under the conditions described, the 25 kDa protein could be extracted from the lamellae. If the extract remained

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Fig. 8. Self-assembly of silicatein; SEM analysis. The lamellae of Monorhaphis had been extracted and the resulting 25 kDa protein was analyzed for the ability to form organizes into a filamentous structures; SEM analysis. (A–D) The silicatein(-containing extract) was allowed to stand in a Tris buffer, supplemented with 1% glycerol, for 2 min up to 120 min, prior to the electron microscopic analysis. (E–H) Formation of filamentous structures in Tris buffer, in the absence of glycerol. They display a smooth surface. (I–L) Generation of rod-like/cuboid particles (pa) on the surface of the filamentous structures formed from the 25 kDa protein in the Tris buffer, supplemented with 10 lM ortho-silicate. The following incubation periods had been chosen: 30 s (I), 2 min (A and E), 30 min (B, F and J), 60 min (C, G and K) or 120 min (D, H and L).

in a glycerol-free buffer dimers and trimers of the 25 kDa protein could be identified by SDS–PAGE at non-denaturing conditions. The process of polymerization of monomeric Monorhaphis silicatein results in the construction of a three-dimensional network. High-resolution SEM analysis highlighted that in the presence of low concentrations of glycerol (1%) the filament formation is almost completely prevented. This observation is attributed to the known effect of glycerol to abolish aggregation of proteins without changing the pH or the dielectric constant in the milieu (Meng et al., 2001). Under our conditions used, no evidence for the formation of an intermediate state that is stabilized by intermolecular disulfide bonds (Murr and Morse, 2005) could be identified for Monorhaphis silicatein. Especially previous electron microscopic imaging studies suggested both in T. aurantium (Murr and Morse, 2005) and in S. domuncula (Müller et al., 2007a), the assembly of oligomeric units from monomers or dimers to fractal-like structures. In a final stage, the filaments condense and organize into filamentous structures. In Monorhaphis the initial fractal stage is short and requires only 2–5 min, while in S. domuncula this process needs 30–60 min (Müller et al., 2007a). The self-assembled 0.5–

2 lm long filaments form linear structures which ultimately cluster together. The surfaces of these aggregates are smooth. If the self-assembly process of the silicatein molecules continues in the present of ortho-silicate, the surfaces of the lobulated structures provoke the generation of 30–60 nm long small rod-like/cuboid particles. Energy dispersive X-ray analysis supports that these structures represent poly-silicate precipitates (not shown here). Similar particles of a size of nanospheres have been identified also after in vitro enzymatic synthesis of poly-silicate by silicatein (Tahir et al., 2004). In future studies dynamic light scattering analyses have to be performed, in analogy to published experience (Murr and Morse, 2005), in order to provide a detailed kinetics of oligomerization of the silicatein monomers. The structural (SEM analyses) and functional data (enzymic reactions) on the 25 kDa protein in Monorhaphis, together with the cloning information from the hexactinellid Crateromorpha meyeri (Müller et al., 2008c), indicate that an enzymatically active silicatein exists also in spicules from hexactinellids which is—like in demosponges—involved in bio-silica formation. This conclusion will help also to close a gap in the understanding of the axial, lon-

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Fig. 9. Immunogold electron microscopy (TEM analysis) of cross-sections through growing spicules in primmorphs from S. domuncula. Sections through growing spicules were prepared and reacted with anti-silicatein antibodies. The antigen–antibody complexes are visualized with nanogold anti-rabbit IgG using TEM. (A) An immature spicule in the extracellular space shows around the growing silica rod (si) an array of concentric string- and net-like rings (ri), displaying strong antigen reaction with anti-silicatein antibodies. (B) Concentric string- and net-like rings (ri) surrounding a lamella (la). (C) At a higher magnification the immunological reaction with the axial filament (af) can be demonstrated as well in addition to the string- and net-like rings (ri). (D) An axial section through a spicule displays the high accumulation of the grains (antigen–antibody complexes), especially around the tip (ti) of the spicule (). (E) A more detailed close-up from the tip region (ti) of a spicule, showing the accumulation of clumps of silicatein molecules (). (F) At a later stage, during which the silica rods of the spicules are well developed, the silicatein molecules are arranged around the spicule and also around the tip (ti) in a string- and net-like ordered pattern as rings (ri). Within a ring and the silica surface a less strong reactivity to the antibodies is seen. Scale bars: 1 lm.

gitudinal growth of the spicules, which had been outlined above. The cone-like silica structures, formed via the silicatein-catalyzed reaction, construct a lamellar organization of the spicules. It is now the aim of future research activities to localize the protein scaffold within the silica matrix/the silica nanospheres. In addition, it has to be elucidated if within one lamella a gradient of different compositions/densities of inorganic silica nanoparticles exists, which builds and maintains the delimitation of the individual lamellae and prevents fusion of the lamellae, especially in hexactinellids. Acknowledgments We thank the Marine Biological Museum of the Chinese Academy of Sciences in the Institute of Oceanography (Qingdao; China) to provide us with the Monorhaphis spicules for our research. We very much acknowledge the ‘‘Fachgruppe Werkstoffe der Elektrotechnik” (Technical University Ilmenau [Prof. Dr. Gerd Jäger]) for the length and angle measurements at the nanoscale level. We thank Mr. Gunnar Glasser (Dr. I. Lieberwirth Research group ‘‘Surface Chemistry” [Prof. Butt] Max Planck Institute for Polymer Research, Mainz) for excellent assistance in electron microscopic analysis. This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung, Germany [projects: Center of Excellence BIOTECmarin and German Chinese cooperation program], the National Natural Science Foundation of China (No. 50402023), the International S&T Cooperation Program of China (Grant No. 2008DFA00980), and the Human Frontier Science Program. References Aizenberg, J., Weaver, J.C., Thanawala, M.S., Sundar, V.C., Morse, D.E., Fratzel, P., 2005. Skeleton of Euplectella sp.: structural hierarchy from nanoscale to the macroscale. Science 309, 275–278. Bäuerlein, E. (Ed.), 2007. Handbook of Biomineralization. Wiley-VCH, Weinheim. Vol. 1. Bergquist, P.R., 1978. Sponges. University of California, Berkeley. Cha, J.N., Shimizu, K., Zhou, Y., Christianssen, S.C., Chmelka, B.F., Stucky, G.D., Morse, D.E., 1999. Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro. Proc. Natl. Acad. Sci. USA 96, 361– 365.

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