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Journal of Structural Biology 159 (2007) 325–334

StructuralBiology

Apposition of silica lamellae during growth of spicules in thedemosponge Suberites domuncula: Biological/biochemical studies

and chemical/biomimetical confirmation

Heinz C. Schroder a, Filipe Natalio a, Ibrahim Shukoor b,Wolfgang Tremel b, Ute Schloßmacher a, Xiaohong Wang c, Werner E.G. Muller a,*

a Institut fur Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universitat, Duesbergweg 6, D-55099 Mainz, Germanyb Institut fur Anorganische Chemie und Analytische Chemie, Universitat, Duesbergweg 10-14, D-55099 Mainz, Germany

c National Research Center for Geoanalysis, 26 Baiwanzhuang Dajie, CHN-100037 Beijing, China

Received 6 December 2006; received in revised form 13 January 2007; accepted 17 January 2007Available online 23 January 2007

Abstract

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 sur-faces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailedstudies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamel-lae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that theappositional growth of the spicules in radial and longitudinal direction proceeds in the extracellular space along hollow cylinders; theirsurfaces 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 hollowcylinder suggests that the newly formed silica lamella originate there; in addition the data are compatible with the view that the silicateinmolecules, attached at the centripetal and centrifugal surfaces, mediate biosilica formation. In a chemical/biomimetical approach silica-tein is linked onto the organic material-free spicules after their functionalization with aminopropyltriethoxysilane [amino groups]–poly(acetoxime methacrylate) [reactive ester polymer]–Ne-benzyloxycarbonyl L-lysine tert-butyl ester–Ni(II); finally His-tagged silicateinis immobilized. The matrix-bound enzyme synthesized a new biosilica lamella. These bioinspired findings are considered as the basis for atechnical use/application/utilization of hollow cylinders formed by matrix-guided silicatein molecules for the biocatalytic synthesis ofnanostructured tubes.� 2007 Elsevier Inc. All rights reserved.

Keywords: Sponges; Porifera; Suberites domuncula; Spicules; Silicasomes; Biomimesis; Silicatein

1. Introduction

Even though silicon and oxygen are, with 28% and 46%,respectively, the two most abundant elements of the earth’scrust, the calcium carbonate minerals dominate the bio-

1047-8477/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.jsb.2007.01.007

* Corresponding author. Fax: +49 6131 39 25243.E-mail address: wmueller@uni-mainz.de (W.E.G. Muller).URL: http://www.biotecmarin.de (W.E.G. Muller).

genic minerals in the living world. Only one animal phy-lum, the Porifera (sponges), which evolved in the silica-rich environment during the Neo-Proterozoic Eon (1000–520 million years ago [MYA]) (Muller, 1998) is extant stilltoday. Two sponge classes, the Demospongiae and Hexac-tinellida, use a biomineralization concept which is uniqueand allows the formation of their skeletal, filigreed ele-ments, the spicules; these spicules are composed of amor-phous biosilica. While bacteria or unicellular algae

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(diatoms) form silica in a matrix-controlled way, the pro-cess of silica deposition in the siliceous sponges is enzymat-ically mediated (Shimizu et al., 1998; Cha et al., 1999;Muller et al., 2003) and matrix-guided (Eckert et al.,2006; Schroder et al., 2006).

The enzyme forming the silica particles in the spiculeshas been termed silicatein (Shimizu et al., 1998) andbelongs—based on its protein sequence similarity—to thefamily of hydrolytic enzymes, the cathepsins (see Weaveret al., 2003). While marine sponges are composed of onlytwo silicatein forms, freshwater sponges, e.g. Lubomirskia

baicalensis, comprise more than four different polypeptides(Muller et al., 2006c). The silicatein molecules ‘‘multimer-ize’’ to the axial filament, which is localized in the axialcanal of the spicules (Shimizu et al., 1998; Murr andMorse, 2005). The growth process of the spicules has beenelucidated in the cell culture system of the demospongeSuberites domuncula (Muller et al., 2005, 2006a). Thegrowth of spicules (megascleres) starts intracellularly andafter having reached about 5–8 lm in length the spiculesare extruded and completed extracellularly up to 450 lmin length and 5–7 lm in diameter. With antibodies againstsilicatein it was disclosed that the spicules grow in twodirections, longitudinal and diametral, in an appositionalmanner (Muller et al., 2005). Silicatein was identified notonly in the axial filament but, notably, also (i) at the sur-face of the growing spicules allowing the appositionalgrowth by lamellated silica layers. Hence, only the firstlamella is formed around the axial filamentous structure,while the additional layers are formed within a cylindersurrounding the first layer. (ii) The outer layer of the cylin-der is formed by galectin molecules which function as amatrix for the silicatein molecules (Schroder et al., 2006).The galectins form strings around the smaller axis of thespicule which allow the association with silicatein (Schro-der et al., 2006); this finding indicates that the formationof the silicatein-mediated concentric silica layers ismatrix-guided. The surface of the spicule functions as theinner layer of the cylinder/collar, while the galectin net withthe associated silicatein molecules provides the outer sur-face of the cylinder. Within these tube-like cylinders, whichare stabilized by collagen fibers, a new silica lamella wasassumed to be formed (Eckert et al., 2006).

The finding of the appositional growth of the spiculesopened a new question for the route of the (ortho)silicicacid within the sponge animal. Silicic acid is taken up bythe animals from the surrounding water (Maldonadoet al., 1999) and pumped into the cells (sclerocytes) via aNaþ=HCO3

�½SiðOHÞ4� (NBCSA) cotransporter (Schroderet al., 2004). X-ray analysis suggested that silicon existsintracellularly in sclerocytes; until now silicon has not yetbeen found extracellularly in the mesohyl (Uriz et al.,2000).

The aim of the present study was to outline the apposi-tional growth pattern in vivo and to use the data for imitat-ing this principle for layered silica formation in vitro.Firstly, studies were performed with sponge tissue to dem-

onstrate the existence of an organic mantel (hollow cylin-der) around the growing spicules composed of twosilicatein layers. In addition, electron microscopical evi-dence is given, suggesting that electron-dense vesicles,which are released by the perispicular sclerocytes are trans-ported into the peri-cylinder space around the spicules;they are termed silicasomes.

In the second part, a biomimetic approach is presentedwhich supports the view of appositional growth of the spic-ules. For these experiments recombinant silicatein waslinked to the surface of the spicule; after incubation withtetraethoxysilane (TEOS) it was obvious that biosilica isdeposited onto the spicules. In the earlier studies (Chaet al., 1999) TEOS was used as substrate for the silicateinenzyme. Since TEOS spontaneously decomposes in wateralso to silicic acid it remains to be studied if the functionof silicatein is a dual one; (i) to convert TEOS to silicic acidand (ii) to cause a polymerization/condensation of silicicacid (in the monomeric or oligomeric form) to biosilica.

These studies became possible after the development ofthe immobilization methods of silicatein onto self-assem-bled monolayers allowing the synthesis of biosilica and alsobiotitania (Tahir et al., 2004, 2005; reviewed in: Tahir et al.,2006). The matrix, e.g., TiO2 nanowires, was functionalizedby coating with a polymeric ligand into which nitrilotriace-tic acid (NTA) groups had been incorporated. In turn his-tidine-tagged silicatein was non-covalently linked to NTA.It could be demonstrated that the immobilized enzymeretained its function and formed biosilica or biotitania(Tahir et al., 2005, 2006).

2. Materials and methods

2.1. Materials

Tetraethoxysilane [TEOS] (cat. Nr. 333859; >99.9%)was obtained from Sigma–Aldrich (Taufkirchen; Ger-many); 3-aminopropyltriethoxysilane (#919-30-2; 99%)and Ne-benzyloxycarbonyl L-lysine tert-butyl ester(#26239-55-4; 99%) from Acros (Geel; Belgium); dimethyl-formamide (DMF) from Riedel-de Haen (Seelze; Ger-many), Cy3-conjugated F(ab 0)2 goat anti-rabbit IgG fromJackson ImmunoResearch (Cambridgshire CB7 5UE;UK) and gallocyanine (#48610) from Fluka (Munchen;Germany).

2.2. Sponges, primmorphs and spicules

Specimens of the marine sponge Suberites domuncula

(Porifera, Demospongiae, Hadromerida) were collected inthe Northern Adriatic near Rovinj (Croatia), and then keptin aquaria in Mainz (Germany) at 17 �C for more than 5months.

The in vitro primmorph cell system was used (Kraskoet al., 2000). Primmorphs were kept at 17 �C in natural sea-water (enriched with 60 lM silicate), supplemented with

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1% RPMI 1640 medium. Approximately 20 days later theprimmorphs were used for analysis.

To obtain spicules, tissue samples were placed into asolution of concentrated sulfuric acid (1 part):concentratednitric acid (0.2 parts) at room temperature for 1 day. Thenthe acid was discarded and the material transferred to n-butanol (1 part)–water (5 parts). After washing, the spic-ules devoid of any organic surface protein were used forthe experiments.

2.3. Recombinant silicatein and preparation of antibodies

Recombinant silicatein was prepared in Escherichia coli

and purified as described (Muller et al., 2003). The recom-binant protein contained a His-tag as described (Kraskoet al., 2000). Polyclonal antibodies (PoAb-aSILIC) wereraised against purified, recombinant silicatein in femalerabbits (White New Zealand); (Wiens et al., 2003; Schroderet al., 2003 and Muller et al., 2005). The titer of the PoAb-aSILIC antibodies were >1:3,000.

2.4. Electron microscopy

2.4.1. TEM analysis

For transmission electron microscopic analysis (TEM)primmorph specimens were cut into pieces and incubatedin glutaraldehyde and finally washed in phosphate buffer.After dehydration with ethanol, the dried samples wereincubated with propylene oxide, fixed in propylene oxide/araldite, embedded in araldite and cut to 60 nm ultrathinslices. The samples were transferred onto coated coppergrids and analyzed with a Tecnai 12 microscope (FEI Elec-tron Optics, Eindhoven; Netherlands). Details have beengiven recently (Schroder et al., 2006). Where indicatedthe spicules where etched by limited exposure to hydroflu-oric acid (HF), as described (Shimizu et al., 1998).

2.4.2. SEM analysis

Non-derivatized spicules, as well as biosilica-layeredspicules were analyzed by scanning electron microscopy(SEM); the samples were mounted on stubs (carbon adhe-sive Leit-Tabs No.: G 3347 [Plano, Wetzlar; Germany])and observed with a Zeiss DSM 962 Digital Scanningmicroscope (Zeiss, Aalen; Germany). Where indicated thesamples had been analyzed in the backscattered mode.

2.4.3. Electron immunogold labelingPrimmorph samples were treated in glutaralde-

hyde:paraformaldehyde, dehydrated in ethanol and embed-ded in LR-White Resin. 60 nm thick slices were cut andblocked with 5% bovine serum albumin (BSA) in PBSand then incubated with the primary polyclonal antibodyPoAb-aSILIC (1:1000) for 12 h at 4 �C. In controls, pre-immune serum was used. After three washes with PBS/1% BSA sections were incubated with 1:100 dilution ofthe secondary antibody (1.4 nm nanogold anti-rabbitIgG; 1:200 diluted) for 2 h. Sections were rinsed in PBS,

treated with 1% glutaraldehyde/ phosphate buffered salinefor 5 min washed and dried. Subsequently, enhancing ofthe immunocomplexes was performed with silver asdescribed (Danscher, 1981). The samples were examinedas above with the Tecnai 12 microscope.

2.5. Immobilization of silicatein on spicules

Spicules (1 mg) from S. domuncula were functionalizedwith 100 ll of 3-aminopropyltriethoxysilane [APS](10 mg/ml) in 35 ml of ethanol, under reflux for 12 h(60 �C). Subsequently, the spicules were washed with etha-nol to remove the excess of the silane solution by centrifu-gation (4000 rpm, 15 min) and then dried under vacuum.

Then both non-derivatized as well as amine functional-ized (with APS) spicules were incubated with 10 ll eachof 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC)(0.1%) and gallocyanine (10 mM) for 20 min at room tem-perature followed by centrifugation at 1000 rpm for 2 minas described (Tahir et al., 2006). These samples of (i) non-derivatized spicules, or (ii) non-derivatized spicules thathad additionally been treated with EDC and gallocyanine,were used as negative controls.

In the next step, the APS-functionalized spicules weretreated with 100 mg of a reactive ester polymer, poly(acet-oxime methacrylate), which specifically reacts with primaryamines (Tahir et al., 2004). Binding of silicatein wasachieved following the described technique (Tahir et al.,2004). Finally, the spicules were washed with 20 mMMOPS [3-(N-morpholino)propanesulfonic acid; pH 7.4]buffer.

The immobilization of silicatein on the functionalizedspicules was checked by incubation of the spicules withthe anti-silicatein antibodies PoAb-aSILIC (1:1000) (Mul-ler et al., 2005) in PBS, supplemented with 0.3% BSA for1 h at room temperature. Subsequently, the samples werewashed three times with TBS-T buffer (Tris–HCl, pH 8.0;150 mM NaCl, 0.05% Tween-20) for 5 min. Then the spic-ules were incubated with the secondary antibody, the Cy-3conjugated goat anti-rabbit IgG, for 1 h at room tempera-ture. Controls were performed using either spicules coveredwith immobilized recombinant silicatein and pre-immuneserum, or with spicules lacking silicatein on their surfacefunctionalized with Ni(II), complexed to NTA only. Fluo-rescence analysis was performed with an Olympus AHBT3light microscope, together with an AH3-RFC reflectedlight fluorescence attachment at the emission wavelengthof 546 nm (filter G) or of 495 nm (filter U).

2.6. Formation of the biosilica layer around the spicules

Silicatein-functionalized spicules were treated with60 lM TEOS in MOPS buffer (20 mM; pH 7.0) for 12 hat room temperature to coat the spicules with polymerizedsilica. The 50 nm ultrathin slices of silica-coated spiculeswere analyzed by scanning TEM (STEM) and then byEDX.

Fig. 1. Zonation of the silica shell of the spicules from Suberites

domuncula. (A and B [etched samples]) SEM analysis of broken spiculesin 20 days old primmorphs. Around the axial canal (ac) two layers oflamellae (l1 [lamella 1] and l2 [lamella 2]) can be distinguished. At highermagnification the nanoparticles, forming the spicules are seen (> in B). (Cand D) Effect of limited etchging of spicules with HF in order to show thetransition from the completely homogenous appearing spicule biosilicashell to the lamellar organization of the siliceous mantel; SEM analysis.(C) Broken section non-etched, untreated spicule. (D) Partially etched,HF-treated spicule. (E and F) Electron immunogold labeling (TEM)analysis. The sections were incubated with antibodies against silicatein(PoAb-aSILIC). In the axial canal (ac) the axial filament is visible (af). InF the gap between the lamellae is less electrodense and comprises antigenicmaterial, reacting with PoAb-aSILIC. (G and H) TEM section though agrowing spicule in a primmorph, at different magnifications. The twolayers (l1 and l2) as well as the gap between these two layers are marked (><). Size bars correspond to 1 lm.

328 H.C. Schroder et al. / Journal of Structural Biology 159 (2007) 325–334

2.7. Analytical techniques

2.7.1. FT-IR

In parallel, the specimens had been analyzed by Fouriertransform infrared spectroscopy (FT-IR) using a Matsson2030 Galaxy-Fourier Transform-InfraRed Spectrometer(Mattson Instruments, Madison, WI; USA). Spectra wererecorded between 400 and 4000 cm�1 wave number rangeas described (Croce et al., 2004). The spectra were analyzedas described (Burneau and Gallas, 1998).

2.7.2. EDX

Energy dispersive X-ray analysis of the extraspiculargranules (silicasomes) were performed on SEM images(backscattered mode) using a Philips XL 30 ESEM micro-scope as described (Ni et al., 2006).

2.7.3. STEM-EDX

Where indicated, the samples were transferred ontocoated copper grids mounted into a single tilt holder andanalyzed by scanning TEM (STEM)-EDX with a Tecnai30 microscope (300 kV microscope with STEM unit andEDX—HAADF detector) as described (Fujikawa et al.,2005).

3. Results

3.1. Lamellar organization of the spicules

Primmorphs, formed after about 20 days, were used forthe analysis. Sections were analyzed by SEM and it wasfound that around the 1–2 lm wide axial canal the siliceousspicules show several fracture planes, which are here oper-ationally termed lamella-1 and lamella-2. The thickness ofthese lamellae varies between 0.5 and <2 lm. This zonationis frequently seen by SEM at broken ends of spicules(Fig. 1A). At higher magnifications the granular composi-tion of the silica shell can be visualized; the diameter of thenanoparticles is around 100–200 nm (Fig. 1B). For thisanalysis the spicules had been exposed to HF for a shortperiod to visualize the zonation of the silica shell surround-ing the axial filament. During this treatment a transitionfrom a homogeneous surface of a fracture through a silicaspicule (Fig. 1C), to a surface displaying concentric rings ofappositionally arranged silica layers can be seen (Fig. 1D).

By electron immunogold labeling (TEM) analysis thedifferent layers could be distinguished also. Sections wereprepared, incubated with antibodies against silicatein andsubjected to the electron immunogold labeling procedure.In Fig. 1E two lamellae, with a thickness of 0.5–1 lm,can be distinguished in the cross sections. At higher magni-fication the organization of the lamellae as well as the com-position of the fractured silica shell is more prominent.Interesting is the finding that between the lamellae a gapof less electrodense material exists in which the labeledantibodies recognize specifically antigenic material(Fig. 1F). The axial filament is located in the axial canal

(Fig. 1E). In a second set of experiments, a section througha growing spicule is shown, displaying again the gapbetween the two silica layers (Fig. 1G and H).

3.2. Formation of the hollow cylinder around a growing

spicule

Immature spicules in primmorphs are surrounded byhollow cylinders, formed from silicatein/galectin/collagen

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(Schroder et al., 2006). If longitudinal, cross and traversesections were subjected to immunogold labeling (withPoAb-aSILIC antibodies) and analyzed by TEM, itbecame obvious that the entire spicule is surrounded byhollow cylindrical structures (Fig. 2A–D). The antibodiesrecognize especially the layers forming the inner and theouter layer of the hollow cylinder (Fig. 2D); the megasclereshown in Fig. 2D is just at the beginning of its formation,since no silica depositions can be identified yet. A cross sec-tion through a spicule, composed of at least one lamella ofbiosilica shows the clear patterning of the hollow cylinderinto an inner and outer surface (Fig. 2E). In a traverse sec-tion, the characteristic organization of a spicule from theaxial canal—silica shell—to the surrounding hollow cylin-der is visible (Fig. 2F). In parallel sections preimmuneserum was used, which gave no reaction with sponge struc-tures (not shown).

3.3. Release of granules into the extraspicular space

If released into the mesohyl, the spicules are surroundedby cells (sclerocytes) which never come into close contact

Fig. 2. Existence of the hollow cylinder around growing spicules inprimmorphs; immunogold labeling (TEM) analysis; the immunoreactionwas performed with PoAb-aSILIC. (A–D) Longitudinal section along animmature megasclere (meg; > <). With increasing magnification theorganization of the hollow cylindrical structures (hc) in an outer (ol) andan inner layer (il) becomes prominent due to the high density of the labeledPoAb-aSILIC grains. (E) A cross section through a spicule, composed ofone lamella of biosilica (si). The outer and the inner layer of the hollowcylinder around the growing spicule are contrasted. (F) The transversesection through a megasclere shows the regional composition from theaxial canal (ac) via the biosilica shell to the hollow cylinder. All size barsmeasure 1 lm with the exception of D (0.5 lm).

with the siliceous surface (Schroder et al., 2006; Eckertet al., 2006); Fig. 3A and B. Very frequently the cells arefilled with 0.2–0.8 lm large electrodense granules. Betweenthe cells collagen fibers are abundantly present (Fig. 3B).Especially in close neighborhood to the spicules, the silica-somes are released into the extracellular mesohyl compart-ment; this release process is shown in Fig. 3C–F. Aftercontrasting with PoAb-aSILIC and subsequent TEM anal-ysis it can be demonstrated that the electrodense silica-somes are surrounded by molecules/structures reactingwith the labeled antibodies (Fig. 3G and H). Frequentlythe fibers oriented in parallel to the surface of the cells con-tain vesicles, surrounded by structures/molecules that reactwith the antibodies (Fig. 3G).

Fig. 3. Release of granules into the extraspicular space. (A and B) TEMimages showing sclerocytes (sc) in close contact with the siliceous surface(si) of the spicules. This contact is never intimate; the gap zone containscollagen (co) fibers. The sclerocytes contain 0.2–0.8 lm large electrodensegranules, termed silicasomes (sis). (C) Immunogold labeling (TEM)analysis of the non-cellular material surrounding a spicule (sp) whichcontains also silicasomes. (D–F) Release of the electrodense granules fromsclerocytes in close neighborhood to the spicules (TEM). Those silica-somes which are in the process of release are marked (+). (G and H)Higher magnification of sections stained with antibodies, showing thedecoration of the silicasomes with material reacting strongly with PoAb-aSILIC.

Fig. 4. SEM/EDX analysis of a sclerocyte, surrounding a spicule. TheSEM image in the backscattered mode is shown. Granules in theneighborhood of a spicule (sp) and existing in a sclerocyte (sc) have beenselected for EDX analysis. Three granules have been selected: one vacuolewhich is electron-poor (va), and two which are electron-dense and termedsilicasome (sis-1 and sis-2). EDX analyses of the electron-poor vacuole (B-1; va) and two electron-dense silicasomes (B-2; sis-1, and B-3; sis-2) areshown. The peaks corresponding to carbon, oxygen and silicon aremarked.

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3.4. Energy dispersive X-ray analysis of the extraspicular

granules: silicasomes

In order to obtain a quantitative evaluation of silicon/silica deposition in the granules, EDX-micro-analyses onsections were performed (Fig. 4A). An SEM image froma sclerocyte, surrounding a spicule, was prepared and ana-lyzed by EDX. Granules in sclerocytes which appearedelectron-dense were selected for analysis and compared toa vacuole in the same cell which was electron-poor(Fig. 4A). The result of a scan through an electron-poorvacuole shows two prominent peaks of carbon and oxygenbut not of silicon (Fig. 4B-1). In contrast, if the analyses ofthe two selected electron-dense particles were performed,the silicon peaks were prominently identified (Fig. 4B-2and B-3). Due to the high silicon content of the particlesthese electron-dense granules were termed silicasomes.

3.5. Functionalization of spicules

In the first step organic material-free spicules were func-tionalized with APS as described under Section 2. The sam-ples were analyzed by Fourier transform infraredspectroscopy (FTIR) to analyze the surface of the spicules.The absorbance spectra of FTIR from the spicules showedthe characteristic pattern for the vibrational mode of silicabetween the 500 and 1300 cm�1 (Fig. 5). The followingpeaks could be distinguished; the symmetric signals fromthe bending of the Si–O–Si linkages (800 cm�1); the asym-metric stretching of the Si–O–Si groups (1200 cm�1); and inthe range between 2400 and 3700 cm�1 the signals comefrom hydroxyl and amine (2400 cm�1). The latter peakwas present only on the APS-derivatized spicules andabsent on the organic-free surface of starting spicule prep-arations (Fig. 5).

After having linked an amino group to the surface of thespicules, the amino (NH2)-functionalized spicules werecoupled to fluorochrome, gallocyanine, applying EDC asdescribed under Section 2. Gallocyanine was linked tothe primary amine groups at the surface of the spicules.Microscopical fluorescence analysis shows a bright appear-ance of the surface of the spicules (Fig. 6B); the corre-sponding Nomarsky image is given (Fig. 6A). In controlsnon-derivatized spicules, or non-derivatized spicules whichhad been treated with EDC and gallocyanine (Fig. 6C andD) were inspected by microscopical fluorescence analysis;these samples did not show any signals.

3.6. Immobilization of silicatein on spicules

In order to immobilize silicatein to the NH2-functional-ized spicules they had been reacted with the reactive poly-mer. Finally, the His-tagged silicatein was linked to thefunctionalized spicules via nickel as outlined under Section2. The immobilization of silicatein onto those functional-ized spicules was checked by incubation of the spicules withthe anti-silicatein antibodies. The spicules which had been

Fig. 5. Fourier transform infrared absorbance/transmittance spectrum ofspicule samples in the 400–4000 wavenumber cm�1 range. Spectra havebeen recorded for (i) spicules with organic-free surface (above) (ii) spiculeswith APS functionalized surface (below). The infrared spectra show thecharacteristic pattern for vibrational mode of silica (500–1300 cm�1

range). The OH stretching and vibration at 3410 and 1623 cm�1,respectively, suggests the coating of OH groups onto the spicules surface.The peaks in the curve below correspond as follows: (1) the symmetricstretching signals originate from the bending of the Si–O–Si linkages(800 cm�1); and (2) the asymmetric stretching (1200 cm�1) reflects likewisethe Si–O–Si groups. Bands in the range 1600–2200 cm�1 range are due tosilica modes. The signals around 2300 cm�1 can be attributed to C–Sistretching. In the 2400–3700 cm�1 range the signals come from hydroxyl(–OH) and amine (–NH2); (3) –NH group; (4) decreasing of the -OHgroups present at the surface; (5) NH2 groups wagging out of the plane.

Fig. 6. Functionalization of the spicules with amine groups using APS.NH2-functionalized spicules were coupled to the fluorochrome, gallocy-anine, applying EDC and analyzed by Nomarsky light system (A), or theywere subjected by fluorescence light microscopy at 495 nm (B). In acontrol, the non[NH2]-derivatized spicules (Nomarsky; C) had beenreacted with EDC and gallocyanine (fluorescence image; D). (E and F)Immobilization of silicatein onto spicules. (E) The NH2-functionalizedspicules had been linked to recombinant silicatein and then incubated withanti-silicatein antibodies (PoAb-aSILIC), as described under Section 2.Inspection was performed under fluorescence light (emission wavelengthof 546 nm). (F) In parallel, the silicatein-linked spicules were treated withpre-immune serum.

Fig. 7. SEM analysis of spicules linked on their surfaces with recombinantsilicatein. (A–C) NH2-functionalized spicules reacted with the polymerand then with the His-tagged silicatein was analyzed. (D) In a control,those functionalized remained non-treated with silicatein.

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linked to silicatein and subsequently treated with the anti-bodies gave bright signals with fluorescence microscopicalanalysis (Fig. 6E), while those which were incubated withpre-immune serum showed only a slight fluorescencearound the surface of the spicule (Fig. 6F).

In a second approach the spicules, linked with silicateinhad been analyzed by SEM analysis. The images show thatthose spicules that are covered with silicatein show a wrin-kled surface indicating the proteinaceous cover (Fig. 7A–C). In the controls, those spicule samples which had beenNH2-functionalized, treated with the polymer but not withreceptor silicatein showed a completely smooth surface(Fig. 7D).

3.7. Biosilica synthesis by silicatein-coated spicules

The derivatized/functionalized spicules which had beencovered with silicatein were incubated with TEOS, as asubstrate for the enzyme. Subsequently the spicule sampleshad been subjected to SEM inspection and subsequently toEDX analysis. Backscattered SEM images showed at thesurface of the silica shell of the broken spicules a 200–500 nm thick more complete additional surface layer(Fig. 8A and B). One spot within this layer was analyzedby EDX and found to contain one distinct silicon peak(Fig. 8B-1), while at reference sample taken from the centerof the spicule, the axial filament, this peak was absent(Fig. 8B-2).

Fig. 8. Scanning TEM (STEM) and EDX analysis of the silica coatformed onto silicatein-functionalized spicules (sp) after incubation withTEOS. (A-1 and A-2) Dark-field image obtained by STEM. The indicatedareas (a) and (b) are the points to be analyzed for their elementalcomposition by STEM-EDX point analysis. (B-1 and B-2) EDX analysisof the selected areas (a) and (b).

332 H.C. Schroder et al. / Journal of Structural Biology 159 (2007) 325–334

4. Discussion

While the elucidation of the morphology of the spiculeswas intensively approached during more than 250 years,starting with the illustrations of Donati (1753), the descrip-tions by Carter (1849) and the comprehensive synthesis byUriz et al. (2000), the mechanism by which silica is concen-trated/deposited within and around the spicules remainedunclear. Focusing on the demosponge Tethya aurantium

(Shimizu et al., 1998; Cha et al., 1999) and a little lateron S. domuncula (Krasko et al., 2000) it had been foundthat silica/biosilica in this class of sponges in formed enzy-matically via the enzyme silicatein. This enzyme belongs tothe group of cathepsins which had been identified andcloned in the course of these studies from Geodia cydonium

(Krasko et al., 1997). Recombinant silicatein protein servedto prepare the powerful tool, antibodies against silicatein(Muller et al., 2005). In the latter study we could demon-strate that the formation of the siliceous spicules in Demo-spongiae proceeds by an initial synthesis of the core silicarod from the central axial filament, located in the axial

canal, and completed by appositional growth via newlyformed layers. In turn, and applying the in vitro cell culturesystem, the primmorphs, it could be unequivocally demon-strated that the initial formation of the spicules starts intra-cellularly; then the spicules are completed extracellularly inthe mesohyl. A further step towards an understanding ofthe components/proteins involved in the intra-organismicformation of the spicules came from the recent studies thatspicule growth is a matrix-guided process in which galectinmolecules orient the silicatein molecules in strings (Schro-der et al., 2006), which are morphogenetically guided bycollagen (Eckert et al., 2006).

The aim here was to study the proposed hollow cylinder,located around the axis of a growing spicule, in moredetail. In a recent, very detailed study (Pisera, 2003), amicroscopical analysis of the inorganic biosilica shellaround the axial filaments of spicules in several lithistidsponges is given. It is outlined that initially silica nanopar-ticles of a size 0.1–1 lm are formed which finally fuse andform the concentric layers. If the spicules are etched, por-ous silica layers become visible which are framed by moredense silica surfaces. In this report the remarkable arrange-ment of the silica nanospheres into strings is illustrated.

At first we could document that the lamellar growth ofthe macroscleres in S. domuncula proceeds by appositionallayering of 0.3–0.6 lm thick silica shells. Using electronimmunogold microscopy it became overt that betweenthe silica layers silicatein polypeptides exist. This findingsupported earlier data which revealed that silicatein is inti-mately associated with the spicule surface. A closer view ofthe growth pattern of spicules in the in vitro primmorphsystem revealed that prior to the formation of a new silicalayer a hollow cylinder is formed with a diameter ofapproximately 0.3 lm (Fig. 2D and E). This hollow cylin-der is formed by silicatein molecules, stringed along thepreviously identified linear matrix of galectin (Schroderet al., 2006). The hollow cylinder guides the growth ofthe silica lamella through silicatein not only in radial butalso in longitudinal direction. The mature spicules are com-posed of concentric silica layers as described earlier(Schwab and Shore, 1971; Simpson, 1984; Sandford,2003; Weaver and Morse, 2003). However, already in somereports SEM images of immature spicules have been pre-sented from tissue cuts, which showed that the surface ofthe spicules is not always regular; even sometime a zona-tion into lamellae can be observed (Garrone et al., 1981).After the development of the primmorph cell culture sys-tem it was possible to study the rapid synthesis of spiculesin S. domuncula thoroughly, focussing also on the veryearly stages of growth. In this cellular model it became pos-sible to distinguish between zones of biosilica in growingspicules with organic layers in between. If cross sectionsare prepared from growing spicules in most cases ring-likesilica layers are seen which had been broken into silicaislands. As shown also in this paper, the broken islandswithin one zone do not correspond to those seen in the fol-lowing one and are separated by an organic layer/gap.

Fig. 9. Schematic outline of the tube-like cylinder (hollow cylinder),composed of two layers of silicatein, which surrounds the spicule. Theinner surface of the cylinder is stabilized by the surface of the spicule ontowhich the silicatein molecules are attached. The outer layer of the cylinderis formed by the galectin net with the associated silicatein molecules.Within these layers silicatein polymerizes (ortho)silicic acid to polymericsilica. The cells surrounding the spicules, the sclerocytes, produce vesiclesin which silicate [or oligomers from orthosilicic acid] has accumulated.After release of these vesicles (termed silicasomes) into the extracellularspace, they channel oligomeric silicate into the hollow cylinder.

H.C. Schroder et al. / Journal of Structural Biology 159 (2007) 325–334 333

Such a morphology had already been presented in animage, but not commented on (Uriz et al., 2003; Weaverand Morse, 2003).

The finding that maturation of the spicules proceeds andis completed extracellularly opened a new question. In aprevious study evidence could be presented that intracellu-larly silica is very likely accumulated through aNaþ=HCO3

�½Sið OHÞ4� cotransporter system (Schroderet al., 2004). After intracellular completion of the firstlayer(s) of an immature spicule and its subsequent extru-sion into the extracellular space, it must be asked for thesupply of silica for the extracellular formation of silica.Electron microscopical studies revealed that the cells, sur-rounding growing spicules, the sclerocytes are filled withgranules of varying electron density. Striking was theobservation that some of the electron-dense granules arereleased into the intimate space around the spicules. Evenmore suggestive had been the finding that some of thesegranules are surrounded by a layer of silicatein. The proofthat these granules contain high levels of silicon came fromSEM–EDX analyses. The spectra showed that within theelectron-dense granules a pronounced elemental peak forsilicon exists compatible with the assumption that in thesclerocytes distinct vesicles exist which function as a reser-voir for accumulated silicate. Therefore, we have termedthese silica-rich and electron-dense vesicles silicasomes.

Taking these cell biological data together it can be pro-posed that the sclerocytes accumulate silica through theNaþ=HCO3

�½SiðOHÞ4� cotransporter within distinct vesi-cles, the silicasomes. After release of these silicasomes intothe extracellular space their content is channeled into thehollow cylinder, which surrounds the newly growing spic-ule. Compatible with the experimental data is also theassumption the hollow cylinder is composed in its adaxialand abaxial surfaces of silicatein/galectin strings. A sche-matic outline of this view is given in Fig. 9. At present itis not possible to decide if the proposed silicasomes arefilled with silicon, what appears to be unlikely since this ele-ment is taken up be cells as silicic acid. Therefore, we pro-pose that the vesicles are filled with silicic acid at anundetermined determination. Hence we adopt the viewproposed by Chisholm et al. (1978) and Sullivan (1986)showing the accumulation of silicic acid in the silica depo-sition vesicles.

In a biomimetic approach the lamellar apposition of thesilica lamellae has been accomplished by immobilization ofsilicatein on the surface of the organic-layer free spicules.The established procedure was used to amino group-func-tionalize the silica surface with aminopropyltriethoxysilaneto allow binding of the reactive ester [poly(acetoxime meth-acrylate)] that is required as matrix for silicatein (Tahir et al.,2004, 2005). The presence of amino groups on the surface ofsilicatein was verified analytically and also by binding of gal-locyanine via EDC. The recombinant silicatein, with its His-tag, was linked to the matrix via Ni(II) (Tahir et al., 2004,2005, 2006). Notable is the fact that silicatein remains func-tionally active after close attachment of the organic

matrix—a biomimetic approach allowing also the applica-tion of the enzymatic biosilica technology for various chem-ical (new materials), physical (electronical) and biomedicalapplications (see Tahir et al., 2006; Wang and Wang,2006; Schroder et al., in press; Muller et al., in press).

In conclusion, these significant results on the apposi-tional formation of silica lamellae around the spicules dur-ing growth opens a new view on the use/application/utilization of hollow cylinders, formed by matrix-guidedsilicatein molecules for biocatalytic synthesis of nanostruc-tured tubes. This bioinspired technology is required inlithography for electronic chips and for two- and three-dimensional semiconductors (Muller et al., 2004).

Acknowledgments

Ms. E. Sehn (Zoological Institute; University of Mainz[Germany]) for the valuable technical assistance and Ms.M. Muller, and G. Hartmann (Max-Planck Institute forPolymer Research, Mainz [Germany]) for the scanningelectron microscope pictures. This work was supportedby grants from the European Commission, the DeutscheForschungsgemeinschaft, the Bundesministerium fur Bil-dung und Forschung Germany, the National Natural Sci-ence Foundation of China (No. 50402023) and theInternational Human Frontier Science Program.

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