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Bioorganic/inorganic hybrid composition of sponge spicules: Matrix of the giant spicules and of the comitalia of the deep sea hexactinellid Monorhaphis

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Bioorganic/inorganic hybrid composition of sponge spicules: Matrix of the giant spicules and of the comitalia of the deep sea hexactinellid Monorhaphis
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  Bioorganic/inorganic hybrid composition of sponge spicules:Matrix of the giant spicules and of the comitalia of the deepsea hexactinellid  Monorhaphis Werner E.G. Mu¨ller  a,* , Xiaohong Wang  b , Klaus Kropf   a , Hiroshi Ushijima  c ,Werner Geurtsen  d , Carsten Eckert  a,e , Muhammad Nawaz Tahir  f  , Wolfgang Tremel  f  ,Alexandra Boreiko  a , Ute Schloßmacher  a , Jinhe Li  g , Heinz C. Schro¨der  a a Institut fu¨r Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universita¨t, Duesbergweg 6, D-55099 Mainz, Germany b National Research Center for Geoanalysis, 26 Baiwanzhuang Dajie, CHN-100037 Beijing, PR China c Department of Developmental Medical Sciences, Institute of International Health, Graduate School of Medicine,The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan d Division of Operative Dentistry, Department of Restorative Dentistry, School of Dentistry, University of Washington, Seattle, WA 98195-7456, USA e Museum fu¨ r Naturkunde, Institut fu¨ r Systematische Zoologie, Invalidenstraße 43, D-10155 Berlin, Germany f  Institut fu¨ r Anorganische Chemie und Analytische Chemie, Universita¨ t, Duesbergweg 10-14, D-55099 Mainz, Germany g Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, CHN-266071 Qingdao, PR China Received 9 June 2007; received in revised form 16 October 2007; accepted 17 October 2007Available online 26 October 2007 Abstract The giant basal spicules of the siliceous sponges  Monorhaphis chuni   and  Monorhaphis intermedia  (Hexactinellida) represent the largestbiosilicastructuresonearth(upto3 mlong).Herewedescribetheconstruction(lamellarorganization)ofthesespiculesandofthecomitaliaandhighlighttheirorganicmatrixinordertounderstandtheirmechanicalproperties.Thespiculesdisplaythreedistinctregionsbuiltofbio-silica:(i)theouterlamellarzone(radius:>300  l m),(ii)thebulkyaxialcylinder(radius:<75  l m),and(iii)thecentralaxialcanal(diameter:<2  l m) with its organic axial filament. The spicules are loosely covered with a collagen net which is regularly perforated by 7–10  l m largeholes;thenetcanbesilicified.Thesilicalayersformingthelamellarzoneare  5  l mthick;thecentralaxialcylinderappearstobecomposedofalmostsolidsilicawhichbecomesporousafteretchingwithhydrofluoricacid(HF).Dissolutionofacompletespiculedisclosesitscomplexstructurewithdistinctlamellaeintheouterzone(lamellarcoating)andamoreresistantcentralpart(axialbarrel).Rapidlyafterthereleaseof theorganiccoatingfromthelamellarzonetheproteinlayersdisintegratetoformirregularclumps/aggregates.Incontrast,theproteinaceousaxialbarrel,hiddeninthesiliceousaxialcylinder,issetupbyrope-likefilaments.Biochemicalanalysisrevealedthatthe(dominant)moleculeof the lamellar coating is a 27-kDa protein which displays catalytic, proteolytic activity. High resolution electron microscopic analysisshowed that this protein is arranged within the lamellae and stabilizes these surfaces by palisade-like pillars. The mechanical behavior of the spicules was analyzed by a 3-point bending assay, coupled with scanning electron microscopy. The load-extension curve of the spiculeshowsabiphasicbreakage/crackingpattern.Theouterlamellarzonecracksinseveraldistinctstepsshowinghighresistanceinconcertwithcomparablylowelasticity,whiletheaxialcylinderbreakswithhighelasticityandlowerstiffness.Thecomplexbioorganic/inorganichybridcompositionandstructure ofthe  Monorhaphis spiculesmight providetheblueprintforthesynthesis ofbio-inspiredmaterial, withunusualmechanical properties (strength, stiffness) without losing the exceptional properties of optical transmission.   2007 Elsevier Inc. All rights reserved. Keywords:  Hybrid composite material; Sponges;  Monorhaphis ; Spicules; Elasticity; Silicatein 1047-8477/$ - see front matter    2007 Elsevier Inc. All rights reserved.doi:10.1016/j.jsb.2007.10.009 * Corresponding author. Tel.: +49 6131 39 25910; fax: +49 6131 39 25243. E-mail address:  wmueller@uni-mainz.de (W.E.G. Mu¨ller). URL:  http://www.biotecmarin.de/ (W.E.G. Mu¨ller). www.elsevier.com/locate/yjsbi  Available online at www.sciencedirect.com Journal of Structural Biology 161 (2008) 188–203 Journal of StructuralBiology  1. Introduction The earth’s surface is composed of more than 55% sili-con and oxygen. Silicon is present mainly as silica and sil-icates. In the inorganic natural state it is found primarily asanhydrous quartz, cristobalite and tridymite; moreover, insoils and hot-springs silica exists as amorphous opal-A andalso as rigid/flexible biological material, e.g. bio-silica, andcombines the properties of strength and stiffness withtoughness (Mayer, 2005). The toughness of rigid biologicalskeletal material, irrespective if made of calcium or silicaminerals, is in the first place caused by the lamellar organi-zation of the inorganic matrix, as seen in nacre of molluskshells (calcareous material) or in spicules in sponges (phy-lum Porifera) of the classes of Demospongiae and Hexacti-nellida (siliceous biomineral) (reviewed in Mayer, 2005).The strength of siliceous skeletal spicules has been studiedin great detail in the hexactinellid sponge  Euplectella asper- gillum  (Aizenberg et al., 2005; Weaver et al., 2007; Woesz et al., 2006). The spicules of hexactinellid sponges gainedadditional attention because of their fiber-optic properties;they can function as single-mode, few-mode, or multimodefibers, with spines serving as illumination points along thespicule shaft (Aizenberg et al., 2004; Mu¨ller et al., 2006b;Wang et al., 2007).For our biochemical, optical, and biomechanical studieswith spicules from Hexactinellida we have used the giantbasal spicules (and the comitalia) of glass sponges,  Hyalo-nema sieboldi   (Mu¨ller et al., 2006b) and two other closelyrelated species,  Monorhaphis chuni   and  Monorhaphis inter-media  (Mu¨ller et al., 2007a; Wang et al., 2007). These spe-cies are grouped to the order of Amphidiscoida, while thehexactinellid  E. aspergillum  that has been primarily studiedby Aizenberg and colleagues (2005) and Weaver and col- leagues (2007) belongs to the order Hexactinosida (Tabach-nick, 2002; Reiswig, 2006). The most outstanding feature of the spicules from  Monorhaphis  is their size; the giant basalspicules are with up to 3 m (diameter of up to 8 mm) thelongest/largest bio-silica structures on earth (Schulze,1925). The morphology of these giant basal spicules hasearlier been thoroughly described (Schultze, 1860, 1904,1925; Levi et al., 1989). Cross sections of the giant spiculeof   Monorhaphis  allow the distinction of three zones; (i) theouter lamellar zone (radius: >300  l m), (ii) the bulky axialcylinder (radius: <75  l m), and (iii) the central axial canal(diameter: <2  l m) that surrounds the organic axial filament(Mu¨ller et al., 2007a; Wang et al., 2007). The outer lamellarzone is composed of 300–500 lamellae that are concentri-cally arranged and are each 3–8  l m thick. Related to thesegiant basal spicules, with respect to their structure, are thecomitalia which reach sizes of 60 mm (length) and 0.5 mm(diameter).An outstanding feature of siliceous spicules, from bothDemospongiae and Hexactinellida, is the formation of the silica shell around the axial canal/axial filament. Thegroup of Morse (Shimizu et al., 1998; Cha et al., 1999)described that the major protein of the axial filament of the demosponge  Tethya aurantium  is an enzyme which theytermed silicatein, that mediates polymerization/condensa-tion of ortho-silicate to polymeric (bio)silicate (Cha et al.,1999). Shortly after, this enzyme was also identified in thedemosponge  Suberites domuncula  (Krasko et al., 2000).The silicateins belong to the cathepsin L-proteinase family,and are distinguished from them by one amino acid in thecatalytic center (catalytic triad Ser-His-Asn (in silicatein)and Cys-His-Asn (in cathepsins)) (Cha et al., 1999; Kraskoet al., 2000; Mu¨ller et al., 2007c). It is well established thatcathepsins can be effectively inhibited by E-64 ( L - trans -epoxysuccinyl-leucylamido(4-guanidino)butane) throughbinding to the active sites of the enzymes (Barrett et al.,1982, 2002).For the demosponge  S. domuncula  a detailed biochemi-cal and cytochemical analysis of the synthesis of the sili-ceous spicules was established (reviewed in Mu¨ller et al.,2006a, 2007b). In brief, growth of the spicules starts intra-cellularly (Mu¨ller et al., 2005). After having reached alength of about 8  l m, the primordial spicules are extrudedfrom the cells and reach in the extracellular space their finalforms by longitudinal and lamellar thickening growth. Thesize-increase of the spicules occurs by lamellar appositionof silica layers, which are separated by sheets of organicmatrices formed of galectin (Schro¨der et al., 2006). Despitetheattractiveness ofbiosilica forbiotechnological(reviewedin Morse, 2001; Patwardhan et al., 2005; Mu¨ller et al.,2007b) and biomimetical applications (Wang and Wang,2006) and despite the fact that only in sponges is biosilicaformed via an enzyme, no detailed analyses of the protein-aceous components of spicules from hexactinellids have yetbeenundertaken (seeLeysetal.,2007).Thespiculesofmosthexactinellids are larger and more luxuriously architecturedthan those in demosponges (Tabachnick, 2002). Very likely,also in hexactinellids growth of spicules starts from an axialfilament (Hartman, 1983 and reviewed in Leys et al., 2007), around which bio-silica with the general formula of SiO 2 • n H 2 O ( n  = 0.3) is formed (Sandford, 2003).From studies with  Monorhaphis , Levi and coworkers(1989) suggested that the layered structure of the spiculeshas a ‘‘beneficial’’ effect on the mechanical properties of the spicules. The concept of natural composite materialin rigid biological systems was fundamentally outlined byMayer (2005). The organic phase controls energy dissipa-tion especially in systems that are interspersed by very thinorganic layers. In continuation of this topic Mayer et al.(2005) proposed from their load–displacement studies thatin  Euplectella  breakages of spicules follow a telescope-likepattern.While the first genes from a hexactinellid sponge,  Rhab-docalyptus dawsoni  , encoding proteins (serine/threoninekinases) had been identified already in 1998 (Kruse et al.,1998), first analyses of proteins in spicules have been pub-lished only recently. For these studies giant basal spiculesfrom  Monorhaphis  were used because of their large sizeand their morphology. These spicules contain one proteinof a size of around 25/27 kDa and, in addition, higher W.E.G. Mu¨ller et al. / Journal of Structural Biology 161 (2008) 188–203  189  molecular weight protein(s) (>60 kDa) (Mu¨ller et al.,2007a; Wang et al., 2007). It could be demonstrated that Monorhaphis  spicules contain two molecules, which haverelated counterparts in demosponges; a lectin (Schro¨deret al., 2006) and a silicatein-like protein (reviewed in Wanget al., 2007). Therefore we proposed that the lamellargrowth of   Monorhaphis  spicules proceeds by appositionallamellar growth involving very likely silicatein-like pro-tein(s) and lectin (Mu¨ller et al., 2007a; Wang et al.,2007). The outer surfaces of the spicules are covered by acollagen net as visualized electron microscopically (Mu¨lleret al., 2007a).In the present study we demonstrate that the proteina-ceous matrix of the  Monorhaphis  spicules (the giant basalspicules and the comitalia) is not evenly distributedthroughout the inorganic shell around the axial canal. Infact, morphological/structural zones can be distinguished;the axial cylinder and the lamellar zone. Earlier wedescribed the morphology of the giant basal spicules of  Monorhaphis  (Mu¨ller et al., 2007b). Now we show, thatthe layers setting up the lamellar zone are composed (exclu-sively) of one protein (size: 27 kDa); based on its bindingproperty to labeled E-64 the 27-kDa molecule can be char-acterized as a protease, a (silicatein-related) polypeptide.Finally, the surface of the spicules is enveloped by a colla-gen net.By load–displacement studies it became also possible tocorrelate the pattern of fractures within the spicules withthe organization of the lamellar zone and the axial cylinder;both areas are characterized by different bioorganic/inor-ganic hybrid compositions. 2. Materials and methods  2.1. Materials Casein and Coomassie Brilliant blue were obtained fromServa (Heidelberg; Germany); 1-ethyl-3-[3-dimethylamino-propyl] carbodiimide (EDC) (#22980, PIERCE, Rockford,IL; USA), E-64 Sirius Red F3BA (Direct Red 80) and Rho-damine 123 from Sigma–Aldrich (Taufkirchen; Germany);EZ-Link Amine-PEO-Biotin labeling reagent (#21346;PIERCE, Rockford, IL; USA);  Z  -Phe-Arg-AMC fromBachem (King of Prussia, PA, USA).  2.2. Sponges Spicules (giant basal spicules and comitalia) from thehexactinellids  M. chuni   (Porifera:Hexactinellida:Amphidis-cosida:Monorhaphididae) and  M. intermedia  were used asdescribed (Mu¨ller et al., 2007a). The  Monorhaphis  spongesreach average sizes of 1.5 m (Fig. 1B). Around the 1.5-mlong giant basal spicule (also termed basalia) the cylindricalbody of the sponge is attached. The largest spicules withinthe body of   M. chuni   are the comitalia, parenchymal diac-tin spicules. The latter spicules are located in the choano-some and reach a maximal size of 60 mm; they have alsobeen termed principalia and comprise the same structurelike the giant basal spicules (Mu¨ller et al., 2007a). For Fig. 1. The two hexactinellid sponge species, used for biophysical,biomechanical and biochemical studies. (A)  Euplectella aspergillum ; theup to 25 cm high specimens live in depths down to 5000 m. Here (one of)the oldest illustrations from Chimmo (1878) is reproduced which shows anon-typical bifurcated specimen found around the Philippine Islands in adepth of 250 m. (B)  Monorhaphis chuni   from the collection of Dr. K.Tabachnick (Institute of Oceanology Moscow, Russia). Around the giantbasal spicules (basalia, ba) the cylindrical body (bo) of the sponge isarranged which opens with its osculi (os) to the surrounding water. (C)  M.chuni   had been described thoroughly by Schulze (1904). The specimenshad been collected during the ‘‘Valdivia’’ expedition (1898–1899) at EastAfrica (Somalia basin) in a depth of 1600 m. The morphology of the giantbasal spicule (diameter of the spicule: 4 mm) is shown with its threesections; (i) the axial canal (ac), (ii) the axial cylinder (cy) and the lamellarzone (la). The axial canal harbors the organic axial filament. (D) Theellipsoid body of   M. chuni   (bo) is shown which is regularly arrangedaround the giant basal spicule (ba) and which opens with its osculi (os);they are arranged in one line.190  W.E.G. Mu¨ller et al. / Journal of Structural Biology 161 (2008) 188–203  the experiments in the 3-point bending assay, giant basalspicules had been used, which had been freshly collectedin the South Chinese Sea (Guangzhou Marine GeologicalSurvey; China). Until the experiments they were storedwet (in seawater).  2.3. Spicules and spicule extracts The spicules were treated with an ultra-sonicator(S3000; IUL Instruments, Ko¨nigswinter; Germany) toremove loosely attached organic material and then usedfor the analysis of the collagen sheet attached to their sur-faces. This collagen net could be completely removed fromthe spicules by overnight treatment with 2% (w/v) sodiumdodecyl sulphate solution. For stepwise dissolution of thesilica, the  Monorhaphis  spicules were treated with hydroflu-oric acid (HF) as described (Shimizu et al., 1998; Mu¨lleret al., 2007a; Wang et al., 2007). Spicules were placed onplastic slides and treated with 6 M HF/8 M NH 4 F untileither the complete silica material had been dissolved, or,until the different organic layers were stepwise exposed.At indicated time points, the HF-mediated dissolution pro-cess was terminated by replacing the HF solution with 2 MCaCl 2 . To visualize the released proteins, Coomassie solu-tion (0.1% (w/v) Coomassie Brilliant blue 250; 40% metha-nol, 10% acetic acid) was added to the HF solution (Mu¨lleret al., 2007a). In a parallel series, the CaCl 2  solution wassupplemented with Sirius Red F3BA (0.1% (w/v) in satu-rated picric acid), to stain the collagen-like fibers asdescribed (Junqueira et al., 1979; Walsh et al., 1992; Taski-ran et al., 1999). Additionally, the organic material in thesilica shell of the spicules was stained with Rhodamine123 (0.01% (w/v) in dimethyl sulfoxide) as described(Schro¨der et al., 2006). At different times of incubation(up to 40 min) photos from Coomassie- and Sirius Red-stained samples were taken with an Olympus AHBT3microscope and/or by Nomarsky differential interferencecontrast (DIC) imaging. The fluorescence of the Rhoda-mine stained specimens was visualized using the filter pair489 nm (excitation)/520 nm (emission).Matrix protein from the lamellar zone of the freshly col-lected spicules was obtained by partial dissolution of thiszone; the HF-treatment was stopped after 3 h. Then theundissolved silica material was removed and the remainingorganic material was collected by centrifugation (5000  g  ;10 min; 4   C). Both, the supernatant and the sediment(approximately 50  l g of protein), were dissolved in 50  l lNaDodSO 4  –PAGE sample buffer and then size-separatedby NaDodSO 4  –PAGE. In a separate series of experimentsthe sediment was dissolved in 6 M urea, supplemented with10% (v/v)  b -mercaptoethanol for 30 min, while heating(60   C), and again size-separated (NaDodSO 4  –PAGE).Samples of individual (single) lamellae were obtained bymechanical treatment of the spicules which chipped off cherts of lamellae from the axial cylinder.In another set of experiments spicules from  Monorhaphis were extracted avoiding HF, as described before for S. domuncula  (Schro¨der et al., 2006). In brief, the spiculeswere ground/pulverized thoroughly in a mortar togetherwith lysis-buffer (1 ·  TBS (Tris-buffered saline); pH 7.5,1 mM EDTA, 1% Nonidet-P40) and subsequently stirredfor 1 h at 4   C.  2.4. Electron microscopy Three millimetres (giant basal spicule) or 500  l m (com-italia) thick spicules were embedded in an epoxy resin(Struble and Stutzman, 1989) and sliced (Mu ¨ller et al.,2007a). The surfaces of the cross sections were polishedwith emery paper (Siliconcarbide; Matador, Hoppenstedt,Darmstadt, Germany) and the quality of the surface wasinspected under a stereomicroscope with an enlargementof about 30 · . Backscattered analysis was performed withthis microscope using 15-keV beam voltage and 50  l Aemission current at a working distance of 6 mm (Holmeset al., 1987).In one experiment the spicule was broken to allow a tan-gential view of the fractured surface. Scanning electronmicroscopy (SEM) analysis of spicules was performed witha Zeiss DSM 962 Digital Scanning Microscope (Zeiss,Aalen; Germany). The samples were mounted onto alumi-num stubs (SEM-Stubs G031Z; Plano, Wetzlar; Germany)that had been covered with adhesive carbon (carbon adhe-sive Leit-Tabs G3347). Then the samples were sputteredwith a 10-nm thin layer of gold in argon plasma (Bal TecMed 020 coating system; Bal Tec, Balzers, Liechtenstein).The high resolution SEM (HRSEM) analysis was carriedout using a broken spicule, which had not been polishedwith emery paper, in a LEO 1530 Gemini field-emissionscanning electron microscope (FESEM). An energy withinthe range 1 and 5 kV of extraction voltage was applied; inthis range no change in the images could be recorded. Thesignals were detected (in-lens annular, secondary electron)and processed (pixel averaging, frame integration/continu-ous averaging). In order to reach high morphological reso-lution, small pieces from spicules were fastened withconductive carbon tabs on aluminum sample holders. Sam-ples were prepared by placing a drop of material on siliconwafers. For better conductivity, the samples were sputteredwith 8 nm of gold using a Baltec MED020 coating system.  2.5. EDX analysis Energy dispersive X-ray (EDX) analysis of the collagenlayer was performed on SEM images (backscattered mode)using a Philips XL 30 ESEM microscope because this isable to image uncoated and hydrated samples by meansof a differential pumping system and a gaseous secondaryelectron detector. ESEM offers full functionality in thethree modes of operation: high vacuum, low vacuum, andESEM mode. A low-vacuum mode is suitable for the exam-ination of uncoated nonconductive samples. ESEM modeallows very high chamber pressures up to 20 Torr (Ra¨dleinand Frischat, 1997; Tahir et al., 2005; Ni et al., 2006). W.E.G. Mu¨ller et al. / Journal of Structural Biology 161 (2008) 188–203  191   2.6. NaDodSO 4  –PAGE  Samples from spicule extracts containing 1–3  l g of pro-tein were dissolved in loading buffer (Roti-Load, Roth,Karlsruhe, Germany), boiled for 5 min, and then subjectedto 10% polyacrylamide gel electrophoresis, containing 0.1%sodium dodecyl sulphate (NaDodSO 4  –PAGE; Laemmli,1970). After separation, the gels were washed in 10% meth-anol (supplemented with 7% acetic acid) for 30 min andthen stained in Coomassie Brilliant blue as described (Mu¨l-ler et al., 2005). In parallel, silver staining was performedaccording to the described procedure (Radojkovic andKusic, 2000).  2.7. Determination of proteolytic activity Spicule extracts from  Monorhaphis  were obtained avoid-ing HF (see above) and assayed for cathepsin L-activity asdescribed (Dvorak et al., 2005). The soluble protein frac-tion was added to the reaction assay (0.2 ml volume) asdescribed (Quian et al., 1989; Mort, 2002) in 96-well plates(Nunc 96 MicrowellTM Plates) at room temperature. Theassay contained 10  l M  Z  -Phe-Arg-AMC as substrate andincubation was performed for 60 min at room temperature.A standard curve was established with 7-amino-4-methyl-coumarin (AMC); the fluorescence of the free AMCreleased was determined using an excitation at 355 nmand an emission at 460 nm in an F-2000 Hitachi fluores-cence spectrophotometer. The activity was calculated andgiven in nmoles AMC released/mg protein  ·  min (Dvoraket al., 2005). Five parallel experiments were performed,and the means and standard deviations were calculated(Sachs, 1984).Where here indicated the  Monorhaphis  extracts werepreincubated (30 min; 20   C) with 1  l M E-64.  2.8. Binding studies with biotinylated E-64 Biotinylated E-64 was obtained from unlabeled E-64 bycoupling the inhibitor via amine–PEO 2  –Biotin (Pierce,Rockford, IL, USA) and using the cross linker 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC), accordingto the instructions of the manufacturer (Pierce). Thislabeled E-64 was used as a probe to detect the enzymati-cally active center.  Monorhaphis  extract (10  l l), obtainedby solubilization with a 50-mM Tris–HCl buffer (pH; 8.5;10% (v/v) glycerol), was supplemented with 10  l l of 50 mM Na-acetate buffer (pH 5.5; 100 mM NaCl, 1 mMEDTA). After an activation period of 10 min at room tem-perature 5  l l (50  l M final concentration) of biotinylated E-64 was added to the activated enzyme and the mixture wasincubated at 22   C for 1 h. This sample was mixed with 5  l lof 6  ·  Laemmli sample buffer and heated for 5 min at95   C. The samples were analyzed by NaDodSO 4  –PAGE(10% gels) according to Laemmli (1970). Then the proteinswere transferred to a PVDF membrane. The biotin-[E-64]protein complex was visualized using the biotin/avidinVECTASTAIN Elite ABC detection system (Hsu et al.,1981; Vector Labs., Burlingame, CA, USA). The signalswere enhanced by the chemiluminescence procedure usingthe ‘‘western horse radish peroxidase substrate’’/LuminolReagent (Millipore, Schwalbach, Germany).  2.9. Mechanical property measurement The measurement for the flexural properties of an indi-vidual, giant basal spicule samples (between 30 and 35 mmin length) were tested as described (Levi et al., 1989; Sari-kaya et al., 2001; Autumn et al., 2006). The samples werecut from the spicule using a disk saw, to avoid cracking.The spicule sample was fixed in a 3-point bending system(WDW 3020, Changchun; China), selecting a fixture witha 20-mm span and an articulated center loading rod at acrosshead rate of 0.2 mm/min. The diameters of the testedspicules ranged from 2.1 to 2.6 mm. Tests were performedwith a computer-controlled MTS servo-hydraulic testframe. Crosshead displacement and load were automati-cally recorded throughout the tests. For these studiesfreshly collected giant basal spicules were used, whichhad remained in wet conditions (in seawater) until theiruse for the experiments.  2.10. Analytical method  For the quantification of protein the Bradford method(Compton and Jones, 1985; Roti-Quant solution—Roth)was used. 3. Results Primarily two hexactinellid species have so far been ana-lyzed to understand the biophysical, biomechanical andbiochemical processes of spicule formation,  Euplectellaaspergillum  (Fig. 1A) and  Monorhaphis chuni  / Monorhaphisintermedia  (Fig. 1B–D). In the present study we used thegiant basal spicules, and—where indicated—comitaliafrom  Monorhaphis , for the analysis. 3.1. Growth of the giant basal spicules: axial cylinder and lamellar sheet One  Monorhaphis  specimen grows around one giantbasal spicule (Fig. 1B). The soft body is arranged in anellipsoid growth form axially along the spicule, and has  5 cm large osculi along the major axis. The giant basalspicule is fixed asymmetrically within the body and is closerto the osculi than to the opposite pore-rich part of the body(Fig. 1B and D). While the giant basal spicule fixes thecomplete soft body to the ground, several comitalia areusually associated with one oscular section. Already in1904, Schulze described the lamellar organization of thespicules from  Monorhaphis , focusing on the giant basalspicules. In the center of the spicule lies the axial cylinderwhich appears to be less foliated; this cylinder surrounds 192  W.E.G. Mu¨ller et al. / Journal of Structural Biology 161 (2008) 188–203
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