INTRODUCTIONOne of the major innovative steps in the evolution of uni- andmulticellular animals was the acquisition of a hard, mineralizedskeleton. The development of skeletal elements facilitated anincrease in size of the organisms – a phyletic trend that is knownin metazoans as Cope’s rule (Nicol, 1966). As changes in body sizeaffect almost every aspect of life (Schmidt-Nielsen, 1984), twostrategies have been developed in animals to circumvent anyconstraints arising from body size increase. First, the acquisition ofa hydrostatic skeleton and, second, the development of rigid solidskeletal elements (Biewener, 2005). Exclusively, the formation ofinorganic structures in uni- and multicellular organisms is guidedby organic templates (Lowenstam and Weiner, 1989). Thosetemplate-induced or controlled mineralization processes have beentermed biomineralization. The ubiquitous occurrence of template-induced biomineralization processes in nature became obvious withthe discovery that even the formation of the polymetallic nodulesand crusts of the deep-sea is initiated and directed by biogenictemplates (Wang and Müller, 2009). In 1924, Schmidt (Schmidt,1924), who was the first scientist to compile template-caused/controlled biomineralization processes (Weiner and Dove,2003), highlighted the importance of an inorganic skeleton in theestablishment of a body plan. Two concepts of biomineralizationwere categorized by Weiner and Dove (Weiner and Dove, 2003),based on earlier systematic studies (Lowenstam and Weiner, 1989).They distinguished between biologically induced mineralization,whereby biological structures act as causative agents for nucleationand subsequent growth of biominerals, and biologically controlledmineralization, a process during which cells/organisms direct boththe nucleation/growth and final location of the minerals within
an organism. The composite biominerals can be depositedextracellularly, as in Foraminifera or in shells of mollusks,intercellularly as in some calcareous algae, or intracellularly, as inbacteria (magnetosome formation), plants or animals (reviewed inWeiner and Dove, 2003). Besides calcium-based skeletons, silica-based skeletal systems arose during the early evolution of uni- andmulticellular eukaryotes in the Precambrian (Proterozoic), more than542 million years ago (Müller et al., 2007).
Focusing on silica, the major taxa that use this monomericinorganic molecule to form solid skeletons through controlledsilica deposition processes are some protozoans, diatoms,choanoflagellates and silicoflagellates (Leadbeater and Jones, 1984),and metazoans, with the siliceous sponges (phylum Porifera) as themost prominent representative, as well as higher plants (see Mülleret al., 2003; Perry, 2003). All of those organisms take up silica intotheir cells as monomeric silicic acid in order to polymerize/polycondensate amorphous and hydrated bio-silica. It is amazingthat those organisms are able to deposit almost pure, amorphousquartz glass at ambient, physiological conditions from monomericsilicate (see Perry, 2003). There are two mechanisms by which bio-silica is formed, first by oversaturation and second by enzymaticsynthesis. Oversaturation of mono-/oligo-silica results inpolycondensation at concentrations above 100mmoll–1 at neutral/physiological pH and body temperature (Iler, 1979; Benning et al.,2005). The rate of aggregation/polycondensation from themonomeric silica increases with temperature at an activation energyof approximately 10kcal/mol (Iler, 1979), a value that is about halfof the average activation energy required for the breaking of anaverage covalent bond (Porter et al., 2009). In the second strategy,the silica-depositing organisms use an enzyme, silicatein, to lower
SUMMARYLoricate choanoflagellates (unicellular, eukaryotic flagellates; phylum Choanozoa) synthesize a basket-like siliceous loricareinforced by costal strips (diameter of approximately 100nm and length of 3m). In the present study, the composition of thesesiliceous costal strips is described, using Stephanoeca diplocostata as a model. Analyses by energy-dispersive X-rayspectroscopy (EDX), coupled with transmission electron microscopy (TEM), indicate that the costal strips comprise inorganic andorganic components. The organic, proteinaceous scaffold contained one major polypeptide of mass 14kDa that reacted withwheat germ agglutinin. Polyclonal antibodies were raised that allowed mapping of the proteinaceous scaffold, the (glyco)proteins,within the costal strips. Subsequent in vitro studies revealed that the organic scaffold of the costal strips stimulatespolycondensation of ortho-silicic acid in a concentration- and pH-dependent way. Taken together, the data gathered indicate thatthe siliceous costal strips are formed around a proteinaceous scaffold that supports and maintains biosilicification. A scheme isgiven that outlines that the organic template guides both the axial and the lateral growth of the strips.
the activation energy required for lower silica concentrations todeposit monomers and oligomers to polymerize amorphous silica(Cha et al., 1999; Krasko et al., 2000). This enzyme shows an affinityconstant (Km value) to the mono-/oligomeric substrate ofapproximately 50moll–1 (Müller et al., 2008b), allowing thepolymerization to proceed at environmental concentrations that inthe sea amount to approximately 5moll–1 (Maldonado et al., 1999).It can be postulated that bio-silica deposition, irrespective of its wayof formation, non-enzymatically or enzymatically, is facilitated ifthe guiding organic template remains surrounded by the inorganicpolymer formed. This assumption stems from the observations that,at interfaces between two phases, sudden and considerable changesof the apparent activation energies occur (Wynn-Williams, 1976;Ben-Shooshan et al., 2002). Until now, only from the spicules ofthe siliceous sponges has a protein-bio-silica hybrid been described(Müller et al., 2008a; Müller et al., 2008c). In sponge spicules, thishybrid composition provides them with unusually high toughnesscombined with extreme flexibility, a feature that pushed sponge bio-silica to the forefront of material sciences (Mayer, 2005) (reviewedin Schröder et al., 2008).
In the present study, the inorganic, siliceous skeletal frameworkof choanoflagellates was studied with the aim of clarifying whethertheir siliceous structures are composed of an inorganic [siliceous]:organic [proteinaceous] composite, as well. Choanoflagellates[phylum Choanozoa (Shalchian-Tabrizi et al., 2008)] are globallydistributed free-living unicellular or colonial flagellate eukaryotesliving in marine and freshwater environments (Thomsen and Larsen,1992; Buck and Garrison, 1988). The choanoflagellates aresubdivided into three families (based on the composition andexistence and/or structure of the extracellular matrix, the periplast)the: Codonosigidae (lacking any periplast), Salpingoecidae(encased/coated into a firm theca composed of organic material),and Acanthoecidae (captured and protected by a basket-like lorica).The lorica comprises siliceous ribs or ‘costae’, termed costal strips.After being taken up by the unicellular eukaryotes, the ortho-silicateis deposited in the fibril-shaped siliceous strips, with a diameter ofapproximately 100nm (Leadbeater, 1989; Leadbeater et al., 2008).Initially, the siliceous strips are formed intracellularly in membrane-sealed vesicles that are located within the peripheral cytoplasm(Leadbeater, 1989). Those strip-containing vesicles are alwaysassociated with the membranes of the Golgi apparatus (Arndt et al.,2000). Once the strips are developed, they are released from thecells, stored at first in the top of the collar, from where they aretaken for the assembly of the lorica; this organelle comprises a two-layer arrangement and is pieced together within a few minutes whilethe cells undergo a rotational movement (Buck and Garrison, 1988;Leadbeater, 1979a; Leadbeater, 1979b; Leadbeater et al., 2008;Leadbeater et al., 2009). Initial observations suggest that aconnection between the strips exists that might contain some kindof organic connective material (Mann and Williams, 1983).
Until now, no biochemical evidence has been presented that couldindicate that organic substances are located within the different zonesof the siliceous cylinder of costal strips. To test this issue, we usedthe choanoflagellate Stephanoeca diplocostata as a model organism.The unicellular S. diplocostata are free-living flagellates that arecommon in coastal water. They take up free silica from theenvironment and use it as a building block for the synthesis of thelorica; their culture conditions are well defined, especially withrespect to the formation of the lorica (Leadbeater, 1979a; Leadbeater,1979b; Leadbeater and Davies, 1984; Leadbeater and Jones, 1984;Leadbeater, 1985; Leadbeater, 1989; Leadbeater et al., 2009). Inthe present study, we outline the ultrastructure of the siliceous strips
and demonstrate the existence of organic components within thestrips. The respective proteins were isolated from costal strips andtheir distribution within those strips was mapped using theimmunogold labeling technique. Moreover, in vitro silicaprecipitation experiments were performed that led to the conclusionthat the organic components exert a silica-inductive effect, resultingin bio-silica polycondensation.
MATERIALS AND METHODSMaterials
The choanoflagellate Stephanoeca diplocostata Ellis was obtainedfrom the ATCC (ATCC50456; Manassas; VA, USA). The followingmaterials were purchased: Percoll, polyvinylpyrrolidone (Mr
360,000), biotinylated wheat germ agglutinin, alkaline phosphataseconjugated avidin, goat-anti-mouse serum coupled to 5nm goldparticles, goat-anti-mouse IgG-conjugated alkaline phosphatase andtetraethylorthosilicate from Sigma-Aldrich (Taufkirchen orSteinheim; Germany); blocking reagent and BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/4-nitro-blue tetrazolium chloride)were from Roche (Mannheim, Germany); peptone and yeast extractwere from Roth (Karlsruhe; Germany). Culture flasks were obtainedfrom Greiner Bio-one (Frickenhausen, Germany). Artificial seawaterwas prepared according to the recipe described by Harrison andcolleagues (Harrison et al., 1980) and autoclaved.
Cell cultureThe choanoflagellate S. diplocostata cells were grown in anartificial-seawater-based medium, supplemented with bacteria(Klebsiella pneumoniae subsp. pneumoniae), at 16°C as described(Harrison et al., 1980; Leadbeater and Davies, 1984; Leadbeaterand Jones, 1984; Leadbeater, 1985). The medium for thisheterotrophic culture comprised 800mg/l peptone, 400mg/l yeastextract, 1% wheatgrass extract and enrichment solution (55.3mgNa2EDTA.2H2O, 466.7mg NaNO3, 300mg Na2SiO3.9H2O, 66.7mg-glycerophosphate Na2 salt, 38mg H3BO3, 0.14mg CoCl2.6H2O,1.5mg ferric citrate, 5.4mg MnSO4.4H2O, 0.73mg ZnSO4.7H2Oand 100ml H2O; pH 7.4). The wheatgrass extract was prepared asproposed by the ATCC, with some modifications. Ten grams ofwheatgrass (Bienenschwarmmm; Emmerich, Germany) were boiledin 1l artificial seawater for 5min; after cooling, the extracts werepassed through a 0.2m filter (Nalgene; Rochester, NY, USA).
Cell harvest and isolation of siliceous costal stripsCultures were grown for 14 days in T-500 flasks (Greiner Bio-one;Frickenhausen, Germany) and then centrifuged at 2000g (15min)to collect the cell pellets. Percoll discontinuous density centrifugationwas used (Gong et al., 2008a) to separate the S. diplocostata cellsfrom the bacteria. In detail, decreasing concentrations of Percoll,60%–40%–15%, were layered on top of each other in a 15ml tube.After centrifugation (2000g; 16°C; 40min), the interface betweenthe 40% and 60% layers was collected by aspiration. The cells werewashed twice in artificial seawater by centrifugation (2000g;10min) and then stored as a pellet at –20°C until the isolation ofthe siliceous costal strips.
For the isolation of the costal strips, a total of approximately 4g(wet mass) of choanoflagellates was broken up by osmotic shockin distilled water. The cell particles were collected (2000g; 15min)and washed twice in distilled water. The resulting pellets containingmost siliceous costal strips and some possible cell debris werecleaned by 4% NaDodSO4 water solution for 24h, or washed byconcentrated sulfuric acid and nitric acid (v/v, 4:1) for 1h to removepossible organic substances mixed in the isolated costal strip
N. Gong and others
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(Schmid, 1980). The pellets thus obtained were thoroughly washedby water and kept at 4°C for further experiments.
Demineralization of siliceous costal stripsTwo protocols were used for demineralization of the costal strips(‘costal strip’ fraction). First, the aliquot (ca. 200mg) was treatedwith buffered fluoric acid [hydrofluoric acid (HF)/0.5 M ammoniumfluoride (NH4F); pH 5], as described by Shimizu and colleagues(Shimizu et al., 1998). By this procedure, a completedemineralization of the bio-silica structure could be expected. Theexposure time to buffered fluoric acid was 20min at 24°C.Subsequently, the material was transferred into dialysis tubes(Spectrum Laboratories; CA, USA) with a molecular mass cut-offof 1000 and then dialyzed against distilled water to remove fluoricacid. Finally, the sample was dialyzed twice against phosphate-buffered saline. The solid HF-insoluble material (ca. 20mg) wascollected by centrifugation (2400g; 10min). Separately, the ‘costalstrip’ fraction was resuspended in 1% SDS (200l) and incubatedfor 30min at 95°C. After centrifugation, the resulting supernatantincluding the total proteins from costal strips was collected andstored at –20°C. The pellets were thoroughly rinsed with water. Thesamples were used for EDX-analysis.
A second protocol was applied to partially demineralize the costalstrips. The ‘costal strip-fraction’ was transferred to the 0.2moll–1
NaOH/Na2PO4 buffer (pH 10.0 or 12.0), and the costal strips weretreated for 2days at 24°C; these conditions are known to partiallydissolve amorphous silica (van Dokkum et al., 2004). Aftertreatment, those partially demineralized strips were collected bycentrifugation (2400g; 10min), suspended in 400l distilled waterand then used in the silica precipitation assays.
Transmission electron microscopy and energy-dispersiveX-ray spectrometry (EDX)
For transmission electron microscopy (TEM) observation, samplesincluding the intact siliceous costal strips as well as the partially tocompletely demineralized strips were dehydrated in an ascendingalcohol series (incubated for 5-min periods in ethanol: 30%, 50%,70%, 80%, 96% and, finally, twice in 100% ethanol). Then, onedrop of the suspension was placed onto a formvar carbon-coatedcopper grid and left to dry on filter paper in air at room temperature.Morphological studies were performed by Philips EM420. Line-scan and spot EDX were performed in scanning transmissionelectron microscopy (STEM) mode by a FEI Tecnai F30 equippedwith an energy-dispersive X-ray spectrometer (Mugnaioli et al.,2009).
SDS-PAGE and immunoblot assayAnalysis by sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE), using a 4–15% discontinuouspolyacrylamide gel, was performed as described previously(Laemmli, 1970). After running, the gels were stained withCoomassie brilliant blue.
The proteins were electrophoretically transferred topolyvinyldifluoride membranes (PVDF; Millipore, MA; USA)using a Trans-Blot SD Semi-dry Transfer Cell (Bio-Rad; HerculesCA, USA). The membranes were blocked with 5% skimmed milkand incubated with primary antibody [diluted 1:2000 fold in 3%BSA (bovine serum albumin), PBS (phosphate buffered saline)] for2h. The membranes were washed with PBST (PBS, supplementedwith 0.05% Tween 20) and incubated with goat-anti-mouse IgG-conjugated alkaline phosphatase (Sigma-Aldrich, Steinheim;Germany); detection was carried out using BCIP/NBT (5-bromo-
4-chloro-3-indolyl-phosphate/nitro blue tetrazolium chloride)solution (Roche) (Gong et al., 2008b).
Wheat germ agglutinin labelingAfter size separation by SDS-PAGE, the proteins were transferredonto PVDF and blocked in 2% (w/v) polyvinylpyrrolidone-360 in50mmoll–1 Tris-HCl buffer (pH 7.5; 0.5 moll–1 NaCl) overnight at4°C. The membranes were transferred into a 2gml–1 biotinylatedwheat germ agglutinin (WGA) solution (Tritium vulgaris) and thenincubated for 2h at 24°C. Finally, the membranes were rinsed threetimes (10min each) in 50mmoll–1 Tris-HCl (pH 7.5; 0.5moll–1
NaCl, 0.1% Triton X-100 [v/v]), followed by an incubation withalkaline-phosphatase-conjugated avidin at a dilution of 5�10–3 for1h at 24°C. The lectin-biotin complexes were visualized with thephosphatase substrate BCIP/NBT (Bédouet et al., 2001).
Immunogold labeling of costal stripsPolyclonal antibodies were raised in one BALB/C mouse againstthe protein band with the apparent molecular mass of 14kDaobtained by loading SDS-PAGE gels with costal strip proteins. Theantiserum, termed Poly-anti-S14, was achieved after three roundsof immunization during a 1.5month period and the titer (1:10,000)was measured by ELISA assay (Mayer and Walker, 1987). Poly-anti-S14 was diluted 2000 fold and then applied for immunogoldlabeling. Three different samples of costal strips were analyzed. First,freshly isolated costal strips; second, costal strips incubated in waterfor 40days; third, costal strips treated with alkaline solution (0.2 MNaOH/Na2PO4; pH 12.0) for 2days.
Before labeling, the costal strips were blocked in 1% westernblocking reagent (Roche, Mannheim, Germany) in 3% BSA/PBSsolution overnight at 4°C and subsequently incubated with eitherPoly-anti-S14 or with preimmune serum, obtained from the sameanimal used for immunization, for 2h at room temperature. Thesamples were then incubated with a 20-fold diluted goat-anti-mouseserum coupled to 5nm gold particles. After each step of incubation,the samples were washed three times with PBST for 5min. Finally,the samples were fixed in 2.5% glutaraldehyde for 15min, washedextensively with distilled water and dehydrated in the ascendingalcohol series and then inspected by Philips EM420 (Gong et al.,2008b).
Silica precipitation assayA solution of orthosilicic acid was freshly prepared by dissolvingtetraethylorthosilicate (TEOS) in 1mmoll–1 HCl to a finalconcentration of 0.9moll–1. The silica precipitation assay wasperformed in a final volume of 190l and composed of one of thetwo buffers, either of a 100mmoll–1 sodium acetate buffer (HAc-NaAc) [acetic acid (HAc), buffered with 1 moll–1 NaOH to pH 5.0,5.5, 6.0 or 6.5] or 100mmoll–1 Hepes buffer (buffered with NaOHto pH 7.0 and 7.5). Routinely, the assays were performed at pH 6.0,a value that had been found to promote optimally the reaction. Thesamples (10l), containing either untreated strips or partiallydemineralized ones, were suspended in water. 10l of the suspendedsamples were transferred into the 190l buffer and thensupplemented with 10l of orthosilicic acid/TEOS solution. Thefinal concentration of silicic acid was adjusted to 40mmoll–1 or9mmoll–1, respectively. The periods of incubation (24°C) were10min and 30min, respectively. Next, the samples were centrifuged(12,000g; 5min) and the pellets were washed twice with distilledwater to remove free silicic acid. The pellets thus obtained wereresuspended in 200l of 1moll–1 NaOH (95°C; 30min) to formmonomeric silica. Silicic acid concentrations were quantitatively
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determined in these solutions with -silicomolybdate using thesilicate colorimetric kit from Merck (Darmstadt, Germany),according to the manufacturer’s instructions (Iler, 1979; Krasko etal., 2002). The OD650nm values were determined, and theconcentration of silicic acid was calculated on the basis of a linearcalibration curve obtained with orthosilicic acid.
A further analytical methodFor protein quantifications, the Bradford method (Compton andJones, 1985) (Roti-Quant solution–Roth) was used.
RESULTSStructural characterization of lorica and costal strips of
S. diplocostataThe cells of S. diplocostata are characterized by their basket-likelorica (Fig.1), which surrounds one single flagellum (Fig.1A,B).The choanoflagellates contain longitudinal as well as vertical costalstrips that are woven into a basket, the lorica (Fig.1B). To obtainsufficient starting material for the isolation of the costal strips (4gof wet mass), cells from 5l cultures were collected. Subsequently,the choanoflagellates were separated from the bacteria bydiscontinuous Percoll density gradient centrifugation. The S.diplocostata cells were collected within the 40 and 60% Percolllayers and then used for the analysis.
The lorica of S. diplocostata was separated from the protoplastby osmotic shock treatment in distilled water. An intact lorica(Fig.2A) is built of longitudinal and transverse costal strips, withdiameters of >75nm (Fig.2B,C); the length of the strips isapproximately 3m. TEM observation revealed that large rod-likestrips forming the base and also the longitudinal basket-like loricafunction as a bracket-like support. The smaller strips (diameter<100nm) are additionally used in the other parts of this basket,especially at the fringe and the intervening space. It appears thatthe links of the costal strips are reinforced by a connective-likesubstance (Fig.2B,C), which can be removed by SDS.
TEM inspection showed that the costal strips have a markedtexture reflecting distinct structures. Depending on their size, thecostal strips can be grouped into large costal strips, with a diameterof >100nm (Fig.2C,D), and smaller strips, with a diameter of<100nm (Fig.2C,E). The larger strips appear as compact rods witha uniform texture, whereas the smaller strips show a less compacttexture and display, especially in the central region, a fluffyappearance. Common to both types of costal strips is the presenceof one tubular-like structure in the central, electron-lucent regionof each strip. In contrast to the center region, the periphery of thestrips is more electron dense (Fig.2D,E). It is striking that especially
the smaller costal strips have a wider electron-lucent central region(diameter of approximately 10nm) than seen in larger rods (diameterof approximately 2nm) (Fig.2E,F). Moreover, another distinctivestructure of smaller costal strips has been observed under the high-resolution of TEM. The outer edge is the most electron-lucent region,which makes it look like a capsule surrounding the surface of thestrips (Fig.3B1).
Element determination by EDX spot analysis showed that costalstrips (Fig.3A1,B1) comprise only silicon and oxygen (Fig.3A2,B2).The copper peaks came from the grids. Furthermore, line EDX scanswere used to measure silicon and oxygen concentrations across alarge and a small costal strip. EDX analysis indicated that the outercapsule-like structure contains the least silicon (Fig.3B3, indicatedby arrow). As the probing continued, the silicon and the oxygenconcentrations increased from the peripheral to the central regionuntil the electron-lucent region was reached. The siliconconcentration in the electron-lucent region was lowest; the oxygenlevels were more uniform throughout a strip (indicated byarrowheads; Fig.3A3, Fig.3B3). These findings revealed that costalstrips have no homogeneous composition; the two distinctivestructures, an outer capsule-like structure and an electron-lucentcentral region, contain lower levels of silicon than the remainingregions. Furthermore, some observations led to the assumption thatthe central region might be filled by filament-like substances. Thebroken costal strips displayed a filament-like structure in the centralcanal (Fig.2B). Even more supportive was the observation that afilament protruded from the central canal (Fig.2E). Frequently, thosefilaments link individual costal strips together, and they can beremoved by SDS. Taken together, we presume that the central silica-poorer region might not be hollow but contain organic substances,besides silica.
Isolation of costal strips and complete demineralization withhydrofluoric acid
In order to obtain pure siliceous costal strips not associated withsurface-bound organic material, 4% SDS was used for 24h to freeisolated lorica from organic materials. The obtained crescent-shaped rods with a length of approximately 3m were used fordemineralization experiments (Fig.4A,B).
For the analysis of the possible organic scaffold in the costal strips,the isolated strips were further cleaned by concentrated sulfuric acidand nitric acid (4:1 v/v) for 1h. After thoroughly washing with water,the obtained costal strips were treated with buffered HF to removethe silica phase. The mass of costal strips was determined beforeand after HF treatment. Approximately 10% of the initial massremained in the insoluble form after HF treatment; no protein was
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Fig.1. The choanoflagellate Stephanoecadiplocostata. (A)Light-microscopic image ofloricates (l) from living cells. (B)TEM picture of alorica and the encaged protoplast (p). The loricasurrounds one flagellum (f).
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detected in the HF-soluble phase after dissolution of the costal strips,checked by SDS-PAGE.
As a first attempt to analyze the chemical nature of the insolubleresidues after HF treatment, the ‘hydrofluoric acid-insoluble’ (HF-IS) fraction was analyzed by EDX. The spectrum obtained from theHF-IS sample showed a prominent peak for sulfur, as well as forphosphorus (Fig.5A), supporting the conclusion that the scaffold
comprises organic material. The HF-IS sample was subsequentlytreated with 1% SDS (95°C; 30min), and the remaining insolublematerial was subjected to EDX analysis. The spectrum obtainedshowed distinct peaks for sulfur and copper, whereas the peak forphosphorus was insignificant (Fig.5B). The copper peaks came fromthe support grid, and calcium is believed to originate from distilledwater.
Fig.2. Lorica and costal strips from S. diplocostata; TEM images. (A)An intact basket-like lorica displaying arrays of costal strips that can be grouped intolarge and small strips. The large, longitudinal costal strips form the base and the strutting of the basket, whereas the small strips fringe and interlink thelarge strips. (B,C) Higher magnifications, showing the two kinds of costal strips, based on their sizes and textures. The large strips (ls) with a diameter of>100nm are more compact than small strips (ss) of a size of <100nm. Frequently, connective substance (cs) exists at the joints of individual strips. Withinthe bio-silica strips, (non-siliceous) filaments (fi) exist. (D)Tubular, canal-like structures (c) exist within the costal strips and harbor the filaments.(E)Protrusion of a filament (fi) from a canal of a costal strip. (F)Cross-section through costal strips, displaying the electron-lucent central canal (c).
Fig.3. Element analysis of costalstrips. (A)Large costal strips and(B) small costal strips.(A1,B1) TEM images. (A2,B2) EDXspectra of both kinds of costal stripsdisplaying the major peaks for thetwo elements silicon (Si) andoxygen (O); copper (Cu) is lessprominent. (A3,B3) EDX linescanning for silicon and oxygenacross a large and a small costalstrip. A capsule-like structure isobserved in the outer-edge of smallcostal strips, in which the siliconconcentration is the lowest (arrow).Silicon and the oxygenconcentrations are highest aroundthe central lucent region, comparedwith the more peripheral zone. Inthe center regions, especially forthe silicon levels, a small, butdistinct, gouge is seen (arrowhead).
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Partial demineralization of costal strips in alkaline solutionBesides the method to demineralize silica skeletal elements withfluoric acid, a second protocol that was based on alkaline treatmentwas applied to achieve (partial) dissolution of silica (van Dokkumet al., 2004). Therefore, the SDS-cleaned ‘costal strip’ fraction wastreated with pH 10.0 or 12.0 NaOH-Na2HPO4 buffer as describedin the Materials and methods. Compared with the intact costal strips(Fig.4B), the costal strips after 2 days of alkaline treatment losttheir smooth surfaces, converted to blurry structures and released,to some extent, the tattered (organic) matrix (Fig.4C). In addition,those flocculated membranous materials remained attached to thepartially demineralized costal strips.
SDS-PAGE analysis of proteinaceous scaffold in the costalstrips
The costal strips after demineralized in pH 12.0 NaOH-Na2HPO4
were extracted with either 8moll–1 urea (pH 7.0) or HAc-NaAc(pH 5.0); likewise, an equal amount of untreated costal strips wasextracted in the same way. The supernatant solutions were analyzedby SDS-PAGE and the gel was stained with Coomassie brilliantblue (Fig.6A). The gel loaded with the 8moll–1 urea extract ofpH12.0 solution-treated strips showed a band with an apparentmolecular mass of 14kDa (Fig.6A, lane c), whereas the band wasabsent in the extracts treated by HAc-NaAc (pH5.0) or 1% HAc(Fig.6A, lanesd and e). Likewise this band was also absent from8moll–1 urea extract of the untreated costal strips (Fig.6A, lanesaand b). These results showed that the proteins only came from alkali-treated costal strips, but not from intact costal strips. 8moll–1 ureacan solubilize these proteins, but neutral or acid solution can not.Meanwhile, the supernatant was also examined after costal stripswere treated with alkaline solution, and no protein was detected
even in the concentrated sample (concentrated by 10-fold; Fig.6A,lane f).
Further experiments were used to test the effects of alkalinesolutions with different pH on the costal strips. The gel loaded withthe 8moll–1 urea extract from pH12.0 alkali-treated costal strips(Fig.6B, lane c and d) showed a denser band at 14kDa than thatthe one from pH10.0 alkali-treated costal strips (Fig.6B, lanesa andb). This result confirmed that an alkaline solution with higher pHcan release more proteins from costal strips, which correlates withthe increasingly pH-dependent demineralization level of amorphoussilica (van Dokkum et al., 2004). The isolated 14kDa protein wasnamed S14.
Wheat germ agglutinin labeling revealed highly positive stainingof S14 in the probing of SDS-PAGE gels, indicating that thisprotein is highly glycosylated (Fig.7, lanesa and b). Furthermore,this protein was sized-separated in a SDS-PAGE gel, and the bandat 14kDa was cut out, ground and suspended in PBS forimmunization of mice to raise polyclonal antibodies. Thespecificity of the achieved antibodies was proved byimmunoblotting assay, and the band at 14kDa was prominentlyrecognized (Fig.7, lane c). A faint band at approximately 28kDawas also recognized. It might be suggested that the band at 28kDarepresents a dimer of the 14kDa monomer. Therefore, theantibodies, named with Poly-anti-S14, were used to map thedistribution of S14 in the costal strips.
Immunogold labeling of protein composition in partiallydemineralized costal strips
For this experiment, three kinds of costal strip preparations wereused for immunogold labeling studies using the polyclonalantibodies Poly-anti-S14 (Fig.8). First, newly isolated costal strips
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Fig.4. Organic scaffold within SDS-exposed costal strips. (A)Light and (B) TEM image of SDS-pretreated costal strips. (C)Treatment of costal strips at highalkalinity (pH 12.0) for 48h. Membranous masses (me) are released from the strips.
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Fig.5. EDX elemental analysis of insoluble residues fromcostal strips cleaned by 4% SDS and mixed concentratedsulfuric acid and nitric acid (v/v, 4:1). (A)Spectrum from theHF-insoluble material. The residue comprises little silicon,but high sulfur as well as phosphorus. (B)The HF-insolublematerial was additionally treated with SDS, and theremaining insoluble residues were analyzed. The spectrum,taken under identical apparatus settings, shows still adistinct peak for sulfur but no phosphorus.
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(Fig.8A); second, costal strips that had been incubated in water for40days (Fig.8B); and, third, alkali-treated costal strips (Fig.8C).
Colloidal gold particles (attached to the secondary antibodies)were used for visualization of the protein S14 and detecting itsdistribution within the costal strips. When newly isolated costal stripswere incubated with Poly-anti-S14, sparse immunocomplexes couldbe identified with gold labeled secondary antibodies on the surfaceof costal strips (Fig.8A). The images from the negative controls(treated with pre-immune serum; Fig.8A-pre) indicated thereliability of the labeling. By contrast, for costal strips that had beenstored for 40days in distilled water, immunogold particles could bedetected in the samples treated with Poly-anti-S14 (Fig.8B-ab),whereas those immunocomplexes were absent in controls (Fig.8B-pre). The complexes accumulated especially on the surfaces of thestrips. The extent of immunogold complexes was drasticallyincreased when the costal strips were treated with alkali (pH12;2days, 24°C). Those strips exposed on their surfaces large numbersof proteins that could be recognized by Poly-anti-S14 and visualizedby colloidal gold particles (Fig.8C-ab-1, 2 and 3). The imagesrevealed that the colloidal gold particles were clustered on the etchedsurfaces of costal strips, suggesting a possible organic scaffoldembedded within a silica matrix. Once the silica structure waspartially dissolved, the embedded organic scaffold could be exposed,approached by the antibodies and labeled by the colloid goldparticles. In the parallel control, no immunoreactions could be seen(Fig.8C-pre), suggesting the good reliability of the positive labeling.
Silica precipitation onto partially demineralized costal stripsFor the above outlined data, it can be summarized that the costalstrips are traversed by a proteinaceous scaffold that is exposed afteralkali treatment. In order to test whether an organic scaffold canaccelerate the silica deposition, partially demineralized strips wereincubated with orthosilicate. The strips were treated for 4days in abuffer of pH12 (see Materials and methods), a period during which
the majority of the strips lost their silica (Fig.9). Those sampleswere incubated with orthosilicate (9mmoll–1 and 40mmoll–1). Afterincubation, the newly polycondensated silica, as well as the residualsiliceous matrix from the strips, was collected by centrifugation.Subsequently, the poly(silicate) was quantified by using the -silicomolybdate assay (after solubilization with NaOH). Theexperiments revealed that, in assays without additional orthosilicate,the level of poly(silicate) in the partially demineralized strips waslow and amounts to 0.025 OD650nm units, whereas the concentrationof poly(silicate) was – as expected – in the intact strips much higher(0.22OD650nm units) (Fig.9). The concentration of poly(silicate) inthe assays with partially demineralized strips strongly increasedconcentration dependently from 0.12OD650nm units (9mmoll–1
orthosilicate) to 0.4OD650nm units in the presence of 40mmoll–1
orthosilicate. In parallel, the concentration of poly(silicate) did notchange significantly in assays containing non-treated strips, and thevalues remained at OD650nm units of 0.24 to 0.26. Moreover, thereaction of poly(silicate) deposition at partially demineralized costalstrips was pH dependent. The maximal newly formed poly(silicate)was seen in reactions at a pH value of 6.0–6.5 (Fig.10).
The newly formed poly(silicate) in assays comprising partiallydemineralized strips could be visualized by TEM inspection. In theassays supplemented with 9mmoll–1 orthosilicate, the organiclobate scaffold became densely associated with strings of beads,which we interpreted as silica particles (Fig.11B). In control assayslacking orthosilicate, none of those structures is seen (Fig.11A).
DISCUSSIONIt is a challenge for the future to disclose the organic scaffolds inbiominerals at the chemical/biochemical level and to understand theirroles in the establishment of the individual morphologies of theinorganic matrixes, which are usually highly elaborated andcomplex. Hence, a first task is to identify the organic scaffold inany kind of inorganic skeletons in organisms. For a few organisms,
Fig.6. SDS-PAGE analysis of extracts from non-treated or from alkali-treated costal strips. (A)Lanes a and b, non-treated (nt) costal strips: theyhad been extracted with either 1% HAc or with 8mol l–1 urea; no bandsbecame visible. Lanes c–e, pH12.0 alkali-treated costal strips (12.0), hadbeen extracted (i) with 8mol l–1 urea, (ii) with HAc-NaAc (pH5.0) or (iii) with1% HAc. After size separation of the extracts, only in the 8mol l–1 urea-extracts could a 14kDa band be visualized (lane c). Lane f, a concentratedsupernatant from costal strips, treated at pH12.0; no band could bedetected. (B)Lanes a and b, pH10.0 alkali-treated costal strips (10.0); theyhad been extracted with (i) 8mol l–1 urea or (ii) 1% SDS; only very faintbands at 14kDa had been visualized. Lanes c and d, pH12.0 alkali-treatedcostal strips (12.0); they had been extracted with either 8mol l–1 urea or 1%SDS. Strong bands at a mass of 14kDa are resolved. M, protein markers(kDa).
Fig.7. Proteinaceous scaffold within costal strips. (Lane a) SDS-PAGEanalysis of the total protein material from demineralized costal strips. Thegel was stained for Coomassie brilliant blue. (Lane b) Probing forglycoprotein(s) in the total scaffold of the strips. After size separation andblot transfer, the proteins were reacted with the WGA agglutinin.(Lane c) Immunoblotting of the total protein material using polyclonalantibodies raised against the 14 kDa protein. This 14 kDa protein (markedwith a short horizontal line) is prominent in all three gels/blots. (Lane d) asa control to the blot shown in lane c, the filters were reacted with the pre-immune serum after size separation and blot transfer. M, protein markers(kDa).
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the structure and the biochemical properties of the organic scaffoldhave been thoroughly studied. From the two taxa the diatoms andthe siliceous sponges, the importance of the organic templates forthe formation of their biomineralic skeletons has been wellelucidated. The ornate and elaborated diatom frustules are formedonto organic templates (Kröger et al., 1994; Kröger et al., 1997;Kröger et al., 1999; Sumper and Brunner, 2008; Hildebrand et al.,2008), the biosilica-associated peptides (silaffins) and the long-chainpolyamines (see Poulsen et al., 2003). Furthermore, it was foundthat those organic molecules accelerate silica formation from a silicicacid solution in vitro. In addition, it had been speculated that thesilica deposition vesicles contain a matrix of organic macromoleculesthat not only regulate silica formation but also act as templates tomediate biosilica nanopatterning (Robinson and Sullivan, 1987;Kröger et al., 1999). The understanding of the formation of spiculesfrom siliceous sponges has been considerably pushed ahead by thefinding that the enzyme silicatein mediates the formation of thepoly(silicate) (Cha et al., 1999; Krasko et al., 2000; Müller et al.,2008b) and also by the experimentally based finding that this proteinremains entrapped in the poly(silicate) product (Müller et al., 2008a;Müller et al., 2009). Hence, the sponge siliceous spicules representintriguing and fascinating model systems allowing investigationsand understanding of biomineral formation from the genomic levelto morphological realization.
A third taxon of organisms forming a silica skeleton, termed silicalorica, are the choanoflagellates. While the genome structure andorganization of those protozoans are well established (Carr et al.,2008; Abedin and King, 2008; King et al., 2008), the nature of theorganic scaffold for the formation of their siliceous structuralelements is not well understood. Based on the pioneering studiesof Mann and Williams (Mann and Williams, 1983) and the detailedstructural and biomechanical investigations of Leadbeater(Leadbeater, 1979a; Leadbeater, 1979b), it could be postulated that
the siliceous loricae with their costal strips are formed along aninterior or exterior organic scaffold. Otherwise the morphology andfunctional interactions of their silica structures cannot be understood.Silica is taken up by the choanoflagellates from the aqueousenvironment very likely by an energy-consuming pumping systemand then stored in special organelles, membrane-enveloped silica-deposition vesicles (SDVs) (Leadbeater, 1979a; Mann and Williams,1983; Leadbeater 1989; Preisig, 1994). Within the cells, the SDVs
N. Gong and others
Fig.8. Localization of protein S14 in costal strips by immunogold labeling. Newly isolated costal strips (A), strips incubated in distilled water for 40 days (B),or strips treated in alkaline solution for 2 days (C), were used for the studies. Control specimens (A-pre to C-pre), treated with preimmune mouse serum andcoupled goat anti-mouse antibodies with gold particles (5nm). In parallel (A-ab to C-ab), the samples were treated with mouse anti-S14 protein antibodies,and then with gold-labeled goat anti-mouse antibodies (5nm). The thus-treated strips were documented at different magnifications (C-ab-1 to C-ab-3).
Without OS
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Fig.9. Silica precipitation on costal strips. For the studies, either partiallydemineralized costal strips (dem) or non-treated costal strips (nt) wereincubated in the absence (‘without’) or presence of 9mmoll–1 or 40mmoll–1
ortho-silicic acid (OS), prepared from TEOS, for 30min or 10min. Theamount of silica precipitated on the costal strips was quantified by the -silicomolybdate assay, as described in the Materials and methods. Thenewly formed bio-silica is indicated in brown, in the assays comprisingdemineralized or non-treated strips.
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are closely associated with the Golgi apparatus and the endoplasmicreticulum, suggesting that there silica formation around the costalstrips proceeds in an intimate neighborhood of a protein-processingmachinery. However, before the present study, only first suggestions(Mann and Williams, 1983) but no clear evidence has beendocumented that the process of strip formation and, in turn, theirsilicification require the presence of a proteinaceous scaffold.
In a first series of experiments, the morphology of the costal stripsis described. We have classified these skeletal elements, buildingthe lorica, into the group of large costal strips (diameter >100nm)and small strips (<100nm), according to their sizes. Both classesof strips disclose a central, electron-lucent canal. It is striking thatthe diameters of these central canals are wider in the smaller strips(diameter approximately 10nm) than in the larger strips (diameterapproximately 2nm). At present, we postulate that the canals in theprimordial strips are wider and become smaller during the ageingand maturation process. This process is difficult to studyexperimentally as the maturation of the strips proceeds rapidly inSDVs in the short period of approximately 12min (Leadbeater,1979a; Leadbeater, 1979b). Cross-sections through the costal strips,as well as breaking studies, supported electron microscopically thatthe central cylinder of the strips is made by a material, differentfrom silica, that is organic in nature. The TEM images showed that,from the center of the strips, filament-like structures protrude, whichmight suggest that those organic structures might be involved in thelongitudinal organization of the strips. The subsequent EDXanalyses, line scanning, supported this assumption by the findingthat the center of the strips is poorer in silica. Again, it should behighlighted that the difference in the chemical composition, adecrease/lowering of the silica concentration in the center of thestrips, is much more pronounced in the smaller strips, comparedwith the larger ones.
Another distinctive structure of the smaller costal strips is thecapsule-like outer edge, which contains least silica and thereforeis distinguished from the inner region – although this structure isnot significant in the large costal strips. It can be proposed that thecapsule-like edge is the growing front of costal strips, followed bya scaffold that has been prebuilt on the surface of costal strips. Thisscaffold is supposed to support the precipitation of silica particles.
With time, additional silica is deposited onto those surfaces,allowing a growth of the costal strips and also a reinforcement ofthe silica materials due to an increased density of this inorganicpolymer. Gentle treatment with alkali (pH 10.0 and 12.0) had beenshown to dissolve the outer edge of the strips and resulted in therelease of a fluffy matrix. This observation can be taken as furtherevidence that a possible organic scaffold exists within the costalstrips. Moreover, our EDX analysis revealed that also otherelements, such as sulfur and phosphorus, exist within the residualmaterials of siliceous strips after complete demineralization. Wealso show that, after treatment of the strips with 1% SDS and byheat, these elements, including also phosphorous, are absent. Byanalogy to the silaffins from diatoms (Kröger et al., 2002), weassume that the organic matrix within the costal strips might bephosphorylated.
SDS-PAGE analyses revealed that the organic scaffold iscomposed of proteins (positive for Coomassie brilliant blue); and
5.5 6.0 6.5 7.0 7.50
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Fig.10. Dependence of the extent of silica precipitation onto demineralizedcostal strips as a function of the pH in the incubation assay. Theexperiments were performed with 40mmoll–1 ortho-silicic acid for 10min.The pH optimum varies from 6.0 to 6.5.
Fig.11. TEM images of silica precipitates formed in assays with partiallydemineralized strips. (A)Control strips not reacted with ortho-silicic acid.(B)Silica deposition (arrowhead) around partially demineralized costal stripsafter incubation with 9mmoll–1 ortho-silicic acid for 30min.
Organic central filament(glyco)protein complex
silica depositionProtein scaffold
Silica matrix
Hybrid: organic/inorganic
Appositional growth
Shrinking of central filament
Fig.12. Schematic outline of the appositional growth of costal strips fromthe loricate choanoflagellate S. diplocostata. A central canal, comprising anorganic filament (brown), arranges protein molecules around it (yellow) andpromotes/facilitates the deposition of silica (green). The promoting proteinsremain entrapped within the deposited poly(silicate). It is highlighted thatboth the axial and the lateral growth of the strips is guided by the organictemplate.
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the amount of released proteins relates to the extent ofdemineralization. It is known that amorphous silica is stable in waterbut increasingly dissolves as pH increases (van Dokkum et al.,2004). Our SDS-PAGE gels showed that few proteins were presentafter water treatment. More proteins were released after treatmentwith pH 12.0 alkali than following a pH 10.0 alkali treatment. Thereleased proteins are naturally in an insoluble form. Urea or SDScan effectively dissolve them, but neutral or acidic solutionscannot. As these proteins were specifically detected after silica hadbeen dissolved, we are convinced that these proteins originated fromcostal strips but not from the surrounding impurities. Furtherblotting studies and subsequent use of WGA agglutinin as a proberevealed that a glycoprotein S14 with an apparent molecular massof 14 kDa was dominant among the released proteins. Thespecificity of this plant lectin had been attributed to the sugarmoieties NeuNAc as well as GlcNAc (Kronis and Carver, 1982).Glycoproteins comprising those sugar residues occur frequently,from plants to metazoa (Karpati et al., 1999). However, this findingis noteworthy, especially in view of earlier findings that revealedthat choanoflagellates (King et al., 2003) are rich in genes codingfor sugar-binding adhesion molecules – lectins and adhesionreceptor tyrosine kinases, of unknown function. These two piecesof data might suggest that those sugar-recognizing proteins inchoanoflagellates are involved in the formation of their skeletalelement. This assumption is based on the fact that, in both classesof siliceous sponges, the Demospongiae and the Hexactinellida,the formation of the extracellular spicule is mediated by silicatein,which is spread in a galectin cylinder laterally around the growingspicules (Schröder et al., 2006; Wang et al., 2009). Future studiesmust show whether in the costal strips of S. diplocostata a galectinexists that can recognize the 14 kDa dominant glycoprotein in thestrips.
This glycoprotein S14 was separated in SDS-PAGE gels andprepared for immunization. The achieved polyclonal antibodieswere specific to recognize S14 as proven by immunoblotting.Using them as a tool, it could be demonstrated that the organicscaffold of the strips became decorated after the rods had been(partially) demineralized, whereas the intact costal strips boundwith few immuno-gold particles. These results confirmed that thecapsule-like structure of costal strips is a protein-silica hybrid, andthe protein S14 is embedded in this structure. Once silica wasdissolved, protein S14 could be detected by the antibodies. Firstsequencing studies by MALDI technique (N.G. and W.E.G.M.,unpublished observations) revealed peptides that, however, didnot match with any protein sequence deduced from thechoanoflagellate Monosiga brevicollis (King et al., 2008). Ourelectron-microscopic studies at the 5 to 20nm scale did not resultin any indication that the organic scaffold, released from the strips,originated exclusively from the central canal. It appears more likelythat the organic fibers and aggregating proteins were released fromthe entire siliceous strip.
In the last series of experiments it was tested whether the scaffoldwithin the costal strips accelerates/facilitates silica deposition. Totest this issue, the costal strips were partially de-mineralized andsubsequently exposed to orthosilicate that had been generated byhydrolysis from TEOS (Matsuoka et al., 2000). The assays todetermine the extent of silica deposition were performed at pH6.0/6.5, which had been found to be optimal for this reaction. Thedata revealed that the deposition of silica on the proteinaceousscaffold increased concentration-dependently; and the firstmeasurable silica formation could be detected with the 9mmoll–1
orthosilicate in the -silicomolybdate-based optical test, under the
incubation conditions used. Electron-microscopic inspectionsrevealed that the newly formed poly(silicate) in vitro in the presenceof the scaffold from the strips becomes associated with the organictemplate in a string-like arrangement of the silica nanoparticles.
ConclusionTaken together, the data presented in this report demonstrate thatthe costal strips are formed of hybrid silica, comprising a silicamatrix and a proteinaceous scaffold. The structural details of thecostal strips revealed that this biomineral is not a single silica rod,but has a more complex construction. The formation and thedevelopment of the costal strips are schematically outlined in Fig.12.It is sketched that, in the center of the strips, a canal exists that isfilled with an organic filament. This axis organizes the longitudinalgrowth of the strips and also facilitates the appositional growth ofthe strips with silica. During the lateral growth of the strips, theprotein molecules become associated with the central organicfilament and form a scaffold that facilitates the deposition of silica.The data gathered might indicate that, also during the lateral growthof the strips, the 14 kDa (glyco)protein plays a crucial role. Thelatter assumption arises from the fact that this protein is prevalentin the protein scaffold from costal strips.
It is clear that the cloning of the genes encoding the 14 kDa proteinwill be of prime importance for us. After having succeeded inpreparing the recombinant protein, a wide array of approaches willopen up. It can be anticipated that this 14 kDa can be used as templatefor the formation of silica-based, and surely also subsequentlytitania- and zirconia-based (Tahir et al., 2005), homogenousnanotubes and nanowires.
ACKNOWLEDGEMENTSThis work was supported by grants from the German Bundesministerium fürBildung und Forschung (project ‘Center of Excellence BIOTECmarin’), theDeutsche Forschungsgemeinschaft (Schr 277/10-1), the International HumanFrontier Science Program, the European Commission [project no. 031541–BIO-LITHO (biomineralization for lithography and microelectronics]) and theconsortium BiomaTiCS at the Universitätsmedizin of the Johannes Gutenberg-Universität Mainz.
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