Journal of Structural Biology 163 (2008)61-75 Contents lists available at ScienceDirect Journal of Structural Biology ELSEVIER journalhomepagewww.elsevier.com/locate/yjsbi Macromolecular structure of the organic framework of nacre in Haliotis rufescens Implications for growth and mechanical behavior Jiddu bezares a, Robert]. Asaro., Marilyn Hawley b of Structural Engineering. University of California, San Diego, Mail Code 0085, La jolla, CA 92093, US Materials Science and Technology Division, Los Alamos National laboratory, Los Alamos, NM 87545,USA ARTICLE IN FO A BSTRACT Article history. Ve have performed a macromolecular structural analysis of the interlamellar and intertabular parts of Received 8 January 2008 he organic framework of the nacreous part of the shell of Haliotis rufescens, including the identification Accepted 9 April 2008 of structural chitin. Using histochemical optical microscopy we have mapped the locations of carboxyl ates and sulfates of proteins and chitin on the surfaces and within the core of the interlamellar layers and Available online 25 April 2008 the intertabular matrix that together form the external organic matrix of site nacre. This extends the earlier work of Nudelmann et al. [ Nudelman, F. Gotliv, B.A. Addadi, L and Weiner, S. 2006. Mollusk Biomineralization tablet in nacre. J. Struct BioL. 153, 176-187] and Crenshaw and Ristedt [Crenshaw, MA, Ristedt, H Mollusk organic framework 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Omori, M, Watabe, N (Eds ) The Mechanisms of Biomineralization in Animals and Plants. Tokai University Press Toyko] on Nautilus pompilius. Our mapping identifies distinct regions, defined by the macromolecule groups, including what is proposed to be the sites of CacO, nucleation and that play a key role in nacre growth Using AFM scanning probe microscopy we have identified a fibrous core within the framework hat we as with chitin. The structural picture that is evolved is then used to develop a simple struc. ral model for the organic framework which is shown to be consistent with mechanical property mea- surements. The role of the intracrystalline matrix within the nacre tablets in mediating nacre's mechanical response is noted within the framework of our model e 2008 Elsevier Inc. All rights reserved. 1 Introduction macromolecular layout so that critical features such as the crystal nucleation site(s)and the chemical structural morphology that Biomineralization is a well regulated process within living control mechanical behavior may be understood in a more quanti- organisms, and involves control over, inter alia, the morphology fied manner. Possible extensions of such understanding include of mineral-biopolymer nano-composite structures, crystal nucle- biomimetic applications to, for example, nano-scale ceramic/poly ation within, and growth of, such structures, along with their poly- mer electronic devices and synthetic bone implants morph type(s) and crystallographic texture (e.g. Lowenstam and Crenshaw and Ristedt( 1976) took a unique approach to study Weiner, 1989: Simkiss and wilbur, 1989: Mann et al., 1989: Bae- ing the macromolecular structure in that they attempted a map- a wide array of other cases, mineralization appears to occur within microscopy. This approach has been recently pursued by nudel- a preformed 3-dimensional organic framework which acts as the man et al. (2006)who identify four different zones on the frame- template that provides the above mentioned control (e.g. Beve- work surface. This information was then shown to be of lander and Nakahara, 1969: Wada, 1972: Schaffer et al, 1997: immediate use for formulating more detailed and defensible mod- Nudelman et al., 2006 ). The organic framework thus mediates els for the biomineralization process. Crenshaw and ristedt's the growth, ie the"fabrication", of the mineralized nano-compos-(1976)and Nudelman et al. s(2006)work was performed on the e and, as it happens, is also key to what is seen to be a rather cephalopod Nautilus pompilius, whereas the latter performed com- cellent array of mechanical properties of the shell (e.g. Sarikaya parative study on the bivalve Atrina rigida. Here, we again et al., 1992: Evans et al, 2001). It is, accordingly, vital to under- the approach, but for the case of the gastropod Haliotis rufescens, stand the structure of the organic framework and in particular its with comparative study on N. pompilius so as to obtain a more comprehensive understanding of the commonality and variances among different members of the mollusk group. We confirm the mail address: asaro@ucsd. edu(r]. Asaro). general findings of Nudelman et al.(2006). now for H. rufescens, 047-8477s-see front matter o 2008 Elsevier Inc. All rights reserved. doi:10.1016/jsb.200804009
Macromolecular structure of the organic framework of nacre in Haliotis rufescens: Implications for growth and mechanical behavior Jiddu Bezares a , Robert J. Asaro a,*, Marilyn Hawley b aDepartment of Structural Engineering, University of California, San Diego, Mail Code 0085, La Jolla, CA 92093, USA b Materials Science and Technology Division, Los Alamos National laboratory, Los Alamos, NM 87545, USA article info Article history: Received 8 January 2008 Received in revised form 8 April 2008 Accepted 9 April 2008 Available online 25 April 2008 Keywords: Mollusk nacre Biomineralization Mollusk organic framework abstract We have performed a macromolecular structural analysis of the interlamellar and intertabular parts of the organic framework of the nacreous part of the shell of Haliotis rufescens, including the identification of structural chitin. Using histochemical optical microscopy we have mapped the locations of carboxylates and sulfates of proteins and chitin on the surfaces and within the core of the interlamellar layers and the intertabular matrix that together form the external organic matrix of composite nacre. This extends the earlier work of Nudelmann et al. [Nudelman, F., Gotliv, B.A., Addadi, L. and Weiner, S. 2006. Mollusk shell formation: mapping the distribution of organic matrix components underlying a single aragonite tablet in nacre. J. Struct. Biol. 153, 176–187] and Crenshaw and Ristedt [Crenshaw, M.A., Ristedt, H. 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Omori, M., Watabe, N. (Eds.) The Mechanisms of Biomineralization in Animals and Plants. Tokai University Press, Toyko] on Nautilus pompilius. Our mapping identifies distinct regions, defined by the macromolecular groups, including what is proposed to be the sites of CaCO3 nucleation and that play a key role in nacre growth. Using AFM scanning probe microscopy we have identified a fibrous core within the framework that we associate with chitin. The structural picture that is evolved is then used to develop a simple structural model for the organic framework which is shown to be consistent with mechanical property measurements. The role of the intracrystalline matrix within the nacre tablets in mediating nacre’s mechanical response is noted within the framework of our model. 2008 Elsevier Inc. All rights reserved. 1. Introduction Biomineralization is a well regulated process within living organisms, and involves control over, inter alia, the morphology of mineral-biopolymer nano-composite structures, crystal nucleation within, and growth of, such structures, along with their polymorph type(s) and crystallographic texture (e.g. Lowenstam and Weiner, 1989; Simkiss and Wilbur, 1989; Mann et al., 1989; Baeuerlein, 2000; Addadi and Weiner, 2001). In mollusk shells, among a wide array of other cases, mineralization appears to occur within a preformed 3-dimensional organic framework which acts as the template that provides the above mentioned control (e.g. Bevelander and Nakahara, 1969; Wada, 1972; Schaffer et al., 1997; Nudelman et al., 2006). The organic framework thus mediates the growth, i.e. the ‘‘fabrication”, of the mineralized nano-composite and, as it happens, is also key to what is seen to be a rather excellent array of mechanical properties of the shell (e.g. Sarikaya et al., 1992; Evans et al., 2001). It is, accordingly, vital to understand the structure of the organic framework and in particular its macromolecular layout so that critical features such as the crystal nucleation site(s) and the chemical/structural morphology that control mechanical behavior may be understood in a more quanti- fied manner. Possible extensions of such understanding include biomimetic applications to, for example, nano-scale ceramic/polymer electronic devices and synthetic bone implants. Crenshaw and Ristedt (1976) took a unique approach to studying the macromolecular structure in that they attempted a mapping of the framework’s components using histochemical light microscopy. This approach has been recently pursued by Nudelman et al. (2006) who identify four different zones on the framework surface. This information was then shown to be of immediate use for formulating more detailed and defensible models for the biomineralization process. Crenshaw and Ristedt’s (1976) and Nudelman et al.’s (2006) work was performed on the cephalopod Nautilus pompilius, whereas the latter performed comparative study on the bivalve Atrina rigida. Here, we again extend the approach, but for the case of the gastropod Haliotis rufescens, with comparative study on N. pompilius so as to obtain a more comprehensive understanding of the commonality and variances among different members of the mollusk group. We confirm the general findings of Nudelman et al. (2006), now for H. rufescens, 1047-8477/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2008.04.009 * Corresponding author. Fax: +1 858 534 6373. E-mail address: rasaro@ucsd.edu (R.J. Asaro). Journal of Structural Biology 163 (2008) 61–75 Contents lists available at ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi
J. Bezares et al /Joumal of structural Biology 163 (2008)61-75 but extend the structural characterization by focusing as well on et al, 2001) but until our assays, to be reported herein, were com the location of chitin within the framework. Our findings concern- pleted no truly definitive and quantitative evidence for chitin had ng chitin provide an important, and previously not understood existed in H rufescens: reasons for this stem from, in particular, dif picture of the organic framework that has specific implications to ficulties in properly hydrolyzing the complete framework prior to the mechanical and structural performance of the shell. analysis. We have, however, recently succeeded in performing an Several mollusk shells, such as that of H. rufescens, contain an accurate analysis with the interesting result that for the as ex- outer prismatic(calcitic) layer(see e.g. Simkiss and wilbur, 1989 tracted insoluble framework we find 6. 4 wt% chitin, whereas in or Zaremba et al., 1996)and an inner nacre. The nacreous portion the extracted framework subjected to digestion with trypsin the is a biomineralized structure in which quasi-hexagonal CaCO3 (ara- content is higher at 6.9 wt%. This is a remarkably high chitin con- gonite) tiles are layered between, and bound to. a thin biopolymer tent even when compared to that found in fungal cell walls protein framework secreted by epithelial cells (eg. Simkiss and (Ruiz-Herrera, 1992: San-Blas and Calderone, 2004), and more spe- Wilbur, 1989; Belcher and Gooch, 2000: Addadi et al, 2006; Rous- cifically as compared with the much lower contents reported by seau et al., 2005). The structure and crystallography of the ceramic Poulicek(1983)in gastropods. Our assay is, however, consistent tiles mollusk nacre have been studied via X-ray diffraction, SEM, with the chitin contents reported by Goffinet and Jeuniaux, 1979 TEM and AFM(see e.g. Wada, 1961, 1968: Wise, 1970: Crenshaw for other mollusk species. There are soluble proteins that are re- and Ristedt, 1976: Mutvei, 1979: Weiner et al., 1983: Sarikaya moved during demineralization and some have been et al, 1995; and Manne et al, 1994). Tiles are crystallographically (see e.g. Belcher et al, 1996). They too are rich in Asp and textured aragonite with a thickness in the range 300-500 nm and Ca, and can play a vital role in mineralization( Falini et al diameters in the range 4-10 um Nacre tiles are arranged in nearly Belcher et al., 1996; Addadi et al., 2001; Gotliv et al, 2003) parallel lamellae separated by thin interlamellar layers of biopoly Of particular interest is the structure of the CaCO3 nucleation mer framework with a thickness in the shell of approximately sites. Crenshaw and Ristedt(1976). following Crenshaw(1972), used mella the tiles grow laterally and meet at what become polygonal the outlines of the tiles, and mapped the location of sulfates, carbox boundaries separated by an intertabular matrix; examples are ylates, and calcium binding sites within them. Wada (1980)later shown below. Nacre tiles often respond, via diffraction, as single confirmed the presence of high concentrations of sulfur in the cen- crystalline tablets but are known to contain an organic intracrys- tral region, thus suggesting that the nucleation site was located talline matrix. Recently, for example, Rousseau et al. (2005)have there. Here, we use AFM methods combined with histochemical performed AFM imaging, in tapping mode, and tEM dark field fluorescent microscopy to map such structure in H rufescens and in- imaging of nacre tablets in the oyster Pinctada maxima and pro- clude chitin within our maps. Our mapping is then used to confirm vided evidence for a continuous intracrystalline matrix surround- and extend models for nacre growth and for developing an approach g coherent nanograins that comprise individual tablets. Their to modeling the shell's mechanical properties. This provides the path ults suggest, among other things, a pathway for modeling the to biomimetics and bio-duplication of synthetic materials. mechanical response of nacre that we use below in suggesting a reliminary model for the interlamellar layers. Likewise, Oaki 2 Materials and methods and Imai (2005)describe a hierarchical structure of nacre in the pearl oyster Pinctada fucata in which individual tiles are seen to 2.1. Materials be composed of nano-scale"building blocks"(ie. nano-crystals) surrounded by an organic matrix(ie the intracrystalline matrix). Fresh shells of both H. rufescens and N. pompilius were obtained The AFM images shown by Rousseau et al. (2005)can be used to from The Abalone Farm Inc. of Monterey, CA and were stored dry at demonstrate that the stiffness of individual tiles should be less 4C. The nacreous portion of the shells were removed by slowly than that of monolithic CaCO3 and, in fact, just on the basis of a grinding off the prismatic (outer) part and then washing the nacre simple rule of mixtures should be on the order of at least 10% less. sections in deionized water(Di). Sections varied in size, but often This estimate is based on the apparent thickness of the intracrys- were up to 20 x 20 mm in cross section and up to 2 mm thick. talline matrix as seen, for example, in the phase image of Fig 3b For the tensile specimens described in Section 4, the sections were f rousseau et al. (2005). At the same time, the intracrystalline ma- larger and up to 40 mm long. Fifteen live H rufescens were also pro- trix would impart increased toughness to the structure via the en- vided by the abalone Farm Inc and were kept in large tanks with ergy absorptive capability of a visco-elastic matrix. continuously running sea water at the Scripps Institution of Ocean- The biopolymer framework has previously been studied primar- ography at UCSD. These were used for the making of"flat pearl ily through optical microscopy (e.g. Crenshaw and Ristedt, 1976: inter alia, as shown in Fig. Ic Flat pearls were prepared by inserting Gregoire, 1957, 1972)and biochemical studies of amino acid com- 5 mm glass slides under the mantle of live H rufescens and allow- position(see e.g. Wheeler and Sykes, 1984: Cariolou and Morse, ing approximately three weeks for nacre to form. The slides were 1988: Simkiss and Wilbur. 1989: Addadi and Weiner. 1985 and then removed and washed in Dl. Aizenberg et al, 1999). Quite limited study using either TEM(Wei- ner et al, 1983; Levi-Kalisman et al, 2001)or AFM( Schaffer et al., 2.2. Demineralization 1997: Manne et al., 1994: Rousseau et al, 2005) has been per formed to date; only the latter was performed on H. rufescens After washing in Dl, shell sections were demineralized either in As is well established, the matrix associated with nacre tissue is Ethylene Diamine Tetraacetic Acid (EDtA, Sigma), or by cation-ex approximately 75-80 wt% protein and balance carbohydrate with change resin( Dowex 50W 50-100 mesh, Sigma). Demineral- me glycoproteins(see e.g. Addadi and Weiner, 1985). Our own ization was carried out at 20C for times that depended on the assays are consistent with this and a typical amino acid analysis method and on the size and thickness of the shell fragments. Decal shows that our H. rufescens insoluble nacre framework contains cification by ion exchange resin was adapted from Gotliv et al residues that are rich in Asp and Glu and are acidic as reported(2003). Nacre fragments were placed in sections of dialysis tubing by others. approximately 20% of the residues are Asp and 4% Glu. (MwCO 3500, 19 mm flat width, Fisher). Unfixed samples were There have been reports of chitin in the framework of several mol- placed in dialysis membranes filled with DI water while fixed sam- ner et al.1983; Poulicek, 1983: Goffinet and Jeuniaux, 1979 zep i- ples were placed in dialysis membranes pc) solution. 10 mm long lusks other than H. rufescer (Weiner and Traub, 1980, 1984; W taining a 4% formalde- hyde, 0.5% cetylpyridinium chloride(CI
but extend the structural characterization by focusing as well on the location of chitin within the framework. Our findings concerning chitin provide an important, and previously not understood picture of the organic framework that has specific implications to the mechanical and structural performance of the shell. Several mollusk shells, such as that of H. rufescens, contain an outer prismatic (calcitic) layer (see e.g. Simkiss and Wilbur, 1989 or Zaremba et al., 1996) and an inner nacre. The nacreous portion is a biomineralized structure in which quasi-hexagonal CaCO3 (aragonite) tiles are layered between, and bound to, a thin biopolymer protein framework secreted by epithelial cells (e.g. Simkiss and Wilbur, 1989; Belcher and Gooch, 2000; Addadi et al., 2006; Rousseau et al., 2005). The structure and crystallography of the ceramic tiles mollusk nacre have been studied via X-ray diffraction, SEM, TEM and AFM (see e.g. Wada, 1961, 1968; Wise, 1970; Crenshaw and Ristedt, 1976; Mutvei, 1979; Weiner et al., 1983; Sarikaya et al., 1995; and Manne et al., 1994). Tiles are crystallographically textured aragonite with a thickness in the range 300–500 nm and diameters in the range 4–10 lm. Nacre tiles are arranged in nearly parallel lamellae separated by thin interlamellar layers of biopolymer framework with a thickness in the shell of approximately 30 nm (e.g. Checa and Rodriguez-Navarro, 2005) . Within each lamella the tiles grow laterally and meet at what become polygonal boundaries separated by an intertabular matrix; examples are shown below. Nacre tiles often respond, via diffraction, as single crystalline tablets but are known to contain an organic intracrystalline matrix. Recently, for example, Rousseau et al. (2005) have performed AFM imaging, in tapping mode, and TEM dark field imaging of nacre tablets in the oyster Pinctada maxima and provided evidence for a continuous intracrystalline matrix surrounding coherent nanograins that comprise individual tablets. Their results suggest, among other things, a pathway for modeling the mechanical response of nacre that we use below in suggesting a preliminary model for the interlamellar layers. Likewise, Oaki and Imai (2005) describe a hierarchical structure of nacre in the pearl oyster Pinctada fucata in which individual tiles are seen to be composed of nano-scale ‘‘building blocks” (i.e. nano-crystals) surrounded by an organic matrix (i.e. the intracrystalline matrix). The AFM images shown by Rousseau et al. (2005) can be used to demonstrate that the stiffness of individual tiles should be less than that of monolithic CaCO3 and, in fact, just on the basis of a simple rule of mixtures should be on the order of at least 10% less. This estimate is based on the apparent thickness of the intracrystalline matrix as seen, for example, in the phase image of Fig. 3b of Rousseau et al. (2005). At the same time, the intracrystalline matrix would impart increased toughness to the structure via the energy absorptive capability of a visco-elastic matrix. The biopolymer framework has previously been studied primarily through optical microscopy (e.g. Crenshaw and Ristedt, 1976; Gregoire, 1957, 1972) and biochemical studies of amino acid composition (see e.g. Wheeler and Sykes, 1984; Cariolou and Morse, 1988; Simkiss and Wilbur, 1989; Addadi and Weiner, 1985 and Aizenberg et al., 1999). Quite limited study using either TEM (Weiner et al., 1983; Levi-Kalisman et al., 2001) or AFM (Schaffer et al., 1997; Manne et al., 1994; Rousseau et al., 2005) has been performed to date; only the latter was performed on H. rufescens . As is well established, the matrix associated with nacre tissue is approximately 75–80 wt% protein and balance carbohydrate with some glycoproteins (see e.g. Addadi and Weiner, 1985). Our own assays are consistent with this and a typical amino acid analysis shows that our H. rufescens insoluble nacre framework contains residues that are rich in Asp and Glu and are acidic as reported by others. Approximately 20% of the residues are Asp and 4% Glu. There have been reports of chitin in the framework of several mollusks other than H. rufescens (Weiner and Traub, 1980, 1984; Weiner et al., 1983; Poulicek, 1983; Goffinet and Jeuniaux, 1979; Zentz et al., 2001) but until our assays, to be reported herein, were completed no truly definitive and quantitative evidence for chitin had existed in H. rufescens; reasons for this stem from, in particular, dif- ficulties in properly hydrolyzing the complete framework prior to analysis. We have, however, recently succeeded in performing an accurate analysis with the interesting result that for the as extracted insoluble framework we find 6.4 wt% chitin, whereas in the extracted framework subjected to digestion with trypsin the content is higher at 6.9 wt%. This is a remarkably high chitin content even when compared to that found in fungal cell walls (Ruiz-Herrera, 1992; San-Blas and Calderone, 2004), and more specifically as compared with the much lower contents reported by Poulicek (1983) in gastropods. Our assay is, however, consistent with the chitin contents reported by Goffinet and Jeuniaux, 1979 for other mollusk species. There are soluble proteins that are removed during demineralization and some have been isolated, (see e.g. Belcher et al., 1996). They too are rich in Asp and Glu, bind Ca++, and can play a vital role in mineralization (Falini et al., 1996; Belcher et al., 1996; Addadi et al., 2001; Gotliv et al., 2003). Of particular interest is the structure of the CaCO3 nucleation sites.Crenshaw and Ristedt (1976), followingCrenshaw (1972), used the fact that once demineralized, the interlamellar matrix reveals the outlines of the tiles, and mapped the location of sulfates, carboxylates, and calcium binding sites within them. Wada (1980) later confirmed the presence of high concentrations of sulfur in the central region, thus suggesting that the nucleation site was located there. Here, we use AFM methods combined with histochemical fluorescent microscopy to map such structure in H. rufescens and include chitin within our maps. Our mapping is then used to confirm and extend models for nacre growth and for developing an approach tomodeling the shell’smechanical properties. This provides the path to biomimetics and bio-duplication of synthetic materials. 2. Materials and methods 2.1. Materials Fresh shells of both H. rufescens and N. pompilius were obtained from The Abalone Farm Inc. of Monterey, CA and were stored dry at 4 C. The nacreous portion of the shells were removed by slowly grinding off the prismatic (outer) part and then washing the nacre sections in deionized water (DI). Sections varied in size, but often were up to 20 20 mm in cross section and up to 2 mm thick. For the tensile specimens described in Section 4, the sections were larger and up to 40 mm long. Fifteen live H. rufescens were also provided by the Abalone Farm Inc. and were kept in large tanks with continuously running sea water at the Scripps Institution of Oceanography at UCSD. These were used for the making of ‘‘flat pearls”, inter alia, as shown in Fig. 1c. Flat pearls were prepared by inserting 5 mm glass slides under the mantle of live H. rufescens and allowing approximately three weeks for nacre to form. The slides were then removed and washed in DI. 2.2. Demineralization After washing in DI, shell sections were demineralized either in Ethylene Diamine Tetraacetic Acid (EDTA, Sigma), or by cation-exchange resin (Dowex 50 W 8 50–100 mesh, Sigma). Demineralization was carried out at 20 C for times that depended on the method and on the size and thickness of the shell fragments. Decalcification by ion exchange resin was adapted from Gotliv et al. (2003). Nacre fragments were placed in sections of dialysis tubing (MWCO 3500, 19 mm flat width, Fisher). Unfixed samples were placed in dialysis membranes filled with DI water while fixed samples were placed in dialysis membranes containing a 4% formaldehyde, 0.5% cetylpyridinium chloride (CPC) solution. 10 mm long 62 J. Bezares et al. / Journal of Structural Biology 163 (2008) 61–75
J. Bezares et al/ Journal of Structural Biology 163(2008)61-75 a b d Fig. 1.(a and b)sEM images of fractured nacre from H rufescens illustrating tiles on nearly parallel lamella. The" terrace consisting of one interlamellar layer of nacre is arls grown on a glass slid image of a cross section of H. rufescens organic matrix, demineralized in EDTA, illustrating individual and apparently porous interlamellar layer p of each stack(d)SEM inserted into the mantle of a live red abalone(described belo sections of tubing containing unfixed specimens were placed in )keer in an Eppendorf centrifuge. The supernatant was removed and re- DI containing 750 ml of pre-washed resin. A stir bar was used to placed by 1 ml of Hepes buffer containing 200 ug/mL proteinase-K the resin in constant suspension at room tempe Sigma). The pellets were sonicated in this solution for 10 min and was replaced once a day. Decalcification was verified by infrared left to incubate for 2 h at room temperature. The samples were then spectroscopy. Fixed samples placed in sections of dialysis tubing centrifuged for 10 min at 12,000 rpm. The supernatant was removed were allowed to demineralize in 50 ml conical tubes filled with and the pellets were washed with Hepes buffer. 15 ml of resin and topped off with the formalyn/CPC solution. The tubes were placed on a tilt platform such that the resin remained 2.3.3. Collagenase in suspension during decalcification. The demineralization was As described in Schaffer et al. (1997). sheets of demineralized complete after 1-4 weeks For starting fragments with a thickness tissue were incubated for 2 h in 1 mL of 200 ug/ mL collagen of approximately 0.5 mm, typically 1 week-10 days was sufficient. combined with 200 HM CaCl2 in 5 mM Hepes buffer, pH 7.5. Sam- Thicker fragments, e.g. those with thickness up to 1 mm, required ples were centrifuged for 10 min at 12,000 rpm and sonicated in DI the longer times. Following Crenshaw and Ristedt (1976), decalcifi- for 10 min cation using EDta was performed by placing nacre fragments in 50 ml conical tubes filled with 1 M EDTA, pH8, at room temperature 2.3. 4 N-Glycosidase F under gentle shaking. Samples were also decalcified in EDTA con- N-Glycosidase F(PNGase F, New England Bio Labs, P0704S) is an taining 4% formaldehyde and 0.5% CPC (Williams and Jackson, amidase that cleaves between the innermost glucosamine and 1956. After demineralization, all samples were extensively washed asparagine residues of numerous N-linked glycoproteins. One cen- in Di water ove any remaining EDTA, formaldehyde or CPC. timetre square sections of demineralized tissue were separated Interlamellar sheets were separated using fine tweezers under an into approximately 1 um thick, interlamellar sheets. The sheets ptical microscope. Demineralization times ranged from 1 week to were incubated in 1 uL of 10 x glycoprotein denaturing buffer 10 days depending on the thickness of the starting fragment. and Di water to make a total reaction volume of 750 HL. The sam- ples and buffer were combined in 1.5 mL centrifuge tubes. The 2.3. Enzym tubes containing the samples were placed in water heated to 100C for 15 min. A total reaction volume of 20 HL was prepared 2.3.1. Trypsin by adding 2 HL of 10 x G7 reaction buffer, 2 uL of 10% NP40, 2 HL Ten microgram of wet interlamellar sheets were incubated for N-Glycosidase F, and 14 HL of DI water. The samples were incu- 24 h at room temperature in 0.1 M ammonium bicarbonate buffer, bated at room temperature in this solution for 2 h. pH 8.0, containing 1 mg/mL trypsin(Invitrogen). 2.4. Staining methods 23.2 Proteinase-K Following Schaffer et al. (1997). interlamellar sheets of deminer- 2. 4. 1. Calcofluor white alized shell were sonicated for 10 min in 1 mLof 5 mM Hepes buffer, Calcofluor white(CW)is a fluorescent brightener that binds to pH7.5.The samples were then centrifuged at 12,000 rpm for 10 min B-1-3 and B-1-4 polysaccharides as are found in cellulose and
sections of tubing containing unfixed specimens were placed in 4 l of DI containing 750 ml of pre-washed resin. A stir bar was used to keep the resin in constant suspension at room temperature and the water was replaced once a day. Decalcification was verified by infrared spectroscopy. Fixed samples placed in sections of dialysis tubing were allowed to demineralize in 50 ml conical tubes filled with 15 ml of resin and topped off with the formalyn/CPC solution. The tubes were placed on a tilt platform such that the resin remained in suspension during decalcification. The demineralization was complete after 1–4 weeks. For starting fragments with a thickness of approximately 0.5 mm, typically 1 week–10 days was sufficient. Thicker fragments, e.g. those with thickness up to 1 mm, required the longer times. Following Crenshaw and Ristedt (1976), decalcifi- cation using EDTA was performed by placing nacre fragments in 50 ml conical tubes filled with 1 M EDTA, pH 8, at room temperature under gentle shaking. Samples were also decalcified in EDTA containing 4% formaldehyde and 0.5% CPC (Williams and Jackson, 1956). After demineralization, all samples were extensively washed in DI water to remove any remaining EDTA, formaldehyde or CPC. Interlamellar sheets were separated using fine tweezers under an optical microscope. Demineralization times ranged from 1 week to 10 days depending on the thickness of the starting fragment. 2.3. Enzymatic digestion 2.3.1. Trypsin Ten microgram of wet interlamellar sheets were incubated for 24 h at room temperature in 0.1 M ammonium bicarbonate buffer, pH 8.0, containing 1 mg/mL trypsin (Invitrogen). 2.3.2. Proteinase-K Following Schaffer et al. (1997), interlamellar sheets of demineralized shell were sonicated for 10 min in 1 mL of 5 mM Hepes buffer, pH 7.5. The samples were then centrifuged at 12,000 rpm for 10 min in an Eppendorf centrifuge. The supernatant was removed and replaced by 1 mL of Hepes buffer containing 200 lg/mL proteinase-K (Sigma). The pellets were sonicated in this solution for 10 min and left to incubate for 2 h at room temperature. The samples were then centrifuged for 10 min at 12,000 rpm. The supernatant was removed and the pellets were washed with Hepes buffer. 2.3.3. Collagenase As described in Schaffer et al. (1997), sheets of demineralized tissue were incubated for 2 h in 1 mL of 200 lg/ mL collagenase combined with 200 lM CaCl2 in 5 mM Hepes buffer, pH 7.5. Samples were centrifuged for 10 min at 12,000 rpm and sonicated in DI for 10 min. 2.3.4. N-Glycosidase F N-Glycosidase F (PNGase F, New England Bio Labs, P0704S) is an amidase that cleaves between the innermost glucosamine and asparagine residues of numerous N-linked glycoproteins. One centimetre square sections of demineralized tissue were separated into approximately 1 lm thick, interlamellar sheets. The sheets were incubated in 1 lL of 10 glycoprotein denaturing buffer and DI water to make a total reaction volume of 750 lL. The samples and buffer were combined in 1.5 mL centrifuge tubes. The tubes containing the samples were placed in water heated to 100 C for 15 min. A total reaction volume of 20 lL was prepared by adding 2 lL of 10 G7 reaction buffer, 2 lL of 10% NP40, 2 lL N-Glycosidase F, and 14 lL of DI water. The samples were incubated at room temperature in this solution for 2 h. 2.4. Staining methods 2.4.1. Calcofluor white Calcofluor white (CW) is a fluorescent brightener that binds to b-1-3 and b-1-4 polysaccharides as are found in cellulose and Fig. 1. (a and b) SEM images of fractured nacre from H. rufescens illustrating tiles on nearly parallel lamella. The ‘‘terrace” consisting of one interlamellar layer of nacre is shown at higher magnification in (b), where the black arrow points to a central region discussed below and referred to in Mutvei (1979). (c) Flat pearls grown on a glass slide inserted into the mantle of a live red abalone (described below). Note the ‘‘stack of coins” arrangement with a smaller tile (or tiles) nucleated at the top of each stack. (d) SEM image of a cross section of H. rufescens organic matrix, demineralized in EDTA, illustrating individual and apparently porous interlamellar layers. J. Bezares et al. / Journal of Structural Biology 163 (2008) 61–75 63
J. Bezares et al /Joumal of structural Biology 163 (2008)61-75 chitin, respectively(Maeda and Ishida, 1967: Peters and Latka, 2.5.3. Silver intensification 1986: Hayat, 1993: Harrington and Hageage, 2003). Staining of Aurion SE-EM silver enhancement reagent(Aurion) was pre- demineralized tissue was performed as follows. A 10% potassium pared immediately before use. Samples treated with WGA-gold hydroxide reagent was prepared by dissolving 10 g of KOH in complexes were rinsed in DI water three times and placed in the 90 mL of DI water to which 10 mL of glycerin were added. a second enhancing reagent for 4 min after which they were washed in five CW reagent was prepared by dissolving 0. 1 g of fluorescent bright- changes of DI water. ener 28(Sigma)in 100 mL of DI water under gentle heating. Inter lamellar sheets, approximately 1-2 um thick were mounted on 2.5.4. Cytochemical controls slides. Two drops of each reagent were added to the samples. After The specificity to chitin of WGA-gold complexes was deter 4 min the samples were rinsed in water, air dried and mounted mined by pre-incubating the complexes in N, N, N-triacetylchitotri- Entellan mounting medium(Merck). ose. Another set of samples was incubated in unlabeled WGA (0.5 mg/mL). Specificity to chitin was further determined by a 2. 4.2. Colloidal iron omparative analysis using FITC-WGA(Molecular Probes)and cw Colloidal iron staining was performed following methods out- which binds specifically to B-1-3 and B-1-4 glucosides ie cellulose lined in Pearse(1968). Stock colloidal iron solution was prepared and chitin, (Maeda and Ishida, 1967; Peters and Latka, 1986; Lukes by stirring in 29% ferric chloride to boiling water. Once the solution et al., 1993). Of these two polysaccharides, chitin is the only one rned dark red it was allowed to cool to 20C. The solution was which is labeled by both WGa and CV then dialyzed three times for 24 h against a volume of DI 5x that of the stock solution. The reagent was stored at 20C. The working 2.5.5. Fluorescein-WGA colloidal iron solution, pH 1.8, had a shelf life of 24 h. This solution Following El Gueddari et al.(2002), interlamellar sheets were consisted of Dl, glacial acetic acid, and stock colloidal iron solution incubated with 2% w/v bovine serum albumin(BSa)in PBS for combined at a ratio of 18: 12: 10. As in Nudelman et al. (2006), the 30 min at 20C. Samples were tissue samples were submerged in the working solution for 1 h, Tween20 and incubated in FITC-WGA(O 1 mg/mL in PBS, 1% w/v rinsed thoroughly with 12% acetic acid, incubated with 5% potas- BSA)for 1 h Samples were washed three times with PBS/Tween20 sium ferrocyanide/5% HCl(1: 1)for 20 min, and washed with water. washed once in DI water, air dried on glass cover slips, and Samples were mounted as described above. mounted in Entellan 2. 43. Aminoacridine 2.6. Scanning electron microscopy Following Nudelman et al. (2006), thin layers of interlamellar tissue were incubated in 1% N-(3 dimethylaminopropyl )-N-ethyl- Samples were dehydrated in an ethanol series and critical point carbodiimide hydrochloride(EDC)in a 20 mM phosphate buffer dried with liquid carbon dioxide. Samples were mounted with ca at pH 4.5. The samples were washed three times with 0.2 M borate bon tabs and sputter coated with 15 nm of chromium. Samples buffer, pH 8.5, and incubated for 12 h in aminoacridone(1 mg/ were immediately examined using a Philips XL30 ESEM operated 100 ml)in the borate buffer. The samples were rinsed with water and mounted as described above 2.7 Immunohistoch .5. Wheat germ agglut Gotliv et al.(2003) produced polyclonal antibodies raised .5.1. Probe preparata against soluble aragonite-nucleating proteins from the bivalve A. Wheat germ agglutinin (WGA)is a lectin that binds to se- rigida. the proteins were earlier found to nucleate aragonite by quences of three B-1-4 linked N-acetyl-D-glucosamine residues as Falini et al. (1996). Nudelman et al. (2006)found positive labeling well as to sialic acid residues. As a result WGa has a high affini with these antibodies in both A rigida and N. pompilius and conse- chitin and to a lesser extent to some glycoproteins. Colloidal quently to explore the possibility of similar effects in H. rufescens gold can be complexed to WGA for the purpose of locating chitin we used similar immunohistochemical labeling procedures. Poly on the biopolymer layer remaining after shell demineralization. clonal antibodies were generously supplied to us by s. Weiner Colloidal gold particles typically used for high resolution TEM stud -(2007). ies can be enhanced using silver enhancement reagents. This al- Interlamellar sheets from H rufescens were incubated in serum lows for the imaging of these particles using standard SEM and containing polyclonal antibodies for 1 h. The serum was diluted adapted from King et al.(1987)and Geoghegan and Ackerman ing The samples were washed twice for 5 min with PBS containing (1997). WGA-gold complexes were prepared as follows. 0.75 mg Tween20(0.05% w/). Samples were then incubated for 40 min in WGA in 1 HL of distilled water were combined with 25 mL of col- the secondary antibody, rhodamine conjugated goat-anti-rab- loidal gold, pH 9.9. The gold particles were of a nominal size of bit(ackson ImmunoResearch, diluted 1: 100 in PBS). Before imag 10 nm(Sigma). The complexes were stirred for 3 min after which ing the specimens were washed three times for 5 min with PBS 1% polyethylene glycol (PEG)was added. After 5 min the reagent and Tween20(0.05% w/v) to remove unbound antibodies. After was centrifuged at 60,000g in a Beckman ultracentrifuge for 1 h rinsing the samples in DI water, the samples were air dried on cov at 4 C The supernatant was removed and the sedimented WGA- er slips and mounted in Entellan. As a control, samples incubated gold complexes were resuspended to 5 mL with phosphate buf- in a pre-immune serum were prepared as above. fered saline(PBS)pH 8, containing 0.2 mg/mL of PEG 2.8. Optical microscopy 2.5.2. Labeling procedure Fixed and unfixed interlamellar tissue was sectioned it Samples were observed using a Nikon Eclipse 80i optical approximately 1 mm thick Samples were incubated in scope equipped with a Photometrics CoolSNAPez digital gold complexes for 30 min. The WGA-gold complexes Calcofluor white and aminoacridone-stained samples wer aI mIcro- t a dilution of 1: 100 in PBS, pH 7.0. After labeling th using a 11003 V3 filter set, while samples labeled with FITC and were washed in six changes of PBS, pH 7.2 rhodamine where viewed using a 41001 filter set(Chroma)
chitin, respectively (Maeda and Ishida, 1967; Peters and Latka, 1986; Hayat, 1993; Harrington and Hageage, 2003). Staining of demineralized tissue was performed as follows. A 10% potassium hydroxide reagent was prepared by dissolving 10 g of KOH in 90 mL of DI water to which 10 mL of glycerin were added. A second CW reagent was prepared by dissolving 0.1 g of fluorescent brightener 28 (Sigma) in 100 mL of DI water under gentle heating. Interlamellar sheets, approximately 1–2 lm thick were mounted on slides. Two drops of each reagent were added to the samples. After 4 min the samples were rinsed in water, air dried and mounted in Entellan mounting medium(Merck). 2.4.2. Colloidal iron Colloidal iron staining was performed following methods outlined in Pearse (1968). Stock colloidal iron solution was prepared by stirring in 29% ferric chloride to boiling water. Once the solution turned dark red it was allowed to cool to 20 C. The solution was then dialyzed three times for 24 h against a volume of DI 5 that of the stock solution. The reagent was stored at 20 C. The working colloidal iron solution, pH 1.8, had a shelf life of 24 h. This solution consisted of DI, glacial acetic acid, and stock colloidal iron solution combined at a ratio of 18:12:10. As in Nudelman et al. (2006), the tissue samples were submerged in the working solution for 1 h, rinsed thoroughly with 12% acetic acid, incubated with 5% potassium ferrocyanide/5% HCl (1:1) for 20 min, and washed with water. Samples were mounted as described above. 2.4.3. Aminoacridone Following Nudelman et al. (2006), thin layers of interlamellar tissue were incubated in 1% N-(3 dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC) in a 20 mM phosphate buffer at pH 4.5. The samples were washed three times with 0.2 M borate buffer, pH 8.5, and incubated for 12 h in aminoacridone (1 mg/ 100 ml) in the borate buffer. The samples were rinsed with water and mounted as described above. 2.5. Wheat germ agglutinin–colloidal gold (WGA–gold) 2.5.1. Probe preparation Wheat germ agglutinin (WGA) is a lectin that binds to sequences of three b-1-4 linked N-acetyl-D-glucosamine residues as well as to sialic acid residues. As a result WGA has a high affinity to chitin and to a lesser extent to some glycoproteins. Colloidal gold can be complexed to WGA for the purpose of locating chitin on the biopolymer layer remaining after shell demineralization. Colloidal gold particles typically used for high resolution TEM studies can be enhanced using silver enhancement reagents. This allows for the imaging of these particles using standard SEM and optical microscopic techniques. Our procedures were closely adapted from King et al. (1987) and Geoghegan and Ackerman (1997). WGA–gold complexes were prepared as follows. 0.75 mg WGA in 1 lL of distilled water were combined with 25 mL of colloidal gold, pH 9.9. The gold particles were of a nominal size of 10 nm(Sigma). The complexes were stirred for 3 min after which 1% polyethylene glycol (PEG) was added. After 5 min the reagent was centrifuged at 60,000g in a Beckman ultracentrifuge for 1 h at 4 C. The supernatant was removed and the sedimented WGA– gold complexes were resuspended to 5 mL with phosphate buffered saline (PBS) pH 8, containing 0.2 mg/mL of PEG. 2.5.2. Labeling procedure Fixed and unfixed interlamellar tissue was sectioned into 1 cm2 approximately 1 mm thick. Samples were incubated in the WGA– gold complexes for 30 min. The WGA–gold complexes were used at a dilution of 1:100 in PBS, pH 7.0. After labeling the samples were washed in six changes of PBS, pH 7.2. 2.5.3. Silver intensification Aurion SE-EM silver enhancement reagent(Aurion) was prepared immediately before use. Samples treated with WGA–gold complexes were rinsed in DI water three times and placed in the enhancing reagent for 4 min after which they were washed in five changes of DI water. 2.5.4. Cytochemical controls The specificity to chitin of WGA–gold complexes was determined by pre-incubating the complexes in N,N0 ,N00-triacetylchitotriose. Another set of samples was incubated in unlabeled WGA (0.5 mg/mL). Specificity to chitin was further determined by a comparative analysis using FITC-WGA(Molecular Probes) and CW which binds specifically to b-1-3 and b-1-4 glucosides i.e. cellulose and chitin, (Maeda and Ishida, 1967; Peters and Latka, 1986; Lukes et al., 1993). Of these two polysaccharides, chitin is the only one which is labeled by both WGA and CW. 2.5.5. Fluorescein-WGA Following El Gueddari et al. (2002), interlamellar sheets were incubated with 2% w/v bovine serum albumin (BSA) in PBS for 30 min at 20 C. Samples were washed three times with PBS/ Tween20 and incubated in FITC-WGA(0.1 mg/mL in PBS, 1% w/v BSA) for 1 h. Samples were washed three times with PBS/Tween20, washed once in DI water, air dried on glass cover slips, and mounted in Entellan. 2.6. Scanning electron microscopy Samples were dehydrated in an ethanol series and critical point dried with liquid carbon dioxide. Samples were mounted with carbon tabs and sputter coated with 15 nm of chromium. Samples were immediately examined using a Philips XL30 ESEM operated at 10 kV. 2.7. Immunohistochemistry Gotliv et al. (2003) produced polyclonal antibodies raised against soluble aragonite-nucleating proteins from the bivalve A. rigida. The proteins were earlier found to nucleate aragonite by Falini et al. (1996). Nudelman et al. (2006) found positive labeling with these antibodies in both A. rigida and N. pompilius and consequently to explore the possibility of similar effects in H. rufescens we used similar immunohistochemical labeling procedures. Polyclonal antibodies were generously supplied to us by S. Weiner (2007). Interlamellar sheets from H. rufescens were incubated in serum containing polyclonal antibodies for 1 h. The serum was diluted 1:25 in PBS containing 0.25% w/v BSA to block non-specific binding. The samples were washed twice for 5 min with PBS containing Tween20(0.05% w/v). Samples were then incubated for 40 min in the secondary antibody, rhodamine conjugated goat-anti-rabbit(Jackson ImmunoResearch, diluted 1:100 in PBS). Before imaging the specimens were washed three times for 5 min with PBS and Tween20(0.05% w/v) to remove unbound antibodies. After rinsing the samples in DI water, the samples were air dried on cover slips and mounted in Entellan. As a control, samples incubated in a pre-immune serum were prepared as above. 2.8. Optical microscopy Samples were observed using a Nikon Eclipse 80i optical microscope equipped with a Photometrics CoolSNAPez digital camera. Calcofluor white and aminoacridone-stained samples were viewed using a 11003 V3 filter set, while samples labeled with FITC and rhodamine where viewed using a 41001 filter set(Chroma). 64 J. Bezares et al. / Journal of Structural Biology 163 (2008) 61–75
J Bezares et al/ Journal of Structural Biology 163(2008)61-75 2.9. Chitin assays 2.11. Mechanical properties and mode Our method was based on the evaluation of glucosamine con tent found in demineralized tissue samples. 1 mg of tissue(dry ished flat sections of the nacreous parts of whole shells. Steel t/- Specimens were"dog bone "type and were machined from weight) was resuspended in 1 ml of 6 M HCl and hydrolyzed at plates were first made of the specimens to ensure dimensional 100C for 17 h, together with N-acetylglucosamine(GlcNAc) as uniformity across the gauge section and the machining was per a standard. Samples were then dried and resuspended in 1 m formed via slow grinding to minimize damage to the edges. The DI water. The quantity of glucosamine released by the hydrolysis gauge sections 14 mm in length, 2 mm wide, and 0.5 mm of 100 HL of this material was determined as follows. An equal thick Axial strain was measured using an extensometer with a res- volume of 4%(v/v)acetylacetone in 1.5 M Na, CO3 was added, olution in strain of approximately 5 x 10-6. Testing was performed and the preparation heated at 100C for 20 min. Samples were in an Instron 5565 material test system, in stroke(ie. displace- then diluted with 700 HL 96% ethanol and incubated for 1 hr at ment) control; this system has a mechanically controlled cross 23C, after 100 HL Ehrlich's reagent(1.6 g/ml p-dimethylamin head and thus is extremely stable. Axial strain rates ranged from benzaldehyde, 6M HCl. 50% ethanol) was added. Samples were 3 to-10-4. Modeling was performed for the purpose incubated for 15 min at 65C before the absorbance was read extracting estimates for the shear stiffness of the interlamellar lay at 520 nm ers using a"shear lag "analytical model that has been verified by finite element analysis 2.10. AFM methods Atomic force microscopy(AFM)techniques were used to 3. Results characterize the protein network structure of decalcified abalone shells.Intermittent contact( tapping)mode of operation was used 3. 1 SEM observations to obtain both surface topographic and phase information In tap- ing mode the"diving board"shaped, low force-constant cantile- The following SEM observations are briefly described most ver, which has a very sharp silicon tip located at the end on the ticularly to provide additional perspective on the structure of nacre bottom side, is oscillated at its resonance frequency by a piezo- in our H. rufescens. Fig. la and b shows SEM images of the tile electric element located on the cantilever holder. which is cross-section of a fractured shell that illustrate the optimal inter mounted on a piezoelectric tube scanner. This intermittent con- digitated nacre brick-wall structure. Additional description of the tact mode of operation and low force-constant cantilevers were structure, including the interlamellar and intertabular layers may riginally developed specifically to minimize damage to soft, eas- be seen in the images and are described in the caption. Interlamel- ily damaged samples. A laser beam, focused onto the backside of regions are identified by the horizontal white arrows in Fig. 1b the cantilever end, is reflected via an adjustable mirror onto an the intertabular regions are identified by the vertical white arrows. ptical sensor. As the cantilever tip is rastered back and forth The black arrow indicates a central region observed in all tiles over the sample surface, variations in sample height result deflection of the cantilever from its rest position. These deflec- an image of individual demineralized interlamellar layers of the tions cause variations in the optical sensor signal that are con- external organic matrix. Such images were obtained from critically verted to a voltage that is applied to the z electrode of the point dried matrix tissue and show thickness in the range 80- scanner, causing it to expand or contract, and thus raise or lower 100 nm; in the shell the interlamellar layers are 30 nm as just the cantilever back to its undeflected position. Since the chang noted. The layers appear porous in Fig. 1d and this will be further in the scanner dimensions are calibrated in nanometers per volt, elucidated when our AFM results are shown later. Fig. 1c shows the the changes in applied voltage are mapped as local variations in growth of"flat pearls"grown on the surface of glass slides inserted sample height superimposed on an x-y grid (scan size)set by under the mantle of live H rufescens; such observations will be re- the user the sharp tip allows one to obtain nanometer resolution ferred to in our discussion of nacre growth. A noteworthy feature of surface fea of the flat pearls is the appearance of more than one tile nucleated Typical resonance frequencies for the commercial AFM cantile- atop a tile growing laterally on a layer just below. An example of vers(Nano Devices, approximately 125 um long x 45 um such multiple tile nucleation is indicated by the black arrow in idex 4 um thick) used in this study were around 300 kHz. In Fig 1c. dition to monitoring changes in the cantilever deflection posi tion, simultaneously phase imaging, which captures the phase 3.2. Optical histochemical microscopy lag angle between the drive signal and actual cantilever oscillation was used to obtain maps of variations in local properties such as 3. 1. Calcofluor white staining stiffness Therefore there is a one-to-one correlation between the Calcofluor white is a fluorophore that binds to glycans and opographic data and the corresponding phase information. fibrillar polysaccharides such as chitin(e.g. Herth, 1980: Ramasw The aFM is equipped with a special cantilever wet cell holder, amy et al. 1997). Organic matrices from H rufescens were stained which was used to image samples under in vitro conditions. Sam- and imaged after demineralization in EDTA and by cation-ex les were mounted on steel pucks that are held in place on the change resin. In both cases strong binding was detected as shown AFM sample stage by a magnet buried in the base. Since the hy in Fig. 2c and d. H perspective, however, we first show SEM were cut large enough to be held in place using the wet cell o-ring plane, Fig. 2a and b. These images may be compared to those re- seal. Only thin continuous sections were used for imaging to pre- ported by Mutvei (1979, 1977), where similarities and differences vent small sections from coming lose during imaging and interfer- may be noted ing with the imaging process. Ambient imaging was used for dried As in Mutvei's(1977)SEM images, the intertabular matrix, su and stained samples. In all cases, tapping mode imaging was used. rounding the tiles, is clearly revealed as is the appearance of an evi- We used Veeco Metrology Nanoscope llla controllers with a D3000 dently organic ring-like structure organized around what was the microscope to image the dried samples and a Multimode micro- tiles'centers. Etching, in the form of"pits", occurs throughout scope for the wet cell imaging the surface of the interlamellar layers, but we do not observe the
2.9. Chitin assays Our method was based on the evaluation of glucosamine content found in demineralized tissue samples. 1 mg of tissue (dry weight) was resuspended in 1 ml of 6 M HCl and hydrolyzed at 100 C for 17 h, together with N-acetylglucosamine (GlcNAc) as a standard. Samples were then dried and resuspended in 1 ml DI water. The quantity of glucosamine released by the hydrolysis of 100 lL of this material was determined as follows. An equal volume of 4% (v/v) acetylacetone in 1.5 M Na2CO3 was added, and the preparation heated at 100 C for 20 min. Samples were then diluted with 700 lL 96% ethanol and incubated for 1 hr at 23 C, after 100 lL Ehrlich’s reagent (1.6 g/ml p-dimethylaminobenzaldehyde, 6 M HCl, 50% ethanol) was added. Samples were incubated for 15 min at 65 C before the absorbance was read at 520 nm. 2.10. AFM methods Atomic force microscopy (AFM) techniques were used to characterize the protein network structure of decalcified abalone shells. Intermittent contact (tapping) mode of operation was used to obtain both surface topographic and phase information. In tapping mode, the ‘‘diving board” shaped, low force-constant cantilever, which has a very sharp silicon tip located at the end on the bottom side, is oscillated at its resonance frequency by a piezoelectric element located on the cantilever holder, which is mounted on a piezoelectric tube scanner. This intermittent contact mode of operation and low force-constant cantilevers were originally developed specifically to minimize damage to soft, easily damaged samples. A laser beam, focused onto the backside of the cantilever end, is reflected via an adjustable mirror onto an optical sensor. As the cantilever tip is rastered back and forth over the sample surface, variations in sample height result in deflection of the cantilever from its rest position. These deflections cause variations in the optical sensor signal that are converted to a voltage that is applied to the z electrode of the scanner, causing it to expand or contract, and thus raise or lower the cantilever back to its undeflected position. Since the changes in the scanner dimensions are calibrated in nanometers per volt, the changes in applied voltage are mapped as local variations in sample height superimposed on an x-y grid (scan size) set by the user. The sharp tip allows one to obtain nanometer resolution of surface features. Typical resonance frequencies for the commercial AFM cantilevers (NanoDevices, approximately 125 lm long 45 lm wide 4 lm thick) used in this study were around 300 kHz. In addition to monitoring changes in the cantilever deflection position, simultaneously phase imaging, which captures the phase lag angle between the drive signal and actual cantilever oscillation, was used to obtain maps of variations in local properties such as stiffness. Therefore there is a one-to-one correlation between the topographic data and the corresponding phase information. The AFM is equipped with a special cantilever wet cell holder, which was used to image samples under in vitro conditions. Samples were mounted on steel pucks that are held in place on the AFM sample stage by a magnet buried in the base. Since the hydrated samples could not be fixed to the sample by tape, samples were cut large enough to be held in place using the wet cell o-ring seal. Only thin continuous sections were used for imaging to prevent small sections from coming lose during imaging and interfering with the imaging process. Ambient imaging was used for dried and stained samples. In all cases, tapping mode imaging was used. We used Veeco Metrology Nanoscope IIIa controllers with a D3000 microscope to image the dried samples and a Multimode microscope for the wet cell imaging. 2.11. Mechanical properties and modeling Specimens were ‘‘dog bone” type and were machined from polished flat sections of the nacreous parts of whole shells. Steel templates were first made of the specimens to ensure dimensional uniformity across the gauge section and the machining was performed via slow grinding to minimize damage to the edges. The gauge sections were 14 mm in length, 2 mm wide, and 0.5 mm thick. Axial strain was measured using an extensometer with a resolution in strain of approximately 5 106 . Testing was performed in an Instron 5565 material test system, in stroke (i.e. displacement) control; this system has a mechanically controlled cross head and thus is extremely stable. Axial strain rates ranged from 103 to 104 . Modeling was performed for the purpose of extracting estimates for the shear stiffness of the interlamellar layers using a ‘‘shear lag” analytical model that has been verified by finite element analysis. 3. Results 3.1. SEM observations The following SEM observations are briefly described most particularly to provide additional perspective on the structure of nacre in our H. rufescens. Fig. 1a and b shows SEM images of the tile cross-section of a fractured shell that illustrate the optimal interdigitated nacre brick-wall structure. Additional description of the structure, including the interlamellar and intertabular layers may be seen in the images and are described in the caption. Interlamellar regions are identified by the horizontal white arrows in Fig. 1b; the intertabular regions are identified by the vertical white arrows. The black arrow indicates a central region observed in all tiles which had been referred to earlier by Mutvei (1979). Fig. 1d shows an image of individual demineralized interlamellar layers of the external organic matrix. Such images were obtained from critically point dried matrix tissue and show thickness in the range 80– 100 nm; in the shell the interlamellar layers are 30 nm as just noted. The layers appear porous in Fig. 1d and this will be further elucidated when our AFM results are shown later. Fig. 1c shows the growth of ‘‘flat pearls” grown on the surface of glass slides inserted under the mantle of live H. rufescens; such observations will be referred to in our discussion of nacre growth. A noteworthy feature of the flat pearls is the appearance of more than one tile nucleated atop a tile growing laterally on a layer just below. An example of such multiple tile nucleation is indicated by the black arrow in Fig. 1c. 3.2. Optical histochemical microscopy 3.2.1. Calcofluor white staining Calcofluor white is a fluorophore that binds to glycans and fibrillar polysaccharides such as chitin (e.g. Herth, 1980; Ramaswamy et al., 1997). Organic matrices from H. rufescens were stained and imaged after demineralization in EDTA and by cation-exchange resin. In both cases strong binding was detected as shown in Fig. 2c and d. For perspective, however, we first show SEM images of demineralized nacre taken normal to the interlamellar plane, Fig. 2a and b. These images may be compared to those reported by Mutvei (1979, 1977), where similarities and differences may be noted. As in Mutvei’s (1977) SEM images, the intertabular matrix, surrounding the tiles, is clearly revealed as is the appearance of an evidently organic ring-like structure organized around what was the tiles’ centers. Etching, in the form of ‘‘pits”, occurs throughout the surface of the interlamellar layers, but we do not observe the J. Bezares et al. / Journal of Structural Biology 163 (2008) 61–75 65