Mollusk shell formation: mapping the distribution of organic matrix components underlying a single aragonitic tablet in nacre

Control over mineral formation in mollusk shells is exerted by the macromolecules of the organic matrix. Using histochemical methods, we mapped the carboxylates and sulfates of proteins and polysaccharides on the surfaces of decalcified interlamellar matrices from the nacreous shell layer of the cephalopod Nautilus pompilius, expanding upon an earlier study by Crenshaw and Ristedt [Crenshaw, M.A., Ristedt, H., 1976. The histochemical localization of reactive groups in septal nacre from Nautilus pompilius. In: Watabe, N., Wilbur, K.M. (Ed.), The Mechanisms of Mineralization in the Invertebrates and Plants. University of South Carolina Press, Colombia, pp. 355-367]. We observed four different zones underlying a single crystal: (1) a central spot rich in carboxylates; (2) a central ring-shaped area rich in sulfates; (3) an area between the central nucleation region and the imprint periphery containing carboxylates, and (4) the intertabular matrix, rich in carboxylates and sulfates. We also mapped matrix functional groups on the nacreous matrix surfaces of the bivalve Atrina rigida, but did not identify well-defined zones. Immuno-mapping of the constituents of the aragonite-nucleating protein fraction from Atrina nacre showed that these macromolecules are located both in the intertabular matrix and in the center of the crystal imprints for both Atrina and Nautilus matrix surfaces. Their presence at the latter location is consistent with their purported role in aragonite nucleation. The observed differentiation in the distribution of matrix components and their functional groups shows that the different stages of single crystal growth are highly controlled by the matrix.     

Macromolecular structure of the organic framework of nacre in Haliotis rufescens: implications for growth and mechanical behavior

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.     

Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus

In molluscs, and more generally in metazoan organisms, the production of a calcified skeleton is a complex molecular process that is regulated by the secretion of an extracellular organic matrix. This matrix constitutes a cohesive and functional macromolecular assemblage, containing mainly proteins, glycoproteins and polysaccharides that, together, control the biomineral formation. These macromolecules interact with the extruded precursor mineral ions, mainly calcium and bicarbonate, to form complex organo-mineral composites of well-defined microstructures. For several reasons related to its remarkable mechanical properties and to its high value in jewelry, nacre is by far the most studied molluscan shell microstructure and constitutes a key model in biomineralization research. To understand the molecular mechanism that controls the formation of the shell nacreous layer, we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of Nautilin-63 by preparative electrophoresis, we demonstrate that this soluble protein is glycine-aspartate-rich, that it is highly glycosylated, that its sugar moieties are acidic, and that it is able to bind chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the morphology of CaCO(3) crystals precipitated in vitro but, unexpectedly, it exhibits an extremely weak ability to inhibit in vitro the precipitation of CaCO(3) . The partial resolution of its amino acid sequence by de novo sequencing of its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like domains. Owing to specific polyclonal antibodies raised against the purified protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre matrix. In conclusion, Nautilin-63 exhibits 'hybrid' biochemical properties that are found both in the soluble and insoluble proteins, rendering it difficult to classify according to the standard view on nacre proteins. DATABASE: The protein sequences of N63 appear on the UniProt Knowledgebase under accession number P86702.     

Ultrastructural Characteristics of the Nacre in Some Gastropods

The nacreous layer in Gibbula, Calliostoma, Trochus and Haliotis is described on the basis of scanning electron microscopic studies. The central part of each nacreous tablet contains a significant amount of calcified organic matrix which is insoluble in a chromium sulphate and a 25% glutaraldehyde solution. In most cases, the tablet is subdivided by radial vertical organic membranes into a varying number (2 to 50) of crystalline sectors. These sectors represent polysynthetically twinned crystal individuals which form cyclic or interpenetrant twins. The nacreous tablets in gastropods are compared with those in bivalves, and with the non-biogenic aragonite. The mechanical properties of the nacre, and the effects of the interlamellar conchiolin membranes upon the nucleation of the tablets, are discussed.     

Structure and composition of the septal nacreous layer of Nautilus macromphalus L. (Mollusca, Cephalopoda)

The nacreous layer of Mollusca is the best-known aragonitic structure and is the usual model for biomineralization. However, data are based on less than 10 species. In situ observations of the septal nacreous layer of the cephalopod Nautilus shell has revealed that the tablets are composed of acicular laths. These laths are composed of round nanograins surrounded by an organic sheet. No hole has been observed in the decalcified interlamellar membranes. A set of combined analytical data shows that the organic matrices extracted from the nacreous layer are glycoproteins. In both soluble and insoluble matrices, S amino acids are rare and the soluble organic matrices have a higher sulfated sugar content than the insoluble matrices. It is possible that the observed differences in the structure and composition of the nacreous layers of the outer wall and septa of the Nautilus shell have a dual origin: evolution and functional adaptation. However, we have no appropriate data as yet to answer this question.     

Dermatopontin, a shell matrix protein gene from pearl oyster Pinctada martensii, participates in nacre formation

Dermatopontin (DPT) is identified as a major component of the shell matrix protein. However, its exact function in the shell formation remains obscure. In this study, we described the characteristic and function of DPT gene from Pinctada martensii. DPT cDNA was 797bp long, containing an open reading fragment (ORF) of 537bp encoding a polypeptide of 178 amino acids with an estimated molecular mass of 21.4kDa and theoretical isoelectric point of 5.97. The 5' untranslated region (UTR) was 11bp and the 3'UTR was 249 with 18bp poly (A) tail. In the peptide, there was a signal sequence, six potential phosphorylation sites, one glycosylation site and eight cysteine residues. Moreover, a sequence motif (D-R-X-W/F/Y-X-F/Y/I/L/M-X(1-2)-C) was contained and repeated itself three times in the entire sequence. DPT mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the mantle, which was nacre formation-related tissue. After decreasing DPT expression using RNA interference (RNAi) technology in the mantle, the nacreous layer showed a disordered growth; whereas the prismatic layer of the shells has no significant changes. These results suggested that DPT obtained in this study was a constitutive matrix protein and participated in nacre formation in P. martensii.     

Dynamics of sheet nacre formation in bivalves

Formation of nacre (mother-of-pearl) is a biomineralization process of fundamental scientific as well as industrial importance. However, the dynamics of the formation process is still not understood. Here, we use scanning electron microscopy and high spatial resolution ion microprobe depth-profiling to image the full three-dimensional distribution of organic materials around individual tablets in the top-most layer of forming nacre in bivalves. Nacre formation proceeds by lateral, symmetric growth of individual tablets mediated by a growth-ring rich in organics, in which aragonite crystallizes from amorphous precursors. The pivotal role in nacre formation played by the growth-ring structure documented in this study adds further complexity to a highly dynamical biomineralization process.     

Geometrical and crystallographic constraints determine the self-organization of shell microstructures in Unionidae (Bivalvia: Mollusca)

Unionid shells are characterized by an outer aragonitic prismatic layer and an inner nacreous layer. The prisms of the outer shell layer are composed of single-crystal fibres radiating from spheruliths. During prism development, fibres progressively recline to the growth front. There is competition between prisms, leading to the selection of bigger, evenly sized prisms. A new model explains this competition process between prisms, using fibres as elementary units of competition. Scanning electron microscopy and X-ray texture analysis show that, during prism growth, fibres become progressively orientated with their three crystallographic axes aligned, which results from geometric constraints and space limitations. Interestingly transition to the nacreous layer does not occur until a high degree of orientation of fibres is attained. There is no selection of crystal orientation in the nacreous layer and, as a result, the preferential orientation of crystals deteriorates. Deterioration of crystal orientation is most probably due to accumulation of errors as the epitaxial growth is suppressed by thick or continuous organic coats on some nacre crystals. In conclusion, the microstructural arrangement of the unionid shell is, to a large extent, self-organized with the main constraints being crystallographic and geometrical laws.     

Sheet nacre growth mechanism: a Voronoi model

Shell nacre (mother of pearl) of Pinctada margaritifera was analyzed by scanning electron microscopy. The originality of this work concerns the sampling performed to observe incipient nacre on the mantle side. The whole animal is embedded in methyl methacrylate followed by separation of the shell from the hardened mantle. It is revealed this way how each future nacre layer pre-exists as a film or compartment. Experimental observations also show for the first time, the progressive lateral crystallization inside this film, finishing under the form of a non-periodic pattern of polygonal tablets of bio-aragonite. It is evidenced that nuclei appear in the film in the vicinity of the zone where aragonite tablets of the underlying layer get in contact to each other. A possible explanation is given to show how nucleation is probably launched in time and space by a signal coming from the underlying layer. Finally, it is evidenced that tablets form a Voronoi tiling of the space: this suggests that their growth is controlled by an "aggregation-like" process of "crystallites" and not directly by the aragonite lattice growth.     

A novel matrix protein participating in the nacre framework formation of pearl oyster,Pinctada fucata

Understanding the molecular composition is of great interest for both nacre formation mechanism and biomineralization in mollusk shell. A cDNA clone encoding an MSI31 relative, termed MSI7 because of its estimated molecular mass of 7.3 kDa, was isolated from the pearl oyster, Pinctada fucata. This novel protein shares similarity with MSI31, a prismatic framework protein of P. fucata. It is peculiar that MSI7 is much shorter in size, harboring only the Gly-rich sequence that has been proposed to be critical for Ca(2+) binding. In situ hybridization result showed that MSI7 mRNA was expressed specifically at the folds and outer epithelia of the mantle, indicating that MSI7 participates in the framework formation of both the nacreous layer and prismatic layer. In vitro experiment on the function of MSI7 suggested that it accelerates the nucleation and precipitation of CaCO(3). Taken together, we have identified a novel matrix protein of the pearl oyster, which may play an important role in determining the texture of nacre.     

Characterization of MRNP34, a novel methionine-rich nacre protein from the pearl oysters

Nacre of the Pinctada pearl oyster shells is composed of 98% CaCO₃ and 2% organic matrix. The relationship between the organic matrix and the mechanism of nacre formation currently constitutes the main focus regarding the biomineralization process. In this study, we isolated a new nacre matrix protein in P. margaritifera and P. maxima, we called Pmarg- and Pmax-MRNP34 (methionine-rich nacre protein). MRNP34 is a secreted hydrophobic protein, which is remarkably rich in methionine, and which is specifically localised in mineralizing the epithelium cells of the mantle and in the nacre matrix. The structure of this protein is drastically different from those of the other nacre proteins already described. This unusual methionine-rich protein is a new member in the growing list of low complexity domain containing proteins that are associated with biocalcifications. These observations offer new insights to the molecular mechanisms of biomineralization.     

A scanning electron microscopic study of the inorganic and organic matrices comprising the mature shell of Amblema, a fresh-water mollusc

The scanning electron microscope has been used to describe the morphology of the mature shell in a fresh-water bivalve. The structure of the organic and inorganic components within the nacre, the myostracum, and the prismatic layer is described. A transitional or intermediate zone, interposed between the prismatic layer and the nacre, was identified. In demineralized samples, the organic component of the nacre was found to consist of parallel matricial sheets interconnected by irregular transverse bridges. The structure of the mineral component of the nacre was found to vary with the method of specimen preparation. With polished-etched samples, brick-like units were seen. When shells were simply broken and fixed in osmium, the layers of nacreous material consisted of fusing rhomboidal crystals of aragonite which demonstrated subconchoidal fractures. On the inner surface of the shell, the rhomboidal crystals showed an apparent spiral growth pattern. The myostracum was characterized by regions of modified nacreous structure consisting of enlarged aragonite crystals with a pyramidal morphology. The peripheral aspect of the muscle scars was characterized by rhomboidal crystals, the latter fusing to form the typical nacreous laminae. The uniqueness of the anterior adductor scar is exemplified by the presence of pores, each pore walled by pyramidal units, for the insertion of adductor fibres. In most regions of the shell, the prismatic layer consisted of one prism unit thickness with a height of approximately 225–250 μm. However, in two specialized regions of the shell, this layer was seen to consist of multiple layers of stacked prisms. The organic matrices of the prismatic layer are arranged in a honeycomb-like arrangement and packed with mineralized spherical subunits.     

Tissue inhibitor of metalloproteinase gene from pearl oyster Pinctada martensii participates in nacre formation

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901 bp long, containing a 5′ untranslated region (UTR) of 51 bp, a 3′ UTR of 169 bp, and an open reading fragment (ORF) of 681 bp encoding 226 amino acids with an estimated molecular mass of 23.37 kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.     

Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: an environmental- and cryo-scanning electron microscopy study

A key to understanding control over mineral formation in mollusk shells is the microenvironment inside the pre-formed 3-dimensional organic matrix framework where mineral forms. Much of what is known about nacre formation is from observations of the mature tissue. Although these studies have elucidated several important aspects of this process, the structure of the organic matrix and the microenvironment where the crystal nucleates and grows are very difficult to infer from observations of the mature nacre. Here, we use environmental- and cryo-scanning electron microscopy to investigate the organic matrix structure at the onset of mineralization in the nacre of two mollusk species: the bivalves Atrina rigida and Pinctada margaritifera. These two techniques allow the visualization of hydrated biological materials coupled with the preservation of the organic matrix close to physiological conditions. We identified a hydrated gel-like protein phase filling the space between two interlamellar sheets prior to mineral formation. The results are consistent with this phase being the silk-like proteins, and show that mineral formation does not occur in an aqueous solution, but in a hydrated gel-like medium. As the tablets grow, the silk-fibroin is pushed aside and becomes sandwiched between the mineral and the chitin layer.     

Ultrastructure of the Interlamellar Membranes of the Nacre of the Bivalve Pteria hirundo,Determined by Immunolabelling

The current model for the ultrastructure of the interlamellar membranes of molluscan nacre imply that they consist of a core of aligned chitin fibers surrounded on both sides by acidic proteins. This model was based on observations taken on previously demineralized shells, where the original structure had disappeared. Despite other earlier claims, no direct observations exist in which the different components can be unequivocally discriminated. We have applied different labeling protocols on non-demineralized nacreous shells of the bivalve Pteria. With this method, we have revealed the disposition and nature of the different fibers of the interlamellar membranes that can be observed on the surface of the nacreous shell of the bivalve Pteria hirundo by high resolution scanning electron microscopy (SEM). The minor chitin component consists of very thin fibers with a high aspect ratio and which are seemingly disoriented. Each fiber has a protein coat, which probably forms a complex with the chitin. The chitin-protein-complex fibers are embedded in an additional proteinaceous matrix. This is the first time in which the sizes, positions and distribution of the chitin fibers have been observed in situ.     

In-depth proteomic analyses of <Emphasis Type="Italic">Haliotis laevigata</Emphasis> (greenlip abalone) nacre and prismatic organic shell matrix

Background

The shells of various Haliotis species have served as models of invertebrate biomineralization and physical shell properties for more than 20 years. A focus of this research has been the nacreous inner layer of the shell with its conspicuous arrangement of aragonite platelets, resembling in cross-section a brick-and-mortar wall. In comparison, the outer, less stable, calcitic prismatic layer has received much less attention. One of the first molluscan shell proteins to be characterized at the molecular level was Lustrin A, a component of the nacreous organic matrix of Haliotis rufescens. This was soon followed by the C-type lectin perlucin and the growth factor-binding perlustrin, both isolated from H. laevigata nacre, and the crystal growth-modulating AP7 and AP24, isolated from H. rufescens nacre. Mass spectrometry-based proteomics was subsequently applied to to Haliotis biomineralization research with the analysis of the H. asinina shell matrix and yielded 14 different shell-associated proteins. That study was the most comprehensive for a Haliotis species to date.

Methods

The shell proteomes of nacre and prismatic layer of the marine gastropod Haliotis laevigata were analyzed combining mass spectrometry-based proteomics and next generation sequencing.

Results

We identified 297 proteins from the nacreous shell layer and 350 proteins from the prismatic shell layer from the green lip abalone H. laevigata. Considering the overlap between the two sets we identified a total of 448 proteins. Fifty-one nacre proteins and 43 prismatic layer proteins were defined as major proteins based on their abundance at more than 0.2% of the total. The remaining proteins occurred at low abundance and may not play any significant role in shell fabrication. The overlap of major proteins between the two shell layers was 17, amounting to a total of 77 major proteins.

Conclusions

The H. laevigata shell proteome shares moderate sequence similarity at the protein level with other gastropod, bivalve and more distantly related invertebrate biomineralising proteomes. Features conserved in H. laevigata and other molluscan shell proteomes include short repetitive sequences of low complexity predicted to lack intrinsic three-dimensional structure, and domains such as tyrosinase, chitin-binding, and carbonic anhydrase. This catalogue of H. laevigata shell proteins represents the most comprehensive for a haliotid and should support future efforts to elucidate the molecular mechanisms of shell assembly.

     

Crystalline structure,orientation and nucleation of the nacreous tablets in the cephalopod <Emphasis Type="Italic">Nautilus</Emphasis>

The nacreous tablets in the Nautilus shell have similar crystalline structure as the tablets in the gastropod Gibbula shell. Etching with Mutvei’s solution reveals that each tablet is composed of vertical crystalline columns that are structurally similar to the acicular crystallites in the outer spherulitic-prismatic layer of the shell wall. The columns are attached to each other to form numerous vertical crystalline lamellae, oriented parallel to the longitudinal axis of the tablet. It is still unknown whether or not the orientation of the vertical lamellae corresponds to that of the crystallographic a- or b-axis. The orientation of the crystalline lamellae in the adjacent tablets is parallel in some nacreous laminae, but random in other laminae. Similar large variation was found in the nacreous tablets of the gastropod and bivalve shells. The nucleation sites of the nacreous tablets are predominantly situated on the peripheral portion of the upper surface of the preceding tablet, both in the shell wall and septa. The “aragonite-nucleating proteins” in the central portion of the crystal imprints of the organic interlamellar sheets, described by several writers, have therefore a negative correlation with the nucleation sites of the nacreous tablets.     

Mineral bridges in nacre

We confirm with high-resolution techniques the existence of mineral bridges between superposed nacre tablets. In the towered nacre of both gastropods and the cephalopod Nautilus there are large bridges aligned along the tower axes, corresponding to gaps (150–200 nm) in the interlamellar membranes. Gaps are produced by the interaction of the nascent tablets with a surface membrane that covers the nacre compartment. In the terraced nacre of bivalves bridges associated with elongated gaps in the interlamellar membrane (>100 nm) have mainly been found at or close to the edges of superposed parental tablets. To explain this placement, we hypothesize that the interlamellar membrane breaks due to differences in osmotic pressure across it when the interlamellar space below becomes reduced at an advanced stage of calcification. In no cases are the minor connections between superimposed tablets (<60 nm), earlier reported to be mineral bridges, found to be such.     

Patterns of Expression in the Matrix Proteins Responsible for Nucleation and Growth of Aragonite Crystals in Flat Pearls of Pinctada fucata

The initial growth of the nacreous layer is crucial for comprehending the formation of nacreous aragonite. A flat pearl method in the presence of the inner-shell film was conducted to evaluate the role of matrix proteins in the initial stages of nacre biomineralization in vivo. We examined the crystals deposited on a substrate and the expression patterns of the matrix proteins in the mantle facing the substrate. In this study, the aragonite crystals nucleated on the surface at 5 days in the inner-shell film system. In the film-free system, the calcite crystals nucleated at 5 days, a new organic film covered the calcite, and the aragonite nucleated at 10 days. This meant that the nacre lamellae appeared in the inner-shell film system 5 days earlier than that in the film-free system, timing that was consistent with the maximum level of matrix proteins during the first 20 days. In addition, matrix proteins (Nacrein, MSI60, N19, N16 and Pif80) had similar expression patterns in controlling the sequential morphologies of the nacre growth in the inner-film system, while these proteins in the film-free system also had similar patterns of expression. These results suggest that matrix proteins regulate aragonite nucleation and growth with the inner-shell film in vivo.     

Electron probe analysis of strontium in mussel (Bivalvia,Mytilidae) shells: Feasibility of estimating water temperature

Electron microprobe step-scan analyses across the inner nacreous layer of a sectionedMytilus edulis shell revealed no long-term periodic (e.g., seasonal) variation in the concentration of strontium. Similarly, no significant difference was found between a specimen sampled in February (water temperature = 1.3 °C) and one sampled in August (water temperature = 18.0 °C) with regard to the concentration of strontium within the most recently deposited aragonite. Correlation of the amount of strontium within various nacreous regions of the shells of living or fossil mytilids with water temperatures (present or past) is probably not possible through the use of an electron probe, at least to the extent that strontium variation within the nacre ofMytilus edulis is representative of that in nacreous layers of all mytilids.