Kazuko Saruwatari

An acidic matrix protein, Pif, is a key macromolecule for nacre formation.

Making Mother of Pearl Nacre is an iridescent layer of calcium carbonate lining the inside of shells of marine mollusks and is commonly known as “mother of pearl.” It is composed of layers of uniformly oriented crystals of aragonite (a metastable form of calcium carbonate) separated by layers of organic matrix. How the ordered structure of aragonite layers is achieved has been unclear. Suzuki et al. (p. 1388, published online 13 August 2009; see the Perspective by Kröger) identified two acidic matrix proteins (Pif 97 and Pif 80) that regulate nacre formation in the Japanese pearl oyster. The proteins appear to form a complex in which Pif 80 binds to aragonite and Pif 97 binds to other macromolecules in the organic matrix. A matrix protein is identified that regulates nacre formation in the Japanese pearl oyster. The mollusk shell is a hard tissue consisting of calcium carbonate crystals and an organic matrix. The nacre of the shell is characterized by a stacked compartment structure with a uniformly oriented c axis of aragonite crystals in each compartment. Using a calcium carbonate–binding assay, we identified an acidic matrix protein, Pif, in the pearl oyster Pinctada fucata that specifically binds to aragonite crystals. The Pif complementary DNA (cDNA) encoded a precursor protein, which was posttranslationally cleaved to produce Pif 97 and Pif 80. The results from immunolocalization, a knockdown experiment that used RNA interference, and in vitro calcium carbonate crystallization studies strongly indicate that Pif regulates nacre formation.

Glycolytic intermediates induce amorphous calcium carbonate formation in crustaceans

It has been thought that phosphorus in biominerals made of amorphous calcium carbonate (ACC) might be related to ACC formation, but no such phosphorus-containing compounds have ever been identified. Crustaceans use ACC biominerals in exoskeleton and gastroliths so that they will have easy access to calcium carbonate inside the body before and after molting. We have identified phosphoenolpyruvate and 3-phosphoglycerate, intermediates of the glycolytic pathway, in exoskeleton and gastroliths and found them important for stabilizing ACC.

Nucleation and growth of aragonite crystals at the growth front of nacres in pearl oyster, Pinctada fucata

The growth front of nacreous layer, which lies just above the outer prismatic layer, is one of the crucial areas to comprehend the formation of nacreous aragonite. The crystallographic properties of aragonite crystals at the growth front in pearl oyster, Pinctada fucata, were investigated using scanning electron microscopy with electron back-scattered diffraction, and transmission electron microscopy with focused ion beam sample preparation technique. Nano-sized aragonite crystals nucleate with random crystallographic orientation inside the dimples on the surface of the organic matrix that covers the outer prismatic columns. The dimples are filled with horn-like aragonite crystals, which enlarge from the bottom to the upper surface to form hemispheric domes. The domes grow concentrically and coalesce together to become the initial nacreous layer. The c-axes of aragonite at the top surface of the domes are preferentially oriented perpendicular to the surface. The horn-like aragonite and its crystallographic orientation are probably attained by geometrical selection with the fastest growth rate of aragonite along the c-axis, until organic sheets are continuously formed and interrupt the crystal growth of aragonite. The further crystal growth along the shell thickness is attained via mineral bridges through discontinuity or holes in the organic sheets. These results indicate that the crystal growth of aragonite at the growth front results from not only biotic process but also inorganic ones such as geometrical selection and mineral bridges.

Microtexture of larval shell of oyster, Crassostrea nippona: a FIB-TEM study.

The initial formation and subsequent development of larval shells in marine bivalve, Crassostrea nippona were investigated using the FIB-TEM technique. Fourteen hours after fertilization (the trochophore stage), larvae form an incipient shell of 100-150nm thick with a columnar contrast. Selected-area electron diffraction analysis showed a single-crystal aragonite pattern with the c-axis perpendicular to the shell surface. Plan-view TEM analysis suggested that the shell contains high density of {110} twins, which are the origin of the columnar contrast in the cross-sectional images. 72h after fertilization (the veliger stage), the shell grows up to 1.2-1.4mum thick accompanying an additional granular layer between the preexisting layer and embryo to form a distinctive two-layer structure. The granular layer is also composed of aragonite crystals sharing their c-axes perpendicular to the shell surface, but the crystals are arranged with a flexible rotation around the c-axes and not restricted solely to the {110} twin relation. No evidence to suggest the existence of amorphous calcium carbonate (ACC) was found through the observation. The well-regulated crystallographic properties found in the present sample imply initial shell formation probably via a direct deposition of crystalline aragonite.

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, Emiliania huxleyi, on Calcite Crystal Formation in In Vitro CaCO3 Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO3 and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO3 crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO3 crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO3 crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.

Crystallographic alignments in a coccolith (Pleurochrysis carterae) revealed by electron back-scattered diffraction (EBSD)

Abstract Crystal orientations of the sub-micrometer-sized calcite units in oval-shaped coccoliths of a coccolithophore, Pleurochrysis carterae, have been investigated using electron back-scattered diffraction (EBSD). Although the contrast of the acquired EBSD patterns was weak due to the small crystal unit size, the orientations could be uniquely determined from the patterns. The crystal orientations for V- and R-units are close to those reported in a previous work using electron diffraction in a TEM. However, more accurate crystal orientations corresponding to the coccolith morphology were obtained by using EBSD in a SEM. In V-units, the c-axis is declined about 35° from the normal of the coccolith plane and one of the ai-axes is roughly parallel to the coccolith plane. The c-axis in R-units is slightly oblique to the radial direction along the coccolith plane and one of the ai-axes is near vertical to the coccolith plane. The projections of the c-axis of V- and R-units on the coccolith plane deviate considerably from the normal of the coccolith circumference, giving a crystallographic chiral property. The atomic arrangements of calcite contacted with the organic base plate are discussed for both units based on the crystallographic orientations derived from EBSD measurements.

Comparison of crystallographic orientations between living (Emiliania huxleyi and Gephyrocapsa oceanica) and fossil (Watznaueria barnesiae) coccoliths using electron microscopes

Abstract Crystallographic orientations of coccoliths produced by the extant species Emiliania huxleyi and Gephyrocapsa oceanica, and of a fossil cocccolith, Watzunaueria barnesiae, all of which are mainly composed of calcite crystals with horizontally oriented c axes (termed R-units), were investigated using two electron diffraction techniques. According to electron backscattered diffraction (EBSD) analyses, the c-axis inclinations of all R-units were oriented about 10-30° from the sample substrate, which is approximately parallel to the organic base plate of the coccolith. However, the directions are toward the coccolith exterior in living coccoliths but toward the interior in fossil coccoliths. The crystallographic orientations of calcite crystals characterized by sub-vertically oriented c axes (termed V-units) were determined only for W. barnesiae and were similar to those of V-units in another living cococlith, Pleurochrysis carterae. Highly regulated crystallographic orientation and consistent chirality were evidenced for all the coccoliths with only a few degrees of variations by Kikuchi patterns analyses from TEM. To determine the indices of the crystallographic plane and edge direction that constitute distal and proximal shield elements, SEM stereo-photogrammetry in combination with EBSD analyses was applied. In the living coccoliths both shields contain the c-axis and the surface is estimated to be {21̅1̅0} with [481] the longer peripheral edge. On the other hand, the two shield surfaces of W. barnesiae are evaluated {101̅4} and the distal shield surface is surrounded by clear [481] edges. This indicates that the crystallography and morphology of R-units were not fixed for the past 230 million years. Based on these results, the calcite nucleation mechanisms are discussed with respect to the atomic arrangements on the organic template through all the coccoliths.

Evidence for the role of organic layers in photoconductivity of organic/inorganic hybrid nanosheets as prepared by Langmuir–Blodgett methods

By measuring the photoconductivity of hybrid LB films of exfoliative layered niobate and octadecylamine, it was evidenced that the film underwent a transition from an insulator to a photosemiconductor during photo-modification treatment by UV light, which was rationalized in terms of the direct contact of inorganic nanosheets achieved by the elimination of organic layers.

Microstructures of the larval shell of a pearl oyster, Pinctada fucata, investigated by FIB-TEM technique

Abstract The structure of the larval shell of a pearl oyster, Pinctada fucata, has been investigated at several growing stages mainly using the focused ion beam (FIB) sample preparation technique and transmission electron microscopy (TEM). Until 12 h from fertilization, the larva does not have any calcified shells. After 18 h from fertilization, the embryo is covered with the first shell made of aragonite with the c-axis normal to the shell. The cross-sectional view of the shell shows a columnar contrast, but plan-view observation revealed that the columnar contrast does not correspond to individual crystals but is related to dense polycyclic {110} twins in the aragonite crystal. After 48 h from fertilization, the larvae formed a new aragonite layer under the initial layer; a homogeneous layer with globular contrast. The c-axis of the globules is normal to the shell. The orientation of the other axes is aligned locally but random in general. Additionally, larvae consisting of monolithic calcite as the inner layer were found at this stage. One to three weeks from fertilization, a new aragonite layer with a prismatic contrast is formed under the homogeneous layer. This layer consists of prismatic grains of aragonite, with their c-axes parallel to the prisms. High-angle annular dark-field (HAADF) images suggest that a considerable amount of organic molecules may be contained in the homogeneous layer but not in the inner prismatic layer, implying that the texture of each layer is related to the amount of organic molecules incorporated.

Textures and polytypes in vermiform kaolins diagenetically formed in a sandstone reservoir: a FIB-TEM investigation

Focused ion beam (FIB) sample preparation was applied to vermiform kaolin minerals to investigate by TEM the morphological evolution and mechanisms of kaolinite-to-dickite transformation during burial diagenesis. A core sample of a K-feldspar bearing arkosic sandstone, collected from the Brent Group sediments of the Froy hydrocarbon reservoir, Norwegian continental shelf, North Sea, was studied. Two types of vermiform aggregates were analyzed: 1) porous aggregates of fine lamellar crystals or intercalations of blocky and lamellar crystals, and 2) aggregates of densely packed hexagonal crystals. Electron diffraction (ED) revealed that aggregates of the first type are composed of pure one-layer kaolinite, whereas those of the second type consist of pure two-layer dickite. The two polytypes never coexist within the same aggregate. Furthermore, ED patterns of kaolinite and dickite are almost streak free, indicating a very low density of stacking faults. These observations suggest that the kaolinite-to-dickite transition in the pores of sandstone reservoir during burial diagenesis takes place by a dissolution-precipitation process rather than by a solid-state process. Blocky kaolinite, grown epitaxially on the (001) surface of muscovite, was also found in the specimen.