Origin and expansion of foliated microstructure in pteriomorph bivalves

The ultrastructure of the calcitic prisms of the prismatic shell layers of pteriomorph bivalves was examined by scanning electronic microscopy and diffraction techniques. Results indicate that the internal structure of the prisms is noticeably different among taxa. In species belonging to the families Pinnidae, Pteriidae, and Isognomonidae (Pterioida), prisms are built up with nanometric calcite crystals. On the other hand, Pectinidae, Propeamussliidae, Anomiidae (order Pectinoida) and the Ostreidae (Ostreoida) have prisms constituted by calcitic laths with micrometric size. These laths are indistinguishable from those constituting the foliated microstructure. In almost all cases, there is mineral continuity from the prisms to the underlying foliated layer, as confirmed by X-ray texture analyses. These findings corroborate a previous assumption that the foliated microstructure derived from calcitic prisms, particularly from those with internal foliated structure. The appearance of the foliated microstructure facilitated drastic mineralogical and microstructural changes in pteriomorph shells-for example, the development of rigid shell margins and the production of largely calcitic shells. Such changes have, no doubt, contributed to the evolutionary success of the groups, which have shown a pronounced diversification over time.     

Bending and branching of calcite laths in the foliated microstructure of pectinoidean bivalves occurs at coherent crystal lattice orientation

Foliated calcite is widely employed by some important pteriomorph bivalve groups as a construction material. It is made from calcite laths, which are inclined at a low angle to the internal shell surface, although their arrangement is different among the different groups. They are strictly ordered into folia in the anomiids, fully independent in scallops, and display an intermediate arrangement in oysters. Pectinids have particularly narrow laths characterized by their ability to change their growth direction by bending or winding, as well as to bifurcate and polyfurcate. Electron backscatter analysis indicates that the c-axes of laths are at a high, though variable, angle to the growth direction, and that the laths grow preferentially along the projection of an intermediate axis between two a-axes, although they can grow in any intermediate direction. Their main surfaces are not particular crystallographic faces. Analyses done directly on the lath surfaces demonstrate that, during the bending/branching events, all crystallographic axes remain invariant. The growth flexibility of pectinid laths makes them an excellent space-filling material, well suited to level off small irregularities of the shell growth surface. We hypothesize that the exceptional ability of laths to change their direction may be promoted by the mode of growth of biogenic calcite, from a precursor liquid phase induced by organic molecules.     

Studies on shell formation. VIII. Electron microscopy of crystal growth of the nacreous layer of the oyster Crassostrea virginica

Electron microscope observations have been made by means of the replica method on growth processes of calcite crystals of the nacreous layer of the shell of the oyster, Crassostrea virginica. Layer formation is initiated by the secretion of a conchiolin matrix and the deposition of rounded crystal seeds on or in this material. In some areas crystal seeds are elongate and within a given area show a similar orientation, probably due to slower deposition. The seeds appear to increase in size by dendritic growth, and smaller seeds become incorporated into larger ones which come into contact to form a single layer. With further growth, crystals overlap, forming a step-like arrangement. The direction of growth is frequently different in neighboring regions. Crystal seeds deposited on crystal surfaces are usually elongate and oriented. Well developed crystals have a tabular idiomorphic form and are parallel in their growth. Rounded and irregular crystals were also observed. The crystals show reticular structure with units of the order of 100 A and striations corresponding with the rhombohedral axes of the crystals. The role of the mantle is discussed in relation to the growth patterns of crystals and shell structure.     

Ultrastructural matrix-mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro

We investigated matrix–mineral relationships in the avian eggshell at the ultrastructural level using scanning and transmission electron microscopy combined with surface-etching techniques to selectively increase topography at the matrix–mineral interface. Moreover, we investigated the distribution of osteopontin (OPN) in the eggshell by colloidal-gold immunolabeling for OPN, and assessed the effects of this protein on calcite crystal growth in vitro. An extensive organic matrix network was observed within the calcitic structure of the eggshell that showed variable, region-specific organization including lamellar sheets of matrix, interconnected fine filamentous threads, thin film-like surface coatings of proteins, granules, vesicles, and isolated proteins residing preferentially on internal {1 0 4} crystallographic faces of fractured eggshell calcite. With the exception of the vesicles and granules, these matrix structures all were immunolabeled for OPN, as were occluded proteins on the {1 0 4} calcite faces. OPN inhibited calcite growth in vitro at the {1 0 4} crystallographic faces producing altered crystal morphology and circular growth step topography at the crystal surface resembling spherical voids in mineral continuity prominent in the palisades region of the eggshell. In conclusion, calcite-occluded and interfacial proteins such as OPN likely regulate eggshell growth by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries to create a biomineralized architecture whose structure provides for the properties and functions of the eggshell.     

Nucleation and growth of calcite on native versus pyrolyzed oyster shell folia

The thin sheets of calcite, termed folia, that make up much of the shell of an oyster are covered by a layer of discrete globules that has been proposed to consist of agglomerations of protein and mineral. Foliar fragments, treated at 475 degrees C for 36 h to remove organic matter, were imaged by atomic force microscopy (AFM) as crystals grew on the foliar surfaces in artificial seawater at calcite supersaturations up to 52-fold. Crystals were also viewed later by scanning electron microscopy. After pyrolysis, the foliar globules persisted only as fragile remnants that were quickly washed away during AFM imaging, revealing an underlying morphology on the foliar laths of a tightly packed continuum of nanometer-scale protrusions. At intermediate supersaturations, crystal formation was seen immediately almost everywhere on these surfaces, each crystal having the same distinctive shape and orientation, even at the outset with crystals as small as a few nanometers. In contrast, nucleation did not occur readily on non-pyrolyzed foliar surfaces, and the crystals that did grow, although slowly at intermediate supersaturations, had irregular shapes. Possible crystallographic features of foliar laths are considered on the basis of the morphology of ectopic crystals and the atomic patterns of various surfaces. A model for foliar lath formation is presented that includes cycles of pulsed secretion of shell protein, removal of the protein from the mineralizing solution upon binding to mineral, and mineral growth at relatively high supersaturation over a time frame of about 1 h for each turn of the cycle.     

Studies on Shell Formation : VIII. Electron Microscopy of Crystal Growth of the Nacreous Layer of the Oyster Crassostrea virginica

Electron microscope observations have been made by means of the replica method on growth processes of calcite crystals of the nacreous layer of the shell of the oyster, Crassostrea virginica. Layer formation is initiated by the secretion of a conchiolin matrix and the deposition of rounded crystal seeds on or in this material. In some areas crystal seeds are elongate and within a given area show a similar orientation, probably due to slower deposition. The seeds appear to increase in size by dendritic growth, and smaller seeds become incorporated into larger ones which come into contact to form a single layer. With further growth, crystals overlap, forming a step-like arrangement. The direction of growth is frequently different in neighboring regions. Crystal seeds deposited on crystal surfaces are usually elongate and oriented. Well developed crystals have a tabular idiomorphic form and are parallel in their growth. Rounded and irregular crystals were also observed. The crystals show reticular structure with units of the order of 100 A and striations corresponding with the rhombohedral axes of the crystals. The role of the mantle is discussed in relation to the growth patterns of crystals and shell structure.     

Crystallographic orientations of structural elements in skeletons of Syringoporicae (tabulate corals,Carboniferous): implications for biomineralization processes in Palaeozoic corals

The crystallographic orientation of structural elements in skeletons of representatives of Carboniferous Syringoporicae (Auloporida) has been analysed by scanning electron microscopy (SEM), petrographic microscopy and electron backscatter diffraction (EBSD) on specimens from the Iberian Peninsula. The skeletons of the tabulate corals of the Syringoporicae consist of biogenic calcite crystals, and their microstructure is composed of lamellae, fibres and granules, or of a combination of these. Independent of the microstructure, the c‐axis is oriented towards the lumen, quasi‐perpendicular to the growth direction of the skeleton (perpendicular to the morphological axis lamellae, parallel to fibres). Most phaceloid taxa have a turbostratic distribution, as a biogenic response to prevent the cleavage of crystals. Cerioid and some phaceloid corals, whose microstructure is conditioned by wall elements, do not exhibit turbostratic distribution. Wall elements are determined by the biology of each taxon. Holacanth septal spines are composed of fibres arranged in a cone‐shape structure, sometimes clamped to the external part of the corallite and show a complex crystallography. Monacanth septal spines are spindle shaped and composed of bundles of fibres. Tabulae are composed of lamellae. Their development and crystallographic orientation depends on the position of the epithelium in each case. Shared walls are formed by a combination of the walls of two independent corallites with a median lamina, composed of granules; these have a crystallographic orientation between that of the two corallites. The growth of the microstructure is derived by a coordinated stepping mode of growth, similar to other groups of organisms such as molluscs and scleractinians. The nucleation and formation of packages of co‐oriented microcrystals suggest a growth mode similar to mineral bridges with a competitive growth mode between each crystal. The growth pattern of corallites suggests that the growth direction is divided into two main components: a horizontal growth direction towards the lumen and a vertical direction towards the top.     

Specific adsorption of osteopontin and synthetic polypeptides to calcium oxalate monohydrate crystals

Protein-crystal interactions are known to be important in biomineralization. To study the physicochemical basis of such interactions, we have developed a technique that combines confocal microscopy of crystals with fluorescence imaging of proteins. In this study, osteopontin (OPN), a protein abundant in urine, was labeled with the fluorescent dye AlexaFluor-488 and added to crystals of calcium oxalate monohydrate (COM), the major constituent of kidney stones. In five to seven optical sections along the z axis, scanning confocal microscopy was used to visualize COM crystals and fluorescence imaging to map OPN adsorbed to the crystals. To quantify the relative adsorption to different crystal faces, fluorescence intensity was measured around the perimeter of the crystal in several sections. Using this method, it was shown that OPN adsorbs with high specificity to the edges between {100} and {121} faces of COM and much less so to {100}, {121}, or {010} faces. By contrast, poly-L-aspartic acid adsorbs preferentially to {121} faces, whereas poly-L-glutamic acid adsorbs to all faces approximately equally. Growth of COM in the presence of rat bone OPN results in dumbbell-shaped crystals. We hypothesize that the edge-specific adsorption of OPN may be responsible for the dumbbell morphology of COM crystals found in human urine.     

Coccolith crystals ofPleurochrysis carterae: Crystallographic faces, organization, and development

Summary The crystallographic and morphological configuration of the mineral ring associated with the coccoliths ofPleurochrysis carterae was determined by transmission electron microscopy and electron diffraction. Mature Pleurochrysis coccoliths consist of an oval organic base plate, a distal rim of interlocking calcite crystals, and a narrow ribbon of organic material which tethers the mineral ring to the base plate. Crystals of two distinct forms (R and V units) alternate about the rim in a quasi regular manner; their crystallographicc-axes are aligned parallel to and inclined about 63° to the coccolith plane, respectively. The mineral ring has four platelike elements: the distal-shield and outer-tube elements which form the V unit, and the proximal-shield and inner-tube elements which form the R units. The platy surfaces of both tube elements correspond to the common (10 4) rhombohedral faces of calcite, and the plates of the proximal-shield element are prismatic (2 0) faces. The plates of the distal-shield element are rather curved and their orientation does not correspond to a favorable calcite face; however, for convenience they are described as approximately ( 108) faces, faces which rarely, if ever, develop in inorganic sources of calcite. During coccolith development the earliest habits observed for both V and R units correspond to rectangular parallelepipeds. Outgrowth from the initial V unit begins by expansion of (10 4) faces which form the platy surfaces of the outer-tube element. Throughout this period of development the mineral ring is flexible, at least in an isolated state. Subsequent outgrowth of the inner-tube and proximal-shield elements from the initial R unit produces a rigid interlocking ring. The unusual ( 108) faces of the distal-shield element develop after the crystals are locked in place. Organic structures in intimate association with the mineral phase during its nucleation and growth include the coccolith ribbon, the calcium-polyanion particles, and the membrane of the coccolith vesicle. These structures are described in reference to their putative functions in regulating the development of V and R units.Abbreviation PS polysaccharide     

Patterns of crystal organization and calcite twin formation in planktonic,rotaliid, foraminifera shells and spines

The foraminiferal order Rotaliida represents one third of the extant genera of foraminifers. The shells of these organisms are extensively used to decipher characteristics of marine ecosystems and global climate events.It was shown that shell calcite of benthic Rotaliida is twinned. We extend our previous work on microstructure and texture characterization of benthic Rotaliida and investigate shell calcite organization for planktonic rotaliid species. Based on results gained from electron backscattered diffraction (EBSD) and field emission electron microscopy (FESEM) imaging of chemically etched/fixed shell surfaces we show for the planktonic species Globigerinoides sacculifer, Pulleniatina obliquiloculata, Orbulina universa (belonging to the two main planktonic, the globigerinid and globorotaliid, clades): very extensive 60°-{0 0 1}-twinning of the calcite and describe a new and specific microstructure for the twinned crystals. We address twin and crystal morphology development from nucleation within a biopolymer template (POS) to outermost shell surfaces. We demonstrate that the calcite of the investigated planktonic Rotaliida forms through competitive growth. We complement the structural knowledge gained on the clade 1 and clade 2 species with EBSD results of Globigerinita glutinata and Candeina nitida shells (clade 3 planktonic species). The latter are significantly less twinned and have a different shell calcite microstructure.We demonstrate that the calcite of all rotaliid species is twinned, however, to different degrees. We discuss for the species of the three planktonic clades characteristics of the twinned calcite and of other systematic misorientations. We address the strong functionalization of foraminiferal calcite and indicate how the twinning affects biocalcite material properties.     

Mechanism of calcite crystal growth inhibition by the N-terminal undecapeptide of lithostathine

Pancreatic juice is supersaturated with calcium carbonate. Calcite crystals therefore may occur, obstruct pancreatic ducts, and finally cause a lithiasis. Human lithostathine, a protein synthesized by the pancreas, inhibits the growth of calcite crystals by inducing a habit modification: the rhombohedral (10 14) usual habit is transformed into a needle-like habit through the (11 0) crystal form. A similar observation was made with the N-terminal undecapeptide (pE(1)R(11)) of lithostathine. We therefore aimed at discovering how peptides inhibit calcium salt crystal growth. We solved the complete x-ray structure of lithostathine, including the flexible N-terminal domain, at 1.3 A. Docking studies of pE(1)R(11) with the (10 14) and (11 0) faces through molecular dynamics simulation resulted in three successive steps. First, the undecapeptide progressively unfolded as it approached the calcite surface. Second, mobile lateral chains of amino acids made hydrogen bonds with the calcite surface. Last, electrostatic bonds between calcium ions and peptide bonds stabilized and anchored pE(1)R(11) on the crystal surface. pE(1)R(11)-calcite interaction was stronger with the (11 0) face than with the (10 14) face, confirming earlier experimental observations. Energy contributions showed that the peptide backbone governed the binding more than did the lateral chains. The ability of peptides to inhibit crystal growth is therefore essentially based on backbone flexibility.     

Colloidal-gold immunocytochemical localization of osteopontin in avian eggshell gland and eggshell.

During mineralization of the avian eggshell, there is a sequential and orderly deposition of both matrix and mineral phases. Therefore, the eggshell is an excellent model for studying matrix-mineral relationships and the regulation of mineralization. Osteopontin, as an inhibitor of crystal growth, potently influences the formation of calcium phosphate and calcium carbonate biominerals. The purpose of this study was to characterize matrix-mineral relationships, specifically for osteopontin, in the avian eggshell using high-resolution transmission (TEM) and scanning (SEM) electron microscopy to gain insight into how calcite crystal growth is structured and compartmentalized during eggshell mineralization. Osteopontin was localized at the ultrastructural level by colloidal-gold immunocytochemistry. In EDTA-decalcified eggshell, an extensive matrix network was observed by TEM and SEM throughout all regions and included interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries that compartmentalized different eggshell structural units. In fractured and undecalcified eggshell, osteopontin was immunolocalized on the {104} crystallographic faces of calcite-its natural cleavage plane. The specific occlusion of osteopontin into calcite during mineralization may influence eggshell structure to modify its fracture resistance.     

CRYSTAL PROPERTY OF THE LARVAL SEA URCHIN SPICULE*

A pair of pluteus skeletal spicules arises from a pair of calcareous granules via the triradiate form. In polarized light, each spicule behaves as though carved out of a single crystal of magnesian calcite. The optic axis lies perpendicular to the plane of the triradiate and parallel to the body rod of the pluteus. However, in the scanning electron microscope, the spicule surface appeared smooth or somewhat spongy and manifested no crystal faces. Neither etching nor fracturing revealed underlying crystalline texture. Nevertheless, rhombohedral calcite crystals could be grown epitaxially onto isolated spicules immersed in a medium containing CaCl₂ and NaHCO₃. The optic axes of all crystals coincided with the optic axis of the spicule on which they were grown. Corresponding faces of the crystals were all aligned parallel to each other despite the complex shape of each spicule. Where the left and right spicules joined, two mutually tilted sets of crystals were observed but not crystals of intermediate orientation. Thus, the sea urchin larval spicule is built from a stack of molecularly contiguous microcrystals but its overall shape is generated by the mesenchyme cells independent of the magnesian calcite crystal habit.     

Atomic force microscopy of crystalline insulins: the influence of sequence variation on crystallization and interfacial structure.

The self-association of proteins is influenced by amino acid sequence, molecular conformation, and the presence of molecular additives. In the presence of phenolic additives, LysB28ProB29 insulin, in which the C-terminal prolyl and lysyl residues of wild-type human insulin have been inverted, can be crystallized into forms resembling those of wild-type insulins in which the protein exists as zinc-complexed hexamers organized into well-defined layers. We describe herein tapping-mode atomic force microscopy (TMAFM) studies of single crystals of rhombohedral (R3) LysB28ProB29 that reveal the influence of sequence variation on hexamer-hexamer association at the surface of actively growing crystals. Molecular scale lattice images of these crystals were acquired in situ under growth conditions, enabling simultaneous identification of the rhombohedral LysB28ProB29 crystal form, its orientation, and its dynamic growth characteristics. The ability to obtain crystallographic parameters on multiple crystal faces with TMAFM confirmed that bovine and porcine insulins grown under these conditions crystallized into the same space group as LysB28ProB29 (R3), enabling direct comparison of crystal growth behavior and the influence of sequence variation. Real-time TMAFM revealed hexamer vacancies on the (001) terraces of LysB28ProB29, and more rounded dislocation noses and larger terrace widths for actively growing screw dislocations compared to wild-type bovine and porcine insulin crystals under identical conditions. This behavior is consistent with weaker interhexamer attachment energies for LysB28ProB29 at active growth sites. Comparison of the single crystal x-ray structures of wild-type insulins and LysB28ProB29 suggests that differences in protein conformation at the hexamer-hexamer interface and accompanying changes in interhexamer bonding are responsible for this behavior. These studies demonstrate that subtle changes in molecular conformation due to a single sequence inversion in a region critical for insulin self-association can have a significant effect on the crystallization of proteins.     

Crystallographic reorganization of the calcitic prismatic layer of oysters

The calcitic columnar prisms of pteriomorphian bivalves have the crystallographic c-axis oriented perpendicular to the shell surface and the a-axes rotated without any preferential orientation. In oysters, SEM, XRD and EBSD analyses show that individual prisms initially have their a-axes randomly oriented but are able to progressively orient them parallel to those of their neighbors. This ability is apparently confined to groups, such as oysters and scallops, in which prisms are internally constituted by smaller lath-like crystal units. We have developed a competition model – not between prisms, but between the lath-like secondary units of prisms – which is based on differences in the inclination of laths relative to the shell growth surface. Units having a growth component which coincides with the growth direction protrude faster from the growth surface and out-compete those which are not favorably oriented, which reduces the overall dispersion of the a-axes of the prismatic lamella. The extent of re-alignment increases with the relative inclination of the growth surface and the length attained by the prisms. Oysters are the only group in which these two characters are pronounced enough to provide a measurable re-alignment. The proposed competition model is unprecedented in biomaterials and reveals how important crystal growth processes are in microstructure organization.     

Microstructure and crystallographic-texture of giant barnacle (Austromegabalanus psittacus) shell

Barnacle shell is a very complex and strong composite bioceramic composed of different structural units which consist of calcite 15 microcrystals of very uniform size. In the study reported herein, the microstructural organization of these units has been examinated in detail with optical and scanning electron microscopy, and X-ray diffraction techniques. These analyses showed that the external part of the shell has a massive microstructure consisting of randomly oriented crystals. Toward the interior, the shell became organized in mineral layers separated by thin organic sheets. Each of these mineral layers has a massive microstructure constituted by highly oriented calcite microcrystals with their c-axes aligned [(001) fibre texture] perpendicular to the organic sheets and the shell surface. Interestingly, in another structural unit, the shell shield, the orientation of the c-axis calcite crystals shifts from being perpendicular to being parallel to the shell surface across its thickness. This study provides evidence that the organic matrix is responsible for the organization of the shell mineral and exterts strong a strict control on the polymorphic type, size and orientation of shell-forming crystals.     

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.     

Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals

We have isolated a new protein from the nacreous layer of the shell of the sea snail Haliotis laevigata (abalone). Amino acid sequence analysis showed the protein to consist of 134 amino acids and to contain three sequence repeats of approximately 40 amino acids which were very similar to the well-known whey acidic protein domains of other proteins. The new protein was therefore named perlwapin. In addition to the major sequence, we identified several minor variants. Atomic force microscopy was used to explore the interaction of perlwapin with calcite crystals. Monomolecular layers of calcite crystals dissolve very slowly in deionized water and recrystallize in supersaturated calcium carbonate solution. When perlwapin was dissolved in the supersaturated calcium carbonate solution, growth of the crystal was inhibited immediately. Perlwapin molecules bound tightly to distinct step edges, preventing the crystal layers from growing. Using lower concentrations of perlwapin in a saturated calcium carbonate solution, we could distinguish native, active perlwapin molecules from denaturated ones. These observations showed that perlwapin can act as a growth inhibitor for calcium carbonate crystals in saturated calcium carbonate solution. The function of perlwapin in nacre growth may be to inhibit the growth of certain crystallographic planes in the mineral phase of the polymer/mineral composite nacre.     

Homologous shell microstructures in Cambrian hyoliths and molluscs

Hyoliths were among the earliest biomineralizing metazoans in Palaeozoic marine environments. They have been known for two centuries and widely assigned to lophotrochozoans. However, their origin and relationships with modern lophotrochozoan clades have been a longstanding palaeontological controversy. Here, we provide broad microstructural data from hyolith conchs and opercula from the lower Cambrian Xinji Formation of North China, including two hyolithid genera and four orthothecid genera as well as unidentified opercula. Results show that most hyolith conchs contain a distinct aragonitic lamellar layer that is composed of foliated aragonite, except in the orthothecid New taxon 1 that has a crossed foliated lamellar microstructure. Opercula are mostly composed of foliated aragonite and occasionally foliated calcite. These blade or lath‐like microstructural fabrics coincide well with biomineralization of Cambrian molluscs rather than lophophorates, as exemplified by the Cambrian members of the tommotiid‐brachiopod linage. Accordingly, we propose that hyoliths and molluscs might have inherited their biomineralized skeletons from a non‐mineralized or weakly mineralized common ancestor rather than as a result of convergence. Consequently, from the view of biomineralization, the homologous shell microstructures in Cambrian hyoliths and molluscs strongly strengthen the phylogenetic links between the two groups.     

Effects of surface-bound water and surface stereochemistry on cell adhesion to crystal surfaces

Crystals of calcium-(R,S)-tartrate trihydrate were used as adhesion substrates (for A6 epithelial cells), to study specific stages in cell adhesion. Events such as surface recognition, cell attachment, spreading, motility, cell-cell aggregation, and cell penetration into the crystal bulk are all shown to depend on the molecular structure of the various crystal faces. These crystals exhibit three chemically equivalent, yet structurally distinct, faces. On the {100}, a layered surface exposing bound water, the cells attach, are motile, and tend to form multicellular aggregates, but do not spread and do not form focal contacts. Following prolonged incubation, single cells attached to the {100} surface undergo apoptosis, while those interacting with other cells are rescued. Macroscopic spiral dislocations emerging on the {100} face of the crystal are highly adhesive for cells. Cells attached to these sites develop long protrusions that penetrate into the crystal. The {011} faces expose mainly hydroxyls attached to the chiral carbons. The cells interact extensively with these faces, are immobilized, do not spread, do not form focal contacts, and subsequently die. The faces belonging to the {0kl}? family are characterized by molecular and topographical steps. The cells attach to these faces, spread, and form focal contacts and stress fibers. Thus the molecular character of the crystal surfaces, including the presence of bound water, the exposure of determinants that promote rapid surface recognition, and the effective association with extracellular adhesive proteins, affect the patterns of cell adhesive behavior and fate.