Hierarchical assembly of cell-matrix adhesion complexes.

The adhesion of cells to the extracellular matrix is a dynamic process, mediated by a series of cell-surface and matrix-associated molecules that interact with each other in a spatially and temporally regulated manner. These interactions play a major role in tissue formation, cellular migration and the induction of adhesion-mediated transmembrane signals. In this paper, we show that the formation of matrix adhesions is a hierarchical process, consisting of several sequential molecular events. One of the earliest steps in surface recognition is mediated, in some cells, by a 1 microm-thick cell-surface hyaluronan coat, which precedes the establishment of stable, cytoskeleton-associated adhesions. The earliest forms of these integrin-mediated contacts are dot-shaped FXs (focal complexes), which are formed under the protrusive lamellipodium of migrating cells. These adhesions recruit, sequentially, different anchor proteins that are involved in binding the actin cytoskeleton to the membrane. Conspicuous in its absence from FXs is zyxin, which is recruited to these sites only on retraction of the leading edge and the transformation of the FXs into a focal adhesion. Continuing application of force to focal adhesions results in the formation of fibrillar adhesions and reorganization of the extracellular matrix. The formation of these adhesions depends on actomyosin contractility and matrix pliability.

Coexistence of amorphous and crystalline calcium carbonate in skeletal tissues.

We describe a new type of composite skeletal tissues in which calcite and stabilized amorphous calcium carbonate (ACC) coexist in well-defined domains. The organisms that form such structures are widely separated in the animal kingdom phylogenetic tree: calcareous sponges and ascidians. This paper compares the microstructures of their composite skeletal elements: The triradiate spicules from the sponge Clathrina are composed of a core of calcite embedded in a thick layer of ACC and covered by a thin calcitic envelope; the tunic spicules from the ascidian Pyura pachydermatina are composed of a core of ACC enveloped by an insoluble organic sheath and covered by a thick calcitic layer. We compare and contrast the macromolecules associated with different amorphous and crystalline phases and their ability to induce the formation of stabilized ACC in vitro.

Chitin-silk fibroin interactions: relevance to calcium carbonate formation in invertebrates.

In mineralized tissues chitin is almost always associated with proteins, many of which are known to have chitin recognition consensus sequences. It has been observed in some mollusk shells that there is a well-defined spatial relation between the crystallographic axes of the crystals and the chitin fibrils. This implies that the chitin functions directly or indirectly as a template for nucleation of the mineral phase. It is thus of much interest to understand the exact nature of the interface between the chitin and the proteins at the molecular level in mineralized tissues. Chitin/silk fibroin interactions were studied in vitro at the molecular level using homogenous films composed of the two macromolecules. The results show that the silk fibroin intercalates between the molecular planes of the chitin, and that the interactions are mainly through the chitin acetyl groups. Published X-ray diffraction patterns and infrared spectra of mineralized tissue organic matrices, as well as infrared spectra reported here of the squid pen and lobster cuticle, all show that in vivo the chitin and protein are not intimately mixed, but exist as two phases. We deduce that there is an interfacial plane between them in which the interactions are through the amide groups.

Structure of the nacreous organic matrix of a bivalve mollusk shell examined in the hydrated state using cryo-TEM.

During mollusk shell formation, the mineral phase forms within an organic matrix composed of beta-chitin, silk-like proteins, and acidic glycoproteins rich in aspartic acid. The matrix is widely assumed to play an important role in controlling mineralization. Thus, understanding its structure is of prime importance. Cryo-transmission electron microscopy (Cryo-TEM) studies of the matrix of the bivalve Atrina embedded in vitrified ice show that the interlamellar sheets are composed mainly of highly ordered and aligned beta-chitin fibrils. The silk, which is quantitatively an important component of the matrix, could not be imaged within the sheets. Organic material was, however, observed between sheets. We infer that this is the location of the silk. As this material reveals no regular structure, we suggest that at least prior to mineralization the silk is in the form of a hydrated gel. This is supported by cryo-TEM structural observations of an artificial assembly of beta-chitin with and without silk. This view of the nacreous organic matrix significantly changes previous models of the matrix structure and hence hypotheses pertaining to the mechanisms by which mineral formation occurs.

Calcitic microlenses as part of the photoreceptor system in brittlestars.

Photosensitivity in most echinoderms has been attributed to 'diffuse' dermal receptors. Here we report that certain single calcite crystals used by brittlestars for skeletal construction are also a component of specialized photosensory organs, conceivably with the function of a compound eye. The analysis of arm ossicles in Ophiocoma showed that in light-sensitive species, the periphery of the labyrinthic calcitic skeleton extends into a regular array of spherical microstructures that have a characteristic double-lens design. These structures are absent in light-indifferent species. Photolithographic experiments in which a photoresist film was illuminated through the lens array showed selective exposure of the photoresist under the lens centres. These results provide experimental evidence that the microlenses are optical elements that guide and focus the light inside the tissue. The estimated focal distance (4-7 micrometer below the lenses) coincides with the location of nerve bundles-the presumed primary photoreceptors. The lens array is designed to minimize spherical aberration and birefringence and to detect light from a particular direction. The optical performance is further optimized by phototropic chromatophores that regulate the dose of illumination reaching the receptors. These structures represent an example of a multifunctional biomaterial that fulfills both mechanical and optical functions.

Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking.

The hypothesis of lipid domains in cellular plasma membranes is well established. However, direct visualization of the domains has been difficult. Here we report direct visualization of plasma membrane cholesterol microdomains modulated by agents that affect cholesterol trafficking to and from the plasma membrane. The cholesterol microdomains were visualized with a monoclonal antibody that specifically detects ordered cholesterol arrays. These unique cholesterol microdomains were induced on macrophages and fibroblasts when they were enriched with cholesterol in the presence of an ACAT inhibitor, to block esterification of excess cellular cholesterol. Induction of the plasma membrane cholesterol microdomains could be blocked by agents that inhibit trafficking of cholesterol to the plasma membrane and by cholesterol acceptors that remove cholesterol from the plasma membrane. In addition, plasma membrane cholesterol microdomains did not develop in mutant Niemann-Pick type C fibroblasts, consistent with the defect in cholesterol trafficking reported for these cells. The induction of plasma membrane cholesterol microdomains on inhibition of ACAT helps explain how ACAT inhibition promotes cholesterol efflux from cells in the presence of cholesterol acceptors such as HDL. The anti-cholesterol monoclonal antibody also detected extracellular cholesterol-containing particles that accumulated most prominently during cholesterol enrichment of less differentiated human monocyte-macrophages. For the first time, cholesterol microdomains have been visualized that function in cholesterol trafficking to and from the plasma membrane.

Calcium oxalate crystals in tomato and tobacco plants: morphology and in vitro interactions of crystal-associated macromolecules.

Plants form calcium oxalate crystals with unique morphologies under well-controlled conditions. We studied the morphology of single calcium oxalate monohydrate (whewellite) crystals extracted from tomato and tobacco leaves. These crystals have a pseudotetrahedral shape. We identified the (101), (101) or (102), (110), and (hk0) faces as stable faces. The morphology is chiral with unique handedness. We also show that calcium oxalate monohydrate crystals isolated from tomato, tobacco, and bougainvillea leaves contain macromolecules rich in Gly, Glx, and Ser. Crystal-associated macromolecules extracted from tomato and tobacco influence the morphology of calcium oxalate monohydrate crystals grown in vitro, promoting preferential development of the [120] faces. Furthermore, crystal-associated macromolecules from tobacco promote nucleation of calcium oxalate monohydrate crystals, whereas model polypeptides do not have any significant effect on nucleation. These results imply an active role of the crystal-associated macromolecules in the formation of pseudotetrahedral shapes in vitro, and these properties may in part be responsible for the unique chiral morphology of the natural pyramidal-shaped crystals.

Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates.

Mechanical forces play a major role in the regulation of cell adhesion and cytoskeletal organization. In order to explore the molecular mechanism underlying this regulation, we have investigated the relationship between local force applied by the cell to the substrate and the assembly of focal adhesions. A novel approach was developed for real-time, high-resolution measurements of forces applied by cells at single adhesion sites. This method combines micropatterning of elastomer substrates and fluorescence imaging of focal adhesions in live cells expressing GFP-tagged vinculin. Local forces are correlated with the orientation, total fluorescence intensity and area of the focal adhesions, indicating a constant stress of 5.5 +/- 2 nNmicrom(-2). The dynamics of the force-dependent modulation of focal adhesions were characterized by blocking actomyosin contractility and were found to be on a time scale of seconds. The results put clear constraints on the possible molecular mechanisms for the mechanosensory response of focal adhesions to applied force.

On how proteins interact with crystals and their effect on crystal formation.

Formation of crystalline materials in biological environments is largely dominated by macromolecules whose complementarity to the surface structure is the basis for molecular pattern recognition. This complementarity may in turn result in regulation of crystal growth and morphology, or in induction of crystal nucleation. The conceptual framework for these effects is illustrated here using examples from biomineralization as well as by the immune responses during pathological crystallizations to crystalline antigens. Antibodies are shown to be a precious tool for discerning the fine details of the interactions between biological macromolecules and ordered molecular arrays, such as are present on the surfaces of crystals, monolayers and possibly cell membranes.

Stereoselective recognition of monolayers of cholesterol, ent-cholesterol, and epicholesterol by an antibody.

The interaction between a monoclonal antibody and four distinct monolayers with varying degrees of structural, chemical, and stereochemical similarity were studied and quantified. The antibody, raised and selected against cholesterol monohydrate crystals, interacts with cholesterol monolayers stereospecifically, but not enantiospecifically. Monolayers of ent-cholesterol molecules, which are chemically identical to cholesterol and whose structure is the exact mirror image of the cholesterol monolayer, interact with the antibody to the same extent as the cholesterol monolayers. The affinity of the antibody for both enantiomeric monolayers is extremely high. However, the antibody does not interact with monolayers of epicholesterol, which is an epimer of cholesterol: The hydroxy group in epicholesterol is in the 3alpha position rather than in the 3beta position, imposing a different angle between the hydroxy group and the rigid steroid backbone, and a different packing of the molecules. Monolayers of triacontanol, a long-chain primary aliphatic alcohol, interact with the antibody to a lesser extent than the cholesterol and ent-cholesterol monolayers, presumably due to the structural flexibility of the triacontanol molecule. The lack of chiral discrimination by the antibody is thus correlated to the level at which the chirality is exposed at the surface of the monolayers.

Stereoselective interactions of a specialized antibody with cholesterol and epicholesterol monolayers.

The stereoselective recognition by monoclonal antibodies of two-dimensional monolayers of cholesterol spread at the air-water interface is presented. Using immunofluorescence, we show that one antibody, raised and selected against crystals of cholesterol monohydrate, specifically recognizes monolayers of cholesterol, but not monolayers of epicholesterol--its epimeric form. This demonstrates that stereoselective recognition also applies to protein-surface interactions.

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.

Cellular control over spicule formation in sea urchin embryos: A structural approach.

The spicules of the sea urchin embryo form in intracellular membrane-delineated compartments. Each spicule is composed of a single crystal of calcite and amorphous calcium carbonate. The latter transforms with time into calcite by overgrowth of the preexisting crystal. Relationships between the membrane surrounding the spiculogenic compartment and the spicule mineral phase were studied in the transmission electron microscope (TEM) using freeze-fracture. In all the replicas observed the spicules were tightly surrounded by the membrane. Furthermore, a variety of structures that are related to the material exchange process across the membrane were observed. The spiculogenic cells were separated from other cell types of the embryo, frozen, and freeze-dried on the TEM grids. The contents of electron-dense granules in the spiculogenic cells were shown by electron diffraction to be composed of amorphous calcium carbonate. These observations are consistent with the notion that the amorphous calcium carbonate-containing granules contain the precursor mineral phase for spicule formation and that the membrane surrounding the forming spicule is involved both in transport of material and in controlling spicule mineralization.

Structural and chemical complementarity between antibodies and the crystal surfaces they recognize.

The sequences of the variable regions of three monoclonal antibodies with different specificities to cholesterol monohydrate and 1,4-dinitrobenzene crystals were determined. The structures of their binding sites were then modeled, based on homology to other antibodies of known structure. Two of these antibodies were previously shown to specifically recognize each one well-defined face of one of the crystals, out of a number of crystal faces of closely related structure. The binding site of the antibody which recognizes the stepped (301) face of the cholesterol crystal is predicted to assume the shape of a step with one hydrophobic and one hydrophilic side, complementary to the corresponding crystal surface. Within the step, the hydroxyl groups of five tyrosines are located such that they can interact with the hydroxyl and water molecules on the cholesterol crystal face, while hydrophobic contacts are made between the cholesterol backbone and hydrophobic amino acid sidechains. In contrast, the modeled binding site of the antibody which recognizes the flat (101) face of 1,4-dinitrobenzene crystals is remarkably flat. It is lined by aromatic and polar residues, that can make favorable contacts with the aromatic ring and nitro groups of the dinitrobenzene molecules, respectively.

Relaxation of micro indentations in calcium oxalate urinary stones.

PURPOSE: We define energy requirements for stone micro indentation as a quantifiable event equivalent to in vivo energy delivery and investigate the change in indentation characteristics with time. MATERIALS AND METHODS: The 7 stones extracted from 7 patients were cut, embedded in resin and polished. Multiple micro indentations were performed on each stone section using a diamond Vickers micro indentor with a 500x light microscope and video system. The resulting indentations were observed by optical and scanning electron microscopy as a function of time. Organic matrix content was determined by dissolving stones in ethylenediaminetetraacetic acid solution. RESULTS: The energy requirement for stone indentation varies among stones (median range 43.6 to 109.9 kg/mm2) and at different locations in the same stone. Indentations relaxed by 10 to 70% during the first 2 weeks after indentation. Stones with a high organic matrix content were ductile and the phenomenon of indentation relaxation was pronounced. Brittle, low matrix stones relaxed to a lesser extent. CONCLUSIONS: The relaxation phenomena may have a practical implementation when considering repeat shock wave lithotripsy. A significant fraction of the energy invested in a stone which did not cause fracture or critical cracks is lost within 1 to 2 weeks after the procedure, particularly in elastic stones with a high organic matrix content. We suggest that the preferred interval for repeat shock wave lithotripsy be less than 2 weeks.

Design strategies of sea urchin teeth: structure, composition and micromechanical relations to function.

The teeth of sea urchins comprise a variety of different structural entities, all of which are composed of magnesium-bearing calcite together with a small amount of organic material. The teeth are worn down continuously, but in such a way that they remain sharp and functional. Here we describe aspects of the structural, compositional and micromechanical properties of the teeth of Paracentrotus lividus using scanning electron microscopy, infrared spectrometry, atomic absorption. X-ray diffraction and microindentation. The S-shaped single crystalline calcitic fibres are one of the main structural elements of the tooth. They extend from the stone part to the keel. The diameter of the fibres increases gradually from less than 1 micron at the stone tip to about 20 microns at the keel end, while their MgCO3 contents decrease from about 13 mol% to about 4.5 mol%. Each fibre is coated by a thin organic sheath and surrounded by polycrystalline calcitic discs containing as much as 35 mol% MgCO3. This structure constitutes a unique kind of gradient fibre-reinforced ceramic matrix composite, whose microhardness and toughness decrease gradually from the stone part to the keel. Primary plates are also important structural elements of the tooth. Each primary plate has a very unusual sandwich-like structure with a calcitic envelope surrounding a thin apparently amorphous CaCO3 layer. This central layer, together with the primary plate/disc interface, improves the toughness of this zone by stopping and blunting cracks. The self-sharpening function of the teeth is believed to result from the combination of the geometrical shape of the main structural elements and their spatial arrangement, the interfacial strength between structural elements, and the hardness gradient extending from the working stone part to the surrounding zones. The sea urchin tooth structure possesses an array of interesting functional design features, some of which may possibly be applicable to materials science.

Cell-adhesion to crystal surfaces. Adhesion-induced physiological cell death.

Cultured epithelial cells interact massively, rapidly and stereospecifically with the ¿011¿ faces of calcium (R,R)-tartrate tetrahydrate crystals. It was suggested that the massive rapid adhesion represents an exaggerated and isolated form of the first initial events in the attachment of cultured cells to conventional tissue culture surfaces (Hanein, et al., Cells and Materials, 5, 197-210; 1995). Attachment is however not followed by normal cell spreading and development of focal adhesions, but results in massive cell death. In this study, the fate of the crystal-bound cells was characterized by electron microscopy, flow cytometry and microscopic morphometry and was found to display the characteristics of physiological cell death. We show that the direct interaction with the highly homogenous and repetitive ¿011¿ faces per se does not trigger the transduction of lethal transmembrane signals. We suggest that the excessive direct interactions between the cell membrane and the crystal, by impairing cell motion, prevent the evolution of RGD-dependent cell adhesion. This implies that the deprivation of proper extracellular matrix (ECM)-receptor contacts of substrate-attached epithelial cells eventually triggers physiological cell death.

Monoclonal antibodies that specifically recognize crystals of dinitrobenzene.

Monoclonal antibodies have been elicited and selected after injection of crystals of 1,4-dinitrobenzene (1,4-DNB) and cholesterol monohydrate in mice. The reactivity of some of these antibodies to 1,4-DNB crystals, cholesterol monohydrate crystals, and other solid substrates has been characterized. Two of the antibodies selectively recognize 1,4-DNB crystal surfaces in an appropriately modified ELISA. They do not interact either with 1,4-DNB/BSA conjugates or with polystyrene and cholesterol monohydrate surfaces. They do interact with 1,2-DNB crystal surfaces, albeit with much lower reactivity. It is consequently suggested that these antibodies are not specific to the DNB molecule but rather to a repetitive motif of molecular moieties exposed at the crystal surfaces. Characterization of their binding regions may help to elucidate the interactions of antibodies with solid substrates and, in general, with antigens exposed on biological and artificial surfaces.