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

International audienceA 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

Structure and morphology of light-reflecting synthetic and biogenic polymorphs of isoxanthopterin: A comparison

Until recently it was thought that the only optical function of pteridines in biology was to act as light-absorbing pigments, but a recent report by some of us revealed that crystalline isoxanthopterin is a reflector in the eyes of decapod crustaceans. Here, we report the formation of crystalline isoxanthopterin synthetically from the polar dimethyl sulfoxide solvent, with X-ray diffraction analysis revealing a crystal structure different from that of biogenic isoxanthopterin. The structure of the new polymorph was determined in two independent ways. In one approach, it was generated and optimized using first-principles calculations, followed by comparison of simulation and experiment for high-resolution powder X-ray diffraction (PXRD) and electron diffraction. In the other approach, the structure was obtained definitively from PXRD data using a direct-space genetic algorithm for structure solution followed by Rietveld refinement. The synthetic structure is different from its biogenic counterpart, especially in having a nonplanar criss-cross H-bonded arrangement. We also rationalized the morphology of the crystals and the effect of the DMSO thereon, via a comparison between observed and theoretical growth morphologies. In addition, we calculated the optical properties of the synthetic structure and found its two dominant refractive indices to be somewhat lower than those of its biogenic counterpart, but still as high as those of reflecting guanine crystals. Synthetic isoxanthopterin therefore emerges as a promising candidate for incorporation in artificial optical systems

The Architecture of the Adhesive Apparatus of Cultured Osteoclasts: From Podosome Formation to Sealing Zone Assembly

BACKGROUND: Osteoclasts are bone-degrading cells, which play a central role in physiological bone remodeling. Unbalanced osteoclast activity is largely responsible for pathological conditions such as osteoporosis. Osteoclasts develop specialized adhesion structures, the so-called podosomes, which subsequently undergo dramatic reorganization into sealing zones. These ring-like adhesion structures, which delimit the resorption site, effectively seal the cell to the substrate forming a diffusion barrier. The structural integrity of the sealing zone is essential for the cell ability to degrade bone, yet its structural organization is poorly understood. PRINCIPAL FINDINGS: Combining high-resolution scanning electron microscopy with fluorescence microscopy performed on the same sample, we mapped the molecular architecture of the osteoclast resorptive apparatus from individual podosomes to the sealing zone, at an unprecedented resolution. Podosomes are composed of an actin-bundle core, flanked by a ring containing adhesion proteins connected to the core via dome-like radial actin fibers. The sealing zone, hallmark of bone-resorbing osteoclasts, consists of a dense array of podosomes communicating through a network of actin filaments, parallel to the substrate and anchored to the adhesive plaque domain via radial actin fibers. SIGNIFICANCE: The sealing zone of osteoclasts cultured on bone is made of structural units clearly related to individual podosomes. It differs from individual or clustered podosomes in the higher density and degree of inter-connectivity of its building blocks, thus forming a unique continuous functional structure connecting the cell to its extracellular milieu. Through this continuous structure, signals reporting on the substrate condition may be transmitted to the whole cell, modulating the cell response under physiological and pathological conditions

Substrate Adhesion Regulates Sealing Zone Architecture and Dynamics in Cultured Osteoclasts

The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data

Initial stages of cell-matrix adhesion can be mediated and modulated by cell-surface hyaluronan.

A conceptual temporal and spatial gap exists between the first encounter of a cell with an adhesive substrate and the advanced stages of focal adhesion formation. Although ample information is available on focal adhesions structure and function, the mechanism of the first interaction events and the nature of the molecules mediating them are largely unknown. In this paper we identify cell-surface-associated hyaluronan as a mediator and modulator of the first steps of adhesion of A6 and other cells to conventional tissue culture substrates as well as to the surfaces of calcium-(R,R)-tartrate tetrahydrate crystals. Treatment of A6 cells with hyaluronidase suppresses their rapid interactions with these adhesive substrates, and incubation of either the hyaluronidase-treated cells or the substrate with hyaluronan restores cell adhesion. In contrast, excess hyaluronan on both the cells and the substrate strongly inhibits adhesion. We thus propose that cell-surface-associated hyaluronan can mediate and modulate cell-matrix adhesion at the very first encounter with the substrate. It may promote it through the establishment of exquisitely stereospecific chemical interactions or inhibit it by virtue of steric exclusion and/or electrostatic repulsion