Crystalline Domain Structure and Cholesterol Crystal Nucleation in Single Hydrated DPPC:Cholesterol:POPC Bilayers

Grazing incidence X-ray diffraction measurements were performed on single hydrated bilayers and monolayers of DPPC:Cholesterol:POPC at varying concentrations. There are substantial differences in the phase and structure behavior of the crystalline domains formed within the bilayers relative to the corresponding monolayers, due to interactions between the opposing leaflets. Depending on the lipid composition, these interactions led to phase separation, changes in molecular tilt angle, or formation of cholesterol crystals. In monolayers, DPPC and cholesterol form a single crystalline phase at all compositions studied. In bilayers, a second crystalline phase appears when cholesterol levels are increased: domains of cholesterol and DPPC form monolayer thick crystals where each of the lipid leaflets diffracts independently, whereas excess cholesterol forms cholesterol bilayer thick crystals at a DPPC:Chol ratio < 46:54 ± 2 mol %. The nucleation of the cholesterol crystals occurs at concentrations relevant to the actual cell plasma membrane composition

The Stabilizing Effect of Silicate on Biogenic and Synthetic Amorphous Calcium Carbonate

Silicate ions increase the thermal stability of the unstable amorphous calcium carbonate (ACC). This effect was observed first by comparing ACC from two different species of cystoliths, small calcified bodies formed in the leaves of some plants. The temperature of crystallization to calcite in the silicate-rich cystoliths from M. alba is 100 °C higher than that of the silicate-poor cystoliths from F. microcarpa. The stabilizing effect is confirmed in vitro with synthetic samples differing in their silicate content. With increasing silicate concentration in ACC, the crystallization temperature to calcite also increases. A mechanism of geometric frustration is suggested, whereby the presence of the tetrahedral silicate ion in the flat carbonate lattice prevents organization into crystalline polymorphs

Supplementary Information from Surface microtopography modulates sealing zone development in osteoclasts cultured on bone

Supplementary Information for the article: “Surface Microtopography Modulates Sealing Zone development in Osteoclasts Cultured on Bone” for publication in the Journal of the Royal Society Interfac

Mineral and Matrix Components of the Operculum and Shell of the Barnacle <i>Balanus amphitrite</i>: Calcite Crystal Growth in a Hydrogel

Sessile barnacles produce two types of mineralized exoskeletons: a cone-shaped shell and an operculum that is used to seal the shell opening. The mineral of both types is calcite. We show that the calcite crystals of the shell and the operculum (specifically the scutum) of the sessile barnacle <i>Balanus amphitrite</i> both fracture with conchoidal cleavage, have surfaces decorated with small rhombohedral shaped calcite crystals, and are poorly oriented. The scutum calcite is significantly more disordered at the atomic level than the shell calcite. We also show that a major component of the intercrystalline organic matrix of the shell and scutum is a nonproteinaceous sulfate-rich polymer that behaves as a hydrogel, and that the intracrystalline matrix contains highly acidic proteins. The crystal properties and microstructure are consistent with the calcite crystals forming in a hydrogel-like environment. The barnacle shell and operculum have many unique properties indicating that the crystal growth conditions are well controlled and possibly adapted to fulfill mechanical functions, which enable the barnacle to survive in the high energy environment of the intertidal zone

Temperature-Sensitive Micrometer-Thick Layers of Hyaluronan Grafted on Microspheres

The giant polyelectrolyte glycosaminoglycan hyaluronan (1−10 MDa) is a major component of the pericellular coat on a variety of cells, where it is an important modulator and mediator of early cell adhesion events. This pericellular layer can reach 5 μm thickness on cells that produce cartilage (chondrocytes), and up to 2 μm on Xenopus laevis kidney epithelial cells (A6). We are interested in generating model systems for the pericellular coat in order to learn more about the structure and function of hyaluronan on biological or artificial surfaces. We report here the synthesis of model systems where a coat of coordinatively cross-linked hyaluronan of up to 2 μm thickness was covalently photografted onto polystyrene microspheres. The hydrated coat was imaged directly by environmental scanning electron microscopy (ESEM) at close to 100% relative humidity. The key feature of the procedure is the reversible reverse-temperature phase transition of hyaluronan induced by trivalent lanthanide cations, which is exploited to achieve sufficient density for grafting of thick layers. The microsphere-grafted coat shows a temperature-dependent swelling when labeled with lanthanide ions (Gd3+ or Tb3+). We directly observed a volume contraction of 20% with increasing temperature between 1 and 11 °C by wet-mode ESEM

Study of Osteoclast Adhesion to Cortical Bone Surfaces: A Correlative Microscopy Approach for Concomitant Imaging of Cellular Dynamics and Surface Modifications

Bone remodeling relies on the coordinated functioning of osteoblasts, bone-forming cells, and osteoclasts, bone-resorbing cells. The effects of specific chemical and physical bone features on the osteoclast adhesive apparatus, the sealing zone ring, and their relation to resorption functionality are still not well-understood. We designed and implemented a correlative imaging method that enables monitoring of the same area of bone surface by time-lapse light microscopy, electron microscopy, and atomic force microscopy before, during, and after exposure to osteoclasts. We show that sealing zone rings preferentially develop around surface protrusions, with lateral dimensions of several micrometers, and ∼1 μm height. Direct overlay of sealing zone rings onto resorption pits on the bone surface shows that the rings adapt to pit morphology. The correlative procedure presented here is noninvasive and performed under ambient conditions, without the need for sample labeling. It can potentially be applied to study various aspects of cell-matrix interactions

Chirality of Amyloid Suprastructures

Amyloids are pathological fibrillar aggregates of proteins related to more than 20 different diseases. Amyloid fibers have a characteristic cross-β structure consisting of a series of β-strands extended perpendicular to the fiber axis and joined by hydrogen bonds parallel to the fiber direction. Fibril aggregation results in helical suprastructures. Here we used high-resolution SEM and cryo-SEM for the study of chirality in the amyloid suprastructure. We found that amyloids of Aβ1-40 and hen lysozyme form at all hierarchical levels always and only left-handed helices. In contrast, amyloid fibers formed by the N-terminal sequence of serum amyloid A (SAA1-12) consist of right-handed helices exclusively. Consistently, the peptide enantiomer, formed of (R)-aminoacids, aggregates exclusively in left-handed helices. We conclude that the opposite handedness of the SAA1-12 amyloids is an intrinsic feature of the peptide structure. The left-handed chirality observed for the Aβ1-40 and hen lysozyme amyloid suprastructures is consistent with the conventional β-sheet structural model. In contrast, the right-handedness observed in (all-S) SAA1-12 fibers indicates that the cross-β structure of SAA1-12 fibers is probably not formed of β-sheets. Whatever the answer to the dilemma of the right-handed helicity of SAA1-12 amyloid fibers is, its existence shows that the supramolecular chirality of amyloid fibers is not only dictated by the molecular chirality of the component molecules but also by their structural organization

The Structural Basis for Enhanced Silver Reflectance in Koi Fish Scale and Skin

Fish have evolved biogenic multilayer reflectors composed of stacks of intracellular anhydrous guanine crystals separated by cytoplasm, to produce the silvery luster of their skin and scales. Here we compare two different variants of the Japanese Koi fish; one of them with enhanced reflectivity. Our aim is to determine how biology modulates reflectivity, and from this to obtain a mechanistic understanding of the structure and properties governing the intensity of silver reflectance. We measured the reflectance of individual scales with a custom-made microscope, and then for each individual scale we characterized the structure of the guanine crystal/cytoplasm layers using high-resolution cryo-SEM. The measured reflectance and the structural-geometrical parameters were used to calculate the reflectance of each scale, and the results were compared to the experimental measurements. We show that enhanced reflectivity is obtained with the same basic guanine crystal/cytoplasm stacks, but the structural arrangement between the stack, inside the stacks, and relative to the scale surface is varied when reflectivity is enhanced. Finally, we propose a model that incorporates the basic building block parameters, the crystal orientation inside the tissue, and the resulting reflectance and explains the mechanistic basis for reflectance enhancement

Formation of 3D Cholesterol Crystals from 2D Nucleation Sites in Lipid Bilayer Membranes: Implications for Atherosclerosis

Atherosclerosis is the major precursor of cardiovascular disease. The formation of cholesterol crystals in atherosclerotic plaques is associated with the onset of acute pathology. The cholesterol crystals induce physical injury in the plaque core, promoting cell apoptosis and triggering an increased inflammatory response. Herein we address the question of how cholesterol crystal formation occurs in atherosclerosis. We demonstrate that three-dimensional (3D) cholesterol crystals can undergo directed nucleation from bilayer membranes containing two-dimensional (2D) cholesterol crystalline domains. We studied crystal formation on supported lipid bilayers loaded with exogenous cholesterol and labeled using a monoclonal antibody that specifically recognizes ordered cholesterol arrays. Our findings show that 3D crystals are formed exclusively on the bilayer regions where there are segregated 2D cholesterol crystalline domains and that they form on the domains. This study has potentially significant implications for our understanding of the crucial step in the mechanism by which atherosclerotic lesions form

Relation between Serum Amyloid A Truncated Peptides and Their Suprastructure Chirality

Amyloids are pathological fibrillar aggregates of proteins related to over 20 diseases. Amyloid fibers are characterized by the cross-β motif, which is minimally defined as a series of β-strands extended perpendicular to the fiber axis, joined by hydrogen bonds parallel to the fiber direction. Several structures, all in agreement with the cross-β definition, have been proposed for specific amyloids. We study the correlation among the suprastructural chirality, molecular structure, and molecular chirality of amyloids. Here we investigate the suprastructure chirality of different (all-S) serum amyloid A (SAA) truncated peptides. We found that the suprastructure chirality of amyloid fibers from segments SAA2−6, SAA1−11 and the majority of those from SAA2−9 is left-handed, which is consistent with the β-sheet protofilament model. In contrast, SAA1−12 and SAA2−12 as well as SAA1−12, where the C-terminal aspartic acid was point mutated to either leucine or alanine, form right-handed helical amyloid fibers. Such a suprastructure switch indicates a molecular change in the protofilament structure. This is supported by the behavior observed in the FTIR spectra, where the amide I peak of all of the right-handed fibers is red shifted relative to the left-handed amyloid fibers. This work is a case study where isolated short fragments of SAA containing the same amyloidogenic core sequence fold into different amyloid structures. We show that core sequences, supposed to start the misfolding aggregation of the full-length amyloid peptides, may have structures different from those assumed by the isolated segments