Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization.

Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca2+) and inorganic phosphate (Pi) within their lumen and forming a nucleation core (NC). After further sequestration of Ca2+ and Pi, the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca2+) and non-mineralizing (-Ca2+) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca2+. This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.

A new functional membrane protein microarray based on tethered phospholipid bilayers.

A new prototype of a membrane protein biochip is presented in this article. This biochip was created by the combination of novel technologies of peptide-tethered bilayer lipid membrane (pep-tBLM) formation and solid support micropatterning. Pep-tBLMs integrating a membrane protein were obtained in the form of microarrays on a gold chip. The formation of the microspots was visualized in real-time by surface plasmon resonance imaging (SPRi) and the functionality of a GPCR (CXCR4), reinserted locally into microwells, was assessed by ligand binding studies. In brief, to achieve micropatterning, P19-4H, a 4 histidine-possessing peptide spacer, was spotted inside microwells obtained on polystyrene-coated gold, and Ni-chelating proteoliposomes were injected into the reaction chamber. Proteoliposome binding to the peptide was based on metal-chelate interaction. The peptide-tethered lipid bilayer was finally obtained by addition of a fusogenic peptide (AH peptide) to promote proteoliposome fusion. The CXCR4 pep-tBLM microarray was characterized by surface plasmon resonance imaging (SPRi) throughout the building-up process. This new generation of membrane protein biochip represents a promising method of developing a screening tool for drug discovery.

New Tethered Phospholipid Bilayers Integrating Functional G-Protein-Coupled Receptor Membrane Proteins.

Membrane proteins exhibiting extra- and intracellular domains require an adequate near-native lipid platform for their functional reconstitution. With this aim, we developed a new technology enabling the formation of a peptide-tethered bilayer lipid membrane (pep-tBLM), a lipid bilayer grafted onto peptide spacers, by way of a metal-chelate interaction. To this end, we designed an original peptide spacer derived from the natural α-laminin thiopeptide (P19) possessing a cysteine residue in the N-terminal extremity for grafting onto gold and a C-terminal extremity modified by four histidine residues (P19-4H). In the presence of nickel, the use of this anchor allowed us to bind liposomes of variable compositions containing a 2% molar ratio of a chelating lipid, 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] so-called DOGS-NTA, and to form the planar bilayer by triggering liposome fusion by an α-helical (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A protein. The formation of pep-tBLMs was characterized by surface plasmon resonance imaging (SPRi), and their continuity, fluidity, and homogeneity were demonstrated by fluorescence recovery after photobleaching (FRAP), with a diffusion coefficient of 2.5 × 10-7 cm2/s, and atomic force microscopy (AFM). By using variable lipid compositions including phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol 4,5-bisphosphate (PIP2), sphingomyelin (SM), phosphatidic acid (PA), and cholesterol (Chol) in various ratios, we show that the membrane can be formed independently from the lipid composition. We made the most of this advantage to reincorporate a transmembrane protein in an adapted complex lipid composition to ensure its functional reinsertion. For this purpose, a cell-free expression system was used to produce proteoliposomes expressing the functional C-X-C motif chemokine receptor 4 (CXCR4), a seven-transmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs). We succeeded in reinserting CXCR4 in pep-tBLMs formed on P19-4H by the fusion of tethered proteoliposomes. AFM and FRAP characterization allowed us to show that pep-tBLMs inserting CXCR4 remained fluid, homogeneous, and continuous. The value of the diffusion coefficient determined in the presence of reinserted CXCR4 was 2 × 10-7 cm2/s. Ligand binding assays using a synthetic CXCR4 antagonist, T22 ([Tyr5,12, Lys7]-polyphemusin II), revealed that CXCR4 can be reinserted in pep-tBLMs with functional folding and orientation. This new approach represents a method of choice for investigating membrane protein reincorporation and a promising way of creating a new generation of membrane biochips adapted for screening agonists or antagonists of transmembrane proteins.