Bastien Seantier

Influence of mono- and divalent ions on the formation of supported phospholipid bilayers via vesicle adsorption.

We have used the quartz crystal microbalance with dissipation monitoring (QCM-D) technique to investigate how mono- and divalent cations influence the formation of supported (phospho)lipid bilayers (SPB, SLB), occurring via deposition of nanosized palmitoyloleoyl phosphatidylcholine (POPC) vesicles on a SiO2 support. This process is known to proceed via initial adsorption of intact vesicles until a critical surface coverage is reached, where the combination of vesicle-surface and vesicle-vesicle interaction causes the vesicles to rupture. New vesicles then rupture and the lipid fragments fuse until a final continuous bilayer is formed. We have explored how this process and the critical coverage are influenced by different mono- and divalent ions and ion concentrations, keeping the anions the same throughout the experiments. The same qualitative kinetics is observed for all cations. However, different ions cause quite different quantitative kinetics. When compared with monovalent ions, even very small added concentrations of divalent cations cause a strong reduction of the critical coverage, where conversion of intact, adsorbed vesicles to bilayer occurs. This bilayer promoting effect increases in the order Sr2+Na+>K+. The results are of practical value for preparation of lipid bilayers and help shed light on the role of ions and on electrostatic effects at membrane surfaces/interfaces.

Influence of nanotopography on phospholipid bilayer formation on silicon dioxide

We have investigated the effect of well-defined nanoscale topography on the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicle adsorption and supported phospholipid bilayer (SPB) formation on SiO2 surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Unilamellar lipid vesicles with two different sizes, 30 and 100 nm, were adsorbed on pitted surfaces with two different pit diameters, 110 and 190 nm, as produced by colloidal lithography, and the behavior was compared to results obtained on flat surfaces. In all cases, complete bilayer formation was observed after a critical coverage of adsorbed vesicles had been reached. However, the kinetics of the vesicle-to-bilayer transformation, including the critical coverage, was significantly altered by surface topography for both vesicle sizes. Surface topography hampered the overall bilayer formation kinetics for the smaller vesicles, but promoted SPB formation for the larger vesicles. Depending on vesicle size, we propose two modifications of the precursor-mediated vesicle-to-bilayer transformation mechanism used to describe supported lipid bilayer formation on the corresponding flat surface. Our results may have important implications for various lipid-membrane-based applications using rough or topographically structured surfaces.

Calcium-induced formation of subdomains in phosphatidylethanolamine-phosphatidylglycerol bilayers: a combined DSC, 31P NMR, and AFM study.

We study the effect of Ca(2+) on the lateral segregation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). Supported lipid bilayers (SLBs) were observed by atomic force microscopy (AFM). Since SLBs are formed from liposomes of POPE:POPG, we examined the effect of calcium on these suspensions by differential scanning calorimetry (DSC) and (31)P nuclear magnetic resonance spectroscopy ((31)P NMR). AFM images revealed the existence of two separated phases, the higher showing a region with protruding subdomains. Force spectroscopy (FS) was applied to clarify the nature of each phase. The values of breakthrough force (F(y)), adhesion force (F(adh)), and height extracted from the force curves were assigned to the corresponding gel (L(beta)) and fluid (L(alpha)) phase. The endotherms obtained by DSC suggest that, in the presence of Ca(2+), phase separation already exists in the suspensions of POPE:POPG used to form SLBs. Due to the temperature changes applied during preparation of SLBs a (31)P NMR study was performed to assess the lamellar nature of the samples before spreading them onto mica. With in situ AFM experiments we showed that the binding of Ca(2+) to POPG-enriched domains only induces the formation of subdomains in the L(beta) phase.

Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM.

In plasma membranes, most glycosylphosphatidylinositol-anchored proteins (GPI proteins) would be associated with ordered microdomains enriched in sphingolipids and cholesterol. Debates on the composition and the nano- or mesoscales organization of these membrane domains are still opened. This complexity of biomembranes explains the use, in the recent years, of both model systems and atomic force microscopy (AFM) approaches to better characterize GPI proteins/membranes interactions. So far, the studies have mainly been focused on alkaline phosphatases of intestinal (BIAP) or placental (PLAP) origins reconstituted in model systems. The data show that GPI-anchored alkaline phosphatases (AP-GPI) molecules inserted in supported membranes can be easily imaged by AFM, in physiological buffer. They are generally observed in the most ordered domains of model membranes under phase separation, i.e. presenting both fluid and ordered domains. This direct access to the membrane structure at a mesoscopic scale allows establishing the GPI protein induced changes in microdomains size. It provides direct evidence for the temperature-dependent distribution of a GPI protein between fluid and ordered membrane domains. Origins of reported differences in the behavior of BIAP and PLAP are discussed. Finally, advantages and limits of AFM in the study of GPI proteins/membrane domains interactions are presented in this review.

Temperature-dependent imaging of living cells by AFM

Characterization of lateral organization of plasma membranes is a prerequisite to the understanding of membrane structure-function relationships in living cells. Lipid-lipid and lipid-protein interactions are responsible for the existence of various membrane microdomains involved in cell signalization and in numerous pathologies. Developing approaches for characterizing microdomains associate identification tools like recognition imaging with high-resolution topographical imaging. Membrane properties are markedly dependent on temperature. However, mesoscopic scale topographical information of cell surface in a temperature range covering most of cell biology experimentation is still lacking. In this work we have examined the possibility of imaging the temperature-dependent behavior of eukaryotic cells by atomic force microscopy (AFM). Our results establish that the surface of living CV1 kidney cells can be imaged by AFM, between 5 and 37 degrees C, both in contact and tapping modes. These first temperature-dependent data show that large cell structures appeared essentially stable at a microscopic scale. On the other hand, as shown by contact mode AFM, the surface was highly dynamic at a mesoscopic scale, with marked changes in apparent topography, friction, and deflection signals. When keeping the scanning conditions constant, a progressive loss in the image contrast was however observed, using tapping mode, on decreasing the temperature.

Characterization of phospholipid bilayer formation on a thin film of porous SiO2 by reflective interferometric Fourier transform spectroscopy (RIFTS).

Classical methods for characterizing supported artificial phospholipid bilayers include imaging techniques such as atomic force microscopy and fluorescence microscopy. The use in the past decade of surface-sensitive methods such as surface plasmon resonance and ellipsometry, and acoustic sensors such as the quartz crystal microbalance, coupled to the imaging methods, have expanded our understanding of the formation mechanisms of phospholipid bilayers. In the present work, reflective interferometric Fourier transform spectrocopy (RIFTS) is employed to monitor the formation of a planar phospholipid bilayer on an oxidized mesoporous Si (pSiO(2)) thin film. The pSiO(2) substrates are prepared as thin films (3 μm thick) with pore dimensions of a few nanometers in diameter by the electrochemical etching of crystalline silicon, and they are passivated with a thin thermal oxide layer. A thin film of mica is used as a control. Interferometric optical measurements are used to quantify the behavior of the phospholipids at the internal (pores) and external surfaces of the substrates. The optical measurements indicate that vesicles initially adsorb to the pSiO(2) surface as a monolayer, followed by vesicle fusion and conversion to a surface-adsorbed lipid bilayer. The timescale of the process is consistent with prior measurements of vesicle fusion onto mica surfaces. Reflectance spectra calculated using a simple double-layer Fabry-Perot interference model verify the experimental results. The method provides a simple, real-time, nondestructive approach to characterizing the growth and evolution of lipid vesicle layers on the surface of an optical thin film.

Patterning colloidal monolayer films using microcontact particle stripping

We combined the basic component of microcontact printing (μCP), the use of an elastomeric stamp to transfer a pattern, with colloidal lithography, a parallel technique to obtain arrays of nanostructures at surfaces. This novel method, entitled microcontact particle stripping (μCPS), uses a stamp to selectively remove the pre-adsorbed particles located in the contact regions, leaving the particles in the non-contact regions unaffected. The particles, in the nanopatterned regions, can further be used as a lithographic mask.

Preferential insertion of lactose permease in phospholipid domains: AFM observations.

We report the insertion of a transmembrane protein, lactose permease (LacY) from Escherichia coli (E. coli), in supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), in biomimetic molar proportions. We provide evidence of the preferential insertion of LacY in the fluid domains. Analysis of the self-assembled protein arrangements showed that LacY: (i) is inserted as a monomer within fluid domains of SLBs of POPE:POPG (3:1, mol/mol), (ii) has a diameter of approx. 7.8nm; and (iii) keeps an area of phospholipids surrounding the protein that is compatible with shells of phospholipids.

Transfer on hydrophobic substrates and AFM imaging of membrane proteins reconstituted in planar lipid bilayers.

The lipid‐layer technique allows reconstituting transmembrane proteins at a high density in microns size planar membranes and suspended to a lipid monolayer at the air/water interface. In this paper, we transferred these membranes onto two hydrophobic substrates for further structural analysis of reconstituted proteins by Atomic Force Microscopy (AFM). We used a mica sheet covered by a lipid monolayer or a sheet of highly oriented pyrolytic graphite (HOPG) to trap the lipid monolayer at the interface and the suspended membranes. In both cases, we succeeded in the transfer of large membrane patches containing densely packed or 2D‐crystallized proteins. As a proof of concept, we transferred and imaged the soluble Shiga toxin bound to its lipid ligand and the ATP‐binding cassette (ABC) transporter BmrA reconstituted into a planar bilayer. AFM imaging with a lateral resolution in the nanometer range was achieved. Potential applications of this technique in structural biology and nanobiotechnology are discussed. Copyright

The gelsolin:calponin complex nucleates actin filaments with distinct morphologies

Gelsolin and calponin are cytoskeletal and signalling proteins that form a tight 1:1 complex (GCC). We show that calponin within the GCC inhibits the rate of gelsolin mediated nucleation of actin polymerization. The actin-binding function of calponin is ablated within the GCC as the actin-binding site overlaps with one of the gelsolin binding sites. The structure of filaments that result from nucleation by GCC are different to those nucleated by gelsolin alone in that they are longer, loosely bundled and stain heterogeneously with phalloidin. GCC nucleated filaments appear contorted and wrap around each to form the loose bundles.