Compression, Rupture, and Puncture of Model Membranes at the Molecular Scale

International audienceElastic properties of biological membranes are involved in a large number of membrane functionalities and activities. Conventionally characterized in terms of Young’s modulus, bending stiffness and stretching modulus, membrane mechanics can be assessed at high lateral resolution by means of atomic force microscopy (AFM). Here we show that the mechanical response of biomimetic model systems such as supported lipid bilayers (SLBs) is highly affected by the size of the AFM tip employed as a membrane indenter. Our study is focused on phase-separated fluid-gel lipid membranes at room temperature. In a small tip radius regime (≈ 2 nm) and in the case of fluid phase membranes, we show that the tip can penetrate through the membrane minimizing molecular vertical compression and in absence of molecular membrane rupture. In this case, AFM indentation experiments cannot assess the vertical membrane Young’s modulus. In agreement with the data reported in the literature, in the case of larger indenters (>2 nm) SLBs can be compressed leading to an evaluation of Young’s modulus and membrane maximal withstanding force before rupture. We show that such force increases with the indenter in agreement with the existing theoretical frame. Finally, we demonstrate that the latter has no influence on the number of molecules involved in the rupture process that is observed to be constant and rather dependent on the indenter chemical composition

Remodelling of ordered membrane domains by GPI-anchored intestinal alkaline phosphatase

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Influence of calcium on direct incorporation of membrane proteins into in-plane lipid bilayer

International audienceReconstitution of transmembrane proteins by direct incorporation into supported lipid bilayers (SLBs) is a new method to provide suitable samples for high-resolution atomic force microscopy (AFM) analysis of membrane proteins. First experiments have reported successful incorporation of proteins into detergent-destabilized SLBs. Here, we analyzed by AFM the incorporation of membrane proteins in the presence of calcium, a divalent cation functionally important for several membrane proteins. Using lipid-phase-separated membranes, we first show that calcium strongly stabilizes the SLBs decreasing the insertion of low cmc detergents, dodecyl-beta-maltoside, dodecyl-beta-thiomaltoside, and N-hexadecylphosphocholine (Fos-Choline-16) and further insertion of proteins. However, high yield of protein insertion is recovered in the presence of calcium by increasing the detergent concentration in the solution. These data revealed the importance of the calcium in the structure of SLBs and provided new insights into the mechanism of protein insertion into these model membranes

Temperature-dependent localization of GPI-anchored Intestinal alkaline Phosphatase in model rafts

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Automatic detection of diffusion modes within biological membranes using back-propagation neural network

International audienceAbstractBackgroundSingle particle tracking (SPT) is nowadays one of the most popular technique to probe spatio-temporal dynamics of proteins diffusing within the plasma membrane. Indeed membrane components of eukaryotic cells are very dynamic molecules and can diffuse according to different motion modes. Trajectories are often reconstructed frame-by-frame and dynamic properties often evaluated using mean square displacement (MSD) analysis. However, to get statistically significant results in tracking experiments, analysis of a large number of trajectories is required and new methods facilitating this analysis are still needed.ResultsIn this study we developed a new algorithm based on back-propagation neural network (BPNN) and MSD analysis using a sliding window. The neural network was trained and cross validated with short synthetic trajectories. For simulated and experimental data, the algorithm was shown to accurately discriminate between Brownian, confined and directed diffusion modes within one trajectory, the 3 main of diffusion encountered for proteins diffusing within biological membranes. It does not require a minimum number of observed particle displacements within the trajectory to infer the presence of multiple motion states. The size of the sliding window was small enough to measure local behavior and to detect switches between different diffusion modes for segments as short as 20 frames. It also provides quantitative information from each segment of these trajectories. Besides its ability to detect switches between 3 modes of diffusion, this algorithm is able to analyze simultaneously hundreds of trajectories with a short computational time.ConclusionThis new algorithm, implemented in powerful and handy software, provides a new conceptual and versatile tool, to accurately analyze the dynamic behavior of membrane components

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

International audienceThe 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

Continuous Planar Phospholipid Bilayer Supported on Porous Silicon Thin Film Reflector

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In‐plane molecular organization of hydrated single lipid bilayers: DPPC:cholesterol

International audienceUnderstanding the physical properties of the cholesterol‐ phospholipid systems is essential to get a better knowledge on the function of each membrane constituent. We present a novel, simple and user‐friendly setup that allows for straightforward grazing incidence X‐rays diffraction characterization of hydrated individual supported lipid bilayers. This configuration minimizes the scattering from the liquid and allows the detection of the extremely weak diffracted signal of the membrane, enabling the differentiation of coexisting domains in DPPC:cholesterol single bilayers. Cell membranes are composed mainly of a mixture of lipids and proteins. Laterally segregation of membrane components into domains of lipids enriched in cholesterol (chol) and sphingolipids are involved in many membrane functions, for instance signaling, remodeling and trafficking. 1, 2 Chol is responsible for controlling the phase behavior as well as the lipid organization, regulating the fluidity and permeability of the membrane while increasing its mechanical resistance. 3‐8 In this context, it is of high interest to understand the physical properties of the chol‐phospholipid systems to get a better knowledge on the role of chol in the membrane. Membranes comprising phospholipids and chol have been extensively studied, including simplified models based on two components. In particular, temperature‐composition phase diagrams of DPPC (1,2‐dipalmitoyl‐sn‐glycero‐3‐ phosphocholine):chol have been defined using different techniques such as nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), neutron and X‐rays (XR) scattering. 9‐14 Yet, discrepancies on the determination of a complete phase diagram able to cover all compositional space and temperature range still remain. Atomic force microscopy (AFM) and force spectroscopy (AFM‐FS) have provided insights into the thermal transition of DPPC:chol supported lipid bilayers (SLBs) at the nanometric scale. AFM has the capability to operate under environmentally controlled conditions, 3, 4 to characterize the topology and nanomechanics, as well as to define the coexistence of different domains, facilitating the linking between the chol content and the lateral organization of the membrane. 3, 4, 15, 16 Similar information about phase segregation in lipid bilayers can be in principle also gathered by XR scattering techniques. XR are very powerful, noninvasive techniques that have been extensively used in lipid bilayer studies to probe length scales ranging from angstroms to microns. A large part of the XR based experiments have been focused so far on determining the electronic vertical structure of lipid monolayers, bilayers and stacks of bilayers (or multi‐bilayers), at the liquid‐air and solid‐ liquid interfaces, respectively, by means of XR reflectivity (XRR), which is a well‐established technique in the field. 17‐21 Information about the lateral in‐plane structure of such systems can be instead obtained by grazing incidence XR diffraction (GIXD). Nevertheless, the requirement of the wetting preservation to guarantee the stability of biological membranes at the solid‐liquid interface makes the in‐plane structural characterization of a single lipid bilayer extremely challenging. The presence of a wetting layer makes necessary the use of high energy XR to increase the transmission through the liquid, resulting into a weaker signal from the organic molecules. 19 Additionally, the scattering generated by the liquid environment, increases the background level complicating the detection of the signal scattered by the bilayer structure. For this reason, most of the reported structural information relative to lipid membranes has been extrapolated from experiment