In JoVE (1)

Other Publications (16)

Articles by Sylvain Trépout in JoVE

Other articles by Sylvain Trépout on PubMed

Membrane Protein Selectively Oriented on Solid Support and Reconstituted into a Lipid Membrane

Langmuir : the ACS Journal of Surfaces and Colloids. Feb, 2007  |  Pubmed ID: 17261039

Mimetic functional membranes on solid support are now emerging for the development of membrane biosensor or for the study of membrane-mediated processes and should have an important impact on biodiagnostics. We established a method to reconstitute a membrane protein into a lipid membrane in a selective orientation on a solid support. Membrane protein OprM, a component of OprM-MexA-MexB multidrug efflux pump, solubilized in detergent was immobilized via its extracellular domain on aminosilane-modified silica surface. The oriented protein was reconstituted into a lipid membrane by detergent removal. The membrane protein reconstitution process carried out on silica nanoparticles and on planar silica surfaces was followed by cryo-electron microscopy (cryo-EM) and quartz crystal microbalance with dissipation monitoring (QCM-D) respectively. The selective protein orientation on aminosilane-modified silica surface was assessed by cryo-EM and was compared to the nonspecific protein deposition on silica surface. Finally, the binding of MexA, a periplasmic component of the tripartite efflux complex, was monitored with QCM-D on the oriented OprM protein monolayer. The large adsorbed mass gave a direct evidence of the high affinity of MexA with the periplasmic helical part of OprM.

Organization of Reconstituted Lipoprotein MexA Onto Supported Lipid Membrane

European Biophysics Journal : EBJ. Nov, 2007  |  Pubmed ID: 17665187

MexA, a periplasmic component of OprM-MexA-MexB tripartite multidrug efflux pump from Pseudomonas aeruginosa, is natively anchored via its fatty acid in the bacteria inner membrane protruding into the periplasm. We used supported lipid bilayer (SLB) to attach the protein to a single leaflet mimicking its perisplamic orientation. For that purpose, we studied the solubilization of DOPC lipid bilayer supported on silica surface with beta-octyl glucoside (betaOG). First we showed that SLBs resist to betaOG concentrations that usually solubilize liposomes. Native form of MexA was directly inserted in the outer leaflet at (betaOG concentrations in a range of 20-25 mM). Second, observations by cryo-electron microscopy (cryoEM) revealed a dense protein layer attached to the surface corresponding to a 13-nm layer of MexA proteins. Analysis of protein densities allows proposing a schematic organization of native MexA inserted in lipid membrane. This structural organization provides further insights with respect to the partially solved structure of the soluble form.

Structure of Artificial and Natural VE-cadherin-based Adherens Junctions

Biochemical Society Transactions. Apr, 2008  |  Pubmed ID: 18363560

In vascular endothelium, adherens junctions between endothelial cells are composed of VE-cadherin (vascular endothelial cadherin), an adhesive receptor that is crucial for the proper assembly of vascular structures and the maintenance of vascular integrity. As a classical cadherin, VE-cadherin links endothelial cells together by homophilic interactions mediated by its extracellular part and associates intracellularly with the actin cytoskeleton via catenins. Although, from structural crystallographic data, a dimeric structure arranged in a trans orientation has emerged as a potential mechanism of cell-cell adhesion, the cadherin organization within adherens junctions remains controversial. Concerning VE-cadherin, its extracellular part possesses the capacity to self-associate in solution as hexamers consisting of three antiparallel cadherin dimers. VE-cadherin-based adherens junctions were reconstituted in vitro by assembly of a VE-cadherin EC (extracellular repeat) 1-EC4 hexamer at the surfaces of liposomes. The artificial adherens junctions revealed by cryoelectron microscopy appear as a two-dimensional self-assembly of hexameric structures. This cadherin organization is reminiscent of that found in native desmosomal junctions. Further structural studies performed on native VE-cadherin junctions would provide a better understanding of the cadherin organization within adherens junctions. Homophilic interactions between cadherins are strengthened intracellularly by connection to the actin cytoskeleton. Recently, we have discovered that annexin 2, an actin-binding protein connects the VE-cadherin-catenin complex to the actin cytoskeleton. This novel link is labile and promotes the endothelial cell switch from a quiescent to an angiogenic state.

Magnetic Multilamellar Liposomes Produced by in Situ Synthesis of Iron Oxide Nanoparticles: "magnetonions"

The Journal of Physical Chemistry. B. Jun, 2009  |  Pubmed ID: 19534563

We report the formation of magnetic onion-type multilamellar vesicles. Iron oxide nanoparticles (np's) were synthesized inside lipidic multilamellar vesicles by coprecipitation of vesicle-encapsulated Fe(2+) and Fe(3+) ions induced by HO(-) diffusion through vesicle lamellae. The iron ion encapsulation efficiency of onions was measured by potentiometry and UV-vis absorbance spectroscopy. Its high value (75 +/- 5% for both Fe(2+) and Fe(3+)) ensures an intravesicular synthesis, as confirmed by cryo-transmission electron microscopy (TEM) imaging. The as-grown nanoparticles are characterized by X-ray diffraction analysis and TEM, and magnetic onions are imaged by cryo-TEM. The np size, controlled by temperature and time, ranges from 3 to 6 nm and is shown to be a key parameter for onion stability.

Cryo-electron Tomography of Nanoparticle Transmigration into Liposome

Journal of Structural Biology. Dec, 2009  |  Pubmed ID: 19596070

Nanoparticle transport across cell membrane plays a crucial role in the development of drug delivery systems as well as in the toxicity response induced by nanoparticles. As hydrophilic nanoparticles interact with lipid membranes and are able to induce membrane perturbations, hypothetic mechanisms based on membrane curvature or hole formation have been proposed for activating their transmigration. We report on the transport of hydrophilic silica nanoparticles into large unilamellar neutral DOPC liposomes via an internalization process. The strong adhesive interactions of lipid membrane onto the silica nanoparticle triggered liposome deformation until the formation of a curved neck. Then the rupture of this membrane neck led to the complete engulfment of the nanoparticle. Using cryo-electron tomography we determined 3D architectures of intermediate steps of this process unveiling internalized silica nanoparticles surrounded by a supported lipid bilayer. This engulfing process was achieved for a large range of particle size (from 30 to 200 nm in diameter). These original data provide interesting highlights for nanoparticle transmigration and could be applied to biotechnology development.

Structure of Reconstituted Bacterial Membrane Efflux Pump by Cryo-electron Tomography

Biochimica Et Biophysica Acta. Oct, 2010  |  Pubmed ID: 20599691

Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.

Structure Determination of Membrane Protein by Both Cryo-electron Tomography and Single Particle Analysis

Methods in Molecular Biology (Clifton, N.J.). 2010  |  Pubmed ID: 20665268

The structure determination of membrane protein in lipid environment can be carried out using cryo-electron microscopy combined with the recent development of data collection and image processing. We describe a protocol to study assemblies or stacks of membrane protein reconstituted into a lipid membrane using both cryo-electron tomography and single particle analysis, which is an alternative approach to electron crystallography for solving 3D structure. We show the organization of the successive layers of OprM molecules revealing the protein-protein interactions between OprM molecules of two successive lipid bilayers.

Multilamellar Liposomes Entrapping Aminosilane-modified Maghemite Nanoparticles: "magnetonions"

Physical Chemistry Chemical Physics : PCCP. Oct, 2010  |  Pubmed ID: 20820553

4.6 nm-sized aminosilane-modified maghemite (γ-Fe(2)O(3)) nanoparticles (aMNPs) were synthesized and encapsulated into onion-type multilamellar vesicles of soybean phosphatidylcholine (90%mol) and monoolein (10%mol). The magnetic multilamellar vesicles were obtained by shearing lipids with an aqueous dispersion of the preformed aMNPs (ferrofluid). The influence of ferrofluid concentration on the stability of the constitutive lamellar phase and the resulting dispersed onions was analyzed by small-angle X-ray diffraction (SAXD) and cryo-TEM imaging, respectively. When [Fe(III)] <60 mM, stable, magnetic onions were produced with aMNPs inserted inside onion water compartments as isolated or aggregated particles. Encapsulation efficiencies were measured by EPR spectroscopy and magnetic measurements: much higher values (up to 75%) than unilamellar liposomes were found. The deduced aMNP-to-onion ratio increased with ferrofluid concentration before reaching a maximal value of ca. 45 as confirmed by cryo-TEM imaging. When [Fe(III)] >60 mM, uni- or oligolamellar vesicles in addition to onions formed, probably because of a two-phase separation between an aMNP-rich phase and an aMNP-containing lamellar phase as revealed by SAXD.

Riboregulation of the Bacterial Actin-homolog MreB by DsrA Small Noncoding RNA

Integrative Biology : Quantitative Biosciences from Nano to Macro. Jan, 2015  |  Pubmed ID: 25407044

The bacterial actin-homolog MreB is a key player in bacterial cell-wall biosynthesis and is required for the maintenance of the rod-like morphology of Escherichia coli. However, how MreB cellular levels are adjusted to growth conditions is poorly understood. Here, we show that DsrA, an E. coli small noncoding RNA (sRNA), is involved in the post-transcriptional regulation of mreB. DsrA is required for the downregulation of MreB cellular concentration during environmentally induced slow growth-rates, mainly growth at low temperature and during the stationary phase. DsrA interacts in an Hfq-dependent manner with the 5' region of mreB mRNA, which contains signals for translation initiation and thereby affects mreB translation and stability. Moreover, as DsrA is also involved in the regulation of two transcriptional regulators, σ(S) and the nucleoid associated protein H-NS, which negatively regulate mreB transcription, it also indirectly contributes to mreB transcriptional down-regulation. By using quantitative analyses, our results evidence the complexity of this regulation and the tangled interplay between transcriptional and post-transcriptional control. As transcription factors and sRNA-mediated post-transcriptional regulators use different timescales, we propose that the sRNA pathway helps to adapt to changes in temperature, but also indirectly mediates long-term regulation of MreB concentration. The tight regulation and fine-tuning of mreB gene expression in response to cellular stresses is discussed in regard to the effect of the MreB protein on cell elongation.

New Insight into the Structure and Function of Hfq C-terminus

Bioscience Reports. Apr, 2015  |  Pubmed ID: 25772301

Accumulating evidence indicates that RNA metabolism components assemble into supramolecular cellular structures to mediate functional compartmentalization within the cytoplasmic membrane of the bacterial cell. This cellular compartmentalization could play important roles in the processes of RNA degradation and maturation. These components include Hfq, the RNA chaperone protein, which is involved in the post-transcriptional control of protein synthesis mainly by the virtue of its interactions with several small regulatory ncRNAs (sRNA). The Escherichia coli Hfq is structurally organized into two domains. An N-terminal domain that folds as strongly bent β-sheets within individual protomers to assemble into a typical toroidal hexameric ring. A C-terminal flexible domain that encompasses approximately one-third of the protein seems intrinsically unstructured. RNA-binding function of Hfq mainly lies within its N-terminal core, whereas the function of the flexible domain remains controversial and largely unknown. In the present study, we demonstrate that the Hfq-C-terminal region (CTR) has an intrinsic property to self-assemble into long amyloid-like fibrillar structures in vitro. We show that normal localization of Hfq within membrane-associated coiled structures in vivo requires this C-terminal domain. This finding establishes for the first time a function for the hitherto puzzling CTR, with a plausible central role in RNA transactions.

Scanning Transmission Electron Microscopy Through-focal Tilt-series on Biological Specimens

Micron (Oxford, England : 1993). Oct, 2015  |  Pubmed ID: 26093182

Since scanning transmission electron microscopy can produce high signal-to-noise ratio bright-field images of thick (≥500 nm) specimens, this tool is emerging as the method of choice to study thick biological samples via tomographic approaches. However, in a convergent-beam configuration, the depth of field is limited because only a thin portion of the specimen (from a few nanometres to tens of nanometres depending on the convergence angle) can be imaged in focus. A method known as through-focal imaging enables recovery of the full depth of information by combining images acquired at different levels of focus. In this work, we compare tomographic reconstruction with the through-focal tilt-series approach (a multifocal series of images per tilt angle) with reconstruction with the classic tilt-series acquisition scheme (one single-focus image per tilt angle). We visualised the base of the flagellum in the protist Trypanosoma brucei via an acquisition and image-processing method tailored to obtain quantitative and qualitative descriptors of reconstruction volumes. Reconstructions using through-focal imaging contained more contrast and more details for thick (≥500 nm) biological samples.

Poly(ε-caprolactone)-block-polysarcosine by Ring-Opening Polymerization of Sarcosine N-Thiocarboxyanhydride: Synthesis and Thermoresponsive Self-Assembly

Biomacromolecules. Oct, 2015  |  Pubmed ID: 26388179

Biocompatible amphiphilic block copolymers composed of polysarcosine (PSar) and poly(ε-caprolactone) (PCL) were synthesized using ring-opening polymerization of sarcosine N-thiocarboxyanhydride initiated by oxyamine-ended PCL and characterized by NMR, SEC, and DSC. Self-assembling of two triblock copolymers PSar8-b-PCL28-b-PSar8 (CS7) and PSar16-b-PCL40-b-PSar16 (CS10) in dilute solution was studied in detail toward polymersome formation using thin-film hydration and nanoprecipitation techniques. A few giant vesicles were obtained by thin-film hydration from both copolymers and visualized by confocal laser scanning microscope. Unilamellar sheets and nanofibers (with 8-10 nm thickness or diameter) were obtained by nanoprecipitation at room temperature and observed by Cryo-TEM. These lamellae and fibrous structures were transformed into worm-like cylinders and spheres (D∼30-100 nm) after heating to 65 °C (>Tm,PCL). Heating CS10 suspensions to 90 °C led eventually to multilamellar polymersomes (D∼100-500 nm). Mechanism II, where micelles expand to vesicles through water diffusion and hydrophilic core forming, was proposed for polymersome formation. A cell viability test confirmed the self-assemblies were not cytotoxic.

Nanobody-functionalized PEG-b-PCL Polymersomes and Their Targeting Study

Journal of Biotechnology. Nov, 2015  |  Pubmed ID: 26433047

We prepared and characterized polymersomes functionalized with nanobodies (VHHs) on the basis of biocompatible, biodegradable and FDA-approved poly(ethylene glycol)-block-poly(ϵ-caprolactone) (PEG-b-PCL). Fluorescein isothiocyanate (FITC) and N-beta-maleimidopropyl-oxysuccinimide ester were allowed reacting with H2N-PEG-b-PCL to produce FITC and maleimide (Mal) functionalized copolymers, Mal-PEG-b-PCL and FITC-PEG-b-PCL. A mixture of MeO-PEG-b-PCL, Mal-PEG-b-PCL and FITC-PEG-b-PCL was used to prepare polymersomes by thin film hydration and nanoprecipitation methods. Morphological studies by cryogenic transmission electron microscopy (Cryo-TEM) showed that the nanoparticles exhibited predominantly vesicular structures (polymersomes). Their mean diameters measured by dynamic light scattering were around 150 nm and the zeta-potentials around -1 mV at pH 7.4. The nanoparticles were functionalized with either anti-HER2 (VHH1) or anti-GFP (VHH2) nanobodies using maleimide-cysteine chemistry. Their particle size and zeta-potential increased slightly after nanobody-functionalization. The specific binding of VHH-functionalized polymersomes and control nanoparticles towards HER2 positive breast cancer cells was analyzed by flow cytometry and confocal microscopy. The collected results represent the first report which experimentally demonstrates that VHH1-functionalized PEO-b-PCL polymersomes can target specifically breast cancer cells expressing HER2 receptors. The detailed morphological and cell-binding studies described herein pave the way for future in vivo studies to evaluate the feasibility to use such nanoparticles for targeted drug delivery.

Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase

PloS One. 2015  |  Pubmed ID: 26714308

Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development.

Polymersomes of Biodegradable Polysarcosine-block-poly(ε-caprolactone)

Journal of Controlled Release : Official Journal of the Controlled Release Society. Sep, 2015  |  Pubmed ID: 27005081

Overview of Chemical Imaging Methods to Address Biological Questions

Micron (Oxford, England : 1993). May, 2016  |  Pubmed ID: 26922256

Chemical imaging offers extensive possibilities for better understanding of biological systems by allowing the identification of chemical components at the tissue, cellular, and subcellular levels. In this review, we introduce modern methods for chemical imaging that can be applied to biological samples. This work is mainly addressed to the biological sciences community and includes the bases of different technologies, some examples of its application, as well as an introduction to approaches on combining multimodal data.

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