Science

Highlights

GEM Members: if you want to show on this page, and in a didactic way, your most recent and significant results, you can submit a text (French & English) and an image to the GEM secretariat: gem@univ-lyon1.fr. It will be published after consultation of the Scientific Committee.

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CLAFEM: A combination of 3 microscopies developped in Lille

 

A Research Group from the Pasteur Institute in Lille has performed an amazing feat developing an original and highly innovative method allowing to observe an inorganic of living sample with unprecedented resolution, at the nanometer scale, using in a correlated manner photonic super-resolution, atomic force and  electron microscopy. This work has been published in Methods in Cell Biology, April 2017.

 

Atomic force microscopy (AFM) is becoming increasingly used in the biology field. It can give highly accurate topography and biomechanical quantitative data, such as adhesion, elasticity, and viscosity, on living samples. Nowadays, correlative light electron microscopy (CLEM) is a must-have tool in the biology field that combines different microscopy techniques to spatially and temporally analyze the structure and function of a single sample. This work describes the combination of AFM with super-resolution light microscopy and electron microscopy. This technique is named correlative light atomic force electron microscopy (CLAFEM) in which AFM can be used on fixed and living cells in association with super-resolution light microscopy and further processed for transmission or scanning electron microscopy (TEM or SEM). This work illustrates this approach to observe cellular bacterial infection and cytoskeleton. It shows that CLAFEM brings complementary information at the cellular level, from on the one hand protein distribution and topography at the nanometer scale and on the other hand elasticity at the pN scales to fine ultrastructural details.

Reference: CLAFEM: Correlative light atomic force electron microscopy. Janel S, Werkmeister E, Bongiovanni A, Lafont F, Barois N. Methods Cell Biol. 2017;140:165-185.

 

Contact: Frank Lafont, UMR8204 CNRS, U1019 Inserm, Institut Pasteur de Lille, Lille, France; Email:  frank.lafont@pasteur-lille.fr

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Structural characterization of the amyloid precursor protein transmembrane domain and its gamma-cleavage site

Alzheimer’s disease is the most common form of dementia that affects about 50 million of sufferers worldwide. A major role for the initiation and progression of Alzheimer’s disease has been associated with the amyloid β-peptide (Aβ), which is a protease cleavage product of the amyloid precursor protein. The amyloid precursor protein is an integral membrane protein with a single transmembrane domain. In the context of a European research and training network together with our collaborators from the Free University of Brussels we investigated the structural integrity of the transmembrane domain within lipid bilayers, and determined the tilt angle distribution and dynamics of various subdomains using oriented solid-state NMR and ATR-FTIR spectroscopies. Importantly, pronounced conformational and topological heterogeneity were observed for the g- and, to a lesser extent, the z-cleavage site, with pronounced implications for the production of Aβ and related peptides.

Reference: Structural characterization of the amyloid precursor protein transmembrane domain and its gamma-cleavage site. Anna Itkin,  Evgeniy S. Salnikov, Christopher Aisenbrey, Jesus Raya, Elise Glattard, Vincent Raussens, Jean-Marie Ruysschaert, Burkhard Bechinger. ACS Omega, 2017, 2 (10), pp 6525–6534.

 

Contact: Burkhard Bechinger, UMR 7177 CNRS, U. Strasbourg,Strasbourg, France; Email: bechinge@unistra.fr

 


Triggering bilayer to inverted-hexagonal nanostructures by thiol-ene click chemistry on cationic lipids: consequences on gene transfection

Many cationic amphiphiles used as vector for nucleic acid delivery feature unsaturated lipid chains. We have used these C-C double bonds to produce branched lipid chains by using click thiol-ene reaction. We report that this ramification induced an outstanding modification of the supramolecular behavior of the amphiphiles. Indeed, the starting amphiphiles (with linear lipid chains)  adopt, in aqueous media, a lamellar phase whereas after ramification inverted hexagonal (HII) phases are produced. This behavior is observed for lipophopshoramidates but also for DOTMA thus demonstrating the general aspect of the method and the similar observations for the supramolecular assemblies in water. The consequences of the type of phases on transfection efficacies and toxicity is also reported.

Reference: Triggering bilayer to inverted-hexagonal nanostructures by thiol-ene click chemistry on cationic lipids: consequences on gene transfection. Afonso, D.; Le Gall, T.; Couthon-Gourvès, H.; Grélard, A.; Prakash, S.; Berchel, M.; Kervarec,N.;  Dufourc,E.J.; Montier, T.; Jaffrès, P.A. Soft Matter, 2016, 12, 4516 – 4520

 

Contact: Paul-Alain Jaffrès, UMR6521 CNRS, U. Brest, Brest, France. Email: pjaffres@univ-brest.fr

 


Mechanism of Shiga Toxin Clustering on Membranes

The team of Ludger Johannes (U1143 INSERM — UMR3666 CNRS at Institut Curie, Paris, France), in collaboration with John Hjort Ipsen (MEMPHYS - Center for Biomembrane Physics, Odense, University of Southern Denmark) and Julian Shillcock (Ecole polytechnique fédérale de Lausanne, Switzerland) proposes a novel mechanism according to which the tight binding to biological membranes of glycosphingolipid ligands (here: Shiga toxin) would cause an attractive force by suppressing lipid fluctuations, leading to their clustering.

The bacterial Shiga toxin interacts with its cellular receptor, the glycosphingolipid globotriaosylceramide (Gb3 or CD77), as a first step to entering target cells. Previous studies have shown that toxin molecules cluster on the plasma membrane, despite the apparent lack of direct interactions between them. The precise mechanism by which this clustering occurs remains poorly defined. Here, we used vesicle and cell systems and computer simulations to show that line tension due to curvature, height or compositional mismatch, and lipid or solvent depletion cannot drive the clustering of Shiga toxin molecules. By contrast, in coarse-grained computer simulations a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface. The most likely interpretation of these findings is that a membrane fluctuation-induced force generates an effective attraction between toxin molecules. Such force would be of similar strength to the electrostatic force at separations around 1 nm, remain strong at distances up to the size of toxin molecules (several nm), and persist even beyond. This force is predicted to operate between manufactured nanoparticles providing they are sufficiently rigid and tightly bound to the plasma membrane, thereby suggesting a novel route for the targeting of nanoparticles to cells for biomedical applications.

Reference: Mechanism of Shiga Toxin Clustering on Membranes. Pezeshkian W, Gao H, Arumugam S, Becken U, Bassereau P, Florent JC, Ipsen JH, Johannes L, Shillcock J (2017) Mechanism of Shiga toxin clustering on membranes. ACS Nano 11: 314-324

 

Contact: Ludger Johannes, U1143 INSERM, UMR3666 CNRS, Institut Curie, Paris, France. Email: ludger.johannes@curie.fr

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