Eric Larquet

Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein

Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

Optical and Topological Characterization of Gold Nanoparticle Dimers Linked by a Single DNA Double Strand

We demonstrate that symmetric or asymmetric gold nanoparticle dimers with substantial scattering cross sections and plasmon coupling can be produced with a perfectly controlled chemical environment and a high purity using a single DNA linker as short as 7 nm. A statistical analysis of the optical properties and morphology of single dimers is performed using darkfield and cryo-electron microscopies. These results, correlated to Mie theory calculations, indicate that the particle dimers are stretched in water by electrostatic interactions.

How ATP Hydrolysis Controls Filament Assembly from Profilin-Actin IMPLICATION FOR FORMIN PROCESSIVITY

Formins catalyze rapid filament growth from profilin-actin, by remaining processively bound to the elongating barbed end. The sequence of elementary reactions that describe filament assembly from profilin-actin at either free or formin-bound barbed ends is not fully understood. Specifically, the identity of the transitory complexes between profilin and actin terminal subunits is not known; and whether ATP hydrolysis is directly or indirectly coupled to profilin-actin assembly is not clear. We have analyzed the effect of profilin on actin assembly at free and FH1-FH2-bound barbed ends in the presence of ADP and non-hydrolyzable CrATP. Profilin blocked filament growth by capping the barbed ends in ADP and CrATP/ADP-Pi states, with a higher affinity when formin is bound. We confirm that, in contrast, profilin accelerates depolymerization of ADP-F-actin, more efficiently when FH1-FH2 is bound to barbed ends. To reconcile these data with effective barbed end assembly from profilin-MgATP-actin, the nature of nucleotide bound to both terminal and subterminal subunits must be considered. All data are accounted for quantitatively by a model in which a barbed end whose two terminal subunits consist of profilin-ATP-actin cannot grow until ATP has been hydrolyzed and Pi released from the penultimate subunit, thus promoting the release of profilin and allowing further elongation. Formin does not change the activity of profilin but simply uses it for its processive walk at barbed ends. Finally, if profilin release from actin is prevented by a chemical cross-link, formin processivity is abolished.

Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy

The joint use of 3 complementary techniques, namely, nanoparticle tracking analysis (NTA), cryo-electron microscopy (Cryo-EM) and Raman tweezers microspectroscopy (RTM), is proposed for a rapid characterisation of extracellular vesicles (EVs) of various origins. NTA is valuable for studying the size distribution and concentration, Cryo-EM is outstanding for the morphological characterisation, including observation of vesicle heterogeneity, while RTM provides the global chemical composition without using any exogenous label. The capabilities of this approach are evaluated on the example of cell-derived vesicles of Dictyostelium discoideum, a convenient general model for eukaryotic EVs. At least 2 separate species differing in chemical composition (relative amounts of DNA, lipids and proteins, presence of carotenoids) were found for each of the 2 physiological states of this non-pathogenic microorganism, that is, cell growth and starvation-induced aggregation. These findings demonstrate the specific potency of RTM. In addition, the first Raman spectra of human urinary exosomes are reported, presumably constituting the primary step towards Raman characterisation of EVs for the purpose of human diseases diagnoses.

Reversible switching of the interparticle distance in DNA-templated gold nanoparticle dimers.

We produce gold nanoparticle dimers with a surface-to-surface distance that varies reversibly by a factor of 3 when hybridizing or removing a single target DNA strand. The dimers are built on one DNA template that features a stem-loop enabling the interparticle distance change. Using electrophoresis, we reach 90% sample purities and demonstrate that this chemical process is reversible in solution at room temperature for a low molar excess of the target DNA strand. The kinetics of the reaction is asymmetric due to steric hindrance in the stem-loop opening process. Furthermore, a statistical analysis of cryo-electron microscopy measurements allows us to provide the first quantitative analysis of distance changes in chemically switchable nanoparticle assemblies.

Controlled growth of core@shell heterostructures based on Prussian blue analogues

This work aims to develop a controlled strategy for the growth of multifunctional core@shell nanoparticles based on Prussian blue analogues. We mainly focussed our attention on Rb0.45Co[Fe(CN)6]0.8·3H2O@Rb0.2Ni[Cr(CN)6]0.7·zH2O particles, which combine a photoswitchable (photo-magnetic/-chromic) core with a ferromagnetic shell. The control of the chemical composition in the heterostructure is a key point to obtain the expected magnetic, optical and structural properties. We found that the removal of the unreacted species by washing after the growth of the primary particles led to an irreversible aggregation attributed to the desorption of stabilizing [Fe(CN)6] surface units. We showed that this difficulty could be overcome by washing the particles in a solution containing chromicyanide ions, which are precursors of the Rb0.2Ni[Cr(CN)6]0.7·zH2O shell phase, thus avoiding a contamination of the shell. Both X-ray diffraction and magnetic measurements confirmed that a controlled shell of the desired composition could be obtained this way.

Nanoscale topography of hepatitis B antigen particles by atomic force microscopy

Hepatitis B virus envelope is mainly composed of three forms of the same protein expressed from different start codons of the same open reading frame. The smaller form named S protein corresponds to the C-terminal common region and represents about 80% of the envelope proteins. It is mainly referred as hepatitis B virus surface antigen (HBsAg). Over expressed in the host cell, this protein can be produced as spherical and tubular self-organized particles. Highly immunogenic, these particles are used in licensed hepatitis B vaccines. In this study we have combined transmission electron microscopy and atomic force microscopy to determine the shape and size of HBsAg particles produced from the yeast Hansenula polymorpha. Tapping mode atomic force microscopy in liquid allows structural details of the surface to be delineated with a resolution in the nanometer range. Particles were decorated by closely packed spike-like structures protruding from particle surface. Protrusions appeared uniformly distributed at the surface and an average number of 75 protrusions per particle were calculated. Importantly, we demonstrated that proteins mainly contribute to the topography of the protrusions.

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-Mda Hexamer by Cryoelectron Microscopy and its Oligomerization Behavior in Solution: Functional Implications.

The three-dimensional structure of the hexameric (αβ)6 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex iron-sulfur flavoprotein. In the hexameric species, interprotomeric α-α and α-β contacts are mediated by the C-terminal domain of the α subunit, which is based on a β helical fold so far unique to glutamate synthases. The αβ protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the β subunit, to the FMN on the α subunit, through the low potential [4Fe-4S]1+/2+ centers on the β subunit and the [3Fe-4S]0/1+ cluster on the α subunit. The (αβ)6 hexamer exhibits a concentration-dependent equilibrium with αβ monomers and (αβ)2 dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP+. Hexamerization seems to decrease the catalytic efficiency of the αβ protomer only 3-fold by increasing the Km values measured for l-Gln and 2-OG. However, it cannot be ruled out that the (αβ)6 hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.

Quantum dot-loaded PEGylated poly(alkyl cyanoacrylate) nanoparticles for in vitro and in vivo imaging

Visible- and near-infrared-emitting quantum-dots (QDs) were readily encapsulated with high yields into PEGylated, poly(alkyl cyanoacrylate) nanoparticles (NPs) by self-assembly in aqueous solution. The resulting QD-loaded NPs were efficiently purified and characterized by transmission electron microscopy, dynamic light scattering and ζ-potential measurements as well as by stability studies and cell viability assays. Concomitant encapsulation of different kinds of QDs led to barcode NPs. They were then used for human brain endothelial cell imaging allowing their uptake to be monitored. Finally, the in vivo fate of near-infrared fluorescent NPs was visualized noninvasively by fluorescence optical imaging.

Nanovesicles released by Dictyostelium cells: A potential carrier for drug delivery

Nanovesicles released by Dictyostelium discoideum cells grown in the presence of the DNA-specific dye Hoechst 33342 have been previously shown to mediate the transfer of the dye into the nuclei of Hoechst-resistant cells. The present investigation extends this work by conducting experiments in the presence of hypericin, a fluorescent therapeutic photosensitizer assayed for antitumoral photodynamic therapy. Nanovesicles released by Dictyostelium cells exhibit an averaged diameter between 50 and 150 nm, as measured by transmission cryoelectron microscopy. A proteomic analysis reveals a predominance of actin and actin-related proteins. The detection of a lysosomal membrane protein (LIMP II) indicates that these vesicles are likely generated in the late endosomal compartment. The use of the hypericin-containing nanovesicles as nanodevices for in vitro drug delivery was investigated by fluorescence microscopy. The observed signal was almost exclusively located in the perinuclear area of two human cell lines, skin fibroblasts (HS68) and cervix carcinoma (HeLa) cells. Studies by confocal microscopy with specific markers of cell organelles, provided evidence that hypericin was accumulated in the Golgi apparatus. All these data shed a new light on in vitro drug delivery by using cell-released vesicles as carriers.