Valérie Marchi-Artzner

Time scales of membrane fusion revealed by direct imaging of vesicle fusion with high temporal resolution

Membrane fusion is a vital process of life involved, for example, in cellular secretion via exocytosis, signaling between nerve cells, and virus infection. In both the life sciences and bioengineering, controlled membrane fusion has many possible applications, such as drug delivery, gene transfer, chemical microreactors, or synthesis of nanomaterials. Until now, the fusion dynamics has been elusive because direct observations have been limited to time scales that exceed several milliseconds. Here, the fusion of giant lipid vesicles is induced in a controlled manner and monitored with a temporal resolution of 50 μs. Two different fusion protocols are used that are based on synthetic fusogenic molecules and electroporation. For both protocols, the opening of the fusion necks is very fast, with an average expansion velocity of centimeters per second. This velocity indicates that the initial formation of a single fusion neck can be completed in a few hundred nanoseconds.

High-Affinity Labeling and Tracking of Individual Histidine-Tagged Proteins in Live Cells Using Ni2+ Tris-nitrilotriacetic Acid Quantum Dot Conjugates

Investigation of many cellular processes using fluorescent quantum dots (QDs) is hindered by the nontrivial requirements for QD surface functionalization and targeting. To address these challenges, we designed, characterized and applied QD-trisNTA, which integrates tris-nitrilotriacetic acid, a small and high-affinity recognition unit for the ubiquitous polyhistidine protein tag. Using QD-trisNTA, we demonstrate two-color QD tracking of the type-1 interferon receptor subunits in live cells, potentially enabling direct visualization of protein-protein interactions at the single molecule level.

Small and stable peptidic PEGylated quantum dots to target polyhistidine-tagged proteins with controlled stoichiometry.

The use of the semiconductor quantum dots (QD) as biolabels for both ensemble and single-molecule tracking requires the development of simple and versatile methods to target individual proteins in a controlled manner, ideally in living cells. To address this challenge, we have prepared small and stable QDs (QD-ND) using a surface coating based on a peptide sequence containing a tricysteine, poly(ethylene glycol) (PEG), and an aspartic acid ligand. These QDs, with a hydrodynamic diameter of 9 +/- 1.5 nm, can selectively bind to polyhistidine-tagged (histag) proteins in vitro or in living cells. We show that the small and monodisperse size of QD-ND allows for the formation of QD-ND/histag protein complexes of well-defined stoichiometry and that the 1:1 QD/protein complex can be isolated and purified by gel electrophoresis without any destabilization in the nanomolar concentration range. We also demonstrate that QD-ND can be used to specifically label a membrane receptor with an extracellular histag expressed in living HeLa cells. Here, cytotoxicity tests reveal that cell viability remains high under the conditions required for cellular labeling with QD-ND. Finally, we apply QD-ND complexed with histag end binding protein-1 (EB1), a microtubule associated protein, to single-molecule tracking in Xenopus extracts. Specific colocalization of QD-ND/EB1 with microtubules during the mitotic spindle formation demonstrates that QD-ND and our labeling strategy provide an efficient approach to monitor the dynamic behavior of proteins involved in complex biological functions.

Selective Adhesion of Endothelial Cells to Artificial Membranes with a Synthetic RGD-Lipopeptide

A constrained cyclic ArgGly-Asp-D-Phe-Lys, abbreviated as cyclo(-RGDfK-), lipopeptide has been synthesized and incorporated into artificial membranes such as giant vesicles with DOPC and solid-supported lipid bilayers. The selective adhesion and spreading of endothelial cells of the human umbilical cord on solids functionalized by membranes with this RGD-lipopeptide have been observed. Furthermore, we have demonstrated strong selective adhesion of giant vesicles to endothelial cells through local adhesion domains by combined application of hydrodynamic flow field and reflection interference contrast microscopy (RICM). The adhesion can be inhibited by competition with a water-soluble RGD peptide. We suggest that this strategy could improve the efficiency of liposomes targeting used as vectors or as drug carriers to cells.

Three-Dimensional Self-Assembling of Gold Nanorods with Controlled Macroscopic Shape and Local Smectic B Order

We describe a method of controlled evaporation on a textured substrate for self-assembling and shaping gold-nanorod-based materials. Tridimensional wall features are formed over areas as large as several square millimeters. Furthermore, analyses by small-angle X-ray scattering and scanning electron microscopy techniques demonstrate that colloids are locally ordered as a smectic B phase. Such crystallization is in fact possible because we could finely adjust the nanoparticle charge, knowledge that additionally enables tuning the lattice parameters. In the future, the type of ordered self-assemblies of gold nanorods we have prepared could be used for amplifying optical signals.

Integrin reconstituted in GUVs: a biomimetic system to study initial steps of cell spreading

A novel in vitro membrane system mimicking the first steps of integrin-mediated cell spreading has been developed and characterized. We have reconstituted the transmembrane alpha(IIb)beta(3) integrin into giant unilamellar vesicles (GUVs). The reconstitution process has been validated by analyzing protein incorporation and biological activity by checking the specific interaction of GUVs containing integrin with quantum dots (QD) or surfaces coated with the integrin receptor tri-peptide RGD.(1) The spreading dynamics of integrin-functionalized GUVs onto fibrinogen-coated surfaces has been monitored by Reflection Interference Contrast Microscopy (RICM). Our results are quantitatively consistent with a theoretical model based on a dewetting process coupled to binder diffusion and provide a comprehensive description of the following sequence: i) nucleation and growth of adhesive patches coupled to the diffusion of the adhesive proteins to these adhesive zones ii) fusion of patches and formation of an adhesive ring iii) complete spreading of the GUV by dewetting of the central liquid film from the border to form an adhesive circular patch that is not significantly enriched in integrins, as compared to the unbound membrane. This finding is consistent with the recognized role of the actin cytoskeleton in stabilizing focal complexes and focal adhesions in a cell-extracellular matrix contact. These very large unilamellar integrin-containing vesicles provide a unique artificial system, which could be further developed towards realistic cell mimic and used to study the complexity of integrin-mediated cell spreading.

Crystallization of fluorescent quantum dots within a three-dimensional bio-organic template of actin filaments and lipid membranes.

Biological molecules and molecular self-assemblies are promising templates to organize well-defined inorganic nanostructures. We demonstrate the ability of a self-assembled three-dimensional crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots. Functionnalized tricystein peptidic quantum dots (QDs) are incorporated during the dynamical self-assembly of this actin/lipid template resulting in the formation of crystalline fibers. The crystal parameters, 26.5×18.9×35.5 nm3, are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. This process ensures the high quality of the crystal and results in unexpected fluorescence properties. This method of preparation offers opportunities to generate crystals with new symmetries and a large range of distance parameters.

Molecular recognition induced aggregation and fusion between vesicles containing lipids bearing complementary hydrogen bonding head-groups

Equimolar mixtures of large unilamellar vesicles, obtained from mixtures of egg lecithin and lipids containing complementary hydrogen bonding head-groups (barbituric acid and triaminopyrimidine), aggregate and fuse generating much larger vesicles.

Synthesis and characterisation of magnetic-luminescent composite colloidal nanostructures

In this communication, we report fluorescent and magnetic bifunctional structures in the nanoscale range. The synthesis and characterisation of various colloidal nanostructures as Y₃F_e5-xAl_xO₁₂-TRITC, gamma-Fe₂O₃-Cs₂Mo₆Br₁₄@SiO₂ and Fe₂O₃-QDs will be presented. At the outset, the first synthesis of aluminium substituted YAIG (Y₃Fe_5-xAl_xO₁₂) by the citrate gel process and its surface modification by TRITC through grafting of 3-aminopropyltrimethoxysilane (g-APS) for biology-related labelling will be reported. Secondly, the synthesis of nanoparticles under restricted environments offered by water-in-oil microemulsions provides excellent control over particle size and shape and interparticle spacing. These environments have been used in the synthesis of silica nanoparticles (50 nm) with magnetic nanocrystals core surrounded by optically active nanoclusters. Finally, we will describe the preparation of composite magnetic QD bioconjugable micelles by simultaneous encapsulation of hydrophobic CdSe/ZnS QDs (2-4 nm) and magnetic gamma-Fe₂O₃ nanoparticles (3-4 nm) into micelles of synthetic functional PEG amphiphiles bearing a bioactivable terminal group.