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.

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.

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.

Interaction between water-soluble peptidic CdSe/ZnS nanocrystals and membranes: formation of hybrid vesicles and condensed lamellar phases.

Due to their tunable optical properties and their well-defined nanometric size, core/shell nanocrystals (quantum dots, QDs) are extensively used for the design of biomarkers as well as for the preparation of nanostructured hybrid materials. It is thus of great interest to understand their interaction with soft lipidic membranes. Here we present the synthesis of water-soluble peptide CdSe/ZnS QDs and their interaction with the fluid lipidic membrane of vesicles. The use of short peptides results in the formation of small QDs presenting both high fluorescence quantum yield and high colloidal stability as well as a mean hydrodynamical diameter of 10 nm. Their interaction with oppositely charged vesicles of various surface charge and size results in the formation of hybrid giant or large unilamellar vesicles covered with a densely packed layer of QDs without any vesicle rupture, as demonstrated by fluorescence resonance energy transfer experiments, zetametry, and optical microscopy. The adhesion of nanocrystals onto the vesicle membrane appears to be sterically limited and induces the reversion of the surface charge of the vesicles. Therefore, their interaction with small unilamellar vesicles induces the formation of a well-defined lamellar hybrid condensed phase in which the QDs are densely packed in the plane of the layers, as shown by freeze-fracture electron microscopy and small-angle X-ray scattering. In this structure, strong undulations of the bilayer maximize the electrostatic interaction between the QDs and the bilayers, as previously observed in the case of DNA polyelectrolytes interacting with small vesicles.