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.
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.
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