ausblenden:
Schlagwörter:
Adhesion; Integrin; Membrane protein reconstitution; Spreading kinetics; RICM; GUV; Fibrinogen; Quantum dots
Zusammenfassung:
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.
