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

Abstract

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.

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

“…The method has already been applied to reconstitute Cx26 in GUVs (24). To obtain larger protein amounts, Streicher et al (46) reconstituted integrins into GUVs by briefly drying preformed proteoliposomes on ITO coated-glass slides. However, the drying process can cause inactivation of the protein.…”

Elucidating the molecular mechanisms underlying cellular response to biophysical cues using synthetic biology approaches

“…Researchers are now going a step further and beginning to create artificial cells in the form of liposomes containing lipid membrane-bound receptors, such as integrin. 95 These synthetic cells have already been utilized to study receptormediated spreading and adhesion 95,96 and actin-loaded liposomes were shown to drive actin polymerization to spontaneously form cytokinetic rings. 97 These synthetic "cells" have huge potential and could help to answer many complex biological questions in several fields of life sciences such as virology (in particular viral entry), and loaded liposomes have already been adapted for clinical applications in drug delivery.…”

Exosome mimetics: a novel class of drug delivery systems

“…However, work in this area is progressing and has yielded promising results. [108][109][110][111][112] For instance, the functional reconstitution of a voltage-gated potassium channel and α bII β 3 integrin in giant unilamellar vesicles was recently described. 108,110 The potential advantages of the incorporation of several exosomal proteins in exosome mimetics are discussed below.…”