Integrin-Functionalised Giant Unilamellar Vesicles via Gel-Assisted Formation: Good Practices and Pitfalls

Abstract

Giant unilamellar vesicles (GUV) are powerful tools to explore physics and biochemistry of the cell membrane in controlled conditions. For example, GUVs were extensively used to probe cell adhesion, but often using non-physiological linkers, due to the difficulty of incorporating transmembrane adhesion proteins into model membranes. Here we describe a new protocol for making GUVs incorporating the transmembrane protein integrin using gel-assisted swelling. We report an optimised protocol, enumerating the pitfalls encountered and precautions to be taken to maintain the robustness of the protocol. We characterise intermediate steps of small proteoliposome formation and the final formed GUVs. We show that the integrin molecules are successfully incorporated and are functional.

Talin and kindlin cooperate to control the density of integrin clusters

“…This F3-NPxY interaction was shown to be sufficient to activate αIIbβ3 integrin in cells (Tadokoro, 2003). Inactive αIIbβ3 integrins, from human platelets, were purified and labelled with Alexa Fluor 647 as previously described (Souissi et al, 2021). Integrins were first inserted into proteoliposomes after removal of detergent from Triton-lipid micelles (Rigaud & Lévy, 2003; Souissi et al, 2021; Streicher et al, 2009).…”

“…Inactive αIIbβ3 integrins, from human platelets, were purified and labelled with Alexa Fluor 647 as previously described (Souissi et al, 2021). Integrins were first inserted into proteoliposomes after removal of detergent from Triton-lipid micelles (Rigaud & Lévy, 2003; Souissi et al, 2021; Streicher et al, 2009). GUVs containing integrins were then prepared by the electroformation technique (Supplementary Fig.…”

“…Integrin α IIb β 3 was purified as previously described (Souissi et al, 2021). Briefly, integrin was extracted from outdated human platelets from French Blood Etablishment (EFS) with 1% Triton X-100 and purified via affinity chromatography over Concanavalin A, Heparin Sepharose, and KYGRGDS-columns (HiPrep 16/60 Sephacryl S-300 HR).…”

Experimental platform for the functional investigation of membrane proteins in giant unilamellar vesicles

“…Second, in our experience with charge mediated fusion, 30% negatively charged lipids have been a good compromise for fast and complete fusion. 46 Variation of the lipid composition during GUV formation has been successfully described, 50,61 and is unlikely to cause problems in similar experiments.…”

“…Among the different substrates used, polyvinyl alcohol (PVA) has established itself as a go-to material due to its wide accessibility and ease of use. However, the method is not without pitfalls, which have been explored in several previous publications, 50,60,61 such as for example contamination of the lipid bilayer with residual PVA. 60 Despite this, we have focused on the use of PVA and compared GUV properties important for MP investigation for vesicles produced by PVA assisted swelling and electroformation.…”