From LUVs to GUVs - how to cover micrometer-sized pores with membranes

Pore-spanning membranes (PSMs) are a versatile tool to investigate membrane-confined processes in a bottom-up approach. Pore sizes in the micrometer range are most suited to visualize PSMs using fluorescence microscopy. However, the preparation of these PSMs relies on the spreading of giant unilamellar vesicles (GUVs). GUV production faces several limitations. Thus, alternative ways to generate PSMs starting from large or small unilamellar vesicles that are more reproducibly prepared, are highly desirable. Here we describe a method to produce PSMs obtained from large unilamellar vesicles making use of droplet-stabilized GUVs generated in a microfluidic device. We analyzed the lipid diffusion in the free-standing and supported parts of the PSMs using z-scan fluorescence correlation spectroscopy and fluorescence recovery after photobleaching experiments in combination with finite element simulations. Employing atomic force indentation experiments, we also investigated the mechanical properties of the PSMs. Both, lipid diffusion constants and lateral membrane tension were compared to those obtained on PSMs derived from electroformed GUVs, which are known to be solvent- and detergent-free, under otherwise identical conditions. Our results demonstrate that the lipid diffusion, as well as the mechanical properties of the resulting PSMs, are almost unaffected by the GUV formation procedure but depend on the chosen substrate. With the new method in hand, we were then able to reconstitute the syntaxin-1A transmembrane domain in microfluidic GUVs and PSMs, which was visualized by fluorescence microscopy.