Combining patch-clamping and fluorescence microscopy for quantitative reconstitution of cellular membrane processes with Giant Suspended Bilayers

Abstract

In vitro reconstitution and microscopic visualization of membrane processes is an indispensable source of information about a cellular function. Here we describe a conceptionally novel free-standing membrane template that facilitates such quantitative reconstitution of membrane remodelling at different scales. The Giant Suspended Bilayers (GSBs) spontaneously swell from lipid lamella reservoir deposited on microspheres. GSBs attached to the reservoir can be prepared from virtually any lipid composition following a fast procedure. Giant unilamellar vesicles can be further obtained by GSB detachment from the microspheres. The reservoir stabilizes GSB during deformations, mechanical micromanipulations, and fluorescence microscopy observations, while GSB-reservoir boundary enables the exchange of small solutes with GSB interior. These unique properties allow studying macro- and nano-scale membrane deformations, adding membrane-active compounds to both sides of GSB membrane and applying patch-clamp based approaches, thus making GSB a versatile tool for reconstitution and quantification of cellular membrane trafficking events.

GTP and lipids control self-assembly and functional promiscuity of Dynamin2 molecular machinery

“…S2). To test this conjecture, we used the Giant Suspended Bilayer (GSB) system recently developed for quantitative analysis of membrane remodeling (Velasco-Olmo et al, 2019). In agreement with earlier observations (Liu et al, 2011), Dyn2 showed limited tubulation activity on GSBs containing a low PE amount even at 4 mol% PI(4,5)P2 (Fig.…”

Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon

“…The tip was then carefully introduced into a home-made humidity chamber and subjected to 10–20 min incubation at 60 °C. Then the beads were transferred to the observation chamber filled with the working buffer, which was pretreated with bovine serum albumin to prevent lipid attachment to the glass 59 . Upon immersion in the working buffer, lipid or proteo-lipid film swelling was followed as spontaneous GUV or proteo-GUV formation.…”

Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

“…A significant drawback of ensemble measurements arises from the compositional heterogeneity of proteoliposome reconstitutions, which hampers quantitative analysis of vesicle properties (including stoichiometry of lipids, sterols, transmembrane proteins) and correlation with protein activity 13 . Giant unilameller vesicles (GUVs, 10-100 µm diameter) are model membrane systems with dimensions comparable to that of a cell, allowing single vesicle analysis directly by microscopy techniques such as fluorescence microscopy, fluorescence correlation spectroscopy or atomic force microscopy [14][15][16][17] . Furthermore, such vesicles can be micro-manipulated for position control or mechanical probing.…”