Fast membrane hemifusion via dewetting between lipid bilayers

Abstract

The behavior of lipid bilayer is important to understand the functionality of cells like the trafficking of ions between cells. Standard procedures to explore the properties of lipid bilayer and hemifused states typically use either supported membranes or vesicles. Both techniques have several shortcoming in terms of bio relevance or accessibility for measurements. In this article the formation of individual free standing hemifused states between model cell membranes is studied using an optimized microfluidic scheme wh ich allows for simultaneous optical and electrophysiological measurements. In a first step, two model membranes are formed at a desired location within a microfluidic device using a variation of the droplet interface bilayer (DiB) technique. In a second st ep, the two model membranes are brought into contact forming a single hemifused state. For all tested lipids, the hemifused state between free standing membranes form within hundreds of milliseconds, i.e. several orders of magnitude faster than reported in literature. The formation of a hemifused state is observed as a two stage process, whereas the second stage can be explained as a dewetting process in no-slip boundary condition. The formed hemifusion states are long living and a single fusion event can be observed when triggered by an applied electric field as demonstrated for monoolein.

Direct proof of spontaneous translocation of lipid-covered hydrophobic nanoparticles through a phospholipid bilayer

“…From that, the corresponding bilayer thickness can be calculated as d = ε L ε 0 / C s ≈ 4.4 nm, where ε L = 2.2 is the dielectric constant of the lipid membrane ( 51 ) and ε 0 = 8.85 × 10 −12 F/m is the vacuum permittivity. These estimates agree with literature values ( 51 – 53 ). Monodisperse AuNPs with diameters of 2, 4, and 6 nm are rendered hydrophobic by a covalent 1-dodecanethiol coating and are additionally covered with a DMPC monolayer to allow their dispersion in an aqueous phase (see Materials and Methods).…”

“…A variant of the droplet interface bilayer technique ( 50 , 51 ) was used to produce a free-standing lipid membrane in a microfluidic chip ( 50 , 52 , 53 ). Using a volume-controlled system with syringe pumps, we injected two fingers of an aqueous phase (100 mM NaCl) face-to-face into microchannels of a cross-geometry (see Fig.…”

“…Once the two liquid fingers are brought into contact, their lipid monolayers interact, forming a lipid bilayer within a short time ( 51 , 52 ). The bilayer is stable and can be analyzed simultaneously by optical microscopy and by electrophysiological experiments ( 53 ). The capacitance of the formed particle-free bilayer in this geometry is measured as C ≈ 140 pF for a pure DMPC bilayer (see Fig.…”

The Multifaceted Role of SNARE Proteins in Membrane Fusion

“…Future studies will aim to further close the gap in spatial and temporal resolution between experiments such as electron tomography and simulation. Additionally, approaches allowing to characterize fusion pore properties using defined membrane compositions e.g., in a microfluidic setup (Vargas et al, 2014) will push our understanding of the intricate balance of protein-lipid interaction in fusion.…”

Towards Dewetting Monoclonal Antibodies for Therapeutical Purposes

“…Lipid bilayers and droplets. The behavior of lipid bilayer is crucial to understand biological processes such as ions trafficking between cells (Vargas et al, 2014). The standard procedures to explore the properties of lipid bilayer and hemifused states, which typically use either supported membranes or vesicles, have several shortcoming in terms of biorelevance or accessibility for measurements.…”