In vitro single-vesicle fusion assays are important tools to analyze the details of SNARE-mediated fusion processes. In this study, we employed planar pore-spanning membranes (PSMs) prepared on porous silicon substrates with large pore diameters of 5 μm, allowing us to compare the process of vesicle docking and fusion on the supported parts of the PSMs (s-PSMs) with that on the freestanding membrane parts (f-PSM) under the exact same experimental conditions. The PSMs harbor the t-SNARE ΔN49-complex to investigate the dynamics and fusogenicity of single large unilamellar vesicles doped with the v-SNARE synaptobrevin 2 by means of spinning-disc confocal microscopy with a time resolution of 10 ms. Our results demonstrate that vesicles docked to the s-PSM were fully immobile, whereas those docked to the f-PSM were mobile with a mean diffusion coefficient of 0.42 μm/s. Despite the different dynamics of the vesicles on the two membrane types, similar fusion kinetics were observed, giving rise to a common fusion mechanism. Further investigations of individual lipid mixing events on the s-PSMs revealed semi-stable post-fusion structures.

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The plasma membrane of animal cells is attached to the cytoskeleton, which significantly contributes to the lateral tension of the membrane. Lateral membrane tension has been shown to be an important physical regulator of cellular processes such as cell motility and morphology as well as exo- and endocytosis. Here, we report on lipid bilayers spanning highly ordered pore arrays, where we can control the lateral membrane tension by chemically varying the surface functionalization of the porous substrate. Surface functionalization was achieved by a gold coating on top of the pore rims of the hexagonal array of pores in silicon nitride substrates with pore radii of 600 nm followed by subsequent incubation with various n-propanolic mixtures of 6-mercapto-1-hexanol (6MH) and O-cholesteryl N-(8'-mercapto-3',6'-dioxaoctyl)carbamate (CPEO3). Pore-spanning membranes composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine were prepared by spreading giant unilamellar vesicles on these functionalized porous silicon nitride substrates. Different mixtures of 6MH and CPEO3 provided self-assembled monolayers (SAMs) with different compositions as analyzed by contact angle and PM-IRRAS measurements. Site specific force-indentation experiments on the pore-spanning membranes attached to the different SAMs revealed a clear dependence of the amount of CPEO3 in the monolayer on the lateral membrane tension. While bilayers on pure 6MH monolayers show an average lateral membrane tension of 1.4 mN m(-1), a mixed monolayer of CPEO3 and 6MH obtained from a solution with 9.1 mol % CPEO3 exhibits a lateral tension of 5.0 mN m(-1). From contact angle and PM-IRRAS results, the mole fraction of CPEO3 in solution can be roughly translated into a CPEO3 surface concentration of 40 mol %. Our results clearly demonstrate that the free energy difference between the supported and freestanding part of the membrane depends on the chemical composition of the SAM, which controls the lateral membrane tension.

SNAREs are known as an important family of proteins mediating vesicle fusion. For various biophysical studies, they have been reconstituted into supported single bilayers via proteoliposome adsorption and rupture. In this study we extended this method to the reconstitution of SNAREs into supported multilamellar lipid membranes, i.e. oriented multibilayer stacks, as an ideal model system for X-ray structure analysis (X-ray reflectivity and diffraction). The reconstitution was implemented through a pathway of proteomicelle, proteoliposome and multibilayer. To monitor the structural evolution in each step, we used small-angle X-ray scattering for the proteomicelles and proteoliposomes, followed by X-ray reflectivity and grazing-incidence small-angle scattering for the multibilayers. Results show that SNAREs can be successfully reconstituted into supported multibilayers, with high enough orientational alignment for the application of surface sensitive X-ray characterizations. Based on this protocol, we then investigated the effect of SNAREs on the structure and phase diagram of the lipid membranes. Beyond this application, this reconstitution protocol could also be useful for X-ray analysis of many further membrane proteins.

conductor (SC) nanorods (NRs) with Type-II heterojunctions that exhibit a large Quantum Confined Stark Effect (QCSE) at room temperature (1). For using these NRs as voltage sensors, however, one needs to impart them with membraneprotein like properties so that they can be stably inserted into the membrane. We report here spontaneous insertion of SC NRs into liposomes and cell membranes by functionalizing them with specially designed peptides. We provide evidences for insertion from cryo transmission electron microscopy (TEM) and polarized light microscopy. We also report on first attempts to sense membrane potential with these particles with single-particle sensitivity. With further improvements, SC NRs could potentially be used to study signals from whole neural networks in a large field-of-view. Moreover, successful implementation of SC NRs would allow for the analysis of voltage signals at the nano-(single synapse-) scale.