Continuous Pore-Spanning Lipid Bilayers on Silicon Oxide-Coated Porous Substrates.

A number of techniques has been developed and analyzed in recent years to generate pore-spanning membranes (PSMs). While quite a number of methods rely on nanoporous substrates, only a few use micrometer-sized pores to be able to individually resolve suspending membranes by means of fluorescence microscopy. To be able to produce PSMs on pores that are micrometer in size, an orthogonal functionalization strategy resulting in a hydrophilic surface is highly desirable. Here, we report on a method to prepare PSMs based on the evaporation of a thin layer of silicon monoxide on top of the porous substrate. PM-IRRAS experiments demonstrate that the final surface is composed of SiOx with 1 < x < 2. The hydrophilic surface turned out to be well suited to spread giant unilamellar vesicles forming PSMs. As the method does not rely on a gold coating as frequently used for orthogonal functionalization, fluorescence micrographs provide information not only from the freestanding membrane areas but also from the supported ones. The observation of the entire PSM area enabled us to observe phase-separation in these membranes on the freestanding and supported parts as well as protein binding and possible lipid reorganization of the membranes induced by binding of the protein Shiga toxin.

Epsin N-terminal Homology Domain (ENTH) Activity as a Function of Membrane Tension.

The epsin N-terminal homology domain (ENTH) is a major player in clathrin-mediated endocytosis. To investigate the influence of initial membrane tension on ENTH binding and activity, we established a bilayer system based on adhered giant unilamellar vesicles (GUVs) to be able to control and adjust the membrane tension σ covering a broad regime. The shape of each individual adhered GUV as well as its adhesion area was monitored by spinning disc confocal laser microscopy. Control of σ in a range of 0.08-1.02 mN/m was achieved by altering the Mg(2+) concentration in solution, which changes the surface adhesion energy per unit area of the GUVs. Specific binding of ENTH to phosphatidylinositol 4,5-bisphosphate leads to a substantial increase in adhesion area of the sessile GUV. At low tension (<0.1 mN/m) binding of ENTH can induce tubular structures, whereas at higher membrane tension the ENTH interaction deflates the sessile GUV and thereby increases the adhesion area. The increase in adhesion area is mainly attributed to a decrease in the area compressibility modulus KA We propose that the insertion of the ENTH helix-0 into the membrane is largely responsible for the observed decrease in KA, which is supported by the observation that the mutant ENTH L6E shows a reduced increase in adhesion area. These results demonstrate that even in the absence of tubule formation, the area compressibility modulus and, as such, the bending rigidity of the membrane is considerably reduced upon ENTH binding. This renders membrane bending and tubule formation energetically less costly.

Driving a planar model system into the 3(rd) dimension: generation and control of curved pore-spanning membrane arrays.

The generation of a regular array of micrometre-sized pore-spanning membranes that protrude from the underlying surface as a function of osmotic pressure is reported. Giant unilamellar vesicles are spread onto non-functionalized Si/SiO(2) substrates containing a highly ordered array of cavities with pore diameters of 850 nm, an interpore distance of 4 µm and a pore depth of 10 µm. The shape of the resulting pore-spanning membranes is controlled by applying an osmotic pressure difference between the bulk solution and the femtoliter-sized cavity underneath each membrane. By applying Young-Laplace's law assuming moderate lateral membrane tensions, the response of the membranes to the osmotic pressure difference can be theoretically well described. Protruded pore-spanning membranes containing the receptor lipid PIP(2) specifically bind the ENTH domain of epsin resulting in an enlargement of the protrusions and disappearance as a result of ENTH-domain induced defects in the membranes. These results are discussed in the context of an ENTH-domain induced reduction of lateral membrane tension and formation of defects as a result of helix insertion of the protein in the bilayer.