Abstract
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.
Highlights
Arrays of lipid membranes attached to a support have attained great attention over the years, as they provide a high-throughput platform to analyse membrane-con ned processes.[1,2,3] In general, three major membrane architectures can be distinguished: (i) solid supported membranes, (ii) freestanding membranes and, (iii) immobilized vesicles
Pore-spanning membranes were prepared on non-functionalized substrates composed of silicon with a native silicon dioxide layer atop containing highly ordered arrays of cavities with pore diameters of 850 nm, an interpore distance of 4 mm (Fig. S1, Electronic supplementary information (ESI)†) and a pore depth of 10 mm by spreading of giant unilamellar vesicles (GUVs) composed of 1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC)
To visualize the speci c binding of the ENTH domain to PIP2-containing pore-spanning membranes and analyse its remodelling properties, the protein was uorescently labelled with Alexa[488] (Fig. 7A) and added to protruded pore-spanning DPhPC membranes doped with 1 mol% PIP2 and labelled with Texas Red DHPE (Fig. 7B)
Summary
Arrays of lipid membranes attached to a support have attained great attention over the years, as they provide a high-throughput platform to analyse membrane-con ned processes.[1,2,3] In general, three major membrane architectures can be distinguished: (i) solid supported membranes, (ii) freestanding membranes and, (iii) immobilized vesicles. The top view uorescence image (x–y-plane, Fig. 2A) shows an array of planar pore-spanning membranes, similar to what has been observed previously on functionalized porous silicon nitride substrates with open pore geometries.[18,33] As there is only a native silicon dioxide layer atop the silicon substrate, the Texas Red DHPE uorescence is partly quenched on the pore rims.[34,35] Each membrane separates a cavity volume of 6.5 fL from the bulk solution as speci ed by the manufacturer.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
