Direct reconstitution of plasma membrane lipids and proteins in nanotube-vesicle networks

We demonstrate here that nanotube-vesicle networks can be constructed directly from plasma membranes of cultured cells. We used a combination of dithiothreitol (DTT) and formaldehyde to produce micron-sized plasma membrane vesicles that were subsequently shaped into networks using micromanipulation methods previously used on purely synthetic systems. Only a single cell is required to derive material sufficient to build a small network. This protocol covers the advantages of reconstitution in vesicles, such as full control over the solution environment, while keeping the proteins in their original surroundings with the proper orientation. Furthermore, control of membrane protein and lipid content in the networks is achievable by employing different cell types, for example, by overexpression of a desired protein or the use of specialized cell-types as sources for rare proteins and lipids. In general, the method provides simple accessibility for functional studies of plasma membrane constituents. Specifically, it provides a direct means to functionalize nanotube-vesicle networks with desired proteins and lipids for studies of transport activity both across membranes (protein-mediated) and across nanotubes (diffusion), and substrate conversion down to the single-molecule limit. Nanotube-vesicle networks can adopt different geometries and topologies and undergo shape changes at will, providing a flexible system for changing the physical and chemical environment around, for example, a membrane protein. Furthermore, the method offers unique possibilities for extracting membrane and protein material for nanotechnological sensor and analytical devices based on lipid membrane networks.