In vitro reconstitution of discrete stages of dynamin-dependent endocytosis.

Many proteins involved in vesicle budding are able to interact with lipids and to deform membranes. In this review we present three in vitro assays that can be used to study the roles of these types of proteins and to reconstitute distinct stages of vesicle budding and tubule formation. In the first assay, the dynamic effect of peripheral membrane proteins on liposome morphology is studied, providing insight into the roles of proteins (e.g., the endocytic proteins; dynamins, epsins, amphiphysins, and endophilins) in deforming membranes and in recognizing membrane curvature. In the second assay, preformed lipid nanotubes are used to mimic the neck of a coated vesicle. These nanotubes form a suitable template to study molecules of the dynamin family, allowing visualization of the different conformations of dynamin that occur during vesicle scission. Finally, lipid monolayers have been used as mimics of the internal leaflet of the plasma membrane to investigate early stages of clathrin-coated pit formation. In principle this can equally be used to study budding mechanisms of other coated vesicle types. A combination of these three assays has given considerable insight into the roles of endocytic proteins and is allowing reconstitution and dissection of the different stages of vesicle formation.