Cell-free assays represent an important complement to studies in living cells for the elucidation of mechanisms underlying the dynamics of biological membranes, such as budding, fission, and fusion reactions. Here we describe a method for the reconstitution of endocytosis, the process through which cells internalize portions of the plasma membrane along with extracellular material, under conditions that allow the visualization of individual budding events with high spatial and temporal resolution. The method, which is based on the generation of planar plasma membrane sheets attached to a glass substrate and their subsequent incubation with a cytosolic extract, results in a very robust formation of endocytic buds, which upon appropriate conditions undergo fission. The synchronization of the endocytic events and the accessibility of the material to a variety of manipulations make this experimental system a powerful tool for the molecular dissection of endocytosis.
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