Biochemical Approaches to Studying Caenorhabditis elegans ESCRT Functions In Vitro

Our fundamental understanding of the roles played by the endosomal sorting complex required for transport (ESCRT) machinery in cells comes from interdisciplinary approaches that combine numerous in vivo and in vitro techniques. Here, we focus on methods used to biochemically characterize Caenorhabditis elegans ESCRT components in vitro, including the production and characterization of recombinant ESCRT complexes and their use in membrane interaction studies. Key methodologies used include gel filtration chromatography, glycerol density gradient analysis, multi-angle light scattering, liposome co-flotation, and single-liposome fluorescence microscopy. Collectively, these studies have enabled us to define subunit stoichiometry of soluble C. elegans ESCRT complexes and to demonstrate that the late-acting ESCRT-III complex facilitates membrane bending and remodeling, at least in part by virtue of its ability to sense the curvature of lipid bilayers.