A Platform to Study Curvature Effects of Proteins on Membranes

Abstract

The topography of a cell membrane is heterogeneous but well regulated. Specific cell processes, such as clathrin-mediated endocytosis, locally induce a large degree of curvature in the cell membrane and require several proteins to bind and shape the membrane. The N-terminal Homology domain of Epsin (ENTH) is a membrane binding domain involved in endocytosis that is believed to induce local curvature in lipid bilayers. Previous investigations, looking at small round vesicles in the presence of the ENTH domain using negative-stained electron microscope images, have shown tubulation of vesicles in the presence of purified ENTH domain. However vesicle dehydration from the staining process makes it difficult to discriminate morphological artifacts from protein-induced curvature.Cryo-electron microscopy (cryo-EM) avoids dehydration artifacts from heavy stains and preserves the round morphology of lipid vesicles. Using cryo-EM, we explore two methods of vesicle preparation: extrusion of lipids through a porous membrane with 100 nm pores and solubilization of lipids in detergent and slow dialysis of the detergent. We show that the extrusion method introduces a small sample of elongated and non-spherical shapes in the vesicle population, whereas the detergent-dialysis method produces perfectly round vesicles.Using the detergent-dialysis method to produce round vesicles, we can add putative curvature-inducing proteins to these vesicles and observe the effects of these proteins on vesicle curvature using cryo-EM. This provides an ideal platform to study the effects of curvature-inducing proteins on vesicle morphology.