Interaction of he Epsin N-Terminal Homology domain (ENTH) with artificial membranes as a function of lateral tension

Abstract

During endocytosis, membrane remodeling of a planar membrane into a highly curved vesicle is controlled by a complex protein machinery. However, the regulatory role of physical properties such as membrane tension is highly debated. At the beginning of the formation of a vesicle during clathrin mediated endocytosis, the protein epsin binds to its receptor lipid phosphatidylinositol-4,5-bisphosphate (PIP₂). Upon binding, a previously unstructured part of the epsin N-terminal homology domain (ENTH) forms an α-helix, which inserts into the membrane. Experiments using artificial lipid bilayers were performed to study the interaction of ENTH and lipid bilayers as a function of lipid composition and membrane tension. The binding affinity of ENTH to PIP₂ was analyzed for different lipid compositions and membrane topologies. Similar affinities of ENTH binding to PIP₂ in the high nanomolar range were measured, independent of the used lipid composition and membrane topology investigated. Protruded pore-spanning membranes were established to investigate the remodeling activity of ENTH as a function of lipid composition. Binding of ENTH to membranes having a large area compressibility modulus and a high lysis tension resulted in growth of the membrane protrusions. Binding of ENTH to membranes with a lower area compressibility modulus and lysis tension resulted in the formation of membrane defects. For all lipid compositions analyzed, no vesiculation or tubulation was observed after binding of ENTH. To analyze whether the high membrane tension of the protruded pore-spanning membranes suppresses any membrane remodeling ability of ENTH, experiments with giant unilamellar vesicles adhering to a solid support were conducted. The vesicles’ adhesion strength and lateral tension was adjusted to analyze the ENTH’s remodeling ability at lateral tensions corresponding to low and high membrane tensions found in cells. The formation of membrane tubes was observed for vesicles having a low membrane tension. Increasing the membrane tension resulted in a suppression of tube formation. Independent of the membrane tension, flattening of the vesicles was monitored after binding of ENTH. The inserted ENTH helix disturbs lipid packing, which reduces the area compressibility modulus and thus the bending rigidity of the membranes. The reduced bending rigidity lowers the energy required for the generation of membrane curvature. As the inserted helix of ENTH also splays the lipid head groups, thereby inducing local curvature, the combination of both mechanisms is expected to efficiently initiate the formation of a vesicle during clathrin mediated endocytosis.