ENTH domain-dependent membrane remodelling

Cellular membranes are anything but flat structures.

Complimentary action of structured and unstructured domains of epsin supports clathrin-mediated endocytosis at high tension

Membrane tension plays an inhibitory role in clathrin-mediated endocytosis (CME) by impeding the transition of flat plasma membrane to hemispherical clathrin-coated structures (CCSs). Membrane tension also impedes the transition of hemispherical domes to omega-shaped CCSs. However, CME is not completely halted in cells under high tension conditions. Here we find that epsin, a membrane bending protein which inserts its N-terminus H0 helix into lipid bilayer, supports flat-to-dome transition of a CCS and stabilizes its curvature at high tension. This discovery is supported by molecular dynamic simulation of the epsin N-terminal homology (ENTH) domain that becomes more structured when embedded in a lipid bilayer. In addition, epsin has an intrinsically disordered protein (IDP) C-terminus domain which induces membrane curvature via steric repulsion. Insertion of H0 helix into lipid bilayer is not sufficient for stable epsin recruitment. Epsin’s binding to adaptor protein 2 and clathrin is critical for epsin’s association with CCSs under high tension conditions, supporting the importance of multivalent interactions in CCSs. Together, our results support a model where the ENTH and unstructured IDP region of epsin have complementary roles to ensure CME initiation and CCS maturation are unimpeded under high tension environments.

Cooperativity of membrane-protein and protein–protein interactions control membrane remodeling by epsin 1 and affects clathrin-mediated endocytosis

Membrane remodeling is a critical process for many membrane trafficking events, including clathrin-mediated endocytosis. Several molecular mechanisms for protein-induced membrane curvature have been described in some detail. Contrary, the effect that the physico-chemical properties of the membrane have on these processes is far less well understood. Here, we show that the membrane binding and curvature-inducing ENTH domain of epsin1 is regulated by phosphatidylserine (PS). ENTH binds to membranes in a PI(4,5)P2-dependent manner but only induces curvature in the presence of PS. On PS-containing membranes, the ENTH domain forms rigid homo-oligomers and assembles into clusters. Membrane binding and membrane remodeling can be separated by structure-to-function mutants. Such oligomerization mutants bind to membranes but do not show membrane remodeling activity. In vivo, they are not able to rescue defects in epidermal growth factor receptor (EGFR) endocytosis in epsin knock-down cells. Together, these data show that the membrane lipid composition is important for the regulation of protein-dependent membrane deformation during clathrin-mediated endocytosis.

MOLECULAR DETERMINANTS OF THE ENDOCYTIC PROTEIN EPSIN CONTROLLING ITS LOCALIZATION AND FUNCTION IN CANCER CELL MIGRATION AND INVASION

ABSTRACTEpsins are endocytic adaptor proteins with signaling and endocytic functions. The three mammalian epsin paralogs are made of an Epsin N-Terminal Homology (ENTH) domain and an unstructured C-terminal region. The highly conserved ENTH domain plays a role in signaling by blocking RhoGAP activity and is required for cell migration in mammalian cells. However, our lab has previously shown that only epsin full length overexpression can enhance cell migration, but the ENTH domain alone cannot. Among the three Epsin paralogs, epsin 3 followed by epsin 2 were able to substantially enhance cell migration. This study is the first one to systematically and comprehensibly address the contribution of different motifs within the epsin C-terminus to enhance protein localization and cell migration. We show that is not the lipid-binding ENTH domain, but the C-terminus of epsin the one playing a major role in epsin association with sites of endocytosis. Further, we dissected the contribution of individual C-terminal endocytic (clathrin-, AP2-, Ubiquitin- and EH domain-binding) motifs for epsin localization. We found that while all motifs show a degree of synergism, the clathrin-binding motifs are the most important for epsin localization. Our study also showed that, these motifs (particularly the clathrin binding site) play an important role in sustaining endocytic site dynamics and cell migration.

Complimentary action of structured and unstructured domains of epsin supports clathrin-mediated endocytosis at high tension

Membrane tension plays an inhibitory role in clathrin-mediated endocytosis (CME) by impeding the transition of flat plasma membrane to hemispherical clathrin-coated structures (CCSs). Membrane tension also impedes the transition of hemispherical domes to omegashaped CCSs, a necessary step before their internalization via dynamin-mediated membrane scission. However, CME is not completely halted in cells under high tension conditions. Here we find that epsin, a membrane bending protein which inserts its N-terminus H0 helix into lipid bilayer, supports flat-to-dome transition and increases the stability of CCSs at high tension. This discovery is supported by molecular dynamic simulation of the epsin N-terminal homology (ENTH) domain that becomes more structured when embedded in a lipid bilayer. In addition, epsin has an intrinsically disordered protein (IDP) C-terminus domain which induces membrane curvature via steric repulsion. Insertion of H0 helix into lipid bilayer is not sufficient for stable epsin recruitment as deleting the IDP domain in epsin renders it cytosolic. Epsin’s binding to adaptor protein 2 and clathrin is critical for epsin’s association with CCSs under high tension conditions, supporting the importance of multivalent interactions in CCSs. Together, our results support a model where the ENTH and IDP domains of epsin have complementary roles to ensure CME initiation and CCS maturation are unimpeded under high tension environments.

Cooperativity of membrane-protein and protein-protein interactions control membrane remodeling by epsin 1 and regulate clathrin-mediated endocytosis

Membrane remodeling is a critical process for many membrane trafficking events, including clathrin-mediated endocytosis. Several molecular mechanisms for protein induced membrane curvature have been described in some detail. Contrary, the effect that the physico-chemical properties of the membrane has on these processes is far less well understood. Here, we show that the membrane binding and curvature-inducing ENTH domain of epsin1 is regulated by phosphatidylserine (PS). ENTH binds to membranes in a PI(4,5)P2-dependent manner but only induces curvature in the presence of PS. On PS-containing membranes, the ENTH domain forms rigid homo-oligomers and assembles into clusters. Membrane binding and membrane remodeling can be separated by structure-to-function mutants. Such oligomerization mutants bind to membranes but do not show membrane remodeling activity. In vivo they are not able to rescue defects in epidermal growth factor receptor (EGFR) endocytosis in epsin knock-down cells. Together, these data show that the membrane lipid composition is important for the regulation of protein-dependent membrane deformation during clathrin-mediated endocytosis.