Abstract
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.
Highlights
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)
Previous work from our lab has shown that retinal pigment epithelial (RPE) cells spread on large fibronectin islands exhibited an increase in the proportion of abortive CCSs and smaller CCPs30,31
Using structured illumination microscopy (SIM)-total internal reflection fluorescence (TIRF) super-resolution imaging of RPE cells stably expressing mCherryclathrin light chain (CLC), we characterized the morphology of CCSs and classified them into three categories: (i) abortive CCSs, which are coated structures which dissemble before they reach maturation[32,33,34], (ii) productive clathrin-coated pits (CCPs), which are coated structures that undergo initiation, assembly, and transition to coated-pits followed by membrane scission and internalization, and (iii) stalled CCSs, which are persistent, non-internalizing coated structures in the imaging field[8,12]
Summary
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). 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. An increase in lipid packing defects at high tension may be key in aiding helix insertion at high tension from theoretical studies[27,28] This evidence points to the existence of a tension-sensitive recruitment mechanism of ENTH domaincontaining proteins. We used the combination of total internal reflection fluorescence (TIRF)
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