Mechanical Regulation of Clathrin-Mediated Endocytosis by Membrane Bending Protein Epsin

Abstract

Cells are complex biomechanical systems that constantly adapt to and modify their physical environment. The cell membrane is the boundary that separates organelles and other molecular machineries in a cell from the extracellular environment and plays a major role in maintaining the biochemical and biomechanical homeostasis of cells. The constant deformation and remodeling of cell membranes is key in biological processes like cell division, polarization and migration. It also enables the trafficking of a diverse ensemble of cargos in and out of cells by endocytosis and exocytosis, respectively. This remodeling process is regulated by mechanical factors like membrane tension, rigidity, substrate stiffness, morphology of the cargo,…etc. Clathrin-mediated endocytosis (CME) is an endocytic pathway by which cells internalize cargos from the extracellular environment by extensive remodeling of the plasma membrane from a flat to a bud morphology supported by clathrin-coat assembly. Epsin is an adaptor protein in CME that aids in membrane bending and is recruited prior to clathrin to clathrin-coated pit (CCP) nucleation sites. Epsin has a structured N-terminal ENTH (epsin N-terminal homology) region and an unstructured C-terminal IDP (intrinsically disordered protein) region that are involved in membrane bending via amphipathic helix insertion and steric repulsion, respectively. Membrane tension plays an inhibitory role in CME by destabilizing CCP assembly. My investigation shows that overexpressing epsin rescues the stability of CCPs under high membrane tension. The recruitment of epsin to CCPs increases with elevated membrane tension. Masking the N-terminus of ENTH with EGFP or deletion of ENTH in epsin delays recruitment of epsin to CCP nucleation sites. Interfering with the activity of H0 helix abrogates the elevated recruitment of epsin at high tension. The ENTH domain, while necessary for tension sensitivity of epsin, is not necessary for epsin puncta formation. Deletion of adaptor protein 2 binding sites and clathrin binding sites in the IDP domain of epsin renders it cytosolic. Further, the IDP domain itself is sufficient to impart stability to CCPs as CCPs recruiting epsin with only IDP domain shows reduction in the fraction of abortive pits. My findings conclude that H0 alpha helix in the ENTH domain of epsin acts as a tension sensor and the IDP domain acts as a tether stabilizing CCPs. Our work reveals the complimentary action of structured ENTH and unstructured IDP domains of epsin supporst CME at high tension. CME is also utilized by pathogens like Influenza A virus (IAV) to gain access to host cells. Epsin is a known cargo-specific adaptor for IAVs. Epsin interacts with ubiquitinated surface receptors bound to IAVs via its ubiquitin-interacting motifs. My investigation shows that the ENTH domain in epsin biomechanically regulates curvature generation around spherical IAVs thereby initiating CME of the virion. Mutations in H0 helix negatively regulate the recruitment of epsin to CCPs containing spherical IAVs and their subsequent internalization. However, internalization of filament-forming IAVs were not affected by the inhibition of H0 helix formation in ENTH domain of epsin. In summary, the findings of this dissertation define the biomechanical role of epsin in CME under high membrane tension conditions and during viral entry. By improving our understanding of biomechanics of endocytosis and viral entry, I hope that this research will lead to development of better therapies targeting abnormal endocytosis pathways and viral diseases, as well as advances in viral vector and nanoparticle-based drug delivery systems.