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Adam, Julia (2016): Cryo-EM studies of tubular membrane shaping by the N-BAR protein amphiphysin. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Membrane remodeling is important in cellular processes like vesicle trafficking, organelle shaping, movement or division. In Drosophila the membrane-shaping N-BAR protein amphiphysin is involved in the biogenesis and stabilization of deep invaginations of the sarcolemma, the T-tubules, crucial for the excitation-contraction coupling machinery in striated muscles. Drosophila amphiphysin mutants are viable but flightless due to a disorganized T-tubule network. However, details of the amphiphysin membrane interaction the underlying molecular mechanism how amphiphysin is remodeling and maintaining tubular membrane shapes and the three dimensional structure of the amphiphysin assembly are unknown. In this study I structurally characterized the helical arrangement of Drosophila amphiphysin on membrane tubes by using cryo-EM. I found that the BAR domain cooperatively self-assembles to a helical arrangement on the membrane surface. The cryo-EM 3D reconstruction of amphiphysin N-BAR-mediated tubes provided first structural insights into the unique and well-connected helical amphiphysin BAR assembly. One tip of the crescent-shaped BAR dimer is immersed into the membrane whereas the other tip is protruding outwards from the tube surface. In addition, it seems that the regulatory domains with a SH3 domain of amphiphysin are not involved in the membrane remodeling process. The helical BAR lattice is well-connected by laterally locking BAR domains in adjacent lattice rows via the H0 helix and by tip-to-center interactions of neighboring BAR domains in one lattice row. The H0 helix is insignificant for the membrane remodeling activity but essential for the fast initiation of the helical BAR polymerization and for the formation of a rigidly organized BAR arrangement on the tube surface. Mutations at the tip region or _H0 helix mutants showed impaired tube remodeling activity and a disorganized helical BAR arrangement. This suggests that the H0 helix as well as the BAR tips are crucial for an efficient and organized BAR assembly on the tube surface. I observed that amphiphysin N-BAR-mediated tubes are more rigid and uniform as endophilin N-BAR-mediated tubes, contributing to the dynamic membrane scission process in endocytosis. The cooperative BAR self-assembly and the unique and well-connected BAR arrangement on membranes give insights into the molecular mechanism of membrane shaping by amphiphysin and its ability to maintain the shape of T-tubules.