Mechanisms of membrane remodeling by bar proteins

Numerous physiological phenomena involve highly curved cell membranes with significant shape diversity. The diversity of cell membrane shape is partly regulated by a large family of BAR proteins, which consists of several different classes, including proteins with N-BAR, F-BAR, and I-BAR domains. The BAR proteins have been hypothesized to bend membranes through several different mechanisms, including scaffolding, wedging, oligomerization, and crowding. However, the contributions from these distinct mechanisms to remodel membranes have remained controversial. In this dissertation, I systematically compare and elucidate the mechanisms of membrane remodeling by BAR proteins via in vitro experimental studies. Firstly, I developed a F�rster resonance energy transfer (FRET) method to investigate the homodimerization mechanism and intradimer molecular interactions in an endocytic accessory N-BAR protein, endophilin. I revealed the high dimerization affinity nature of endophilin and found experimental evidence for the presence of an auto-inhibition mechanism, which arises from an intra-dimer, inter-monomer cross-interaction between the H0 helix and the SH3 domain from different subunits within a homodimer, for its membrane binding. Secondly, I quantitatively demonstrated that the amphipathic N-terminal H0 helix of endophilin is important for recruiting this protein to the membrane, but it does not contribute significantly to its intrinsic membrane curvature generation capacity by means of a GUV shape stability assay. Meanwhile, my study suggests that formation of a stable lattice is not necessary for the membrane remodeling of N-BAR proteins. I next revealed that at the same membrane tension, the crowding effect requires far higher protein coverage to induce curvature changes compared to the endocytic accessory proteins. These observations elevate the importance of scaffolding for the membrane curvature generation by N-BAR domains. Furthermore, I studied the regulation mechanism of membrane curvature induced by I-BAR proteins and revealed that I-BAR proteins exploit similar mechanisms as N-BAR proteins in membrane remodeling. Thirdly, to elucidate the functional differences and redundancies of endocytic BAR proteins, I systematically compared the membrane binding, curvature sensing, and curvature generation of endocytic BAR proteins. In the comparison of the N-BARs: endophilin, amphiphysin and SNX9, I observed substantially different abilities in generating (endophilin < amphiphysin < SNX9) and sensing (endophilin > amphiphysin > SNX9) membrane curvatures. In the comparison of FCHo2 and endophilin, I observed interesting competitive membrane binding and distinct membrane curvature generation abilities between them. These observations offer a basis for further understanding the similarities and differences of the physiological functions of BAR proteins. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemistry This dissertation is available at ScholarlyCommons: https://repository.upenn. edu/edissertations/2215 First Advisor Tobias Baumgart Second Advisor Jeffery G. Saven