Date of Award
Doctor of Philosophy (PhD)
Biological membranes undergo constant shape remodeling involving the formation of highly curved structures. As one of the most extensively studied membrane remodeling events, endocytosis is a ubiquitous eukaryotic membrane budding, vesiculation, and internalization process fulfilling numerous roles including compensation of membrane area increase after bursts of exocytosis. There are multiple independent endocytic pathways which differ by their speed as well as the proteins that are involved in. Bin/Amphiphysin/Rvs (BAR) domain proteins, such as endophilin, are responsible for sensing or generating membrane curvature in multiple endocytic pathways. In this dissertation, I elucidate the mechanisms of membrane remodeling through in vitro experimental studies with synthetic lipid bilayers.
Firstly, I investigated the binding and assembly of endophilin on planar membranes. Endophilin was found to be attracted to the membrane through electrostatic forces and to subsequently oligomerize on the membrane with the help of the protein’s N-terminal helices.
Next, I studied the mechanisms that govern membrane shape transitions induced by BAR domain proteins. The initiation of membrane curvature occurs at well-defined membrane tensions and protein surface densities. Importantly, the membrane budding and tubulation initiated by membrane tension reduction provides a mechanistic explanation for high speed endocytic pathways. The experimentally determined membrane shape stability diagram shows remarkable consistency with a three-parameter curvature instability model. Comparing different BAR domain proteins, the ability of three BAR domain proteins to generate high membrane curvature increases significantly from endophilin to amphiphysin, and to SNX9. The protein-protein attraction strength was identified as one of the most important factors that leads to the dramatic difference among these structurally similar proteins.
Furthermore, I examined membrane interactions of α-synuclein, an intrinsically disordered protein whose aggregation is a hallmark of Parkinson’s disease. The binding of α-synuclein monomers was found to linearly expand the membrane area before the protein is able to induce membrane curvature. The area expansion is achieved by thinning of the bilayer. As I experimentally demonstrate, these features make α-synuclein a reporter of membrane tension as well as a promoter for endocytosis.
Finally, I found that Ca2+ ions can induce membrane invaginations through the clustering of charged lipids, albeit less efficiently than BAR domain proteins. As I will discuss, this suggests an intriguing role of Ca2+ ions in the evolution of life.
Shi, Zheng, "Mechanisms of Membrane Remodeling by Peripheral Proteins and Divalent Cations" (2015). Publicly Accessible Penn Dissertations. 2011.