Membrane shape transition control in cellular membrane trafficking phenomena

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PROJECT SUMMARY Dynamic changes in membrane shape are at the heart of cellular membrane trafficking phenomena such as endocytosis, where intracellular transport vehicles form from plasma membrane invaginations. Progress in research aimed to mechanistically understand endocytosis has been challenged in part by the existence of several different endocytic pathways. Clathrin-mediated endocytosis is the best characterized internalization route – it is responsible for the bulk of endocytic trafficking and is comparatively slow. Alternative pathways enable cells to rapidly respond to signals at the plasma membrane. A recently discovered pathway achieves fast responsiveness trough the assembly of transient complexes containing endophilin: a BAR domain protein, and lamellipodin: a multivalent adaptor protein. In this process termed fast endophilin-mediated endocytosis (FEME), dynamic complexes are formed through multivalent interactions that are stabilized through interactions with activated receptors, including members of the receptor tyrosine kinase (RTK), and G-protein coupled receptor (GPCR) classes. Whereas RTKs interact with endophilin only indirectly, involving additional adaptor proteins, several GPCRs show direct endophilin interactions, mediated by the receptors’ third intracellular loop (TIL), which becomes exposed upon receptor engagement by ligand. The combination of GPCR, endophilin, and lamellipodin, therefore represents an ideal system upon which to build an in-depth biophysical description of the mechanisms behind receptor internalization. Our goal is to investigate such mechanisms with the help of model membranes consisting primarily of giant unilamellar vesicles (GUVs) which allow the study of membrane shape transitions under precise control of membrane tension, using techniques that we have developed and refined over the course of this project. A second challenge towards a complete mechanistic understanding of plasma membrane internalization transitions is that membrane shape changes involve interactions in several different layers, all of which must receive due attention. We address this challenge in three aims that each are focused on a single layer, roughly defined by their distance from the membrane. The first aim furthers the understanding of mechanisms that determine the spontaneous bending preference (spontaneous curvature) of the bilayer itself – a prerequisite for rigorous design of the following two aims. A second aim asks how TIL binding modulates endophilin function on the membrane. The third aim considers multivalent interactions occurring distal from the membrane. Specifically, we will test the hypothesis that multivalent interactions involving endophilin’s SH3 domain and lamellipodin’s proline-rich domains, could give rise to critical density fluctuations near a protein-protein (demixing) phase transition, that couples with membrane shape transitions after stabilization through receptor engagement. We believe that findings from this project could translate to the understanding of additional membrane trafficking phenomena involving cooperative interactions under healthy and pathological conditions.