Intracellular Membrane Remodeling Mechanisms Revealed by Cryo-EM

Sammanfattning: Endophilin B1 (EnB1) is a BAR protein located in the cytosol that controls membrane dynamics of different organelles such as the mitochondria and the Golgi, as well as autophagosomes. It has been suggested that this protein coordinates membrane remodeling events during essential cell death processes. For instance, previous studies show that knockdown of EnB1 leads to dysregulation of mitochondrial dynamics and inhibition of apoptosis. This protein could thereby have a critical tumor suppressor role in the cell. Despite the important role of EnB1 in many intracellular signaling processes, some parts of its underlying mechanisms of function are still unknown, more specifically, what drives the protein to bind to the membrane and what the protein structure looks like when bound.  Since EnB1 plays an important role in many intracellular trafficking events, it is of interest to obtain more information about this protein, both about its structure and membrane binding interactions. New information on this subject will contribute to a better understanding of how EnB1 modulates intracellular membranes to control several critical trafficking processes that contribute to neuron degradation and carcinogenesis.  This specific project aims at designing membrane templates that support EnB1 membrane- binding and bending for evaluation of binding capacity and for structural characterization by cryo-EM, and other associated methods.  To study the binding interactions of EnB1, different protein constructs were first expressed and purified. Membrane templates (liposomes and nanodiscs) were then created to enable structural characterization as well as studying the binding capacity of EnB1 to different lipids. A lipid binding assay with multiple variants of liposomes were created to study the biding capacity of EnB1, and negative stain transmission electron microscopy, as well as cryo-electron microscopy was used for visualization of the templates. By analyzing the data from the lipid binding assay, it can be concluded that both lipid composition and membrane curvature affects EnB1 membrane binding. The cryo-EM visualization also confirm that EnB1 is involved in membrane remodeling.