Transportation of proteins and lipids between membrane-enclosed organelles by vesicle formation is a fundamental task in eukaryote cells. The least understood protein complex involved in membrane remodelling for vesiculation, but with a wide range of medical implications, is the retromer complex.The objective of this proposal is:First, to identify the mechanistic principles of membrane bending and scission by retromer, and second to characterize temporal and morphological aspects of retromer and cargo molecules (Shiga toxin (STx) and Alzheimer’s amyloid-beta-cleaving enzyme (BACE)).I will exploit two approaches:First, in vitro reconstitution of the retromer complex using giant unilamellar vesicles to deploy biophysical methods.Second, I will provide a molecular choreography and anatomy of retrograde transport of STx, BACE and retromer by implementing a new imaging technique, structured-illumination microscopy combined with single particle tracking.The proposed interdisciplinary research will break new ground for membrane mechanics and trafficking of disease related cargo molecules.