Fast chemical synaptic transmission is at the basis of neuronal communication. It is mediated by the fusion of synaptic vesicles (SVs) containing neurotransmitter molecules with the presynaptic plasma membrane at the so-called exocytic active zone (AZ) facing post-synaptic sites, where post-synaptic neurotransmitter receptors are located. Exocytic SV fusion is tightly coupled in space and time to the endocytic retrieval of SV proteins and lipids and the reformation of release-ready SVs to enable sustained neurotransmission and thereby brain function. The basis for the coupling of SV exo- and endocytosis is poorly understood. The proposed collaborative project aims to define the structural and molecular basis of exo-endocytic coupling at synapses. We will develop new fluorescent probes and protocols to determine the location of SV exocytosis and endocytosis events and define a putative endocytic zone (EZ) relative to the AZ. We will use rat and transgenic mouse primary hippocampal neurons, as well as in human neurons differentiated from induced pluripotent stem cells (hiPSCs) that are easily amenable for genome editing strategies. Furthermore, we will use advanced live and super-resolution imaging including live gated stimulated emission depletion (gSTED) microscopy to define the molecular markers of the EZ and test how SV proteins regulate its formation. Finally, we will uncouple endocytosis from exocytosis by timed changes in membrane tension or by downregulating the expression of SV or associated proteins that may link the EZ to the AZ, and determine their consequences on synaptic physiology.

Golgi-catalyzed glycosylation of proteins and lipids is essential for life and is generally perceived as a static type of post-translational modification. At variance with this preconceived view, our program will determine mechanisms by which glycans undergo post-Golgi cycles of dynamically triggered modifications that regulate biological functions akin to phosphorylation or ubiquitinylation. We have termed these the GlycoSwitches. We expect that our project will lead to a paradigm shift in glycobiology by establishing a previously unrecognized post-Golgi dynamics of glycosylation, and the molecular mechanisms by which such dynamically tunable glycosylation is translated into biological functions. Our proposal builds on 2 groundbreaking findings that we have recently made: The first is termed the desialylation GlycoSwitch hypothesis. It is based on our observation of a highly dynamic (sec to min time scales) regulation of specific biological functions, including receptor-ligand interaction, endocytosis and cell migration, by growth factor-induced reversible removal of sialic acid sugars from complex glycans on glycoproteins and glycolipids. The second, termed conformational GlycoSwitch hypothesis, is based on our finding that protein conformation determines function in a carbohydrate-specific manner. However, the available protein structural and glycobiological information is insufficient to solve the important mechanistic questions that arise on how glycan information can be tuned in such a rapid and reversible manner to provide a switch-like control over protein activity. Furthermore, the consequences of dysfunction of these GlycoSwitches in signaling, membrane trafficking, and disease are at this stage poorly understood at the molecular, cellular and organismic levels. We have therefore assembled a network of competence that is unique in the world with unmatched conceptual and technological expertise in: (i) cellular and chemical biology of glycan functions in endocytic trafficking in the Johannes team, (ii) analysis and engineering of cellular glycosylation pathways in the Clausen team, (iii) structural biology and biophysics of protein-glycan interaction and large-scale conformational changes of membrane proteins in the Roderer team, and (iv) intravital imaging in mice at subcellular resolution in the Weigert team. In addition to the groundbreaking GlycoSwitch concept, our program holds the promise for various other high impact discoveries in the life sciences: the first full-length high-resolution structures of an integrin and an oligomeric galectin, a mechanism by which epidermal growth factor (EGF) controls nutrient uptake in the intestine, and previously unrecognized multi-omics signatures for EGF receptor family-dependent cancers. Our approaches are designed to generate transferrable tools, including a cell-based platform to display human glycans individually in an arrayable format, DNA origamis for endocytic carrier purification and long-term imaging by lattice light sheet microscopy, a toolbox-like kit for glycosphingolipid construction in living cells, and mouse strains to universally dissect GlycoSwitch functions in mice. This moderate high risk / very high reward program is bound to rewrite a central paradigm in glycobiology, i.e., challenging the concept that glycosylation is a rather static post-translational modification and introducing dynamic regulation to the field. With newly developed easy-to-use tools and the discovery of generic GlycoSwitch mechanisms that operate in various normal and diseased tissue contexts, we expect to transform glycobiology from a field that was conceptually and experimentally largely only approachable by specialists to one that takes center stage in the life sciences. Our program also has the notable potential for improving general public health by identifying novel principles of therapeutic intervention against cancer based on the previously unrecognized GlycoSwitches.