Functional coupling between Synaptic Vesicle exocytosis and recycling in mammalian synapses – couplingSVcycling

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.