Project Summary Neurotransmitter release at synapses critically depends on the precise assembly of the secretory machine. Within a presynaptic nerve terminal, synaptic vesicles fuse at the active zone, a protein scaffold that forms release sites apposed to postsynaptic receptors. This protein complex contains RIM, ELKS, Munc13, RIM-BP, Liprin-α and Bassoon/Piccolo as central components. Recent work provides ground for new models of how these proteins assemble into functional release sites. First, the active zone is remarkably resilient and ablation of individual genes has at most modest effects on its assembly. Instead, combined deletions of RIM, ELKS, or RIM-BP strongly disrupt active zone assembly, establishing scaffolding redundancy. Second, current studies have led to a working model of assembly through liquid-liquid phase separation, with robust contributions of multivalent low-affinity interactions to assembly. Regardless of exact mechanisms, an overarching model that arises from these and other studies is that the active zone is a dynamic protein network that is held together by redundant, low-affinity protein binding. This is different from conventional models in which master organizers mediate assembly through rigid complexes with well-defined stoichiometries. Here, we build on our and other’s recent progress with the goal to identify what mechanisms mediate assembly of the initial active zone scaffold, and how opposing surfaces of these active zone protein networks interact with the target plasma membrane and with the synaptic vesicle cluster, respectively. We will use a three-pronged approach to answer these questions. Aim 1 defines roles and mechanisms of RIM in active zone assembly. We build on our finding that RIM drives recruitment of interacting proteins after removing scaffolding redundancy through RIM+ELKS knockout. We test the model that RIM organizes active zones through a two-step process that mechanistically separates RIM-targeting to active zones from RIM’s activity in recruiting other active zone proteins. Aim 2 dissects how synaptic vesicle clusters and active zones, two presynaptic sub- compartments, interact with one another. We rely on a new, “in-synapse” reconstitution approach and test parallel models to define which binding activities are sufficient to mediate vesicle docking. Aim 3 determines active zone anchoring mechanisms at the target plasma membrane. This aim makes use of our unique collection of conditional and compound mutants to solve the long-standing question of how the active zone scaffolds are physically attached to the right place at the target membrane. We use state-of-the-art methodology including conditional gene knockout, stimulated emission depletion (STED) microscopy, fluorescence recovery after photobleaching (FRAP), high pressure freezing- and correlative light-electron microscopy (CLEM), and electrophysiology to answer these questions. Our work will establish mechanistic models on how the targ...