Mechanisms of assembly of endocytic machinery at the periactive zone and coupling to the presynaptic active zone - Project summary The computational power of the brain relies on the accuracy of synapses to transmit information. At presynaptic terminals, this accuracy depends on the coordinated function between two protein machineries. First, the active zone generates exocytic sites where neurotransmitters are released from synaptic vesicles. Second, the endocytic apparatus restores vesicles for subsequent rounds of neurotransmission. These machineries are assembled in very close proximity, with the endocytic apparatus localized at the plasma membrane region that surrounds the active zone, called periactive zone. This spatial organization, a hallmark of synaptic architecture, ensures sustained neurotransmission. Genetic studies have linked components of both machineries to multiple brain disorders, highlighting their relevance. Our goal is to understand the mechanisms that direct the coordinated assembly of the active zone and the periactive zone, a process that remains poorly understood. We have generated three lines of evidence that indicate that the large GTPase Dynamin is an organizer of both the active zone and the periactive zone. First, deletion of all Dynamin proteins from neurons results in a selective loss of the active zone proteins Munc13-1 and RIM, and decreased neurotransmitter release, which indicates Dynamin roles in active zone assembly. Second, our previous work showed that several endocytic proteins are constitutively deployed to the periactive zone. We have now observed in pilot experiments that these proteins are strictly segregated into different clusters, in line with a disassembled machinery, and that this strict spatial segregation is lost in Dynamin mutants. Third, preliminary data show that Dynamin-1 is localized both at the periactive and at the active zone, consistent with roles bridging compartments. Here, we will dissect mechanisms through which Dynamin organizes the active zone and the periactive zone. We will use neurons cultured from Dynamin-1/2/3 triple mutant mice and human stem cell-derived neurons as models, and further integrate 10X Expansion Microscopy, electrophysiological and live imaging analyses. In Aim-1, we will focus on the active zone. We will define the roles that each of the three Dynamin proteins play in active zone assembly and their nanoscale localization at synaptic terminals. Next, through structure-function rescue experiments, we will identify the sequences that determine the localization of Dynamin proteins and mediate their role in active zone assembly. In Aim-2, we will focus on the periactive zone. We will first characterize the molecular organization of this compartment and how it is modified by synaptic activity. Next, we will establish the role of Dynamin proteins in the organization of the periactive zone and whether impaired membrane fission cause periactive zone disorganization. Finally, we will also test whether disease-associated missense variants of Dynamin-1 result in disrupted active zone or periactive zone organization. Overall, this work will provide insight into the organization of presynaptic protein machinery whose dysfunction is connected to brain disorders, advancing our understanding of synaptic function in health and disease.
