PROJECT SUMMARY Electrical synapses are complex cellular and biochemical structures with important roles in health and disease. They are composed of neuronal gap junctions that link the cytoplasm of synapsing neurons through channels composed of transmembrane Connexin proteins (Cx). However, there are striking gaps in knowledge surrounding the identification of non-Connexin electrical synapse proteins and the characterization of molecular mechanisms driving electrical synapse formation. Electron micrograph images first revealed the now well-characterized molecular assemblies of the chemical synapse, showing large electron dense regions beneath pre- and postsynaptic membranes now known as the Active Zone (AZ) and the Postsynaptic Density (PSD). Similar cytoplasmic electron dense regions have been observed at neuronal gap junctions, suggesting the presence of additional machinery regulating electrical synapses. The Miller lab recently identified ZO1b as being necessary and sufficient for Cx localization and electrical synapse function in the zebrafish Mauthner neural circuit. ZO1b is a multidomain molecular scaffold known for organizing cytosolic and transmembrane proteins at epithelial tight junctions. Interestingly, ZO proteins at tight junctions display the fascinating biochemical property known as liquid-liquid phase separation (LLPS) allowing them to create a non- membrane-bound compartments within the cell and concentrate binding partners to build fluid molecular assemblies. Indeed, ZO1b’s chemical synapse analog PSD95, as well as other synaptic scaffolds of the AZ and PSD, have also been suggested to organize chemical synapse architecture through LLPS. However, functional characterization for LLPS in vivo has been difficult due to the absence of an assessable model system. This proposal uses a combination of protein dynamics, binding assays, and structure/function mutants in cell culture to first determine the functional domains involved in Cx-scaffolding and LLPS of ZO1b and then translates those findings in vivo to the optically transparent, genetically tractable Mauthner cell circuit. Together the results will provide a foundational model for the molecules required for electrical synapse development and the biochemical interactions that drive it, as well as accelerated training for the applicant in neurodevelopment, protein biochemistry, and zebrafish genetics.