Project Summary/Abstract In both invertebrate and vertebrate nervous systems, cell recognition molecules control assembly of synaptic circuits during development. We discovered a network of interacting cell surface proteins (CSPs) through an in vitro binding (“interactome”) screen for interactions among all Drosophila immunoglobulin superfamily (IgSF) proteins. In this network, 11 DIP proteins in one IgSF subfamily interact with 21 Dpr proteins in another subfamily, with affinities ranging from 1 µM to 200 µM. Each DIP and Dpr is expressed by a unique subset of neurons in each area of the developing brain. The connectome of the Drosophila pupal optic lobe (OL) is assembled by activity-independent mechanisms. DIP::Dpr interactions are important for OL and neuromuscular system wiring, and loss of individual DIPs or Dprs can alter synaptic connectivity and cause neuronal death. In this proposal, we address the functions of affinity variation and avidity by examining how changes in DIP::Dpr binding affinity and expression level affect synaptic terminal development in the neuromuscular system and synaptic connectivity in the OL. We also examine other interaction networks that may be involved in determination of the optic lobe connectome. Two such networks are the Beat/Side network of 22 IgSF proteins, which we also discovered in the interactome screen, and a network of ligands for receptor tyrosine phosphatases (RPTPs), which are neuronal signaling receptors that regulate axon guidance and synaptogenesis. We will examine how these three networks work together in vivo to control synaptic connectivity between specific lamina and medulla neurons in the OL. We will generate a comprehensive map of interactions and measure binding affinities for all of the Beat/Side proteins using surface plasmon resonance. To validate interactions in the RPTP network and identify inter-network interactions, we will develop a method for multiplexed interactome screening using high-avidity 60-mer nanoparticles that may be able to identify lower-affinity interactions missed in earlier screens. The objectives of the present application are to define how the affinities of DIP::Dpr interactions affect synaptic connection patterns, to examine how DIPs and Dprs work together with other families of cell recognition molecules, and to develop improved methods to detect and characterize in vitro interactions among CSPs. We plan to attain these objectives through three Specific Aims. Aim 1: Define the functions of DIP::Dpr affinity variation in control of muscle innervation. Aim 2: Examine the roles of DIP::Dpr affinity and of interplay among cell adhesion and signaling molecules in determination of synaptic connectivity in the OL. Aim 3: Map in vitro interactions among Beats, Sides, RPTPs, and other CSPs. The expected outcome of the proposed research will be the acquisition of new insights into the mechanisms by which interactions among cell recognition molecules control the assembly ...