ABSTRACT Transmembrane proteins, which constitute 20%-30% of human genes, play essential roles in coupling cells and in sensing mechanical and biochemical signals from the environment. However, it is extremely challenging to map transmembrane protein interactions using traditional biochemical methods. The first goal of this proposal is to develop Binding Assay for Interacting Transmembrane proteins (BAIT), a molecular technology to discover novel transmembrane protein interactions in cells. BAIT will be performed in two steps. First, we will screen for transmembrane proteins that are located proximal to a target protein, by dual tagging both the extracellular and cytoplasmic region of the target with a proximity labeling enzyme. Next, we will directly test binding interactions between proximal proteins and the target protein using single molecule Atomic Force Microscopy (AFM) and also visualize co-localization of the target and binding partner using super-resolution fluorescence microscopy. We anticipate that BAIT will have a game changing impact in discovering novel transmembrane junctional proteins interactions on the cell surface. The second goal of our proposal is to use BAIT, along with other biophysical tools, to resolve the assembly and organization of desmosomes, an essential intercellular adhesive organelle that mediates the integrity of tissues like the epidermis and heart. While mutations in desmosomal proteins are common in hereditary heart diseases and in skin pathologies, the molecular mechanisms by which these proteins assemble at the plasma membrane are unknown. Since previous studies show that desmosome formation requires E- cadherin (Ecad), a ubiquitous cell-cell adhesion protein, we developed a prototype BAIT assay using Ecad as the target and discovered that two obligate desmosomal adhesive proteins, Desmocollin (Dsc) and Desmoglien (Dsg), bind to Ecad extracellular regions. Using biophysical experiments and cellular structure function studies we showed that Ecad recruits Dsg to intercellular contacts, and triggers desmosome formation. In Aim 2 of the proposal, we will characterize binding interfaces and kinetics of Ecad and Dsc/Dsg interactions and determine their binding conformations using single molecule AFM binding assays, single molecule Fluorescence Resonance Energy Transfer and computer simulations. We will also introduce mutant Ecad, Dsc and Dsg in epithelial cells and monitor desmosome assembly and ultrastructure using super-resolution fluorescence microscopy. These studies will provide key molecular insights into desmosomal integrity in both healthy tissues and in disease states.