PROJECT SUMMARY / ABSTRACT Adherens junctions, desmosomes and endothelial junctions are fundamental adhesive junctions crucial to animal physiology. Mutations and autoimmune diseases targeting them cause severe disorders in humans. Adherens junctions are found in all solid tissues and link actin cytoskeletons of adjacent cells, while desmosomes form strong linkages between intermediate filaments to provide resistance to mechanical stress. Endothelial junctions are distinct, specialized adherens junctions that maintain the integrity of vessels. Members of the cadherin superfamily of Ca2+-dependent adhesion receptors form the core transmembrane components of each of these junctions. Remarkably, our preliminary data show that cadherins of desmosomal and endothelial junctions spontaneously form highly ordered, intricate junctional architectures between reconstituted membranes through adhesive trans interactions and putative lateral cis interactions between cadherins on the same membrane. We have previously characterized their adhesive trans interactions in detail, but the mechanisms by which they assemble ordered extracellular architectures, including the identity of lateral interfaces, remain unknown. Two projects in this proposal aim to illuminate this potentially critical, but poorly understood level of structural organization in molecular detail using a cryo-ET approach in reconstituted and native junctions. At the core of our approach is a method I developed to overcome the difficulties of studying lateral interactions, which tend to be weak and poorly detectable outside of a membrane environment. Purified ectodomains are used to reconstitute junctions on liposomes for direct visualization of assemblies using cryo-ET, providing domain-level resolution of these large structures and identifying specific interfaces for functional validation by mutagenesis. We will employ this system to determine the assembly and lateral interactions of vertebrate desmosomal and endothelial junctions, then extend our findings to native cellular junctions using in situ cryo-ET. Two further projects investigate how cadherin adhesive and lateral interaction properties translate into function at the level of tissue organization, integrity and cell sorting using the power of model organism genetics. Direct assessment of the effects of modifying cadherin properties on morphogenesis in whole organisms has been prevented by functional redundancy and the high cost, difficulty and time of mouse models. These barriers are overcome in model organisms, Drosophila and C. elegans, which have highly restricted cadherin repertoires and far superior genetic tractability. We aim to define the molecular interactions of the adherens junction cadherins of these model organisms by a combined x-ray crystallography, cryo-EM, cryo-ET and biophysical approach to open up the use of these facile model systems for structure-function studies testing mutations that modify adhesive binding specific...