Defining domain-specific orientational order in the desmosomal cadherins

Desmosomes are large, macromolecular protein assemblies that mechanically couple the intermediate filament cytoskeleton to sites of cadherin-mediated cell adhesion, thereby providing structural integrity to tissues that routinely experi-ence large forces. Proper desmosomal adhesion is necessary for the normal development and maintenance of vertebrate tis-sues, such as epithelia and cardiac muscle, while dysfunction can lead to severe disease of the heart and skin. Therefore, it is important to understand the relationship between desmosomal adhesion and the architecture of the molecules that form the ad-hesive interface, the desmosomal cadherins (DCs). However, desmosomes are embedded in two plasma membranes and are linked to the cytoskeletal networks of two cells, imposing extreme difficulty on traditional structural studies of DC architecture, which have yielded conflicting results. Consequently, the relationship between DC architecture and adhesive function remains unclear. To overcome these challenges, we utilized excitation-resolved fluorescence polarization microscopy to quantify the orientational order of the extracellular and intracellular domains of three DC isoforms: desmoglein 2, desmocollin 2, and desmo-glein 3. We found that DC ectodomains were significantly more ordered than their cytoplasmic counterparts, indicating a drastic difference in DC architecture between opposing sides of the plasma membrane. This difference was conserved among all DCs tested, suggesting that it may be an important feature of desmosomal architecture. Moreover, our findings suggest that the or-ganization of DC ectodomains is predominantly the result of extracellular adhesive interactions. We employed azimuthal orien-tation mapping to show that DC ectodomains are arranged with rotational symmetry about the membrane normal. Finally, we performed a series of mathematical simulations to test the feasibility of a recently proposed antiparallel arrangement of DC ec-todomains, finding that it is supported by our experimental data. Importantly, the strategies employed here have the potential to elucidate molecular mechanisms for diseases that result from defective desmosome architecture.