Cadherin Organization in Desmosomes Probed using Fluorescence Polarization Microscopy

Abstract

Desmosomes are intercellular junctions found in epithelia and cardiac muscle that resist mechanical stress by linking the intermediate filaments of neighboring cells. Disruption of the desmosome-intermediate filament complex (DIFC) can cause a loss of tissue integrity as well as abnormalities in tissue differentiation, leading to a variety of diseases. Understanding these diseases will require expanding our knowledge of the relationship between structure and function in desmosomes. Of particular interest is the arrangement, or order, of the desmosomal cadherins. Cadherins are calcium-dependent, transmembrane adhesion proteins. In a single desmosome, many copies of these proteins engage in trans-binding with the cadherins of neighboring cells to form the adhesive interface. Large macromolecular complexes and membrane-associated proteins are difficult to study using traditional structural techniques. To overcome this, we pioneered excitation-resolved fluorescence polarization microscopy (FPM) to study the organization of desmosomes. After placing fluorescent tags in proteins of interest, FPM allows for the quantification of their order within a macromolecular assembly. Unlike methods that require sample fixation, FPM can be performed on living cells, providing insights into DIFC organization under physiologically relevant conditions, while preserving dynamic information. Through this methodology, our lab has shown that the most membrane-proximal extracellular domain of desmoglein 3 is ordered in desmosomes and that this order is rapidly lost after calcium removal. Here, we show that this behavior is conserved in another cadherin isoform, desmolgein 2. We also designed constructs to measure the order of the cadherin cytoplasmic domains, allowing correlation of order across the membrane. Finally, we use FPM to examine the effect of mutations that abrogate binding between intracellular DIFC components. This approach provides a unique tool to study desmosomal architecture and elucidate the roles of specific protein-protein interactions as they relate to desmosome structure and function.