Protein Order in the Desmosome Investigated with Fluorescence Polarization Microscopy

Abstract

Desmosomes are macro-molecular cell-cell junctions critical for conferring mechanical stability to epithelia and cardiac muscle. The size and complexity of the desmosome presents experimental challenges. Desmosomes are comprised of transmembrane cadherin proteins and intracellular plaque proteins that interact with the intermediate filament cytoskeleton. Desmosomes are required to maintain tissue integrity; disruption of desmosomes in a variety of human diseases leads to tissue fragility, skin blistering, and cardiomyopathy. We hypothesize that the order of the proteins in the desmosome is critical for its function: when desmosomes are strongly adhesive the proteins are highly ordered and conversely proteins are disordered in states of weak adhesion. Fluorescence polarization measurements can yield information about the mobility, orientation, and proximity of fluorophores and can be used to study the order of proteins in vivo. We developed a theoretical framework to assess protein order in the desmosome based on geometry, mirror symmetry, and organization relative to the plasma membrane. Using this framework, we can resolve the relative order and disorder of individual protein domains within the complex. We employed in silico modeling to evaluate the impact of protein orientation, the percentage of ordered proteins, and signal to noise. The order of specific desmosomal protein domains was examined using fluorescent tags and fluorescence polarization microscopy. We subsequently characterized protein organization in the desmosome in states of strong and weak adhesion. This approach allows the collection of structural information in vivo with the ability to probe the organization of proteins within the desmosome in living cells.