Protein exchange is reduced in calcium-independent epithelial junctions.

Desmosomes are cell-cell junctions that provide mechanical integrity to epithelial and cardiac tissues. Desmosomes have two distinct adhesive states, calcium-dependent and hyperadhesive, which balance tissue plasticity and strength. A highly ordered array of cadherins in the adhesive interface is hypothesized to drive hyperadhesion, but how desmosome structure confers adhesive state is still elusive. We employed fluorescence polarization microscopy to show that cadherin order is not required for hyperadhesion induced by pharmacologic and genetic approaches. FRAP experiments in cells treated with the PKCα inhibitor Gö6976 revealed that cadherins, plakoglobin, and desmoplakin have significantly reduced exchange in and out of hyperadhesive desmosomes. To test whether this was a result of enhanced keratin association, we used the desmoplakin mutant S2849G, which conferred reduced protein exchange. We propose that inside-out regulation of protein exchange modulates adhesive function, whereby proteins are "locked in" to hyperadhesive desmosomes while protein exchange confers plasticity on calcium-dependent desmosomes, thereby providing rapid control of adhesion.

Bridging the gap: Super-resolution microscopy of epithelial cell junctions.

Cell junctions are critical for cell adhesion and communication in epithelial tissues. It is evident that the cellular distribution, size, and architecture of cell junctions play a vital role in regulating function. These details of junction architecture have been challenging to elucidate in part due to the complexity and size of cell junctions. A major challenge in understanding these features is attaining high resolution spatial information with molecular specificity. Fluorescence microscopy allows localization of specific proteins to junctions, but with a resolution on the same scale as junction size, rendering internal protein organization unobtainable. Super-resolution microscopy provides a bridge between fluorescence microscopy and nanoscale approaches, utilizing fluorescent tags to reveal protein organization below the resolution limit. Here we provide a brief introduction to super-resolution microscopy and discuss novel findings into the organization, structure and function of epithelial cell junctions.

Desmoglein 3 Order and Dynamics in Desmosomes Determined by Fluorescence Polarization Microscopy.

Desmosomes are macromolecular cell-cell junctions that provide adhesive strength in epithelial tissue. Desmosome function is inseparably linked to structure, and it is hypothesized that the arrangement, or order, of desmosomal cadherins in the intercellular space is critical for adhesive strength. However, due to desmosome size, molecular complexity, and dynamics, the role that order plays in adhesion is challenging to study. Herein, we present an excitation resolved fluorescence polarization microscopy approach to measure the spatiotemporal dynamics of order and disorder of the desmosomal cadherin desmoglein 3 (Dsg3) in living cells. Simulations were used to establish order factor as a robust metric for quantifying the spatiotemporal dynamics of order and disorder. Order factor measurements in keratinocytes showed the Dsg3 extracellular domain is ordered at the individual desmosome, single cell, and cell population levels compared to a series of disordered controls. Desmosomal adhesion is Ca2+ dependent, and reduction of extracellular Ca2+ leads to a loss of adhesion measured by dispase fragmentation assay (λ = 15.1 min). Live cell imaging revealed Dsg3 order decreased more rapidly (λ = 5.5 min), indicating that cadherin order is not required for adhesion. Our results suggest that rapid disordering of cadherins can communicate a change in extracellular Ca2+ concentration to the cell, leading to a downstream loss of adhesion. Fluorescence polarization is an effective bridge between protein structure and complex dynamics and the approach presented here is broadly applicable to studying order in macromolecular structures.

Molecular organization of the desmosome as revealed by direct stochastic optical reconstruction microscopy.

Desmosomes are macromolecular junctions responsible for providing strong cell-cell adhesion. Because of their size and molecular complexity, the precise ultrastructural organization of desmosomes is challenging to study. Here, we used direct stochastic optical reconstruction microscopy (dSTORM) to resolve individual plaque pairs for inner and outer dense plaque proteins. Analysis methods based on desmosomal mirror symmetry were developed to measure plaque-to-plaque distances and create an integrated map. We quantified the organization of desmoglein 3, plakoglobin and desmoplakin (N-terminal, rod and C-terminal domains) in primary human keratinocytes. Longer desmosome lengths correlated with increasing plaque-to-plaque distance, suggesting that desmoplakin is arranged with its long axis at an angle within the plaque. We next examined whether plaque organization changed in different adhesive states. Plaque-to-plaque distance for the desmoplakin rod and C-terminal domains decreased in PKP-1-mediated hyperadhesive desmosomes, suggesting that protein reorganization correlates with function. Finally, in human epidermis we found a difference in plaque-to-plaque distance for the desmoplakin C-terminal domain, but not the desmoplakin rod domain or plakoglobin, between basal and suprabasal cells. Our data reveal the molecular organization of desmosomes in cultured keratinocytes and skin as defined by dSTORM.