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

Abstract

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 Cterminal 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.

Protein exchange is reduced in calcium-independent epithelial junctions

“…Desmosome architecture is maintained in Gö6976-induced hyperadhesion Given that cadherin order did not correlate with adhesive state, we hypothesized that changes in the nanoscale architecture of the desmosomal plaque may provide additional insight toward the mechanism of hyperadhesion. We used superresolution direct stochastic optical reconstruction microscopy (dSTORM; Heilemann et al, 2008) to quantify desmosome protein architecture in different adhesive states (Stahley et al, 2016). We conducted dSTORM on HaCaT cells with calcium-dependent (mock-treated) or hyperadhesive (Gö6976-treated) desmosomes.…”

Proteomic analysis of the desmosome identifies novel components required for skin integrity

“…BioID circumvents this problem by biotinylating proteins localized at desmosomes, thereby making them accessible for purification as they transit the soluble fraction and under harsh conditions. The cytoplasmic face of desmosomes contains two ultrastructurally resolvable units, the inner and outer dense plaques (33). In order to target each of these regions we constructed fusions of a mutant biotin ligase, BirA R118G (hereafter called BirA) (32) to truncated variants of the core desmosome protein, desmoplakin, which spans these regions (34).…”

“…The cytoplasmic face of desmosomes contains two ultrastructurally resolvable units, the inner and outer dense plaques (33). In order to target each of these regions we constructed fusions of a mutant biotin ligase, BirA R118G (hereafter called BirA) (32) to truncated variants of the core desmosome protein, desmoplakin, which spans these regions (34). Fusion to the end of desmoplakin's head domain (Dsp N -BirA) is expected to place BirA in the outer dense plaque while fusion of BirA to the end of the coiled-coil domain (Dsp CC -BirA) should place it in the inner dense plaque where desmosomes associate with keratin filaments (Fig.…”

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

“…Structurally characterizing desmosomes presents a challenge, due to their biochemical intractability, complex structure spanning two cells, and functional dynamics. The organization of proteins in the plaque has been determined by electron microscopy (EM) and superresolution stochastic optical reconstruction microscopy (14,15). Through these methods, differences in protein spatial organization have been correlated with changes in adhesion.…”

“…The intracellular plaque proteins create a dense network linking the cadherin cytoplasmic tails to the intermediate filament cytoskeleton (19). Each desmosome contains many copies of each of these proteins, resulting in an overall structure $0.5 mm in diameter (20) with the plaque extending between 100 and 115 nm (14,15) into the cytoplasm.…”