The three-dimensional molecular structure of the desmosomal plaque

Abstract

The cytoplasmic surface of intercellular junctions is a complex network of molecular interactions that link the extracellular region of the desmosomal cadherins with the cytoskeletal intermediate filaments. Although 3D structures of the major plaque components are known, the overall architecture remains unknown. We used cryoelectron tomography of vitreous sections from human epidermis to record 3D images of desmosomes in vivo and in situ at molecular resolution. Our results show that the architecture of the cytoplasmic surface of the desmosome is a 2D interconnected quasiperiodic lattice, with a similar spatial organization to the extracellular side. Subtomogram averaging of the plaque region reveals two distinct layers of the desmosomal plaque: a low-density layer closer to the membrane and a high-density layer further away from the membrane. When combined with a heuristic, allowing simultaneous constrained fitting of the high-resolution structures of the major plaque proteins (desmoplakin, plakophilin, and plakoglobin), it reveals their mutual molecular interactions and explains their stoichiometry. The arrangement suggests that alternate plakoglobin-desmoplakin complexes create a template on which desmosomal cadherins cluster before they stabilize extracellularly by binding at their N-terminal tips. Plakophilins are added as a molecular reinforcement to fill the gap between the formed plaque complexes and the plasma membrane.

Fig. 1.

Ultrastructure of the desmosome from human epidermis. (A) Two-dimensional projection image of a straight desmosome shows the periodic structure in extracellular and intracellular regions. The extracellular desmosomal cadherins appear as electron-dense protrusions extending from the plasma membrane (PM) to a dense midline (DM), showing a pronounced periodicity (purple boxed area). At the intracellular region (IC), two layers with different levels of intensity (green and blue highlighted area) can be observed, corresponding to the plakophilin and plakoglobin layers. (B) A 6-nm-thick tomographic slice shows details of the desmosomal interactions in extracellular and intracellular spaces. cis interactions (cadherin emanating from the same cell) are highlighted by the orange arrows; trans interactions (cadherins emanating from juxtaposed cells), with the characteristic “W” shape, are boxed in the orange areas. The density profile of C is indicated on the image. (C) Graph of relative density as projected orthogonally to the normal of the plasma membrane (in three different colors corresponding to the different regions of the desmosome). The plasma membranes have the highest density, followed by the plakoglobin (PG) layer with a similar density to the dense midline, and finally the plakophilin (PP) layer with the lowest density. The comparable densities between the plakoglobin layer and the dense midline indicate a similar number of proteins clustering at these regions. (D) Superimposed autocorrelation function calculated at various positions of the desmosomal plaque (indicated with blue box in B) shows that the plaque has a periodicity of approximately 7 nm, which matches that of the extracellular region. (Scale bars: A and B, 40 nm.)

Fig. 2.

Three-dimensional average of desmosomal plaque. (A) Illustration shows isosurface of the 3D average (350 subtomograms) of the intracellular part of the desmosome and the computational sections along the three axes visualized in B–E. (B) Coronal slice (0.6 nm thick) through the subtomogram average (as indicated by the letters in the illustration) show that the molecules in the plakoglobin layer form a zigzag pattern. The plakoglobin layer does not have obvious connections to the plakophilin layer. The area between the two regions appears smooth, electron-lucent, and unstructured. (C) Sagittal slice (0.6 nm thick) through the subtomogram average. The cross-sections of two molecules are shown, showing a remarkable similarity to the cross-sections of plakoglobin molecules. (D and E) Two 0.6-nm-thick axial slices through the plakophilin and the plakoglobin layers, respectively (E and F). A clear periodicity can be observed in both layers. Superposition of the two layers shows that the molecules in the plakophilin layer are shifted with respect to the plakoglobin layer, and are hence accessible from the cytoplasmic side. (E) Interconnections between the various molecules are visible. (Scale bars: 7 nm.) (B) The isosurface of the subtomogram average shows three distinct density layers, colored in three different colors: the plasma membrane (PM) and the plakophilin (PP) and plakoglobin layers (PG). The threshold is chosen so that the thickness of the central densities corresponding to the plakoglobin molecules is approximately 3.5 nm. The color coding varies as a function of the depth from red to blue. Individual layers are labeled with similar color. The interacting molecules form a zigzag pattern and are arranged in consecutive layers.

Fig. 3.

Fitting of plakoglobin X-ray structures onto the symmetrized plakoglobin layer. (A) Crossed-eye stereo representation of the isosurface of the symmetrized subtomogram average of the plakoglobin layer (in beige) on which the X-ray structures of an alternating plakoglobin/desmoplakin (green/blue) complex has been placed. The cytoplasmic region of the cadherins is indicated in red. Five individual plakoglobin/desmoplakin complexes were accommodated into the density map. The zigzag structure of the plakoglobin layer is clearly visible. The desmoplakin molecules connect to the plakoglobin molecules at two positions: on the upper part close to the N terminus and in the central armadillo repeats. (B) The same representation as in A rotated 90°. Next to the zigzag structure shown in A, an additional connection between plakoglobins or other densities can be observed, indicating lateral connections in two directions. (C) The same representation as in A rotated 90° around a different axis. This is the typical view observed on 2D EM images. (D) Arbitrary perspective view of the same structure.

Fig. 4.

The complete desmosome structure. The drawing combines the complete knowledge of the interactions of the various desmosomal components on the basis of the cryo-ET data. The interactions of both plaque components and the extracellular cadherins (16) are visualized in the map. Two molecular layers representing five to six unit cells in the extracellular and intracellular parts are shown. The plakoglobin molecules are depicted in green, the desmoplakin in light blue, plakophilins in purple, and the desmosomal cadherins (Dsg and Dsc) in two shades of red. The periodicity of the extracellular layer matches the periodicity of the plaque components remarkably well, suggesting that they are connected by their flexible cytoplasmic tails to the plakoglobin molecules.