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 lowdensity 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.vitreous sectioning | image processing | genetic algorithms | multi-protein fitting C ell junctions are widely distributed in animal tissues and are most abundant in tissues that are subjected to considerable mechanical stress, such as heart, epidermis, and muscle. These junctions are composed of two main classes of proteins: transmembrane adhesion proteins with an extracellular domain that interacts with either the ECM (e.g., focal adhesion and hemidesmosome) or the extracellular domains of specific transmembrane adhesion proteins from the juxtaposed cell (e.g., adherens junctions and desmosomes); and a cytoplasmic tail that binds to one or more intracellular anchor proteins. These form a distinct plaque on the cytoplasmic face of the plasma membrane and connect the junctional complexes to actin filaments or intermediate filaments (IFs). When they have been assembled, these junctions play essential roles in tissue morphogenesis and maintenance; plaque disruption is a hallmark of many blistering and cancer diseases (1, 2). Biochemical and mutagenesis studies have revealed many interactions between the plaque components and their connections to the extracellular space (3-8). However, the organization and interactions of the constituent proteins in the context of the assembled junctions remain poorly understood. For example, whether or how actin filaments bind in situ to the adherens junctions and how the anchoring proteins in hemidesmosome plaque connect integrin to the IFs is still unclear.In the current study, we present the 3D cytoplasmic surface of the desmosome-the desmosomal plaque-fr...

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

“…2G for reference). The dSTORM distributions of Dsg3 Cterminal and plakoglobin N-terminal domains overlap with the corresponding distributions of gold labels in immunoelectron microscopy studies (North et al, 1999), and the dSTORM plakoglobin distribution overlaps with the plakoglobin layer identified by cryoelectron tomography (Al-Amoudi et al, 2011). In contrast to immunogold studies, dSTORM indicates that desmoplakin is localized further from the plasma membrane.…”

“…Many desmosomal protein interactions have been characterized by biochemical studies (Bass-Zubek and Green, 2007;Green and Simpson, 2007;Thomason et al, 2010), and desmosomal ultrastructure has been studied by immunogold and tomographic electron microscopy (Al-Amoudi et al, 2011;Al-Amoudi and Frangakis, 2008;North et al, 1999;Owen et al, 2008;Stokes, 2007). Yet, studying desmosomes on a molecular level remains challenging due to their size, insolubility and molecular complexity.…”

Structural and Functional Diversity of Desmosomes

“…Limited only by restricted expression patterns, the collection of interactions schematically presented in Figure 3 illustrates the potential fl exibility with which cells could construct desmosomes. Existing immunoelectron microscopy data combined with emerging, high-resolution tomography studies promise to shed further light upon the in vivo arrangement of these interactions in steady state desmosomes (North et al, 1999;He et al, 2003;Al-Amoudi et al, 2011;Al-Amoudi et al, 2007).…”

Different roles of cadherins in the assembly and structural integrity of the desmosome complex

“…Desmosomes form punctate structures at cell-cell contacts (Pasdar and Nelson, 1988a;Pasdar and Nelson, 1988b) that comprise an intercellular midline corresponding to sites of trans interactions between opposing cadherins, and a dense plaque structure in the cytoplasm that connects to intermediate filaments (Al-Amoudi et al, 2011;Al-Amoudi et al, 2007;He et al, 2003;North et al, 1999).…”