Discovering the molecular components of intercellular junctions--a historical view

Abstract

The organization of metazoa is based on the formation of tissues and on tissue-typical functions and these in turn are based on cell-cell connecting structures. In vertebrates, four major forms of cell junctions have been classified and the molecular composition of which has been elucidated in the past three decades: Desmosomes, which connect epithelial and some other cell types, and the almost ubiquitous adherens junctions are based on closely cis-packed glycoproteins, cadherins, which are associated head-to-head with those of the hemi-junction domain of an adjacent cell, whereas their cytoplasmic regions assemble sizable plaques of special proteins anchoring cytoskeletal filaments. In contrast, the tight junctions (TJs) and gap junctions (GJs) are formed by tetraspan proteins (claudins and occludins, or connexins) arranged head-to-head as TJ seal bands or as paracrystalline connexin channels, allowing intercellular exchange of small molecules. The by and large parallel discoveries of the junction protein families are reported.

Figure 1.

Electron microscopic aspects of intercellular junctions, in particular desmosomes. (A) Characteristic subapical trias of tight junction (TJ), zonula adhaerens (ZA), and desmosome (D) of murine intestinal epithelial cells in a longitudinal section. (B) Higher magnification of two adjacent desmosomes in fetal (20 wk) human foot-sole epidermis, showing desmosomal substructures (black arrows: midline structure; white arrows: trilaminar “unit membrane” structure of the plasma membrane domain proper, also pointing to electron dense cross-bridge structures; arrowheads: secondary dense layer of the plaque; P: electron-dense primary plaques; IF; anchoring structures of intermediate-sized filaments). (C) Freeze-fracture image of spinous layer of murine epidermis, showing the intramembranous fracture plane of the plasma membrane with two desmosomes (arrows) and a gap junction (arrowhead). Note the typical paracrystalline packing of the connexin substructures in the gap junction in comparison with the loosely and irregularly arranged transmembrane elements of the desmosomes. (D) Immunoelectron microscopy of an ultrathin section through an epithelium, showing the immunogold decoration (5-nm particles) of desmoplakin at—or near—the desmosomal plaque structures. (E) Immunoelectron microscopy of an ultrathin cross-section through the zonula adhaerens of plasma membranes connecting two endothelial cells of a cardiac capillary, showing the specific 5-nm immunogold decoration of the junctional plaque with plakoglobin antibodies, thus demonstrating that plakoglobin can also occur in the plaques of nondesmosomal adhering junctions. Bars denote 0.1 µm (A, B) and 0.5 µm (C–E). For details, see Cowin et al. (1985a), Franke et al. (1987b), Kapprell et al. (1987), and Schlüter et al. (2007).

Figure 2.

Double-label immunofluorescence microscopy of monolayer cultures of epithelial cells derived from human multilayered (A, keratinocytes of line HaCaT; B, squamous cell carcinoma-derived line A-431) or one-layered (C, D: liver carcinoma cells of line PLC) tissues. Two forms of attachment of bundles of keratin IFs (green, mouse monoclonal antibody mAb lu-5) to the plaques of desmosomes (A, B: red, desmoplakin, guinea pig antibodies) represent a continuous transcellular cytoskeletal system (the chromatin in the nuclei of A, C, and D is stained blue with DAPI reagent): The cells in A are connected by cell-to-cell bridges with near-centrally located desmosomes, whereas in B the bodies of the cells are directly and tightly associated with each other via numerous, closely spaced desmosomes. In contrast, no specific anchorage of keratin IF bundles is seen at cell junctions of the zonula or punctum adherens type, which are seen by immunoreaction for β-catenin (C, red, guinea pig antibodies) or protein p0071 (D, red, murine mAb). Bars: 20 µm.

Figure 3.

High-resolution double-label immunofluorescence (A–C) and immunoelectron (D) microscopy of monolayer cell cultures of human breast-carcinoma cells of line MCF-7, as seen after reactions with antibodies to the desmosomal plaque component, desmoplakin (green, guinea pig antibodies), or to the adherens plaque protein, p0071 (red, as in Fig. 2B), as seen on the background of differential interference contrast (DIC) (A), allowing for the most part to distinguish the small puncta adhaerentia from the similarly small desmosomes, located side-by-side. (B, C) Higher magnification micrographs of cells as shown in A, presenting details of cell–cell junctions along close membrane contact regions, allowing to distinguish in many places the alternating pairs of symmetrical plaques of desmosomes (green) and puncta adhaerentia (red). (D) Equivalent comparison at the electron microscopic level, showing an ultrathin section through junctions after immunogold reaction of puncta adhaerentia with antibodies to protein p0071 (brackets, immunogold granules enhanced by secondary silver reaction), in comparison with the negative small desmosomes (D). For details, see Hofmann et al. (2008, 2009). Bars: 20 µm (A), 5 µm (B, C), and 0.1 µm (D).

Figure 4.

Double-label immunofluorescence microscopy showing the tight junction (TJ) systems of monolayer cell cultures of human hepatocellular carcinoma-derived cells of line PLC (A) and of cultures of human keratinocytes of line HaCaT beginning to form the first suprabasal cell layer (B, C), using antibodies to keratin (A, green, mAb lu-5), in comparison with the TJ protein, occludin (A, red), or comparing the localization of the protein tricellulin (B, green, rat antibodies) with the TJ plaque protein, ZO-1 (B, red, rabbit antibodies), or the transmembrane TJ protein, occludin (C, red, rabbit antibodies). Note that these cells are totally interconnected by the TJ system (zonula occludens), which is completed at the tricellular corners by the occludin-related, but very specifically located protein, tricellulin, presenting complete colocalization with protein ZO-1 and occludin at the tricellular corners. For details, see also Schlüter et al. 2007. Bars: 20 µm.

Figure 5.

Electron (A) and immunofluorescence (B–D) microscopy, showing the tight junction (TJ) system in the granular layer (stratum granulosum) of stratified epithelia. (A) The uppermost living layer of human epidermis (the first cornified layer, stratum corneum, is labeled SC) contains a subapical system of TJ structures (horizontal bars and pairs of arrows denote distinct membrane contact and fusion points: “kisses”). Note that the four lowermost TJs (arrows) are interspersed between desmosomes. (B–D) Near-vertical cryostat section through bovine gingiva, showing the TJ system in the uppermost living layers by immunostaining with antibodies to occludin (B, bracket; C, corresponding phase contrast image), whereas the cell–cell junction structures positive for the TJ protein, claudin-1, are not limited to the granular layer but extend basally into a number of stratum spinosum cell layers as well, indicating that there exist further, not yet fully characterized, partly punctate structures containing claudin-1 but not occludin (for methods used, see also Brandner et al. 2002; Schlüter et al. 2007). Bars: 0.2 µm (A), 50 µm (B–D).