3.5. An ultrastructural side view of a tight junction.
The image above reveals the way the tight junction looks at the ultrastructural level (Figure 3.5). A model of a tight junction structure is shown below (Figure 3.6). Tightly aligned rows of tight junction proteins serve to stitch the membrane together effectively sealing the association between adjacent cells. This serves to block the movement of materials through the intercellular space. While we will only cover a couple of the major players in the structure and function of tight junctions, it’s important to understand that the protein complexes that stitch up the membrane are quite complex and form a protein scaffold which can interact with various components in the cytoplasm especially the cytoskeleton.
Figure 3.6. A diagrammatic representation of a tight junction.
Tight Junction Proteins: Claudins, Occludins and ZO1
The four major proteins found in tight junctions are called claudins, occludins, junctional adhesion molecule-1 (JAM-1) and zonula occludin proteins 1-3 (ZO1-3). These are shown in the following figure (Figure 3.7). The claudins are small 26 kDa (kiloDalton) proteins that are critical to the structural integrity of tight junctions. Occludins are larger at 65 kDa and are the major proteins found in these junctional complexes. These integral proteins are not essential since knockout mutants for occludin proteins do not prevent tight junction formation. It has been suggested that they are accessory proteins that don’t provide any structural basis. Knocking out ZO1 interferes with tight junction formation indicating it is a critical protein.
Figure 3.7. A diagrammatic representation of the organization of tight junction proteins.
The zonula occludin protein ZO1 was the first cloned tight junction protein. It lies at the inner side of the cell membrane where it organizes the other junctional adhesion molecules. It is indicated as one of the linker proteins in the figure. ZO1 and occludin both share an interesting and important function: they signal the integrity of the junctional adhesion complex. Thus they make the cell aware of its status regarding its adhesion with other cells.
Adherens Junctions
Adherens junctions are mainly found in epithelial cells where they lie just below tight junctions. Cadherins are the central proteins found in these junctions. As with other cadherins involved in cell adhesion, these proteins bind to ß-catenins in the cytoplasm which in turn associate with vinculin. These proteins are involved in binding to actin filaments. Thus there is a direct link between adherens junctions and the cytoskeleton. These junctions are most often organized as a “belt” that surrounds the complete cell.
Figure 3.8. The structure of an adherens junction.
Adherens junctions are important in keeping endothelial cells tightly bound as well. In blood vessels the tight association between endothelial cells maintains blood vessel integrity, keeping tissue cells out and blood cells contained within the blood vessel. The current view of adherens junctions is shown in Figure 3.8. Vinculin plays a key role in maintaining the tight association between endothelial cells and significant changes occur in the association of vinculin and actin in various diseases such as atherosclerosis and during cancer metastasis. The interplay between vinculin and actin as well as issues of metastasis are all covered in later chapters. The above and the following image show the homotypic binding between cadherins of two cells joined by an adherens junction. The alpha- and beta-catenins and vinculin mediate interaction with the actin cytoskeleton (Figure 3.9).
Figure 3.9. A diagram of the organization of adherens junction proteins.
Proteins That Move Between the Nucleus and Junctional Adhesion Complexes
When they were originally discovered cell junctions were considered to be relatively static structures. This was likely because they appeared to have a consistent, unchanging structure when viewed with the electron microscope. New techniques have revealed that proteins can move in and out of these junctions allowing the cell to sense the status of its intercellular adhesions. For example, occludin and ZO1, two proteins from tight junctions have been shown to move into the nucleus to regulate gene activity. Proteins that move between adhesion complexes have been termed NACos (Nucleus and Adhesion Complexes). β-catenins are the most well-studied NACos. The following graphics show what happens when an adherens junction is disrupted (Figure 3.10).
Figure 3.10. Beta-catenins can move from disrupted tight junctions into the nucleus to regulate gene transcription.
A. Intact cells, B. One cell (left side) is damaged. C. β-catenins enter nucleus to regulate gene transcription.
If epithelial tissues become disrupted such as by a cut in the skin or tear in the intestinal lining, it would lead to the disruption of adherens junctions in adjacent cells in turn releasing ß-catenins as well as other junctional proteins. The ß-catenins can then travel to the nucleus to initiate gene transcription to stimulate cell cycle events or other processes required in wound healing. A similar event happens when tight junctions are disrupted by a bacterial infection as discussed in the next section.
Gastric Ulcers: H. pylori Infection Alters ZO1 Localization
For many years the cause of gastric ulcers was always attributed to poor diet. Several years ago, scientists revealed that a common intestinal bacterium called Helicobacter pylori was actually the causative agent. These bacterial cells attach via adhesions to the cell surface of epithelial cells in the gastric mucosa. Through the secretion of various cytoxins they disrupt the intestinal epithelium causing gastritis and in some individuals gastric cancer. Research into how the H. pylori infects cells has shown that it alters the junctional adhesion complexes of the gastric epithelium of humans. As shown in the following diagram, ZO1 localizes to the surface of all of the cells showing a relatively consistent pattern (Figure 3.11). This localization is altered in infected cells with some of the protein not only disappearing from the cell surface junctions but also appearing within the cytoplasm. Some of the protein can also be seen in some of the nuclei. Based on the function of ZO1 as a NACo, the release of ZO1 proteins from tight junctions results in their movement as soluble proteins in the cytoplasm and from there into the nucleus where they can alter gene activity.
Figure 3.11. A diagrammatic representation of the Localization of ZO1 (green = ZO1; blue = nuclei) in normal (A) human gastric epithelial cells and those infected with H. pylori (B).
Desmosomes
Desmosomes were the first junctional adhesion complex members to be recognized. This is because they are the most distinct component appearing as paired dark and dense masses adjacent to the cell membrane of adjacent epithelial cells. Desmosomes provide strong adhesion between cells. They are typically found in epithelial
