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Tight junctions prevent the diffusion of macromolecules and to varying degrees impede the diffusion of small water-soluble molecules and ions across an epithelial sheet in the spaces between cells. They also maintain the polarity of epithelial cells by preventing the diffusion of membrane proteins and glycolipids (lipids with covalently attached sugars) between the apical and the basolateral regions of the plasma membrane, ensuring that these regions contain different membrane components. As a consequence, movement of many nutrients across the intestinal epithelium is in large part through the transcellular pathway. In this pathway, specific transport proteins in the apical membrane import small molecules from the intestinal lumen into cells; other transport proteins located in the basolateral membrane then export these molecules into the extracellular space. Such transcellular transport is covered in detail in Chapter 7.

Tight junctions are composed of thin bands of plasmamembrane proteins that completely encircle a polarized cell and are in contact with similar thin bands on adjacent cells. When thin sections of cells are viewed in an electron microscope, the lateral surfaces of adjacent cells appear to touch each other at intervals and even to fuse in the zone just below the apical surface (see Figure 6-5b). In freeze-fracture preparations, tight junctions appear as an interlocking network of ridges in the plasma membrane (Figure 6-9a). More specifically, there appear to be ridges on the cytosolic face of the plasma membrane of each of the two contacting cells. Corresponding grooves are found on the exoplasmic face.

Very high magnification reveals that rows of protein particles 3-4 nm in diameter form the ridges seen in freeze-fracture micrographs of tight junctions. In the model shown in Figure 6-9b, the tight junction is formed by a double row of these particles, one row donated by each cell. The two principal integral-membrane proteins found in tight junctions are occludin and claudin. Initially, investigators thought that occludin was the only essential protein component of tight junctions. However, when investigators engineered mice with mutations inactivating the occludin gene, the mice still had morphologically distinct tight junctions. (This technique, called gene knockout, is described in Chapter 9.) Further analysis led to the discovery of claudin. Each of these proteins has four membrane-spanning a helices (Figure 6-9c). The claudin multigene family encodes numerous homologous proteins (isoforms) that exhibit distinct tissue-specific patterns of expression. Recently, a group of junction adhesion molecules (JAMs) have been found to contribute to homophilic adhesion and other functions of tight junctions. These molecules, which contain a single transmembrane a helix, belong to the Ig superfamily of CAMs. The extracellular domains of rows of occludin, claudin, and JAM proteins in the plasma membrane of one cell apparently form extremely tight links with similar rows of the same proteins in an adjacent cell, creating a tight seal. Treatment of an epithelium with the protease trypsin destroys the tight junctions, supporting the proposal that proteins are essential structural components of these junctions.

The long C-terminal cytosolic segment of occludin binds to PDZ domains in certain large cytosolic adapter proteins. These domains are found in various cytosolic proteins and mediate binding to the C-termini of particular plasmamembrane proteins. PDZ-containing adapter proteins associated with occludin are bound, in turn, to other cytoskele-tal and signaling proteins and to actin fibers. These interactions appear to stabilize the linkage between occludin and claudin molecules that is essential for maintaining the integrity of tight junctions.

A simple experiment demonstrates the impermeability of certain tight junctions to many water-soluble substances. In this experiment, lanthanum hydroxide (an electron-dense colloid of high molecular weight) is injected into the pancreatic blood vessel of an experimental animal; a few minutes later, the pancreatic acinar cells, which are specialized epithelial cells, are fixed and prepared for microscopy. As shown in Figure 6-10, the lanthanum hydroxide diffuses from the blood into the space that separates the lateral surfaces of adjacent acinar cells, but cannot penetrate past the tight junction.

The importance of Ca2+ to the formation and integrity of tight junctions has been demonstrated in studies with MDCK cells in the experimental system described previously (see Figure 6-7). If the growth medium in the chamber contains very low concentrations of Ca2 + , MDCK cells form a mono-layer in which the cells are not connected by tight junctions. As a result, fluids and salts flow freely across the cell layer. When sufficient Ca2+ is added to the medium, tight junctions form within an hour, and the cell layer becomes impermeable

▲ EXPERIMENTAL FIGURE 6-10 Tight junctions prevent passage of large molecules through extracellular space between epithelial cells. This experiment, described in the text, demonstrates the impermeability of tight junctions in the pancreas to the large water-soluble colloid lanthanum hydroxide. [Courtesy of D. Friend.]

Apical surface Apical surface of left cell of right cell

Apical surface Apical surface of left cell of right cell

Lanthanum hydroxide (between cells)

to fluids and salts. Thus Ca2+ is required for the formation of tight junctions as well as for cell-cell adhesion mediated by cadherins.

Plasma-membrane proteins cannot diffuse in the plane of the membrane past tight junctions. These junctions also restrict the lateral movement of lipids in the exoplasmic leaflet of the plasma membrane in the apical and basolateral regions of epithelial cells. Indeed, the lipid compositions of the exoplasmic leaflet in these two regions are distinct. Essentially all glycolipids are present in the exoplasmic face of the apical membrane, as are all proteins linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor (see Figure 5-15). In contrast, lipids in the cytosolic leaflet in the apical and ba-solateral regions of epithelial cells have the same composition and can apparently diffuse laterally from one region of the membrane to the other.

Differences in Permeability of Tight Junctions Can Control Passage of Small Molecules Across Epithelia

The barrier to diffusion provided by tight junctions is not absolute. Owing at least in part to the varying properties of the different isoforms of claudin located in different tight junctions, their permeability to ions, small molecules, and water varies enormously among different epithelial tissues. In ep-ithelia with "leaky" tight junctions, small molecules can move from one side of the cell layer to the other through the paracellular pathway in addition to the transcellular pathway (Figure 6-11).

The leakiness of tight junctions can be altered by intra-cellular signaling pathways, especially G protein-coupled pathways entailing cyclic AMP and protein kinase C (Chapter 13). The regulation of tight junction permeability is often

▲ FIGURE 6-11 Transcellular and paracellular pathways of transepithelial transport. Transcellular transport requires the cellular uptake of molecules on one side and subsequent release on the opposite side by mechanisms discussed in Chapters 7 and 17. In paracellular transport, molecules move extracellularly through parts of tight junctions, whose permeability to small molecules and ions depends on the composition of the junctional components and the physiologic state of the epithelial cells. [Adapted from S. Tsukita et al., 2001, Nature Rev.: Mol. Cell Biol. 2:285.]

studied by measuring ion flux (electrical resistance) or the movement of radioactive or fluorescent molecules across monolayers of MDCK cells.

HI The importance of paracellular transport is illus-I trated in several human diseases. In hereditary hy-31 pomagnesemia, defects in the claudinl6 gene prevent the normal paracellular flow of magnesium through tight junctions in the kidney. This results in an abnormally low blood level of magnesium, which can lead to convulsions. Furthermore, a mutation in the claudin14 gene causes hereditary deafness, apparently by altering transport around hair cells in the cochlea of the inner ear.

Toxins produced by Vibrio cholerae, which causes cholera, and several other enteric (gastrointestinal tract) bacteria alter the permeability barrier of the intestinal epithelium by altering the composition or activity of tight junctions. Other bacterial toxins can affect the ion-pumping activity of membrane transport proteins in intestinal epithelial cells. Toxin-induced changes in tight junction permeability (increased paracellular transport) and in protein-mediated ion-pumping proteins (increased transcellular transport) can result in massive loss of internal body ions and water into the gastrointestinal tract, which in turn leads to diarrhea and potentially lethal dehydration.I

Many Cell-Matrix and Some Cell-Cell Interactions Are Mediated by Integrins

The integrin family comprises heterodimeric integral membrane proteins that function as adhesion receptors, mediating many cell-matrix interactions (see Figure 6-2). In vertebrates, at least 24 integrin heterodimers, composed of 18 types of a subunits and 8 types of p subunits in various combinations, are known. A single p chain can interact with any one of multiple a chains, forming integrins that bind different ligands. This phenomenon of combinatorial diversity, which is found throughout the biological world, allows a relatively small number of components to serve a large number of distinct functions.

In epithelial cells, integrin a6p4 is concentrated in hemidesmosomes and plays a major role in adhering cells to matrix in the underlying basal lamina, as discussed in detail in Section 6.3. Some integrins, particularly those expressed by certain blood cells, participate in heterophilic cell-cell interactions. The members of this large family play important roles in adhesion and signaling in both epithelial and nonepithelial tissues.

Integrins typically exhibit low affinities for their ligands with dissociation constants KD between 10"6 and 10"8 mol/L. However, the multiple weak interactions generated by the binding of hundreds or thousands of integrin molecules to their ligands on cells or in the extracellular matrix allow a cell to remain firmly anchored to its ligand-expressing target. Moreover, the weakness of individual integrin-mediated interactions facilitates cell migration.

Parts of both the a and the p subunits of an integrin molecule contribute to the primary extracellular ligand-binding site (see Figure 6-2). Ligand binding to integrins also requires the simultaneous binding of divalent cations (positively charged ions). Like other cell-surface adhesive molecules, the cytosolic region of integrins interacts with adapter proteins that in turn bind to the cytoskeleton and intracellular signaling molecules. Although most integrins are linked to the actin cytoskeleton, the cytosolic domain of the p4 chain in the a6p4 integrin in hemidesmosomes, which is much longer than those of other p integrins, binds to specialized adapter proteins (e.g., plectin) that in turn interact with keratin-based intermediate filaments.

In addition to their adhesion function, integrins can mediate outside-in and inside-out transfer of information (signaling). In outside-in signaling, the engagement of integrins with their extracellular ligands can, through adapter proteins bound to the integrin cytosolic region, influence the cy-toskeleton and intracellular signaling pathways. Conversely, in inside-out signaling, intracellular signaling pathways can alter, from the cytoplasm, the structure of integrins and consequently their abilities to adhere to their extracellular lig-ands and mediate cell-cell and cell-matrix interactions. Integrin-mediated signaling pathways influence processes as diverse as cell survival, cell proliferation, and programmed cell death (Chapter 22). Many cells express several different integrins that bind the same ligand. By selectively regulating the activity of each type of integrin, these cells can fine-tune their cell-cell and cell-matrix interactions and the associated signaling processes.

We will consider various integrins and the regulation of their activity in detail in Section 6.5.

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