Tight Junctions

Tight junctions, also called zonula occludens, occlude the extracellular space between epithelial cells, forming a tight, belt-like adhesive seal that selectively limits the diffusion of water, ions, and larger solutes as well as migration of cells (Fig. 31-2). Thus, they separate the interior of the body from the external world. Tight junctions also define the boundary between the biochemically distinct apical and basolateral domains of the plasma membrane of polarized epithelial cells. Many physiological processes (see Figs. 11-2 through 11-4) depend on the selective permeability of the two pathways across an epithelium: passive “paracellular” diffusion through tight junctions and transcellular movement made possible by the action of pumps, carriers, and channels located selectively in the apical and basolateral domains of the plasma membrane.

Figure 31-2 epithelial tight junctions. A, Electron micrograph of a thin section of endothelial cells, showing a point of contact between the plasma membranes at a tight junction (arrow).B, Electron micrograph of a replica of a freeze-fractured cell. This method exposes proteins within the lipid bilayer and reveals strands aligned along the points of contact between the plasma membranes. C, Interpretive drawing, showing the strands at points of contact as rows of transmembrane proteins.

(A, Courtesy of George Palade, University of California, San Diego.B, Courtesy of Don W. Fawcett, Harvard Medical School, Boston, Massachusetts.)

In electron micrographs of thin sections of tight junctions, the plasma membranes of adjacent cells appear to fuse together in a series of one or more contacts (Fig. 31-2). Early models of tight junctions proposed a fusion between the lipid bilayers of the two membranes to account for the barrier to ion diffusion, but freeze-fracture images revealed that the strands consist of integral membrane proteins. The contacts correspond to continuous strands of intramembranous particles that form a branching network in the plane of the lipid bilayer.

Transmembrane proteins forming the strands ob-served by freeze-fracture were difficult to identify until investigators found a monoclonal antibody that bound to the cytoplasmic side of the plasma membrane at tight junctions. Using this antibody, they isolated an integral membrane protein and named it occludin. The amino acid sequence of occludin suggested four transmembrane strands and two hydrophobic extracellular loops that are extremely rich in tyrosine and glycine residues (Fig. 31-3). The same group then discovered that mice lacking the single occludin gene survive with normal tight junctions, revealing the existence of other tight junction proteins. It is now believed that a family of more than 20 proteins, called claudins, constitutes the main structural proteins of tight junction strands. Claudins have four transmembrane sequences, but they are not related in sequence to occludin. Close association of claudins limits diffusion in the extracellular space between cells as well as diffusion of lipids and proteins in the plane of the membrane.

Figure 31-3 tight junction proteins: occludin and claudin. A, Preliminary model of tight junction structure with claudin linking the two membranes together and peripheral protein ZO-1 linking the cytoplasmic tail of claudin to actin filaments. B–C, Transmembrane topology of claudin and occludin.

The extracellular domains of claudins form rows of pores along the tight junction contacts. Each claudin has a unique selectivity for cations or anions, and the selection of claudins expressed in various epithelia creates their distinct selectivities. The pores of all claudins probably restrict diffusion of solutes larger than about 1 nm in diameter. Larger solutes may cross the barrier by traversing transient breaks in the strands of claudins.

The cytoplasmic tails of claudins interact with numerous proteins with roles as scaffolds and in actin binding, signaling, and cell polarity. ZO-1, ZO-2, and ZO-3, peripheral membrane adapter proteins containing PDZ protein-interaction domains (see Fig. 25-11), interact with the long C-terminal cytoplasmic tail of occludin and JAM (junctional adhesion molecule). These ZO-adapters link the transmembrane proteins to cytoplasmic proteins including actin filaments, cingulin, and a small GTPase. ZO-2 and cingulin are specific for tight junctions, whereas ZO-1 also associates with cadherins in adherens junctions and connexins in gap junctions.

Tight junctions are the barrier that segregates different pumps, carriers, receptors, and lipids in the apical and basolateral domains of the plasma membrane. These domains allow polarized cells to create different extracellular environments in the basal and apical compartments. For example, intestinal epithelial cells take up nutrients from the lumen of the intestine and transport them into the extracellular space beneath the cells (see Fig. 11-2). Tight junctions and epithelial polarity are required for many physiological functions (see Figs. 11-3 and 11-4). Separating the apical and basal compartments also regulates some types of signaling. For instance, airway epithelial cells release the growth factor heregulin from the apical surface, but it activates cell growth only by activating the receptor tyrosine kinase erbB2 (see Fig. 25-4) on the basolateral surface when the epithelium is damaged or the tight junctions compromised.

Circumferential tight junctions account for the electrical resistance across sheets of epithelial cells. The quality of the seal, reflected in the electrical resistance, varies by several orders of magnitude depending on the cell type and is matched to the physiological function of the epithelium. Permeability in the two directions across the junction is identical. The number and continuity of claudin/occludin strands as well as the specific claudin proteins that are expressed determine the tightness of the barrier to diffusion of ions in the extracellular space. Most tight junctions are more permeable to cations than to anions and generally restrict the diffusion of all solutes larger than about 1 nm in diameter. Along with variations in ion and solute permeability, the ability for water to flow through tight junctions also appears to differ among epithelia. Extremely tight barriers with many strands and distinct claudin forms are found where epithelia must maintain high ion gradients, such as in the distal tubules of the kidney, where urine is concentrated. Leaky tight junctions with fewer strands and different claudins are found where ion gradients across epithelia are small but a barrier is required for large solutes, proteins, and leukocytes (e.g., in most blood vessels).

The transepithelial barrier that is established by tight junctions is regulated by extracellular stimuli (e.g., hormones such as vasopressin and aldosterone and cytokines such as tumor necrosis factor; see Chapter 27), their downstream second messengers (e.g., Ca²⁺ and cyclic adenosine monophosphate [cAMP]; see Chapter 26), and effectors (e.g., protein kinases A and C; see Chapter 25). The mechanisms are not yet well understood, but posttranslational modifications of tight junctions might modulate their assembly. Another possibility is that tension on associated actin filaments might physically open passages through tight junctions. The metabolic state of the cell also influences tight junctions; depletion of ATP causes tight junctions to leak without destroying the barrier between the apical and basolateral domains of the plasma membrane. Cells migrating across epithelia, such as white blood cells moving from the blood to the connective tissue, open tight junctions locally without disrupting the tight seal across the epithelium (see Fig. 30-13). Migratory cells induce a localized increase in cytoplasmic Ca²⁺ in the epithelial cells that is required for opening the tight junctions.

Several bacterial toxins affect the tight junction barrier. The ZO-toxin of Vibrio cholerae induces diarrhea by loosening tight junctions, independent of the classic cholera toxin, which induces secretion. Helicobacter pylori injects a protein toxin into the cells lining the stomach. This toxin disrupts tight junctions, breaking the barrier that protects the underlying tissues and predisposing to ulcers. Mutation of individual human claudin genes can result in highly selective defects in epithelial barriers, such as reduced ability of the kidney to reabsorb potassium (claudin-16) or deafness due to loss of ion gradients in the inner ear (claudin-14).

Gap Junctions

For many years, the dominance of the cell theory in biology, which suggested that isolation of cells was a general principle, discouraged curiosity about the possibility of direct intercellular communication. Early electrophysiological experiments on nerves and skeletal muscles reinforced the widespread belief that cells were autonomous. By chance, the cells that were used in these experiments were electrically isolated and communicated exclusively by secreting chemical messengers that bound to receptors on target cells (see Figs 11-8 and 11-9). However, nerve and skeletal muscle cells were found to be exceptions to the general principle, which emerged only later, that cells in animal tissues communicate with each other by gap junctions. The generality of gap junctional communication means that sharing cytoplasmic components is common in animal cell biology. Cells in plant tissues also communicate with each other, but they use direct cytoplasmic connections, called plasmodesmata, rather than gap junctions (Box 31-1 and Fig. 31-4).