Leukocyte activation (PAF activates integrin)

Firm adhesion via integrin/ICAM binding

synthesis of platelet-activating factor (PAF) and ICAM-1, both expressed on the cell surface. PAF and other usually secreted activators, including chemokines, then induce changes in the shapes of the leukocytes and activation of leukocyte integrins such as aLp2, which is expressed by T lymphocytes ( 3). The subsequent tight binding between activated integrins on leukocytes and CAMs on the endothelium (e.g., ICAM-2 and ICAM-1) results in firm adhesion ( 4) and subsequent movement (extravasation) into the underlying tissue ( 5).See text for further discussion. [Adapted from R. O. Hynes and A. Lander, 1992, Cell 68:303.]

dothelial cells: ICAM-2, which is expressed constitutively, and ICAM-1. ICAM-1, whose synthesis along with that of E-selectin and P-selectin is induced by activation, does not usually contribute substantially to leukocyte endothelial cell adhesion immediately after activation, but rather participates at later times in cases of chronic inflammation. The resulting tight adhesion mediated by the Ca2+-independent integrin-ICAM interactions leads to the cessation of rolling and to the spreading of leukocytes on the surface of the endothelium; soon the adhered cells move between adjacent endothelial cells and into the underlying tissue.

The selective adhesion of leukocytes to the endothelium near sites of infection or inflammation thus depends on the sequential appearance and activation of several different CAMs on the surfaces of the interacting cells. Different types of leukocytes express specific integrins containing the subunit: for example, aL^2 by T lymphocytes and aM^2 by monocytes, the circulating precursors of tissue macrophages. Nonetheless, all leukocytes move into tissues by the same general mechanism depicted in Figure 6-30.

Many of the CAMs used to direct leukocyte adhesion are shared among different types of leukocytes and target tissues. Yet often only a particular type of leukocyte is directed to a particular tissue. A three-step model has been proposed to account for the cell-type specificity of such leukocyte-endothelial cell interactions. First, endothelium activation promotes initial relatively weak, transient, and reversible binding (e.g., the interaction of selectins and their carbohydrate ligands). Without additional local activation signals, the leukocyte will quickly move on. Second, cells in the immediate vicinity of the site of infection or inflammation release or express on their surfaces chemical signals (e.g., chemokines, PAF) that activate only special subsets of the transiently attached leukocytes. Third, additional activation-dependent CAMs (e.g., integrins) engage their binding partners, leading to strong sustained adhesion. Only if the proper combination of CAMs, binding partners, and activation signals are engaged in the right order at a specific site will a given leukocyte adhere strongly. This additional example of combinatorial diversity and cross talk allows parsimonious exploitation of a small set of CAMs for diverse functions throughout the body.

Leukocyte-adhesion deficiency is caused by a genetic defect in the synthesis of the integrin ^2 subunit. Persons with this disorder are susceptible to repeated bacterial infections because their leukocytes cannot extravasate properly and thus fight the infection within the tissue.

Some pathogenic viruses have evolved mechanisms to exploit for their own purposes cell-surface proteins that participate in the normal response to inflammation. For example, many of the RNA viruses that cause the common cold (rhinoviruses) bind to and enter cells through ICAM-1, and chemokine receptors can be important entry sites for human immunodeficiency virus (HIV), the cause of AIDS. I

Gap Junctions Composed of Connexins Allow Small Molecules to Pass Between Adjacent Cells

Early electron micrographs of virtually all animal cells that were in contact revealed sites of cell-cell contact with a characteristic intercellular gap (Figure 6-31a). This feature prompted early morphologists to call these regions gap junctions. In retrospect, the most important feature of these junctions is not the gap itself but a well-defined set of cylindrical particles that cross the gap and compose pores connecting the cytoplasms of adjacent cells—hence their alternate name of intercytoplasmic junctions. In epithelia, gap junctions are distributed along the lateral surfaces of adjacent cells (see Figures 6-1 and 6-5).

In many tissues (e.g., the liver), large numbers of individual cylindrical particles cluster together in patches. This property has enabled researchers to separate gap junctions from other components of the plasma membrane. When the plasma membrane is purified and then sheared into small fragments, some pieces mainly containing patches of gap junctions are generated. Owing to their relatively high protein content, these fragments have a higher density than that of the bulk of the plasma membrane and can be purified on an equilibrium density gradient (see Figure 5-37). When these

▲ EXPERIMENTAL FIGURE 6-31 Gap junctions have a characteristic appearance in electron micrographs. (a) In this thin section through a gap junction connecting two mouse liver cells, the two plasma membranes are closely associated for a distance of several hundred nanometers, separated by a "gap" of 2-3 nm. (b) Numerous roughly hexagonal particles are visible in this perpendicular view of the cytosolic face of a region of plasma membrane enriched in gap junctions. Each particle aligns with a similar particle on an adjacent cell, forming a channel connecting the two cells. [Part (a) courtesy of D. Goodenough. Part (b) courtesy of N. Gilula.]

preparations are viewed in cross section, the gap junctions appear as arrays of hexagonal particles that enclose water-filled channels (Figure 6-31b). Such pure preparations of gap junctions have permitted the detailed biophysical and functional analysis of these structures.

The effective pore size of gap junctions can be measured by injecting a cell with a fluorescent dye covalently linked to molecules of various sizes and observing with a fluorescence microscope whether the dye passes into neighboring cells. Gap junctions between mammalian cells permit the passage of molecules as large as 1.2 nm in diameter. In insects, these junctions are permeable to molecules as large as 2 nm in diameter. Generally speaking, molecules smaller than 1200 Da pass freely, and those larger than 2000 Da do not pass; the passage of intermediate-sized molecules is variable and limited. Thus ions, many low-molecular-weight precursors of cellular macromolecules, products of intermediary metabolism, and small intracellular signaling molecules can pass from cell to cell through gap junctions.

In nervous tissue, some neurons are connected by gap junctions through which ions pass rapidly, thereby allowing very rapid transmission of electrical signals. Impulse transmission through these connections, called electrical synapses, is almost a thousandfold as rapid as at chemical synapses (Chapter 7). Gap junctions are also present in many non-neuronal tissues where they help to integrate the

► FIGURE 6-32 Molecular structure of gap junctions. (a) Schematic model of a gap junction, which comprises a cluster of channels between two plasma membranes separated by a gap of about 2-3 nm. Both membranes contain connexon hemichannels, cylinders of six dumbbell-shaped connexin molecules. Two connexons join in the gap between the cells to form a gap-junction channel, 1.5-2.0 nm in diameter, that connects the cytosols of the two cells. (b) Electron density of a recombinant gap-junction channel determined by electron crystallography. Shown here are side views of the complete structure (top) and the same structure with several chains removed to show the channel's interior (center); on the bottom are perpendicular cross sections through the gap junction within and between the membrane bilayers. There appear to be 24 transmembrane a helices per connexon hemichannel, consistent with each of the six connexin subunits having four a helices. The narrowest part of the channel is =1.5 nm in diameter. M = membrane bilayer; E = extracellular gap; C = cytosol. [Part (b) from V.M. Unger et al., 1999, Science 283:1176.]

electrical and metabolic activities of many cells. In the heart, for instance, gap junctions rapidly pass ionic signals among muscle cells and thus contribute to the electrically stimulated coordinate contraction of cardiac muscle cells during a beat. As discussed in Chapter 13, some extracellular hormonal signals induce the production or release of small in-tracellular signaling molecules called second messengers (e.g., cyclic AMP and Ca2+) that regulate cellular metabolism. Because second messengers can be transferred between cells through gap junctions, hormonal stimulation of one cell can trigger a coordinated response by that same cell and many of its neighbors. Such gap junction-mediated signaling plays an important role, for example, in the secretion of digestive enzymes by the pancreas and in the coordinated muscular contractile waves (peristalsis) in the intestine. Another vivid example of gap junction-mediated transport is the phenomenon of metabolic coupling, or metabolic cooperation, in which a cell transfers nutrients or intermediary metabolites to a neighboring cell that is itself unable to synthesize them. Gap junctions play critical roles in the development of egg cells in the ovary by mediating the movement of both metabolites and signaling molecules between an oocyte and its surrounding granulosa cells as well as between neighboring granulosa cells.

A current model of the structure of the gap junction is shown in Figure 6-32. Vertebrate gap junctions are composed

of connexins, a family of structurally related transmembrane proteins with molecular weights between 26,000 and 60,000. A completely different family of proteins, the innexins, forms the gap junctions in invertebrates. Each vertebrate hexagonal particle consists of 12 connexin molecules: 6 of the molecules are arranged in a connexon hemichannel—a hexagonal cylinder in one plasma membrane—and joined to a connexon hemichannel in the adjacent cell membrane, forming the continuous aqueous channel between the cells. Each connexin molecule spans the plasma membrane four times; one conserved transmembrane a helix from each subunit apparently lines the aqueous channel.

There are probably more than 20 different connexin genes in vertebrates, and different sets of connexins are expressed in different cell types. Some cells express a single connexin; consequently their gap-junction channels are homotypic, consisting of identical connexons. Most cells, however, express at least two connexins; these different proteins assemble into hetero-oligomeric connexons, which in turn form heterotypic gap-junction channels. This diversity in channel composition leads to differences in the permeability of channels to various molecules. For example, channels made from a 43-kDa connexin isoform, Cx43, are more than a hundredfold as permeable to ADP and ATP as those made from Cx32 (32 kDa). Moreover, the permeability of gap junctions can be altered by changes in the intracellular pH and Ca2+ concentration, as well as by the phosphorylation of connexin, providing numerous mechanisms for regulating transport through them.

The generation of mutant mice with inactivating mutations in connexin genes has highlighted the importance of connexins in a wide variety of cellular systems. For instance, Cx43-defective mice exhibit numerous defects including defective oocyte maturation due to decreased gap-junctional communication between granulosa cells in the ovary.

Mutations in several connexin genes are related to human diseases, including neurosensory deafness (Cx26 and Cx31), cataract or heart malformations (Cx43, Cx46, and Cx50), and the X-linked form of Charcot-Marie-Tooth disease (Cx32), which is marked by progressive degeneration of peripheral nerves. I

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