Cell Junctions and the Extracellular Matrix PDF

How animal cells are bound together in two major tissue types. In connective tissue, the extracellular matrix is the main stress- bearing component. In epithelial tissue, the cytoskeleton of the cell is the main stress-bearing component: the cytoskeletons of cells are linked from cell to cell by adh...

How animal cells are bound together in two major tissue types. In connective tissue, the extracellular matrix is the main stress- bearing component. In epithelial tissue, the cytoskeleton of the cell is the main stress-bearing component: the cytoskeletons of cells are linked from cell to cell by adhesive junctions and transmit mechanical stresses across the interiors of the cells. Cell matrix attachments bond epithelial tissue to the connective tissue beneath it. Various cell junctions found in a vertebrate epithelial cell. In the apical region of the cell, the relative positions of the junctions are the same in nearly all vertebrate epithelia. The tight junction occupies the most apical position, followed by the adherens junction (adhesion belt) and then by a special parallel row of desmosomes; together, these three junctions form a structure called a junctional complex. Gap junctions and additional desmosomes are less regularly organized. Two types of cell matrix anchoring junctions tether the basal surface of the cell to the basal lamina. The drawing is based on epithelial cells of the small intestine. Transmembrane adhesion proteins link the cytoskeleton to extracellular structures. The external linkage may be either to other cells (cell cell junctions, mediated typically by cadherins) or to extracellular matrix (cell matrix junctions, mediated typically by integrins). The internal linkage to the cytoskeleton is generally indirect, via intracellular adaptor proteins. The cadherin superfamily The diagram shows some of the diversity among cadherin superfamily members. These proteins all have extracellular portions containing multiple copies of the extracellular cadherin domain (green ovals). In the classical cadherins of vertebrates there are 5 of these domains, and in desmogleins and desmocollins there are 4 or 5, but some nonclassical cadherins have more than 30. The intracellular portions are more varied, reflecting interactions with a wide variety of intracellular ligands, including signaling molecules and adaptor proteins that connect the cadherin to the cytoskeleton. In some cases, such as T-cadherin, a transmembrane domain is not present, and the protein is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. The differently colored motifs in Fat, Flamingo, and Ret represent conserved domains that are also found in other protein families. Homophilic versus heterophilic binding. Cadherins in general bind homophilically; some other cell adhesion molecules bind heterophilically. The extracellular region of a classical cadherin contains five copies of the extracellular cadherin domain separated by flexible hinge regions. At a typical extracellular Ca2+ concentration (>1 mM), Ca2+ ions (red dots) bind in the neighborhood of each hinge, preventing it from flexing. To generate cell cell adhesion, the cadherin domain at the N- terminal tip of one cadherin molecule binds the cadherin domain at the N-terminal tip of a cadherin molecule on another cell. The structure was determined by x-ray diffraction of the crystallized C-cadherin extracellular region. The two cadherins shown here, although identical, are colored differently for clarity. If the extracellular Ca2+ concentration is decreased artificially in an experiment, Ca2+ binding decreases. As a result, increased flexibility in the hinge regions results in a floppier molecule that is no longer oriented correctly to interact with a cadherin on another celle and adhesion fails. At a typical cell cell junction, an organized array of cadherin molecules functions like Velcro to hold cells together. Cadherins on the same cell are thought to be coupled by side-to-side interactions between their N-terminal head regions, resulting in a linear array like the alternating green and light green cadherins on the lower cell shown here. These arrays are thought to interact with perpendicular arrays on an adjacent cell (blue cadherin molecules, top cell). Multiple perpendicular arrays on both cells interact to form a tight-knit mat of cadherin proteins. Sorting out. Cells from different layers of an early amphibian embryo will sort out according to their origins. In the classical experiment shown here, mesoderm cells (green), neural plate cells (blue), and epidermal cells (red) have been disaggregated and then reaggregated in a random mixture. They sort out into an arrangement reminiscent of a A2A:AXA1aA+eA1bAXA:AeAaA2eAjAXaA+AeAjA2be AeeAXA2aA+A+eAUAdeXA1A\APee eAXA2aA+A+aAA 2A1d eA\A:deAXA1A2beAeeA2MA:AX de A:A1PL Townes and J. Holtfreter, J. Exp. Zool. 128:53 120, 1955. With permission from John Wiley & Sons.) Changing patterns of cadherin expression during construction of the vertebrate nervous system. The figure shows cross sections of the early chick embryo, as the neural tube detaches from the ectoderm and then as neural crest cells detach from the neural tube. (A, B) Immunofluorescence micrographs showing the developing neural tube labeled with antibodies against (A) E-cadherin (blue) and (B) N-cadherin (yellow). (C) As the patterns of gene expression change, the different groups of cells segregate from one another according to the cadherins they express. Cadherin-dependent cell sorting. Cells in culture can sort themselves out according to the type and level of cadherins they express. This can be visualized by labeling different populations of cells with dyes of different colors. (A) Cells expressing N-cadherin sort out from cells expressing E-cadherin. (B) Cells expressing high levels of E-cadherin sort out from cells expressing low levels of E-cadherin. The cells expressing high levels adhere more strongly and congregate internally. Local changes in cortical tension help promote the initial formation of an adherens junction. (A) Surface tension of a water droplet results from binding among water molecules at the air water interface, pulling the surface inward. (B) In an unattached cell, the contraction of actin myosin bundles at the cell cortex (red) creates cortical tension, drawing the surface inward. (C) When two epithelial cell precursors first interact, small cadherin clusters (green) assemble at the contact site. Cortical tension prevents this initial interaction site from spreading. The cadherins generate local signals to inhibit the GTPase Rho and activate the GTPase Rac, leading to localized disassembly of actin myosin fibers, loss of cortical tension, and formation of branched actin networks and cell protrusions all of which allow the recruitment of more cadherins and the spreading of the cell cell junction over a greater surface area. In the long term (not shown), the large adherens junction inhibits Rac and stimulates Rho, The linkage of classical cadherins to actin filaments. The cadherins are coupled indirectly to actin filaments through an adaptor protein complex containing p120-caAeeA2A2 -caAeeA2A2aA2d -catenin. OAeAeXAUAXA:AeeA2A\A2cA+AjdA2A}A2cAjA+A2aA\A\A:cAaAeAee A - caAeeA2A2aA2dAe+AUAUAXA:A}Aede Ae+A2A’aAAee A:acAeA2 - Catenin has a second, and very important, function in intracellular signaling. Mechanotransduction in an adherens junction. Cell cell junctions are able to sense increased tension and respond by strengthening their actin linkages. When actin filaments are pulled from within the cell by non-muscle myosin II, the resulting force AjA2A:A+dA\adA:A1aAA2A2 -catenin, thereby exposing an otherwise hidden binding site for the adaptor protein vinculin. Vinculin then promotes additional actin recruitment, strengthening the linkages between the junction and the cytoskeleton. Adherens junctions between epithelial cells in the small intestine. These cells are specialized for absorption of nutrients; at their apical surfaces, facing the lumen of the gut, they have many microvilli (protrusions that increase the absorptive surface area). The adherens junction takes the form of an adhesion belt, encircling each of the interacting cells. Its most obvious feature is a contractile bundle of actin filaments running along the cytoplasmic surface of the junctional plasma membrane. The actin filament bundles are tethered by adaptor proteins to cadherins, which bind to cadherins on the adjacent cell. In this way, the actin filament bundles in adjacent cells are tied together. For clarity, this drawing does not show most of the other cell cell and cell matrix junctions of epithelial cells The folding of an epithelial sheet to form an epithelial tube. The oriented contraction of the bundles of actin and myosin filaments running along adhesion belts causes the epithelial cells to narrow at their apical surfaces, thereby helping the epithelial sheet to roll up into a tube. An example is the formation of the neural tube in early vertebrate development Remodeling of cell cell adhesions in embryonic Drosophila epithelium. Depicted at left is a group of cells in the outer epithelium of a Drosophila embryo. During germ-band extension, cells converge toward each other (middle) on the dorsal ventral axis and then extend (right) along the anterior posterior axis. The result is intercalation: cells that were originally far apart along the dorsal ventral axis (green) are inserted between the cells (gray) that separated them. These rearrangements depend on the spatial regulation of actin myosin contractile bundles, which are localized primarily at the vertical cell boundaries (red, left). Contraction of these bundles is accompanied by removal of E- cadherin (not shown) at the same cell boundaries, resulting in shrinkage and loss of adhesion along the vertical axis (middle). New cadherin-based adhesions (blue, right) then form and expand along horizontal boundaries, resulting in extension of the cells in the anterior posterior dimension. Desmosomes. (A) The structural components of a desmosome. On the cytoplasmic surface of each interacting plasma membrane is a dense plaque composed of a mixture of intracellular adaptor proteins. A bundle of intermediate filaments is attached to the surface of each plaque. Transmembrane nonclassical cadherins bind to the plaques and interact through their extracellular domains to hold the adjacent membranes together. Desmosomes. (B) Some of the molecular components of a desmosome. Desmoglein and desmocollin are nonclassical cadherins. Their cytoplasmic tails bind plakoglobin -catenin) and plakophilin (a distant relative of p120-catenin), which in turn bind to desmoplakin. Desmoplakin binds to the sides of intermediate filaments, thereby tying the desmosome to these filaments. (C) An electron micrograph of desmosome junctions between three epidermal cells in the skin of a baby mouse. (D) Part of the same tissue at higher magnification, showing a single desmosome, with intermediate filaments attached to it. Desmosomes, hemidesmosomes, and the intermediate filament network. The keratin intermediate filament networks of adjacent cellse in this example, epithelial cells of the small intestinee are indirectly connected to one another through desmosomes, and to the basal lamina through hemidesmosomes. The role of tight junctions in transcellular transport. For clarity, only the tight junctions are shown. Transport proteins are confined to different regions of the plasma membrane in epithelial cells of the small intestine. This segregation results in the one-way transfer of nutrients across the epithelium from the gut lumen to the blood. In the example shown, glucose is actively transported into the cell by Na+-driven glucose transporters at its apical surface, and it leaves the cell through passive glucose transporters in its basolateral membrane. Tight junctions are thought to confine the transport proteins to their appropriate membrane domains by acting as diffusion baAXAXAeXA\A:AXeA2ceA\eA2AeAe+AUbA+ d aAeAXA:AeAeUA+aA\A1a membrane; these junctions also block the backflow of glucose from the basal side of the epithelium into the gut lumen (see Movie 11.2). The role of tight junctions in allowing epithelia to serve as barriers to solute diffusion. (A) The drawing shows how a small extracellular tracer molecule added on one side of an epithelium is prevented from crossing the epithelium by the tight junctions that seal adjacent cells together. Adherens junctions and other cell junctions are not shown for clarity. (B) Electron micrographs of cells in an epithelium in which a small, extracellular, electron- dense tracer molecule has been added to either the apical side (on the left) or the basolateral side (on the right). The tight junction blocks passage of the tracer in both directions. (B, courtesy of Daniel Friend, by permission of E.L. Bearer.) The structure of a tight junction between epithelial cells of the small intestine. The junctions are shown (A) schematically, (B) in a freeze-fracture electron micrograph, and (C) in a conventional electron micrograph. In B, the plane of the micrograph is parallel to the plane of the membrane, and the tight junction appears as a band of branching sealing strands that encircle each cell in the epithelium. In C, the junction is seen in cross section as a series of focal connections between the outer leaflets of the two interacting plasma membranes, each connection corresponding to a sealing strand in cross section. A model of a tight junction. (A) The sealing strands hold adjacent plasma membranes together. The strands are composed of transmembrane proteins that make contact across the intercellular space and create a seal. (B) The molecular composition of a sealing strand. The major extracellular components of the tight junction are members of a family of proteins with four transmembrane domains. One of these proteins, claudin, is the most important for the assembly and structure of the sealing strands, whereas the related protein occludin governs junction permeability. The two termini of these proteins are both on the cytoplasmic side of the membrane, where they interact with large scaffolding proteins that organize the sealing strands and link the Scaffold proteins at the tight junction. The scaffold proteins ZO-1, ZO-2, and ZO-3 are concentrated beneath the plasma membrane at tight junctions. Each of the proteins contains multiple protein-binding domains, including three PDZ domains, an SH3 domain, a GK domain, and a proline-rich domain (P), linked together like beads on a flexible string. These domains enable the proteins to interact with each other and with numerous other partners, as indicated here by arrows, to generate a tightly woven protein network that organizes the sealing strands of the tight junction and links them to the actin cytoskeleton. Scaffold proteins with similar structure help organize other junctional complexes, including those at neural synapses. Gap junctions as seen in the electron microscope. (A) Thin-section and (B) freeze-fracture electron micrographs of a large and a small gap-junction plaque between fibroblasts in culture. In B, each gap junction is seen as a cluster of homogeneous intramembrane particles. Each intramembrane particle corresponds to a connexon Determining the size of a gap-junction channel. When fluorescent molecules of various sizes are injected into one of two cells coupled by gap junctions, molecules with a mass of less than about 1000 daltons can pass into the other cell, but larger molecules cannot. Thus, the coupled cells share their small molecules (such as inorganic ions, sugars, amino acids, nucleotides, vitamins, and the intracellular signaling molecules cyclic AMP and inositol trisphosphate) but not their macromolecules (proteins, nucleic acids, and polysaccharides). Gap junctions. (A) A drawing of the interacting plasma membranes of two adjacent cells connected by gap junctions. Each lipid bilayer is shown as a gray sheet. Protein assemblies called connexons (green), each of which is formed by six connexin subunits, penetrate the apposed lipid bilayers. Two connexons join across the intercellular gap to form a continuous aqueous channel connecting the two cells. (B) The organization of connexins into connexons, and connexons into intercellular channels. The connexons can be homomeric or heteromeric, and the intercellular channels can be homotypic or heterotypic. (C) The high-resolution structure of a homomeric gap- junction channel, determined by x-ray crystallography of human connexin 26. In this view, we are looking down on the pore, formed from six connexin subunits. The structure illustrates the general features of the channel and suggests a pore size of about 1.4 nm, as predicted from studies of gap-junction permeability with molecules of various sizes. Plasmodesmata. (A) The cytoplasmic channels of plasmodesmata pierce the plant cell wall and connect cells in a plant together. (B) Each plasmodesma is lined with plasma membrane that is common to the two connected cells. It usually also contains a fine tubular structure, the desmotubule, derived from smooth endoplasmic reticulum. The structure and function of selectins. (A) Diagram of P-selectin, which attaches to the actin cytoskeleton through adaptor proteins. (B) How selectins and integrins mediate the cell cell adhesions required for a white blood cell to migrate out of the bloodstream into a tissue. Selectins on endothelial cells bind weakly to oligosaccharides on the white blood cell, so that it becomes loosely attached and rolls along the vessel wall. The white blood cell then activates a cell-surface integrin called LFA1, which binds to a protein called ICAM1 (belonging to the Ig superfamily) on the membrane of the endothelial cell. The white blood cell adheres to the vessel wall and then crawls out of the Members of the Ig superfamily of cell cell adhesion molecules. NCAM is expressed on neurons and many other cell types, and it mediates homophilic binding. ICAM is expressed on endothelial cells and some other cell types and binds heterophilically to an integrin on white blood cells. Nectin is expressed in many cell types and is often found at adherens junctions, where it interacts with cadherins to help establish and strengthen specific cell cell interactions during tissue formation. Fibroblasts in connective tissue. This scanning electron micrograph shows tissue from the cornea of a rat. The extracellular matrix surrounding the fibroblasts is here composed largely of collagen fibrils. The glycoproteins, hyaluronan, and proteoglycans, which normally form a hydrated gel filling the interstices of the fibrous network, have been removed by enzyme and acid treatment. (Courtesy of T. Nishida.) The comparative shapes and sizes of some of the major extracellular matrix macromolecules. Protein is shown in green, and glycosaminoglycan (GAG) in red. The repeating disaccharide sequence of a heparin glycosaminoglycan (GAG) chain. These chains can consist of as many as 75 disaccharide units but are typically less than half that size. There is a high density of negative charges along the chain because of the presence of both carboxyl and sulfate groups; indeed, heparin is the most densely charged biological molecule known. The most common form of heparin carries three sulfate groups in each disaccharide, as shown here. In vivo, the proportion of sulfated and nonsulfated groups is highly variable. Heparin has an average of about 2.7 sulfates per disaccharide. Heparan sulfate is a closely related GAG that is generally about twice the length of heparin and less charged, with an average of about 1 sulfate per disaccharide. The relative dimensions and volumes occupied by various macromolecules. Several proteins and a single hydrated molecule of hyaluronan are shown, with molecular mass in daltons. The repeating disaccharide sequence in hyaluronan, a relatively simple GAG. This ubiquitous molecule in vertebrates consists of a single long chain of up to 25,000 disaccharides. Note the absence of sulfate groups. The linkage between a GAG chain and its core protein in a proteoglycan molecule. A specific linkage tetrasaccharide is first assembled on a serine side chain. The rest of the GAG chain, consisting mainly of a repeating disaccharide unit, is then synthesized, with one sugar added at a time. An aggrecan aggregate from fetal bovine cartilage. (A) An electron micrograph of an aggrecan aggregate shadowed with platinum. Many free aggrecan molecules are also visible. (B) A drawing of the giant aggrecan aggregate shown in A. It consists of about 100 aggrecan monomers noncovalently bound through the N-terminal domain of the core protein to a single hyaluronan molecule. A link protein binds both to the core protein of the proteoglycan and to the hyaluronan molecule, thereby stabilizing the aggregate. The molecular mass of such a complex can be 108 daltons or more, and it A:ccAjAUAe\aA}A:A+AjA1eAWAjA}aA+eA2AeAeA:AeAaAeA:AabacAeeAXAjA1AcAA\abA:AjAeQA1 3. Proteoglycans in the extracellular matrix of rat cartilage. The tissue was rapidly frozen at 1965C and fixed and stained while still frozen (a process called freeze substitution) to prevent the GAG chains from collapsing. In this electron micrograph, the proteoglycan molecules are seen to form a fine filamentous network in which a single striated collagen fibril is embedded. The more darkly stained parts of the proteoglycan molecules are the core AUAXA:AeeA2A\AeaAAe A2AeA+A\AeaAA2edAeAXeadA\aAXAee AeGAGcAaAA2A\0PXWSEB Hunziker and R.K. Schenk. Originally published in J. Cell Biol. https://doi.org/10.1083/jcb.98.1.277. With permission from Rockefeller University Press.) A fibroblast surrounded by collagen fibrils in the connective tissue of embryonic chick skin. In this electron micrograph, the fibrils are organized into bundles that run approximately at right angles to one another. Therefore, some bundles are oriented longitudinally, whereas others are seen in cross section. The collagen fibrils are produced by fibroblasts. The structure of a typical collagen molecule. (A) A model A:AUaAXAeA:aAA \A2A+cA e :A+A+aAeA2cAaAA2A2AcAeacAaA1A2A: acid is represented by a sphere. The chain is about 1000 amino acids long. It is arranged as a left-handed helix, with three amino acids per turn and with glycine as every third aA1A2A:acATAeX d A:e AXaAe 2cAaAA2A\cA:A1AUA:A\A:ed aAA \eAXAe\A:A triplet Gly-X-Y sequences, in which X and Y can be any amino acid (although X is commonly proline and Y is commonly hydroxyproline, a form of proline that is chemically modified during collagen synthesis in the cell). (B) A model of part of a collagen molecule, in which eXee chains, each shown in a different color, are wrapped around one another to form a triple-stranded helical rod. Glycine is the only amino acid small enough to occupy the crowded interior of the triple helix. Only a short length of the molecule is shown; the entire molecule is 300 nm long. (From a model by B.L. Trus.) TABLE 19-2 Some Types of Collagen and Their Properties Type Polymerized form Tissue distribution Mutant phenotype Fibril-forming Fibril Bone, skin, tendons, ligaments, Severe bone defects, fractures (fibrillar) cornea, internal organs (osteogenesis imperfecta) (accounts for 90% of body collagen) Fibril Cartilage, intervertebral disc, Cartilage deficiency, dwarfism notochord, vitreous humor of (chondrodysplasia) the eye Il Fibril Skin, blood vessels, internal Fragile skin, loose joints, blood vessels organs prone to rupture (vascular Ehlers-Danlos syndrome)" Fibril (with type )1 As for type I Fragile skin, loose joints (classical Ehlers- Danlos syndrome) X I Fibril (with type lI) As for type lI Myopia, blindness Fibril-associated XI Lateral association Cartilage Osteoarthritis with type lI fibrils XI Lateral association Tendons Skeletal and muscle abnormalities with type I fibrils Network-forming I Sheetlike network Basal lamina Kidney disease (glomerulonephritis, deafness VI Anchoring fibrils Beneath stratified squamous Skin blistering epithelia Transmembrane XVI Nonfibrillar Hemidesmosomes Skin blistering Proteoglycan XVII Nonfibrillar Basal lamina Myopia, detached retina, hydrocephalus core protein Note that types I, IV, V I are each composed of two or three types of a chains (distinct, nonoverlapping sets ni each case), whereas , IX, and X types I, III, VI, XI, XVI, and XVI are composed of only one type of a chain each. Hydroxylysine and hydroxyproline. These modified amino acids are common in collagen. They are formed by enzymes that act after the lysine and proline have been incorporated into procollagen molecules. Cross-links formed between modified lysine side chains within a collagen fibril. Covalent intramolecular and intermolecular cross-links are formed in several steps. First, the extracellular enzyme lysyl oxidase deaminates certain lysines and hydroxylysines to yield highly reactive aldehyde groups. The aldehydes then react spontaneously to form covalent bonds with each other or with other lysines or hydroxylysines. Most of the cross-links form between the short nonhelical segments at each end of the collagen molecules. Collagen fibrils in the tadpole skin. This electron micrograph shows the plywoodlike arrangement of the fibrils: successive layers of fibrils are laid down nearly at right angles to each other. This organization is also found in mature bone and in the cornea. (Courtesy of Jerome Gross.) Type IX collagen. (A) Type IX collagen molecules binding in a periodic pattern to the surface of a fibril containing type II collagen. (B) Electron micrograph of a rotary-shadowed type II collagen containing fibril in cartilage, decorated by type IX collagen molecules. (CAA2A2dA}AdjaA+AeAUeIXcA:A+A+aAeA2A1A:A+ecAjA+aAB e 20PXWWLVaA dC jAaA2 et al. Originally published in J. Cell Biol. https://doi.org/10.1083/jcb.106.3.991. With permission from Rockefeller University Press.) Elastic fibers. These scanning electron micrographs show (A) a low-AUA:eAXA}A:e aAA \eA1eA2AeA:adA A:A\aA:AXAeaaA2d B) a high-power view of the dense network of longitudinally oriented elastic fibers in the outer layer of the same blood vessel. All the other components have been digested away with enzymes and formic acid. (From K.S. Haas et al., Anat. Rec. 230:86 96, 1991. With permission from Wiley-Liss.) Stretching a network of elastin molecules. The molecules are joined together by covalent bonds (red) to generate a cross-linked network. In this model, each elastin molecule in the network can extend and contract in a manner resembling a random coil, so that the entire assembly can stretch and recoil like a rubber band. Complex glycoproteins of the extracellular matrix. Many matrix glycoproteins are large scaffold proteins containing multiple copies of specific protein-interaction domains. Each domain is folded into a discrete globular structure, and many such domains are arrayed along the protein like beads on a string. This diagram shows four representative proteins among the roughly 200 matrix glycoproteins that are found in mammals. Each protein contains multiple repeat domains, with the names listed in the key at the bottom. Fibronectin, for example, contains numerous copies of three different fibronectin repeats (types I III, labeled here as FN1, FN2, and FN3). Two type III repeats near the center of the protein contain important binding sites for cell-surface integrins, whereas three nearby type III repeats form a binding site for heparin or heparan sulfate proteoglycans. FN repeats at the N-terminus are involved in binding fibrin or collagen. Other matrix proteins contain repeated sequences resembling those of epidermal growth factor (EGF), a major regulator of cell growth and proliferation; these repeats might serve a similar signaling function in matrix proteins. Other proteins contain domains, such as the insulin-like growth factor binding protein (IGFBP) repeat, that bind and regulate the function of soluble growth factors. To add more structural diversity, many of these proteins are encoded by RNA transcripts that can be spliced in different ways, adding or removing exons, such as those in fibronectin. Finally, the scaffolding and regulatory functions of many matrix proteins are further expanded by assembly into multimeric forms, as shown at the right: fibronectin forms dimers linked at the C- termini, whereas tenascin and thrombospondin form N-terminally linked hexamers and trimers, respectively. Other domains include four repeats from thrombospondin (TSPN, TSP1, TSP3, TSP_C). VWC, von Willebrand type C; FBG, fibrinogen-like. The structure of a fibronectin dimer. (B) The two polypeptide chains are similar but generally not identical (being made from the same gene but from differently spliced mRNAs). They are joined by two disulfide bonds near the C- termini. Each chain is almost 2500 amino acids long and is folded into multiple domains (see Figure 19 47). As indicated, some domains are specialized for binding to a particular molecule. For simplicity, not all of the known binding sites are shown. (C) The three-dimensional structure of the ninth and tenth type III fibronectin repeats, as determined by x-ray crystallography. Both the Arg-Gly-AA\AUGDaA R 2Aed A\e A2eAXA\eAWAjA2e ceA\A\A:A2A2 red are important for binding to integrins on cell surfaces. (C, from D.J. Leahy, Annu. Rev. Cell Dev. Biol. 13:363 393, 1997. Organization of fibronectin into fibrils at the cell surface. This fluorescence micrograph shows the front end of a migrating mouse fibroblast. Extracellular fibronectin is stained green, and intracellular actin filaments are stained red. The fibronectin is initially present as small dotlike aggregates near the leading edge of the cell. It accumulates at focal adhesions (sites of anchorage of actin filaments, discussed later) and becomes organized into fibrils parallel to the actin filaments. Integrin molecules spanning the cell membrane link the fibronectin outside the cell to the actin filaments inside it. Tension exerted on the fibronectin molecules through this linkage is thought to stretch them, exposing binding sites that promote fibril formation. (Courtesy of Roumen Pankov and Kenneth Yamada.) Three ways in which the basal lamina is organized. Sheets of basal lamina (yellow) surround certain cells (such as skeletal muscle cells), underlie epithelia, and are interposed between two cell sheets (as in the kidney glomerulus). Note that, in the kidney glomerulus, both cell sheets have gaps in them, and the basal lamina has a filtering as well as a supportive function, helping to determine which molecules will pass into the urine from the blood. The filtration also depends on other protein-based structures, called slit diaphragms, that span the intercellular gaps in the epithelial sheet. The basal lamina supports a sheet of epithelial cells. In this light micrograph of a cross section of the small intestine, the sheet of columnar epithelial cells rests on the basal lamina (red arrowheads). A network of collagen fibrils and other fibers in the underlying connective tissue interacts with the lower face of the basal lamina. (Jose Luis Calvo/Shutterstock.) The structure of laminin. (A) The best-understood family member is laminin-111, shown here with some of its binding sites for other molecules (gray boxes). Laminins are multidomain glycoproteins composed of three AUA:A+AUeAUAeAde\aA2Aed AaAeaAXedA\AjA+Ade -bonded into an asymmetric crosslike structure. Each of the polypeptide cAaAA2A\A\A1A:AXAee AaA2PTOOaA1A2A:acAdA\A+A:A2FA A A}Aee AUeA\A:cA A aAA2A\A:AjAXAeAUeA\A:cA A aAA2A\aA2Aed AXeAeAUeA\A:cA A aAA2A\ are known, and various combinations of these subunits can assemble to form a large variety of different laminins, which are named according to numbers assigned to each of their three subunits: laminin-111, for example, contains PPaA2PA d \AjbAjA2AeA\EacAA\A:A:AXA1AeeA2dA\AeA:AaA}aAe \AUecAAcAeA\A\AjdA\e AeAXAbjAeA:A2ÆA+aA1A2A2 -332 is found in skin, laminin-211 in muscle, and laminin-411 in endothelial cells of blood vessels. Through their binding sites for other proteins, laminin molecules play a central part in organizing the basal lamina and anchoring it to cells. A model of the molecular structure of a basal lamina. (A) The basal lamina is formed by specific interactions (B) between the proteins laminin, type IV collagen, and nidogen and the proteoglycan perlecan. Arrows in B connect molecules that can bind directly to each other. There are various isoforms of type IV collagen and laminin, each with a distinctive tissue distribution. Transmembrane laminin receptors (integrins and dystroglycan) in the plasma membrane are thought to organize the assembly of the basal lamina; only the integrins are shown. (Based on H. Colognato and P.D. Yurchenco, Dev. Dyn. 218:213 234, 2000. With permission from John Wiley & Sons.) Regeneration experiments demonstrating the special character of the junctional basal lamina at a neuromuscular junction. If a frog muscle and its motor nerve are destroyed, the basal lamina around each muscle cell remains intact, and the sites of the old neuromuscular junctions are still recognizable. When the nerve, but not the muscle, is allowed to regenerate (upper right), the junctional basal lamina directs the regenerating nerve to the original synaptic site. When the muscle, but not the nerve, is allowed to regenerate (lower right), the junctional basal lamina causes newly made acetylcholine receptors (blue) to accumulate at the original synaptic site. These experiments show that the junctional basal lamina controls the localization of synaptic components on both sides of the lamina. Some of the molecules responsible for these effects have been identified. Motor neuron axons, for example, deposit agrin in the junctional basal lamina, where it regulates the assembly of acetylcholine receptors and other proteins in the junctional plasma membrane of the muscle cell. Reciprocally, muscle cells deposit a particular isoform of laminin in the junctional basal lamina, and this molecule is likely to interact with specific ion channels on the presynaptic membrane of The subunit structure of an active integrin molecule, linking extracellular matrix to the actin cytoskeleton. The N-terminal heads of the integrin chains attach directly to an extracellular protein such as fibronectin; the C-terminal A2AeAXaceA+A+AjA+aAXAeaAA+A:AeAe2AeeAXA2A\AjbAjA2AebA2dA\AeA: adaptor proteins that interact with filamentous actin. The best-understood adaptor is a giant protein called talin, which contains a string of multiple domains for binding actin and other proteins, such as vinculin, that help reinforce and regulate the linkage to actin filaments. One end of talin binds to a specific site on the integrin -subunit cytoplasmic tail; other regulatory proteins, such as kindlin, bind at another site on the tail. Hemidesmosomes. (A) Hemidesmosomes spot-weld epithelial cells to the basal lamina, linking laminin outside the cell to keratin filaments inside it. (B) Molecular components A:aAA eA1Ade\A1A:A\A:A1A\e AUecAaA+A2ed AeeAXA2 6 4 integrin) spans the membrane, attaching to keratin filaments intracellularly via adaptor proteins called plectin and BP230, and attaching to laminin extracellularly. The adhesive complex also contains, in parallel with the integrin, an unusual collagen family member known as collagen type XVII; this has a membrane-spanning domain attached to its extracellular Defects in any collagen of theseregion. components can give rise to a blistering disease of the skin. One such disease, called bullous pemphigoid, is an autoimmune disease in which the immune system develops antibodies against collagen XVII or BP230. TABLE 19-3 Some Types of Integrins Phenotype when a subunit is Phenotype when & subunit is Integrin Ligand* Distribution mutated mutated a,B, Fibronectin Ubiquitous Death of embryo; defects in Early death of embryo (at blood vessels, somites, neural implantation) crest a.B, Laminin Ubiquitous Severe skin blistering; defects in Early death of embryo (at other epithelia also implantation) a, B , Laminin Muscle Muscular dystrophy; defective Early death of embryo (at myot endin ous junctions implantation) a, B, (LFA1) gI superfamily White blood cells Impaired recruitment of Leukocyte adhesion deficiency counterreceptors leukoc ytes (LAD); impaired inflammatory (ICAM1) responses; recurrent life- threatening infections Fibrinogen Platelets Bleeding; no platelet aggregation Bleeding; no platelet aggregation (Glanzmann disease) (Glanzmann disease); mild osteopetrosis Laminin Hemidesmosomes Severe skin blistering; defects in Severe skin blistering; defects in ni epithelia other epithelia also other epithelia also *Not all ligands are listed. Integrins exist in two major activity states. Inactive (folded) and active (extended) structures of an integrin molecule, based on data from x-ray crystallography and other methods. Activation of integrins by intracellular signaling. Signals received from outside the cell can act through intracellular signaling proteins to stimulate integrin activation. In platelets, as illustrated here, the extracellular signal protein thrombin activates a G-protein coupled receptor on the cell surface, thereby initiating a signaling pathway that leads to activation of Rap1, a member of the monomeric GTPase family. Activated Rap1 interacts with the protein RIAM, which then recruits talin to the plasma membrane. Prior to this recruitment, talin is held in an inactive state by an interaction between its C-terminal actin-binding domain and its N-terminal integrin-binding domain. When it is recruited by RIAM to the plasma membrane, talin unfolds to expose its binding sites for integrin and actin. Together with another protein called kindlin, talin A2AeeAXacAeA\AeAeAe2AeeAXA2cAaAA2AeA:AeAXeAXA2AeeAXA2acAeA}aAeA:A2 Talin then interacts with actin and with adaptor proteins such as vinculin, resulting in the formation of multiple actin linkages Tyrosine phosphorylation at focal adhesions. A fibroblast cultured on a fibronectin-coated substratum and stained with fluorescent antibodies: actin filaments are stained green and proteins that contain phosphotyrosine are red, giving orange where the two components overlap. The actin filaments terminate at focal adhesions, where the cell attaches to the substratum by means of integrins. Proteins containing phosphotyrosine are also concentrated at these sites, reflecting the local activation of FAK and other protein kinases. Signals generated at such adhesion sites help regulate cell division, growth, and survival. (Courtesy of Keith Burridge.) Talin is a tension sensor at cell matrix junctions. Tension across cell matrix junctions stimulates the local recruitment of vinculin and other actin-AXeAjA+aAeA:AXAUAXA:AeeA2A\AeAeXA\eb AeAXeA2AeAe2A2AeAe$AjA2cAeA:A2A\aAeAeacAA1eA2AeAeA:AeAe cytoskeleton. The experiment presented here tested the hypothesis that tension is sensed by the talin adaptor protein that links integrins to actin filaments. (A) The long, flexible talin protein is divided into a series of folded domains, some of which contain vinculin-binding sites (dark green lines) that are thought to be hidden and therefore inaccessible. One domain near the N-AeeAXA1A2AjA\A:AXaAAPe 1AUA+cA e :A1AUAXA\eA\aAA:A+debAjA2dA+A:e APQAeA+AceA\ containing five vinculin-binding sites. (B) This experiment tested the hypothesis that tension stretches the 12-helix domain, thereby exposing vinculin-binding sites. A fragment of talin containing this domain was attached to an apparatus in which the domain could be stretched, as shown here. The fragment was labeled at its N-terminus with a tag that sticks to the surface of a glass slide on a microscope stage. The C-terminal end of the fragment was bound to a tiny magnetic bead, so the talin fragment could be stretched using a small magnetic electrode. The solution around the protein contained fluorescently tagged vinculin proteins. After the talin protein was stretched, excess vinculin solution was washed away, and the microscope was used to determine if any fluorescent vinculin proteins were bound to the talin protein. In the absence of stretching (top), most talin molecules did not bind vinculin. When the protein was stretched (bottom), two or three vinculin molecules were bound (only one is shown here for clarity).