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Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.
Tissues are not made up solely of cells. A substantial part of their volume is extracellular space, which is largely filled by an intricate network of macromolecules constituting the extracellular matrix (Figure 19-33). This matrix is composed of a variety of proteins and polysaccharides that are secreted locally and assembled into an organized meshwork in close association with the surface of the cell that produced them.
Cells surrounded by spaces filled with extracellular matrix. The particular cells shown in this low-power electron micrograph are those in an embryonic chick limb bud. The cells have not yet acquired their specialized characteristics. (Courtesy of Cheryll (more...)
Whereas we have discussed cell junctions chiefly in the context of epithelial tissues, our account of the extracellular matrix concentrates on connective tissues (Figure 19-34). The extracellular matrix in connective tissue is frequently more plentiful than the cells it surrounds, and it determines the tissue's physical properties. Connective tissues form the framework of the vertebrate body, but the amounts found in different organs vary greatly—from cartilage and bone, in which they are the major component, to brain and spinal cord, in which they are only minor constituents.
The connective tissue underlying an epithelium. This tissue contains a variety of cells and extracellular matrix components. The predominant cell type is the fibroblast, which secretes abundant extracellular matrix.
Variations in the relative amounts of the different types of matrix macromolecules and the way in which they are organized in the extracellular matrix give rise to an amazing diversity of forms, each adapted to the functional requirements of the particular tissue. The matrix can become calcified to form the rock-hard structures of bone or teeth, or it can form the transparent matrix of the cornea, or it can adopt the ropelike organization that gives tendons their enormous tensile strength. At the interface between an epithelium and connective tissue, the matrix forms a basal lamina (see Figure 19-34), which is important in controlling cell behavior.
The vertebrate extracellular matrix was once thought to serve mainly as a relatively inert scaffold to stabilize the physical structure of tissues. But now it is clear that the matrix has a far more active and complex role in regulating the behavior of the cells that contact it, influencing their survival, development, migration, proliferation, shape, and function. The extracellular matrix has a correspondingly complex molecular composition. Although our understanding of its organization is still incomplete, there has been rapid progress in characterizing many of its major components.
We focus on the extracellular matrix of vertebrates, but the origins of the extracellular matrix are very ancient and virtually all multicellular organisms, make it; examples include the cuticles of worms and insects, the shells of mollusks, and, as we discuss later, the cell walls of plants.
The macromolecules that constitute the extracellular matrix are mainly produced locally by cells in the matrix. As we discuss later, these cells also help to organize the matrix: the orientation of the cytoskeleton inside the cell can control the orientation of the matrix produced outside. In most connective tissues, the matrix macromolecules are secreted largely by cells called fibroblasts (Figure 19-35). In certain specialized types of connective tissues, such as cartilage and bone, however, they are secreted by cells of the fibroblast family that have more specific names: chondroblasts, for example, form cartilage, and osteoblasts form bone.
Fibroblasts in connective tissue. This scanning electron micrograph shows tissue from the cornea of a rat. The extracellular matrix surrounding the fibroblasts is composed largely of collagen fibrils (there are no elastic fibers in the cornea). The glycoproteins, (more...)
Two main classes of extracellular macromolecules make up the matrix: (1) polysaccharide chains of the class called glycosaminoglycans (GAGs), which are usually found covalently linked to protein in the form of proteoglycans, and (2) fibrous proteins, including collagen, elastin, fibronectin, and laminin, which have both structural and adhesive functions. We shall see that the members of both classes come in a great variety of shapes and sizes.
The proteoglycan molecules in connective tissue form a highly hydrated, gel-like “ground substance” in which the fibrous proteins are embedded. The polysaccharide gel resists compressive forces on the matrix while permitting the rapid diffusion of nutrients, metabolites, and hormones between the blood and the tissue cells. The collagen fibers both strengthen and help organize the matrix, and rubberlike elastin fibers give it resilience. Finally, many matrix proteins help cells attach in the appropriate locations.
Glycosaminoglycans (GAGs) are unbranched polysaccharide chains composed of repeating disaccharide units. They are called GAGs because one of the two sugars in the repeating disaccharide is always an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), which in most cases is sulfated. The second sugar is usually a uronic acid (glucuronic or iduronic). Because there are sulfate or carboxyl groups on most of their sugars, GAGs are highly negatively charged (Figure 19-36). Indeed, they are the most anionic molecules produced by animal cells. Four main groups of GAGs are distinguished according to their sugars, the type of linkage between the sugars, and the number and location of sulfate groups: (1) hyaluronan, (2) chondroitin sulfate and dermatan sulfate, (3) heparan sulfate, and (4) keratan sulfate.
The repeating disaccharide sequence of a dermatan sulfate glycosaminoglycan (GAG) chain. These chains are typically 70–200 sugars long. There is a high density of negative charges along the chain resulting from the presence of both carboxyl and (more...)
Polysaccharide chains are too stiff to fold up into the compact globular structures that polypeptide chains typically form. Moreover, they are strongly hydrophilic. Thus, GAGs tend to adopt highly extended conformations that occupy a huge volume relative to their mass (Figure 19-37), and they form gels even at very low concentrations. Their high density of negative charges attracts a cloud of cations, most notably Na+, that are osmotically active, causing large amounts of water to be sucked into the matrix. This creates a swelling pressure, or turgor, that enables the matrix to withstand compressive forces (in contrast to collagen fibrils, which resist stretching forces). The cartilage matrix that lines the knee joint, for example, can support pressures of hundreds of atmospheres in this way.
The relative dimensions and volumes occupied by various macromolecules. Several proteins, a glycogen granule, and a single hydrated molecule of hyaluronan are shown.
The GAGs in connective tissue usually constitute less than 10% of the weight of the fibrous proteins. But, because they form porous hydrated gels, the GAG chains fill most of the extracellular space, providing mechanical support to the tissue. In one rare human genetic disease, there is a severe deficiency in the synthesis of the dermatan sulfate disaccharide shown in Figure 19-36. The affected individuals have a short stature, prematurely aged appearance, and generalized defects in their skin, joints, muscles, and bones.
It should be emphasized, however, that, in invertebrates and plants, other types of polysaccharides often dominate the extracellular matrix. Thus, in higher plants, as we discuss later, cellulose (polyglucose) chains are packed tightly together in ribbonlike crystalline arrays to form the microfibrillar component of the cell wall. In insects, crustaceans, and other arthropods, chitin (poly-N-acetylglucosamine) similarly forms the main component of the exoskeleton. Together, cellulose and chitin are the most abundant biopolymers on Earth.
Hyaluronan (also called hyaluronic acid or hyaluronate) is the simplest of the GAGs (Figure 19-38). It consists of a regular repeating sequence of up to 25,000 nonsulfated disaccharide units, is found in variable amounts in all tissues and fluids in adult animals, and is especially abundant in early embryos. Hyaluronan is not typical of the majority of GAGs. In contrast with all of the others, it contains no sulfated sugars, all its disaccharide units are identical, its chain length is enormous (thousands of sugar monomers), and it is not generally linked covalently to any core protein. Moreover, whereas other GAGs are synthesized inside the cell and released by exocytosis, hyaluronan is spun out directly from the cell surface by an enzyme complex embedded in the plasma membrane.
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 sugars. Note the absence of sulfate groups.
Hyaluronan is thought to have a role in resisting compressive forces in tissues and joints. It is also important as a space filler during embryonic development, where it can be used to force a change in the shape of a structure, as a small quantity expands with water to occupy a large volume (see Figure 19-37). Hyaluronan synthesized from the basal side of an epithelium, for example, often serves to create a cell-free space into which cells subsequently migrate; this occurs in the formation of the heart, the cornea, and several other organs. When cell migration ends, the excess hyaluronan is generally degraded by the enzyme hyaluronidase. Hyaluronan is also produced in large quantities during wound healing, and it is an important constituent of joint fluid, where it serves as a lubricant.
Many of the functions of hyaluronan depend on specific interactions with other molecules, including both proteins and proteoglycans—molecules consisting of GAG chains covalently linked to a protein. Some of these molecules that bind to hyaluronan are constituents of the extracellular matrix, while others are integral components of the surface of cells.
Except for hyaluronan, all GAGs are found covalently attached to protein in the form of proteoglycans, which are made by most animal cells. The polypeptide chain, or core protein, of a proteoglycan is made on membrane-bound ribosomes and threaded into the lumen of the endoplasmic reticulum. The polysaccharide chains are mainly assembled on this core protein in the Golgi apparatus. First, a special link tetrasaccharide is attached to a serine side chain on the core protein to serve as a primer for polysaccharide growth; then, one sugar at a time is added by specific glycosyl transferases (Figure 19-39). While still in the Golgi apparatus, many of the polymerized sugars are covalently modified by a sequential and coordinated series of reactions. Epimerizations alter the configuration of the substituents around individual carbon atoms in the sugar molecule; sulfations increase the negative charge.
The linkage between a GAG chain and its core protein in a proteoglycan molecule. A specific link tetrasaccharide is first assembled on a serine side chain. In most cases, it is unclear how the particular serine is selected, but it seems that a specific (more...)
Proteoglycans are usually easily distinguished from other glycoproteins by the nature, quantity, and arrangement of their sugar side chains. By definition, at least one of the sugar side chains of a proteoglycan must be a GAG. Whereas glycoproteins contain 1–60% carbohydrate by weight in the form of numerous relatively short, branched oligosaccharide chains, proteoglycans can contain as much as 95% carbohydrate by weight, mostly in the form of long, unbranched GAG chains, each typically about 80 sugars long. Proteoglycans can be huge. The proteoglycan aggrecan, for example, which is a major component of cartilage, has a mass of about 3 × 106 daltons with over 100 GAG chains. Other proteoglycans are much smaller and have only 1–10 GAG chains; an example is decorin, which is secreted by fibroblasts and has a single GAG chain (Figure 19-40).
Examples of a small (decorin) and a large (aggrecan) proteoglycan found in the extracellular matrix. These two proteoglycans are compared with a typical secreted glycoprotein molecule, pancreatic ribonuclease B. All three are drawn to scale. The core (more...)
In principle, proteoglycans have the potential for almost limitless heterogeneity. Even a single type of core protein can vary greatly in the number and types of attached GAG chains. Moreover, the underlying repeating pattern of disaccharides in each GAG can be modified by a complex pattern of sulfate groups. The heterogeneity of these GAGs makes it difficult to identify and classify proteoglycans in terms of their sugars. The sequences of many core proteins have been determined with the aid of recombinant DNA techniques, and they, too, are extremely diverse. Although a few small families have been recognized, no common structural feature clearly distinguishes proteoglycan core proteins from other proteins, and many have one or more domains that are homologous to domains found in other proteins of the extracellular matrix or plasma membrane. Thus, it is probably best to regard proteoglycans as a diverse group of highly glycosylated glycoproteins whose functions are mediated by both their core proteins and their GAG chains.
Given the great abundance and structural diversity of proteoglycan molecules, it would be surprising if their function were limited to providing hydrated space around and between cells. Their GAG chains, for example, can form gels of varying pore size and charge density; one possible function, therefore, is to serve as selective sieves to regulate the traffic of molecules and cells according to their size, charge, or both. Evidence suggests that a heparan sulfate proteoglycan called perlecan has this role in the basal lamina of the kidney glomerulus, which filters molecules passing into the urine from the bloodstream (discussed below).
Proteoglycans are thought to have a major role in chemical signaling between cells. They bind various secreted signal molecules, such as certain protein growth factors, and can enhance or inhibit their signaling activity. For example, the heparan sulfate chains of proteoglycans bind to fibroblast growth factors (FGFs), which stimulate a variety of cell types to proliferate; this interaction oligomerizes the growth factor molecules, enabling them to cross-link and activate their cell-surface receptors, which are transmembrane tyrosine kinases (see Figure 15-50B). Whereas in most cases the signal molecules bind to the GAG chains of the proteoglycan, this is not always so. Some members of the transforming growth factor β (TGF-β) family bind to the core proteins of several matrix proteoglycans, including decorin; binding to decorin inhibits the activity of the growth factors.
Proteoglycans also bind, and regulate the activities of, other types of secreted proteins, including proteolytic enzymes (proteases) and protease inhibitors. Binding to a proteoglycan could control the activity of a secreted protein in any of the following ways: (1) it could immobilize the protein close to the site where it is produced, thereby restricting its range of action; (2) it could sterically block the activity of the protein; (3) it could provide a reservoir of the protein for delayed release; (4) it could protect the protein from proteolytic degradation, thereby prolonging its action; (5) it could alter or concentrate the protein for more effective presentation to cell-surface receptors.
Proteoglycans are thought to act in all these ways to help regulate the activities of secreted proteins. An example of the last function occurs in inflammatory responses, in which heparan sulfate proteoglycans immobilize secreted chemotactic attractants called chemokines (discussed in Chapter 24) on the endothelial surface of a blood vessel at an inflammatory site. In this way, the chemokines remain there for a prolonged period, stimulating white blood cells to leave the bloodstream and migrate into the inflamed tissue.
GAGs and proteoglycans can associate to form huge polymeric complexes in the extracellular matrix. Molecules of aggrecan, for example, the major proteoglycan in cartilage (see Figure 19-40), assemble with hyaluronan in the extracellular space to form aggregates that are as big as a bacterium (Figure 19-41).
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 (more...)
Moreover, besides associating with one another, GAGs and proteoglycans associate with fibrous matrix proteins such as collagen and with protein meshworks such as the basal lamina, creating extremely complex structures. Decorin, which binds to collagen fibrils, is essential for collagen fiber formation; mice that cannot make decorin have fragile skin that has reduced tensile strength. The arrangement of proteoglycan molecules in living tissues is generally hard to determine. As the molecules are highly water-soluble, they may be washed out of the extracellular matrix when tissue sections are exposed to aqueous solutions during fixation. In addition, changes in pH, ionic, or osmotic conditions can drastically alter their conformation. Thus, specialized methods must be used to visualize them in tissues (Figure 19-42).
Proteoglycans in the extracellular matrix of rat cartilage. The tissue was rapidly frozen at -196°C, and fixed and stained while still frozen (a process called freeze substitution) to prevent the GAG chains from collapsing. In this electron micrograph, (more...)
Not all proteoglycans are secreted components of the extracellular matrix. Some are integral components of plasma membranes and have their core protein either inserted across the lipid bilayer or attached to the lipid bilayer by a glycosylphosphatidylinositol (GPI) anchor. Some of these plasma membrane proteoglycans act as co-receptors that collaborate with conventional cell-surface receptor proteins, in both binding cells to the extracellular matrix and initiating the response of cells to some extracellular signal proteins. In addition, some conventional receptors have one or more GAG chains and are therefore proteoglycans themselves.
Among the best-characterized plasma membrane proteoglycans are the syndecans, which have a membrane-spanning core protein. The extracellular domains of these transmembrane proteoglycans carry up to three chondroitin sulfate and heparan sulfate GAG chains, while their intracellular domains are thought to interact with the actin cytoskeleton in the cell cortex.
Syndecans are located on the surface of many types of cells, including fibroblasts and epithelial cells, where they serve as receptors for matrix proteins. In fibroblasts, syndecans can be found in focal adhesions, where they modulate integrin function by interacting with fibronectin on the cell surface and with cytoskeletal and signaling proteins inside the cell. Syndecans also bind FGFs and present them to FGF receptor proteins on the same cell. Similarly, another plasma membrane proteoglycan, called betaglycan, binds TGF-β and may present it to TGF-β receptors.
The importance of proteoglycans as co-receptors is illustrated by the severe developmental defects that can occur when specific proteoglycans are inactivated by mutation. In Drosophila, for example, signaling by the secreted signal protein Wingless depends on the protein's binding to a specific heparan sulfate proteoglycan co-receptor called Dally on the target cell. In mutant flies deficient in Dally, Wingless signaling fails, and the severe developmental defects that result are similar to those that result from mutations in the wingless gene itself. In some tissues, inactivation of Dally also inhibits signaling by a secreted protein of the TGF-β family called Decapentaplegic (DPP).
Some of the proteoglycans discussed in this chapter are summarized in Table 19-4.
Some Common Proteoglycans.
The collagens are a family of fibrous proteins found in all multicellular animals. They are secreted by connective tissue cells, as well as by a variety of other cell types. As a major component of skin and bone, they are the most abundant proteins in mammals, constituting 25% of the total protein mass in these animals.
The primary feature of a typical collagen molecule is its long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains, called α chains, are wound around one another in a ropelike superhelix (Figure 19-43). Collagens are extremely rich in proline and glycine, both of which are important in the formation of the triple-stranded helix. Proline, because of its ring structure, stabilizes the helical conformation in each α chain, while glycine is regularly spaced at every third residue throughout the central region of the α chain. Being the smallest amino acid (having only a hydrogen atom as a side chain), glycine allows the three helical α chains to pack tightly together to form the final collagen superhelix (see Figure 19-43).
The structure of a typical collagen molecule. (A) A model of part of a single collagen α chain in which each amino 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 (more...)
So far, about 25 distinct collagen α chains have been identified, each encoded by a separate gene. Different combinations of these genes are expressed in different tissues. Although in principle more than 10,000 types of triple-stranded collagen molecules could be assembled from various combinations of the 25 or so α chains, only about 20 types of collagen molecules have been found. The main types of collagen found in connective tissues are types I, II, III, V, and XI, type I being the principal collagen of skin and bone and by far the most common. These are the fibrillar collagens, or fibril-forming collagens, with the ropelike structure illustrated in Figure 19-43. After being secreted into the extracellular space, these collagen molecules assemble into higher-order polymers called collagen fibrils, which are thin structures (10–300 nm in diameter) many hundreds of micrometers long in mature tissues and clearly visible in electron micrographs (Figure 19-44; see also Figure 19-42). Collagen fibrils often aggregate into larger, cablelike bundles, several micrometers in diameter, which can be seen in the light microscope as collagen fibers.
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 (more...)
Collagen types IX and XII are called fibril-associated collagens because they decorate the surface of collagen fibrils. They are thought to link these fibrils to one another and to other components in the extracellular matrix. Types IV and VII are network-forming collagens. Type IV molecules assemble into a feltlike sheet or meshwork that constitutes a major part of mature basal laminae, while type VII molecules form dimers that assemble into specialized structures called anchoring fibrils. Anchoring fibrils help attach the basal lamina of multilayered epithelia to the underlying connective tissue and therefore are especially abundant in the skin.
There are also a number of “collagen-like” proteins, including type XVII, which has a transmembrane domain and is found in hemidesmosomes, and type XVIII, which is located in the basal laminae of blood vessels. Cleavage of the C-terminal domain of type XVIII collagen yields a peptide called endostatin, which inhibits new blood vessel formation and is therefore being investigated as an anticancer drug. Some of the collagen types discussed in this chapter are listed in Table 19-5.
Some Types of Collagen and Their Properties.
Many proteins that contain a repeated pattern of amino acids have evolved by duplications of DNA sequences. The fibrillar collagens apparently arose in this way. Thus, the genes that encode the α chains of most of these collagens are very large (up to 44 kilobases in length) and contain about 50 exons. Most of the exons are 54, or multiples of 54, nucleotides long, suggesting that these collagens arose by multiple duplications of a primordial gene containing 54 nucleotides and encoding exactly 6 Gly-X-Y repeats (see Figure 19-43).
Individual collagen polypeptide chains are synthesized on membrane-bound ribosomes and injected into the lumen of the endoplasmic reticulum (ER) as larger precursors, called pro-α chains. These precursors not only have the short amino-terminal signal peptide required to direct the nascent polypeptide to the ER, they also have additional amino acids, called propeptides, at both their N- and C-terminal ends. In the lumen of the ER, selected prolines and lysines are hydroxylated to form hydroxyproline and hydroxylysine, respectively, and some of the hydroxylysines are glycosylated. Each pro-α chain then combines with two others to form a hydrogen-bonded, triple-stranded, helical molecule known as procollagen.
Hydroxylysines and hydroxyprolines (Figure 19-45) are infrequently found in other animal proteins, although hydroxyproline is abundant in some proteins in the plant cell wall. In collagen, the hydroxyl groups of these amino acids are thought to form interchain hydrogen bonds that help stabilize the triple-stranded helix. Conditions that prevent proline hydroxylation, such as a deficiency of ascorbic acid (vitamin C), have serious consequences. In scurvy, the disease caused by a dietary deficiency of vitamin C that was common in sailors until the nineteenth century, the defective pro-α chains that are synthesized fail to form a stable triple helix and are immediately degraded within the cell. Consequently, with the gradual loss of the preexisting normal collagen in the matrix, blood vessels become extremely fragile and teeth become loose in their sockets, implying that in these particular tissues the degradation and replacement of collagen occur relatively rapidly. In many other adult tissues, however, the turnover of collagen (and other extracellular matrix macromolecules) is thought to be very slow. In bone, to take an extreme example, collagen molecules persist for about 10 years before they are degraded and replaced. By contrast, most cell proteins have half-lives of hours or days.
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.
After secretion, the propeptides of the fibrillar procollagen molecules are removed by specific proteolytic enzymes outside the cell. This converts the procollagen molecules to collagen molecules, which assemble in the extracellular space to form much larger collagen fibrils. The propeptides have at least two functions. First, they guide the intracellular formation of the triple-stranded collagen molecules. Second, because they are removed only after secretion, they prevent the intracellular formation of large collagen fibrils, which could be catastrophic for the cell.
The process of fibril formation is driven, in part, by the tendency of the collagen molecules, which are more than a thousandfold less soluble than procollagen molecules, to self-assemble. The fibrils begin to form close to the cell surface, often in deep infoldings of the plasma membrane formed by the fusion of secretory vesicles with the cell surface. The underlying cortical cytoskeleton can therefore influence the sites, rates, and orientation of fibril assembly.
When viewed in an electron microscope, collagen fibrils have characteristic cross-striations every 67 nm, reflecting the regularly staggered packing of the individual collagen molecules in the fibril. After the fibrils have formed in the extracellular space, they are greatly strengthened by the formation of covalent cross-links between lysine residues of the constituent collagen molecules (Figure 19-46). The types of covalent bonds involved are found only in collagen and elastin. If cross-linking is inhibited, the tensile strength of the fibrils is drastically reduced; collagenous tissues become fragile, and structures such as skin, tendons, and blood vessels tend to tear. The extent and type of cross-linking vary from tissue to tissue. Collagen is especially highly cross-linked in the Achilles tendon, for example, where tensile strength is crucial.
Cross-links formed between modified lysine side chains within a collagen fibril. Covalent intramolecular and intermolecular cross-links are formed in several steps. First, certain lysines and hydroxylysines are deaminated by the extracellular enzyme lysyl (more...)
Figure 19-47 summarizes the various steps in the synthesis and assembly of collagen fibrils. Given the large number of enzymatic steps involved, it is not surprising that there are many human genetic diseases that affect fibril formation. Mutations affecting type I collagen cause osteogenesis imperfecta, characterized by weak bones that fracture easily. Mutations affecting type II collagen cause chondrodysplasias, characterized by abnormal cartilage, which leads to bone and joint deformities. Mutations affecting type III collagen cause Ehlers-Danlos syndrome, characterized by fragile skin and blood vessels and hypermobile joints.
The intracellular and extracellular events in the formation of a collagen fibril. (A) Note that collagen fibrils are shown assembling in the extracellular space contained within a large infolding in the plasma membrane. As one example of how collagen (more...)
In contrast to GAGs, which resist compressive forces, collagen fibrils form structures that resist tensile forces. The fibrils have various diameters and are organized in different ways in different tissues. In mammalian skin, for example, they are woven in a wickerwork pattern so that they resist tensile stress in multiple directions. In tendons, they are organized in parallel bundles aligned along the major axis of tension. In mature bone and in the cornea, they are arranged in orderly plywoodlike layers, with the fibrils in each layer lying parallel to one another but nearly at right angles to the fibrils in the layers on either side. The same arrangement occurs in tadpole skin (Figure 19-48).
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 (more...)
The connective tissue cells themselves must determine the size and arrangement of the collagen fibrils. The cells can express one or more genes for the different types of fibrillar procollagen molecules. But even fibrils composed of the same mixture of fibrillar collagen molecules have different arrangements in different tissues. How is this achieved? Part of the answer is that cells can regulate the disposition of the collagen molecules after secretion by guiding collagen fibril formation in close association with the plasma membrane (see Figure 19-46). In addition, as the spatial organization of collagen fibrils at least partly reflects their interactions with other molecules in the matrix, cells can influence this organization by secreting, along with their fibrillar collagens, different kinds and amounts of other matrix macromolecules.
Fibril-associated collagens, such as types IX and XII collagens, are thought to be especially important in this regard. They differ from fibrillar collagens in several ways.
Their triple-stranded helical structure is interrupted by one or two short nonhelical domains, which makes the molecules more flexible than fibrillar collagen molecules.
They are not cleaved after secretion and therefore retain their propeptides.
They do not aggregate with one another to form fibrils in the extracellular space. Instead, they bind in a periodic manner to the surface of fibrils formed by the fibrillar collagens. Type IX molecules bind to type-II-collagen-containing fibrils in cartilage, the cornea, and the vitreous of the eye (Figure 19-49), whereas type XII molecules bind to type-I-collagen-containing fibrils in tendons and various other tissues.
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, sheathed in type IX collagen (more...)
Fibril-associated collagens are thought to mediate the interactions of collagen fibrils with one another and with other matrix macromolecules. In this way, they have a role in determining the organization of the fibrils in the matrix.
Cells interact with the extracellular matrix mechanically as well as chemically, with dramatic effects on the architecture of the tissue. Thus, for example, fibroblasts work on the collagen they have secreted, crawling over it and tugging on it—helping to compact it into sheets and draw it out into cables. When fibroblasts are mixed with a meshwork of randomly oriented collagen fibrils that form a gel in a culture dish, the fibroblasts tug on the meshwork, drawing in collagen from their surroundings and thereby causing the gel to contract to a small fraction of its initial volume. By similar activities, a cluster of fibroblasts surrounds itself with a capsule of densely packed and circumferentially oriented collagen fibers.
If two small pieces of embryonic tissue containing fibroblasts are placed far apart on a collagen gel, the intervening collagen becomes organized into a compact band of aligned fibers that connect the two explants (Figure 19-50). The fibroblasts subsequently migrate out from the explants along the aligned collagen fibers. Thus, the fibroblasts influence the alignment of the collagen fibers, and the collagen fibers in turn affect the distribution of the fibroblasts. Fibroblasts presumably have a similar role in generating long-range order in the extracellular matrix inside the body—in helping to create tendons and ligaments, for example, and the tough, dense layers of connective tissue that ensheathe and bind together most organs.
The shaping of the extracellular matrix by cells. This micrograph shows a region between two pieces of embryonic chick heart (rich in fibroblasts as well as heart muscle cells) that were cultured on a collagen gel for 4 days. A dense tract of aligned (more...)
Many vertebrate tissues, such as skin, blood vessels, and lungs, need to be both strong and elastic in order to function. A network of elastic fibers in the extracellular matrix of these tissues gives them the required resilience so that they can recoil after transient stretch (Figure 19-51). Elastic fibers are at least five times more extensible than a rubber band of the same cross-sectional area. Long, inelastic collagen fibrils are interwoven with the elastic fibers to limit the extent of stretching and prevent the tissue from tearing.
Elastic fibers. These scanning electron micrographs show (A) a low-power view of a segment of a dog's aorta and (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 (more...)
The main component of elastic fibers is elastin, a highly hydrophobic protein (about 750 amino acids long), which, like collagen, is unusually rich in proline and glycine but, unlike collagen, is not glycosylated and contains some hydroxy-proline but no hydroxylysine. Soluble tropoelastin (the biosynthetic precursor of elastin) is secreted into the extracellular space and assembled into elastic fibers close to the plasma membrane, generally in cell-surface infoldings. After secretion, the tropoelastin molecules become highly cross-linked to one another, generating an extensive network of elastin fibers and sheets. The cross-links are formed between lysines by a mechanism similar to the one discussed earlier that operates in cross-linking collagen molecules.
The elastin protein is composed largely of two types of short segments that alternate along the polypeptide chain: hydrophobic segments, which are responsible for the elastic properties of the molecule; and alanine- and lysine-rich α-helical segments, which form cross-links between adjacent molecules. Each segment is encoded by a separate exon. There is still controversy, however, concerning the conformation of elastin molecules in elastic fibers and how the structure of these fibers accounts for their rubberlike properties. In one view, the elastin polypeptide chain, like the polymer chains in ordinary rubber, adopts a loose “random coil” conformation, and it is the random coil structure of the component molecules cross-linked into the elastic fiber network that allows the network to stretch and recoil like a rubber band (Figure 19-52).
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 expand and contract as a random coil, so that the entire assembly (more...)
Elastin is the dominant extracellular matrix protein in arteries, comprising 50% of the dry weight of the largest artery—the aorta. Mutations in the elastin gene causing a deficiency of the protein in mice or humans result in narrowing of the aorta or other arteries as a result of excessive proliferation of smooth muscle cells in the arterial wall. Apparently, the normal elasticity of an artery is required to restrain the proliferation of these cells.
Elastic fibers are not composed solely of elastin. The elastin core is covered with a sheath of microfibrils, each of which has a diameter of about 10 nm. Microfibrils are composed of a number of distinct glycoproteins, including the large glycoprotein fibrillin, which binds to elastin and is essential for the integrity of elastic fibers. Mutations in the fibrillin gene result in Marfan's syndrome, a relatively common human genetic disease affecting connective tissues that are rich in elastic fibers; in the most severely affected individuals, the aorta is prone to rupture. Microfibrils are thought to be important in the assembly of elastic fibers. They appear before elastin in developing tissues and seem to form a scaffold on which the secreted elastin molecules are deposited. As the elastin is deposited, the microfibrils become displaced to the periphery of the growing fiber.
The extracellular matrix contains a number of noncollagen proteins that typically have multiple domains, each with specific binding sites for other matrix macromolecules and for receptors on the surface of cells. These proteins therefore contribute to both organizing the matrix and helping cells attach to it. The first of them to be well characterized was fibronectin, a large glycoprotein found in all vertebrates. Fibronectin is a dimer composed of two very large subunits joined by disulfide bonds at one end. Each subunit is folded into a series of functionally distinct domains separated by regions of flexible polypeptide chain (Figure 19-53A and B). The domains in turn consist of smaller modules, each of which is serially repeated and usually encoded by a separate exon, suggesting that the fibronectin gene, like the collagen genes, evolved by multiple exon duplications. All forms of fibronectin are encoded by a single large gene that contains about 50 exons of similar size. Transcription produces a single large RNA molecule that can be alternatively spliced to produce the various isoforms of fibronectin. The main type of module, called the type III fibronectin repeat, binds to integrins. It is about 90 amino acids long and occurs at least 15 times in each subunit. The type III fibronectin repeat is among the most common of all protein domains in vertebrates.
The structure of a fibronectin dimer. (A) Electron micrographs of individual fibronectin dimer molecules shadowed with platinum; red arrows mark the C-termini. (B) The two polypeptide chains are similar but generally not identical (being made from the (more...)
One way to analyze a complex multifunctional protein molecule like fibronectin is to chop it into pieces and determine the function of its individual domains. When fibronectin is treated with a low concentration of a proteolytic enzyme, the polypeptide chain is cut in the connecting regions between the domains, leaving the domains themselves intact. One can then show that one of its domains binds to collagen, another to heparin, another to specific receptors on the surface of various types of cells, and so on (see Figure 19-53B). Synthetic peptides corresponding to different segments of the cell-binding domain have been used to identify a specific tripeptide sequence (Arg-Gly-Asp, or RGD), which is found in one of the type III repeats (see Figure 19-53C), as a central feature of the binding site. Even very short peptides containing this RGD sequence can compete with fibronectin for the binding site on cells, thereby inhibiting the attachment of the cells to a fibronectin matrix. If these peptides are coupled to a solid surface, they cause cells to adhere to it.
The RGD sequence is not confined to fibronectin. It is found in a number of extracellular proteins, including, for example, the blood-clotting factor fibrinogen. Fibrinogen peptides containing this RGD sequence have been useful in the development of anti-clotting drugs that mimic these peptides. Snakes use a similar strategy to cause their victims to bleed: they secrete RGD-containing anti-clotting proteins called disintegrins into their venom.
RGD sequences are recognized by several members of the integrin family of cell-surface matrix receptors. Each integrin, however, specifically recognizes its own small set of matrix molecules, indicating that tight binding requires more than just the RGD sequence.
There are multiple isoforms of fibronectin. One, called plasma fibronectin, is soluble and circulates in the blood and other body fluids, where it is thought to enhance blood clotting, wound healing, and phagocytosis. All of the other forms assemble on the surface of cells and are deposited in the extracellular matrix as highly insoluble fibronectin fibrils. In these cell-surface and matrix forms, fibronectin dimers are cross-linked to one another by additional disulfide bonds.
Unlike fibrillar collagen molecules, which can be made to self-assemble into fibrils in a test tube, fibronectin molecules assemble into fibrils only on the surface of certain cells. This is because additional proteins are needed for fibril formation, especially fibronectin-binding integrins. In the case of fibroblasts, fibronectin fibrils are associated with integrins at sites called fibrillar adhesions. These are distinct from focal adhesions, in that they are more elongated and contain different intracellular anchor proteins. The fibronectin fibrils on the cell surface are highly stretched and under tension. The tension is exerted by the cell and is essential for fibril formation, as we discuss below. Some secreted proteins function to prevent fibronectin assembly in inappropriate places. Uteroglobin, for example, binds to fibronectin and prevents it from forming fibrils in the kidney. Mice that have a mutation in the uteroglobin gene accumulate insoluble fibronectin fibrils in their kidneys.
The importance of fibronectin in animal development is dramatically demonstrated by gene inactivation experiments. Mutant mice that are unable to make fibronectin die early in embryogenesis because their endothelial cells fail to form proper blood vessels. This defect is thought to result from abnormalities in the interactions of these cells with the surrounding extracellular matrix, which normally contains fibronectin.
The fibronectin fibrils that form on or near the surface of fibroblasts are usually aligned with adjacent intracellular actin stress fibers (Figure 19-54). In fact, intracellular actin filaments promote the assembly of secreted fibronectin molecules into fibrils and influence fibril orientation. If cells are treated with the drug cytochalasin, which disrupts actin filaments, the fibronectin fibrils dissociate from the cell surface (just as they do during mitosis when a cell rounds up).
Coalignment of extracellular fibronectin fibrils and intracellular actin filament bundles. (A) The fibronectin is revealed in two rat fibroblasts in culture by the binding of rhodamine-coupled anti-fibronectin antibodies. (B) The actin is revealed by (more...)
The interactions between extracellular fibronectin fibrils and intracellular actin filaments across the fibroblast plasma membrane are mediated mainly by integrin transmembrane adhesion proteins. The contractile actin and myosin cytoskeleton thereby pulls on the fibronectin matrix to generate tension. As a result, the fibronectin fibrils are stretched, exposing a cryptic (hidden) binding site in the fibronectin molecules that allows them to bind directly to one another. In addition, the stretching exposes more binding sites for integrins. In this way, the actin cytoskeleton promotes fibronectin polymerization and matrix assembly.
Extracellular signals can regulate the assembly process by altering the actin cytoskeleton and thereby the tension on the fibrils. Many other extracellular matrix proteins have multiple repeats similar to the type III fibronectin repeat, and it is possible that tension exerted on these proteins also uncovers cryptic binding sites and thereby influences their polymerization.
Fibronectin is important not only for cell adhesion to the matrix but also for guiding cell migrations in vertebrate embryos. Large amounts of fibronectin, for example, are found along the pathway followed by migrating prospective mesodermal cells during amphibian gastrulation (discussed in Chapter 21). Although all cells of the early embryo can attach to fibronectin, only these migrating cells can spread and migrate on fibronectin. The migration is inhibited by an injection into the developing amphibian embryo of various ligands that disrupt the ability of the cells to bind to fibronectin.
Many matrix proteins are believed to have a role in guiding cell movements during development. The tenascins and thrombospondins, for example, are composed of several types of short amino acid sequences that are repeated many times and form functionally distinct domains. They can either promote or inhibit cell adhesion, depending on the cell type. Indeed, anti-adhesive interactions are as important as adhesive ones in guiding cell migration, as we discuss in Chapter 21.
As mentioned earlier, basal laminae are flexible, thin (40–120 nm thick) mats of specialized extracellular matrix that underlie all epithelial cell sheets and tubes. They also surround individual muscle cells, fat cells, and Schwann cells (which wrap around peripheral nerve cell axons to form myelin). The basal lamina thus separates these cells and epithelia from the underlying or surrounding connective tissue. In other locations, such as the kidney glomerulus, a basal lamina lies between two cell sheets and functions as a highly selective filter (Figure 19-55). Basal laminae have more than simple structural and filtering roles, however. They are able to determine cell polarity, influence cell metabolism, organize the proteins in adjacent plasma membranes, promote cell survival, proliferation, or differentiation, and serve as specific highways for cell migration.
Three ways in which basal laminae are organized. Basal laminae (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, (more...)
The basal lamina is synthesized largely by the cells that rest on it (Figure 19-56). In some multilayered epithelia, such as the stratified squamous epithelium that forms the epidermis of the skin, the basal lamina is tethered to the underlying connective tissue by specialized anchoring fibrils made of type VII collagen molecules. The term basement membrane is often used to describe the composite of the basal lamina and this layer of collagen fibrils. In one type of skin disease, these connections are either absent or destroyed, and the epidermis and its basal lamina become detached from the underlying connective tissue, causing blistering.
The basal lamina in the cornea of a chick embryo. In this scanning electron micrograph, some of the epithelial cells (E) have been removed to expose the upper surface of the matlike basal lamina (BL). A network of collagen fibrils (C) in the underlying (more...)
Although its precise composition varies from tissue to tissue and even from region to region in the same lamina, most mature basal laminae contain type IV collagen, the large heparan sulfate proteoglycan perlecan, and the glycoproteins laminin and nidogen (also called entactin).
Type IV collagens exist in several isoforms. They all have a more flexible structure than the fibrillar collagens; their triple-stranded helix is interrupted in 26 regions, allowing multiple bends. They are not cleaved after secretion, but interact via their uncleaved terminal domains to assemble extracellularly into a flexible, sheetlike, multilayered network.
Early in development, basal laminae contain little or no type IV collagen and consist mainly of laminin molecules. Laminin-1 (classical laminin) is a large, flexible protein composed of three very long polypeptide chains (α, β, and γ) arranged in the shape of an asymmetric cross and held together by disulfide bonds (Figure 19-57). Several isoforms of each type of chain can associate in different combinations to form a large family of laminins. The laminin γ-1 chain is a component of most laminin heterotrimers, and mice lacking it die during embryogenesis because they are unable to make a basal lamina. Like many other proteins in the extracellular matrix, the laminin in basement membranes consists of several functional domains: one binds to perlecan, one to nidogen, and two or more to laminin receptor proteins on the surface of cells.
The structure of laminin. (A) The subunits of a laminin-1 molecule. This multidomain glycoprotein is composed of three polypeptides (α, β, and γ) that are disulfide-bonded into an asymmetric crosslike structure. Each of the polypeptide (more...)
Like type IV collagen, laminins can self-assemble in vitro into a feltlike sheet, largely through interactions between the ends of the laminin arms. As nidogen and perlecan can bind to both laminin and type IV collagen, it is thought that they connect the type IV collagen and laminin networks (Figure 19-58). In tissues, laminins and type IV collagen preferentially polymerize while bound to receptors on the surface of the cells producing the proteins. Many of the cell-surface receptors for type IV collagen and laminin are members of the integrin family. Another important type of laminin receptor is the transmembrane protein dystroglycan, which, together with integrins, may organize the assembly of the basal lamina.
A model of the molecular structure of a basal lamina. (A) The basal lamina is formed by specific interactions (B) between the proteins type IV collagen, laminin, and nidogen, and the proteoglycan perlecan. Arrows in (B) connect molecules that can bind (more...)
The shapes and sizes of some of the extracellular matrix molecules discussed in this chapter are compared in Figure 19-59.
The comparative shapes and sizes of some of the major extracellular matrix macromolecules. Protein is shown in green, and glycosaminoglycan in red.
As we have mentioned, in the kidney glomerulus, an unusually thick basal lamina acts as a molecular filter, preventing the passage of macromolecules from the blood into the urine as urine is formed (see Figure 19-55). The heparan sulfate proteoglycan in the basal lamina seems to be important for this function: when its GAG chains are removed by specific enzymes, the filtering properties of the lamina are destroyed. Type IV collagen also has a role, as a human hereditary kidney disorder (Alport syndrome) results from mutations in type IV collagen α-chain genes.
The basal lamina can also act as a selective barrier to the movement of cells. The lamina beneath an epithelium, for example, usually prevents fibroblasts in the underlying connective tissue from making contact with the epithelial cells. It does not, however, stop macrophages, lymphocytes, or nerve processes from passing through it. The basal lamina is also important in tissue regeneration after injury. When tissues such as muscles, nerves, and epithelia are damaged, the basal lamina survives and provides a scaffold along which regenerating cells can migrate. In this way, the original tissue architecture is readily reconstructed. In some cases, as in the skin or cornea, the basal lamina becomes chemically altered after injury—for example, by the addition of fibronectin, which promotes the cell migration required for wound healing.
A particularly striking example of the instructive role of the basal lamina in regeneration comes from studies on the neuromuscular junction, the site where the nerve terminals of a motor neuron form a chemical synapse with a skeletal muscle cell (discussed in Chapter 11). The basal lamina that surrounds the muscle cell separates the nerve and muscle cell plasma membranes at the synapse, and the synaptic region of the lamina has a distinctive chemical character, with special isoforms of type IV collagen and laminin and a heparan sulfate proteoglycan called agrin.
This basal lamina at the synapse has a central role in reconstructing the synapse after nerve or muscle injury. 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. If the motor nerve, but not the muscle, is allowed to regenerate, the nerve axons seek out the original synaptic sites on the empty basal lamina and differentiate there to form normal-looking nerve terminals. Thus, the junctional basal lamina by itself can guide the regeneration of motor nerve terminals.
Similar experiments show that the basal lamina also controls the localization of the acetylcholine receptors that cluster in the muscle cell plasma membrane at a neuromuscular junction. If the muscle and nerve are both destroyed, but now the muscle is allowed to regenerate while the nerve is prevented from doing so, the acetylcholine receptors synthesized by the regenerated muscle localize predominantly in the region of the old junctions, even though the nerve is absent (Figure 19-60). Thus, the junctional basal lamina apparently coordinates the local spatial organization of the components in each of the two cells that form a neuromuscular junction. Some of the matrix proteins have been identified. Motor neuron axons, for example, deposit agrin in the junctional basal lamina, where it triggers the assembly of acetylcholine receptors and other proteins in the junctional plasma membrane of the muscle cell. Conversely, muscle cells deposit a particular isoform of laminin in the junctional basal lamina. Both agrin and this isoform of laminin are essential for the formation of normal neuromuscular junctions.
Regeneration experiments demonstrating the special character of the junctional basal lamina at a neuromuscular junction. When the nerve, but not the muscle, is allowed to regenerate after both the nerve and muscle have been damaged (upper part of figure), (more...)
The extracellular matrix can influence the organization of a cell's cytoskeleton. This can be vividly demonstrated by using transformed (cancerlike) fibroblasts in culture (discussed in Chapter 23). Transformed cells often make less fibronectin than normal cultured cells and behave differently. They adhere poorly to the culture substratum, for example, and fail to flatten out or develop the organized intracellular bundles of actin filaments known as stress fibers. The decrease in fibronectin production and adhesion may contribute to the tendency of cancer cells to break away from the primary tumor and spread to other parts of the body.
In some cases, fibronectin deficiency seems also to be at least partly responsible for this abnormal morphology of cancer cells: if the cells are grown on a matrix of organized fibronectin fibrils, they flatten out and assemble intracellular stress fibers that are aligned with the extracellular fibronectin fibrils. This interaction between the extracellular matrix and the cytoskeleton is reciprocal in that intracellular actin filaments can promote the assembly and influence the orientation of fibronectin fibrils, as described earlier. Since the cytoskeleton can exert forces that orient the matrix macromolecules the cell secretes and the matrix macromolecules can in turn organize the cytoskeleton of the cells they contact, the extracellular matrix can in principle propagate order from cell to cell (Figure 19-61), creating large-scale oriented structures, as described earlier (see Figure 19-50). The integrins serve as the main adaptors in this ordering process, mediating the interactions between cells and the matrix around them.
How the extracellular matrix could, in principle, propagate order from cell to cell within a tissue. For simplicity, the figure represents a hypothetical scheme in which one cell influences the orientation of its neighboring cells. It is more likely, (more...)
Most cells need to attach to the extracellular matrix to grow and proliferate—and, in many cases, even to survive. This dependence of cell growth, proliferation, and survival on attachment to a substratum is known as anchorage dependence, and it is mediated mainly by integrins and the intracellular signals they generate. The physical spreading of a cell on the matrix also has a strong influence on intracellular events. Cells that are forced to spread over a large surface area survive better and proliferate faster than cells that are not so spread out, even if in both cases the cells have the same area making contact with the matrix directly (Figure 19-62). This stimulatory effect of cell spreading presumably helps tissues to regenerate after injury. If cells are lost from an epithelium, for example, the spreading of the remaining cells into the vacated space will stimulate them to proliferate until they fill the gap. It is still uncertain, however, how a cell senses its extent of spreading so as to adjust its behavior accordingly.
Anchorage dependence and the importance of cell spreading. For many cells, contact with the extracellular matrix is essential for survival, growth, and proliferation. In this experiment, the extent of cell spreading on a substratum, rather than the number (more...)
The regulated turnover of extracellular matrix macromolecules is crucial to a variety of important biological processes. Rapid degradation occurs, for example, when the uterus involutes after childbirth, or when the tadpole tail is resorbed during metamorphosis (see Figure 17-36). A more localized degradation of matrix components is required when cells migrate through a basal lamina. This occurs when white blood cells migrate across the basal lamina of a blood vessel into tissues in response to infection or injury, and when cancer cells migrate from their site of origin to distant organs via the bloodstream or lymphatic vessels—the process known as metastasis. Even in the seemingly static extracellular matrix of adult animals, there is a slow, continuous turnover, with matrix macromolecules being degraded and resynthesized.
In each of these cases, matrix components are degraded by extracellular proteolytic enzymes (proteases) that are secreted locally by cells. Thus, antibodies that recognize the products of proteolytic cleavage stain matrix only around cells. Many of these proteases belong to one of two general classes. Most are matrix metalloproteases, which depend on bound Ca2+ or Zn2+ for activity; the others are serine proteases, which have a highly reactive serine in their active site. Together, metalloproteases and serine proteases cooperate to degrade matrix proteins such as collagen, laminin, and fibronectin. Some metalloproteases, such as the collagenases, are highly specific, cleaving particular proteins at a small number of sites. In this way, the structural integrity of the matrix is largely retained, but cell migration can be greatly facilitated by the small amount of proteolysis. Other metalloproteases may be less specific, but, because they are anchored to the plasma membrane, they can act just where they are needed.
The importance of proteolysis in cell migration can be shown by using protease inhibitors, which often block migration. Moreover, cells that migrate readily on type I collagen in culture can no longer do so if the collagen is made resistant to proteolysis by mutating the collagenase-sensitive cleavage sites. The proteolysis of matrix proteins can contribute to cell migration in several ways: (1) it can simply clear a path through the matrix; (2) it can expose cryptic sites on the cleaved proteins that promote cell binding, cell migration, or both; (3) it can promote cell detachment so that a cell can move onward, or (4) it can release extracellular signal proteins that stimulate cell migration.
Three basic mechanisms operate to ensure that the proteases that degrade the matrix components are tightly controlled.
Local activation: Many proteases are secreted as inactive precursors that can be activated locally when needed. An example is plasminogen, an inactive protease precursor that is abundant in the blood. It is cleaved locally by other proteases called plasminogen activators to yield the active serine protease plasmin, which helps break up blood clots. Tissue-type plasminogen activator (tPA) is often given to patients who have just had a heart attack or thrombotic stroke; it helps dissolve the arterial clot that caused the attack, thereby restoring bloodflow to the tissue.
Confinement by cell-surface receptors: Many cells have receptors on their surface that bind proteases, thereby confining the enzyme to the sites where it is needed. A second type of plasminogen activator called urokinase-type plasminogen activator (uPA) is an example. It is found bound to receptors on the growing tips of axons and at the leading edge of some migrating cells, where it may serve to clear a pathway for their migration. Receptor-bound uPA may also help some cancer cells metastasize (Figure 19-63).
The importance of proteases bound to cell-surface receptors. (A) Human prostate cancer cells make and secrete the serine protease uPA, which binds to cell-surface uPA receptor proteins. (B) The same cells have been transfected with DNA that encodes an (more...)
Secretion of inhibitors: The action of proteases is confined to specific areas by various secreted protease inhibitors, including the tissue inhibitors of metalloproteases (TIMPs) and the serine protease inhibitors known as serpins. These inhibitors are protease-specific and bind tightly to the activated enzyme, blocking its activity. An attractive idea is that the inhibitors are secreted by cells at the margins of areas of active protein degradation in order to protect uninvolved matrix; they may also protect cell-surface proteins required for cell adhesion and migration. The overexpression of TIMPs inhibits the migration of some cell types, indicating the importance of metalloproteases for the migration.
Cells in connective tissues are embedded in an intricate extracellular matrix that not only binds the cells together but also influences their survival, development, shape, polarity, and behavior. The matrix contains various protein fibers interwoven in a hydrated gel composed of a network of glycosaminoglycan (GAG) chains.
GAGs are a heterogeneous group of negatively charged polysaccharide chains that (except for hyaluronan) are covalently linked to protein to form proteoglycan molecules. They occupy a large volume and form hydrated gels in the extracellular space. Proteoglycans are also found on the surface of cells, where they function as co-receptors to help cells respond to secreted signal proteins.
Fiber-forming proteins strengthen the matrix and give it form. They also provide surfaces for cells to adhere to. Elastin molecules form an extensive cross-linked network of fibers and sheets that can stretch and recoil, imparting elasticity to the matrix. The fibrillar collagens (types I, II, III, V, and XI) are ropelike, triple-stranded helical molecules that aggregate into long fibrils in the extracellular space. The fibrils in turn can assemble into a variety of highly ordered arrays. Fibril-associated collagen molecules, such as types IX and XII, decorate the surface of collagen fibrils and influence the interactions of the fibrils with one another and with other matrix components.
In contrast, type IV collagen molecules assemble into a sheetlike meshwork that is a crucial component of all mature basal laminae. All basal laminae are based on a mesh of laminin molecules. The collagen and laminin networks in mature basal laminae are bridged by the protein nidogen and the large heparan sulfate proteoglycan perlecan. Fibronectin and laminin are examples of large, multidomain matrix glycoproteins. By means of their multiple binding domains, such proteins help organize the matrix and help cells adhere to it.
Matrix proteins such as collagens, laminins, and fibronectin are assembled into fibrils or networks on the surface of the cells that produce them by a process that depends on the underlying actin cortex. The organization of the matrix can reciprocally influence the organization of the cell's cytoskeleton and can mechanically influence cell spreading. The matrix also influences cell behavior by binding to cell-surface receptors that activate intracellular signaling pathways.
Matrix components are degraded by extracellular proteolytic enzymes. Most of these are matrix metalloproteases, which depend on bound Ca2+ or Zn2+ for activity, while others are serine proteases, which have a reactive serine in their active site. Various mechanisms operate to ensure that the degradation of matrix components is tightly controlled. Cells can, for example, cause a localized degradation of matrix components to clear a path through the matrix.
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