Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993

Cell adhesion, cell junctions, and the extracellular matrix
Extracellular matrix

Tissues consist of more than just Cells. A significant portion of their volume is occupied by the extracellular space, which is filled with a complex network of macromolecules known as the extracellular matrix (Fig. 14-20). This matrix comprises a variety of Polysaccharides and Proteins secreted by the cells themselves and organized into an ordered network. While our Structure/133.html">Discussion of Intercellular junctions focused primarily on Epithelial Tissues, our examination of the extracellular matrix will deal mainly with Connective Tissues (Fig. 14-21). In such tissues, the matrix typically occupies a larger volume than the cells, surrounds them on all sides, and determines the mechanical Properties of the tissue. In vertebrates, connective tissues form the structural framework of the body, though their proportion varies greatly across different Organs: in the Skin and bones, for instance, they are the primary component, whereas in the Brain AND SPINAL cord, they constitute only a minor fraction.

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Fig. 14-20. Low-magnification electron micrograph showing cells surrounded by the extracellular matrix. In this case, these are undifferentiated limb bud cells of an early chick embryo. (Courtesy of Cheryll Tickle.)

Fig. 14-21. Connective Tissue underlying a layer of epithelial cells.

Variations in the proportions of different macromolecular types and their Organization within the extracellular matrix give rise to an extraordinary diversity of forms, each exceptionally well adapted to the functional needs of a given tissue. The matrix may become calcified to form rock-hard structures such as bone or Teeth, form the transparent substance of the cornea, or take on a rope-like configuration that grants tendons enormous tensile strength. At the interface between the epithelium and connective tissue, the matrix forms the basal lamina—an extremely thin yet dense sheet that plays a crucial role in regulating Cell behavior. Although we will limit our discussion to the vertebrate extracellular matrix, fascinating and unique structures of the same general type are found in many other organisms, such as bacterial and Plant Cell Walls, worm and insect cuticles, and molluscan shells. Plant cell walls will be discussed in detail in Chapter 20.

Until recently, the vertebrate extracellular matrix was viewed as a relatively inert scaffold that merely stabilizes the Physical Structure of tissues. However, it is now clear that it plays a far more active and complex role in regulating The behavior of the cells in contact with it, influencing their development, migration, proliferation, shape, and METABOLISM. The molecular COMPOSITION OF THE extracellular matrix is quite complex, and although our understanding of its organization remains fragmented, rapid progress is being made in elucidating its principal components.

14.2.1. The extracellular matrix consists mainly of Fibrous proteins embedded in a hydrated polysaccharide gel

Extracellular matrix macromolecules are primarily secreted in situ by the cells residing within it. In most connective tissues, this process is carried out mainly by fibroblasts (Fig. 14-22). In certain specialized connective tissues, such as Cartilage and bone, this function is performed by specialized fibroblast-like cells with their own specific names: for instance, chondroblasts form cartilage, and osteoblasts form bone. The two primary classes of macromolecules that make up the matrix are 1) polysaccharide glycosaminoglycans, which are typically covalently linked to protein in the form of Proteoglycans, and 2) fibrous proteins of two functional types: predominantly structural (e.g., Collagen and Elastin) and predominantly adhesive (e.g., fibronectin and laminin). Glycosaminoglycan and proteoglycan molecules form a highly hydrated, gel-like "ground substance" in which the fibrous proteins are embedded. The aqueous phase of the polysaccharide gel facilitates the diffusion of nutrients, metabolites, and Hormones between the Blood and tissue cells; collagen fibers strengthen and organize the matrix, while rubber-like elastin fibers impart resilience. Adhesive proteins promote cell attachment to the extracellular matrix: fibronectin assists in attaching fibroblasts and similar cells to the matrix in connective tissues, whereas laminin AIDS in attaching epithelial cells to the basal lamina.

Fig. 14-22. Scanning electron micrograph of fibroblasts (indicated by arrows) in the connective tissue of the embryonic chick cornea. The extracellular matrix surrounding the fibroblasts consists primarily of collagen fibers (elastic fibers are absent in the cornea). Glycosaminoglycans, which form a hydrated gel in the spaces of the fibrous network, precipitated onto The surface of the collagen fibers during specimen drying. (Courtesy of Robert Trelstad.)

14.2.2. Glycosaminoglycan chains occupy large volumes of space and form hydrated gels [10]

Glycosaminoglycans are long, unbranched polysaccharide chains composed of repeating disaccharide units. They are called glycosaminoglycans because one of the two residues in the repeating disaccharide is always an amino sugar (N-acetylglucosamine or N-acetylgalactosamine). In most cases, one of these amino sugars is sulfated, while the other is a uronic acid. The presence of sulfate or carboxyl groups on many of the sugar residues imparts a high negative charge to glycosaminoglycans (Fig. 14-23). Based on the type of sugar residues, the type of linkage between them, and the number and position of sulfate groups, glycosaminoglycans are divided into four main groups: 1) hyaluronic acid, 2) chondroitin sulfate and dermatan sulfate, 3) heparan sulfate and heparin, and 4) keratan sulfate (Table 14-2).

Polysaccharide chains are not flexible enough to fold into compact globular structures, unlike many polypeptide chains. Furthermore, they are highly hydrophilic. Consequently, glycosaminoglycans tend to adopt the conformation of a highly extended, random coil that occupies a massive volume relative to its mass (Fig. 14-24), forming gels even at very low concentrations. Due to their high density of negative charges, their molecules attract numerous osmotically active ions such as Na⁺, which drives the influx of large amounts of Water into the matrix. This creates a Swelling pressure (turgor) that enables the matrix to resist compressive forces (in contrast to collagen fibers, which counteract tension). This is precisely how the cartilage matrix, for example, resists compression.

Fig. 14-23. Glycosaminoglycans are long, linear polymers composed of a repeating disaccharide sequence. A small portion of a dermatan sulfate chain is shown here; these chains typically contain 70 to 200 sugar residues. The very high density of negative charges along the chain is due to the presence of carboxyl and sulfate groups.

Table 14-2. Glycosaminoglycans

¹⁾ L-iduronic acid is a product of the epimerization of D-glucuronic acid at the carbon atom carrying the carboxyl group. Thus, dermatan sulfate is a modified form of chondroitin sulfate, and the Two Types of repeating Disaccharides typically alternate within the same glycosaminoglycan chain.

The amount of glycosaminoglycans in connective tissue is typically less than 10% of the fibrous protein content. However, because they form a loose, hydrated gel, glycosaminoglycan chains fill the majority of the intercellular space, providing mechanical support to the tissue while permitting the rapid Diffusion of Water-soluble molecules and cell migration.

14.2.3. Hyaluronic acid appears to facilitate cell migration during morphogenesis and tissue regeneration [11]

Hyaluronic acid (also referred to as hyaluronate or hyaluronan), which can contain up to several thousand sugar residues, is a relatively simple molecule composed of a repeating sequence of unsulfated disaccharide units (Fig. 14-25). This substance is found in varying amounts in all tissues and Body Fluids of adult animals, and is particularly abundant in early embryos. Because of its structural simplicity, hyaluronic acid is presumed to be the evolutionarily earliest form of glycosaminoglycan; however, it is atypical compared to most other glycosaminoglycans. All other glycosaminoglycans 1) contain sulfated sugars, 2) most frequently contain several different disaccharide units forming more complex sequences, 3) have much shorter chains containing fewer than 300 sugar residues, and 4) are covalently linked to proteins.

A growing body of evidence indicates that hyaluronic acid performs a specialized function wherever cell migration takes place, such as during embryonic development and wound healing. It is produced in large quantities during periods of cell migration, and once migration ceases, the excess is degraded by the enzyme hyaluronidase. This sequence of events has been observed in A wide variety of tissues. This suggests that a local increase in the synthesis of hyaluronic acid—which attracts water and thereby swells the matrix—serves as a general strategy to facilitate cell migration during morphogenesis and regeneration. Hyaluronic acid is also an essential component of synovial (joint) fluid, where it acts as a lubricant.

14.2.4. Proteoglycans consist of long glycosaminoglycan chains covalently linked to a core protein [12]

With the exception of hyaluronic acid, all glycosaminoglycans are covalently linked to protein in the form of proteoglycans. As with Glycoproteins (Section 8.6.6), the polypeptide chain (core protein) of a proteoglycan is synthesized on membrane-bound Ribosomes and translocated across the membrane into the lumen of The Endoplasmic reticulum. Polysaccharide chains are added to the core protein primarily in the Golgi apparatus: first, a specific linkage trisaccharide is attached to a Serine residue of the protein, serving as a "primer" for polysaccharide growth; then, sugar residues are added one by one with the aid of specific Glycosyltransferases (Fig. 14-26). As the chain elongates in the Golgi apparatus, many of the polymerized sugar residues are modified covalently through a series of sequential and coordinated sulfation (Section 8.7.4) and epimerization reactions, which alter the configuration of functional groups around one of the carbon atoms in the sugar molecule. Sulfation greatly increases the negative charge of proteoglycans.

In general, proteoglycans differ significantly from glycoproteins in the nature, number, and arrangement of their side sugar chains. Glycoproteins typically contain from 1 to 60% of a carbohydrate component in the form of numerous, relatively short (usually fewer than 15 sugar residues) branched oligosaccharide chains linked to oxygen and nitrogen atoms; these chains have a variable composition and often terminate with sialic acid (Section 8.7.1). Although the core protein of a proteoglycan may itself be a glycoprotein, proteoglycans can contain up to 95% carbohydrate by mass, most of which consists of a varying number (from one to several hundred) of unbranched glycosaminoglycan chains, typically comprising about 80 sugar residues each, usually without sialic acid. Furthermore, while glycoproteins rarely have a molecular weight greater than 3 × 10⁵, proteoglycans can be considerably larger. For example, one of the most fully characterized proteoglycans—the major component of cartilage—typically contains about 100 chondroitin sulfate chains and approximately 50 keratan sulfate chains linked to a core protein that is rich in serine and consists of over 2,000 Amino Acids. Its total molecular mass is about 3 × 10⁶, which corresponds to approximately one glycosaminoglycan chain for every 20 amino acid residues (Fig. 14-27). On the other hand, many proteoglycans are much smaller and possess only 1 to 10 glycosaminoglycan chains.

Fig. 14-24. Relative volumes of various protein molecules, a Glycogen granule, and a single hydrated hyaluronic acid molecule with a molecular mass of about 8 × 10⁶.

Fig. 14-25. The repeating disaccharide sequence of hyaluronic acid—a relatively simple glycosaminoglycan consisting of a single very long chain that may contain up to several thousand sugar residues. Note the absence of sulfate groups.

In principle, The structure of proteoglycans allows for an almost unlimited diversity. They can vary substantially in protein content, molecular size, and the number and type of glycosaminoglycan chains per molecule. Moreover, although they are always characterized by repeating disaccharide sequences, the length and composition of the glycosaminoglycan chains can vary widely, as can the spatial arrangement of hydroxyl, sulfate, and carboxyl groups along the chain. Therefore, identifying and classifying proteoglycans based on their constituent sugars is extremely complex. To date, many core proteins have been sequenced using Recombinant DNA technology, and in the future, the Classification of proteoglycans will likely become more meaningful once it is based on the structure of their core proteins rather than their glycosaminoglycan chains.

Fig. 14-26. Schematic representation of the attachment of a glycosaminoglycan chain to a serine residue of the core protein in a proteoglycan molecule. A specific "linker trisaccharide" is attached to the serine [which is often located within the sequence Asp (or Glu)-Asp-(or Glu)-X-Ser-Gly-X-Gly, where X is any amino acid]. The remainder of the glycosaminoglycan chain, built primarily from repeating disaccharide units (consisting in turn of two Monosaccharides A and B listed in Table 14-2), is synthesized later through the sequential addition of sugar residues.

Fig. 14-27. Molecule of the major cartilage proteoglycan. It consists of numerous glycosaminoglycan chains covalently linked to a core protein. In addition to the glycosaminoglycan chains, the core protein also contains oligosaccharide chains attached to nitrogen or oxygen atoms (not shown in the figure). Most proteoglycans are smaller than the one depicted here, and their glycosaminoglycan chains are often restricted to specific Regions of the core protein polypeptide chain. At the bottom, for comparison at the same scale, is a molecule of a typical glycoprotein (pancreatic Ribonuclease B).

14.2.5. Glycosaminoglycan chains can be arranged in a highly ordered manner within the extracellular matrix [13]

Given the structural heterogeneity of proteoglycan molecules, it seems unlikely that their role is merely to create a hydrated space around and between cells. It has been shown that in vitro proteoglycans bind various secreted signaling molecules, and it is reasonable to assume that they perform this function in tissues as well, thereby localizing the action of signaling ligands; for example, fibroblast growth factor (Section 13.3.1, Table 13-1) binds to the heparan sulfate proteoglycan both in vitro and in tissues. Proteoglycans can form gels with varying pore sizes and charge densities, serving as filters that regulate the movement of molecules and cells based on their size and/or charge. Some evidence suggests that proteoglycans perform a similar function in the glomerular basement membrane of the Kidney, which filters molecules from the bloodstream into the urine (Section 14.2.16).

The manner in which Glycosaminoglycans and Proteoglycans are organized in the extracellular matrix remains poorly understood. Biochemical studies indicate that within the matrix, these molecules are specifically bound to one another and to Fibrillar Proteins. It would be surprising if such interactions played no role in matrix organization. It has been established that the major cartilage proteoglycan containing keratan sulfate and chondroitin sulfate (see above) is organized in the extracellular matrix into large aggregates, noncovalently linked via their core proteins to a hyaluronic acid macromolecule. Approximately 100 proteoglycan monomers are attached to a single hyaluronic acid chain, forming a giant complex with a molecular mass of 100 million or more, occupying a volume equivalent to that of a bacterium. When isolated from tissue, this complex is clearly visible under the Electron microscope (Fig. 14-28).

Fig. 14-28. A. Electron micrograph of a proteoglycan aggregate from embryonic bovine cartilage (platinum shadowing). Many free proteoglycan molecules are also visible. B. Cytology/cytology/92.html">SCHEMATIC STRUCTURE OF the giant proteoglycan aggregate shown in micrograph A. It consists of approximately a hundred proteoglycan monomers (such as the one depicted in Fig. 14-27) noncovalently attached to a single hyaluronic acid molecule via two link proteins, which bind simultaneously to both the proteoglycan core protein and the hyaluronic acid chain, thereby stabilizing the aggregate. The molecular mass of such a complex can reach 10⁸ or more, and the volume it occupies is equal to that of a bacterial cell (about 2 × 10^-12 cm³). (A—courtesy of Lawrence Rosenberg.)

However, attempts to determine the spatial arrangement of proteoglycan molecules while they are in the tissue have proven unsuccessful. Because these molecules are highly water-soluble, they are readily washed out of the extracellular matrix during tissue Processing in aqueous fixative solutions. Recently, proteoglycans have been successfully visualized in an almost Native State within cartilage that was rapidly frozen at a very low Temperature (— 196 °C) and high pressure, followed by freeze-substitution fixation and staining (Fig. 14-29). Alternatively, a cationic dye with a relatively low charge density can be used in combination with more traditional fixation. With this staining method, proteoglycans from rat tail tendon appear as filamentous structures wrapping around collagen fibrils at regular intervals of about 65 nm (Fig. 14-30). This interval corresponds to the longitudinal stagger of parallel collagen molecules relative to one another within these fibrils (Section 14.2.8). Such an ordered arrangement of molecules is likely quite common in the extracellular matrix, and given the diversity inherent to collagen and proteoglycan molecules, complex and varied structures can be generated.

It is known that certain polysaccharide chains form highly ordered helical or ribbon-like structures. For instance, in higher plants, the microfibrillar component of cell walls is constructed of Cellulose chains (polyglucose) packed tightly into a ribbon-like crystalline structure (see Fig. 20-5). In vitro, two different polysaccharide chains can specifically interact with each other to form regions with a regular helical structure (Fig. 14-31); such interpolysaccharide interactions could also occur within the extracellular matrix. If the Conformations of proteoglycan molecules are as diverse as their chemical structures, we are only just beginning to understand their organization.

Fig. 14-29. Electron micrograph of extracellular matrix proteoglycans from rat cartilage. The tissue was rapidly frozen at — 196 °C, fixed, and stained in the frozen state (a process known as freeze-substitution) to prevent the collapse of proteoglycan chains. It can be seen that the proteoglycan molecules form a fine fibrous network in which a single cross-banded collagen fibril is embedded. The darker regions of the proteoglycan molecules are the core proteins; the lighter filaments are the glycosaminoglycan chains. (E.B. Hunziker, R.K. Schenk, J. Cell Biol. 98: 277-282, 1985 by copyright permission of the Rockefeller Univ. Press.)

Fig. 14-30. Electron micrograph of a Longitudinal section of rat tail tendon. The specimen was contrasted with a copper-containing stain to reveal proteoglycan molecules. The tendon consists of densely packed collagen fibrils; several such fibrils are visible in the photograph. The proteoglycan molecules appear as fine filaments surrounding each collagen fibril at regular intervals of approximately 65 nm (e.g., indicated by arrows b); this points to a specific interaction between collagen and proteoglycan molecules. In areas where proteoglycan filaments crossing the collagen fibrils are not visible (such as the region marked by the double arrow a), the plane of sectioning apparently passed directly through the fibril. (J.E. Scott, Biochem. J. 187: 887-891, 1980. Copyright 1980. Amer. Chem. Soc.)

Fig. 14-31. Some of the ordered conformations that two different polysaccharide chains, A and B, can adopt during gel formation in vitro. Since the interaction between molecules is restricted to specific regions of their chains (so-called junction zones) and does not extend over the entire molecule, each chain can associate with more than one partner and thereby form a gel lattice. Gel-forming polysaccharides include, in particular, agars (from Algae) and Pectins (from higher plants).

Not all proteoglycans are secreted Components of the extracellular matrix. Some are integral components of The Plasma Membrane, and in some cases, these proteoglycans contain a core protein that spans The Lipid Bilayer. Cell-surface proteoglycans typically consist of only a small number of glycosaminoglycan chains and appear to play a role in cell attachment to the extracellular matrix and in the organization of matrix macromolecules secreted by the cells.

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14.2.6. Collagen: The Major Protein of the Extracellular Matrix

Collagens are a family of highly distinctive fibrous proteins found in all Multicellular animals. They are secreted mainly by connective tissue cells and, in mammals, represent the most abundant proteins, accounting for about 25% of total protein mass. A hallmark of collagen molecules is their rigid, triple-helical structure. Three polypeptide chains, designated a-chains (each roughly 1,000 amino acids long), are wound around one another in a regular rope-like superhelix to form a collagen molecule approximately 300 nm long and 1.5 nm thick. Collagens are exceptionally rich in Proline and Glycine, both of which play vital roles in forming the triple helix. Proline, owing to its ring structure, stabilizes the left-handed helical conformation of each a-chain, which features three amino acid residues per turn. Glycine, the smallest amino acid (possessing only a single hydrogen atom as its side chain), recurs at every third position along the entire central portion of the a-chain. This allows the three helical a-chains to pack closely together to form the complete collagen superhelix (Fig. 14-32).

Figure 14-32. (A) Model of a single collagen a-chain in which each amino acid is represented by a sphere. The chain forms a left-handed helix with three amino acid residues per turn and with glycine (highlighted in dark) at every third position. Thus, the a-chain consists of a long series of Gly-X-Y triplets, where X and Y can be any amino acid (although typically one of them is proline). (B) Model of a segment of a collagen molecule in which three a-chains are wound into a helical cable. One a-helix is highlighted in light red, another in gray, and the third in white. Glycine is the only amino acid small enough to fit into the restricted axial space of the triple helix. Only a small portion of the molecule is shown; the entire molecule is about 300 nm long, and each chain consists of approximately 1,000 amino acid residues. (Sketches based on a model by W. L. Trus.)

Table 14-3. Four Major Types of Collagen and Their Properties

¹⁾ Note that types I and IV collagens contain two types of a-chains, whereas types II and III contain only a single type of a-chain. This table presents only the four major types of collagen, but more than 10 types of collagen and about 20 types of a-chains are currently known.

To date, nearly 20 different collagen chains have been identified, each encoded by a separate Gene. Different combinations of these genes are expressed in various tissues. Although in principle more than 1,000 types of triple-helical collagen molecules could be assembled from these twenty a-chains, only about 10 types have actually been found. Types I, II, III, and IV are the most thoroughly studied (Table 14-3). Types I, II, and III are fibrillar collagens, representing the major collagen types found in connective tissues, with Type I being particularly widespread. Once these Three types of collagen molecules pass from cells into the intercellular space, they organize into ordered polymers called collagen fibrils. These are slender (10–300 nm thick), rope-like structures many micrometers in length that are clearly visible in electron micrographs (Fig. 14-33). These fibrils often bundle together into larger aggregates several micrometers thick, which can be seen under a Light Microscope as collagen fibers. Type IV collagen molecules are found exclusively in the basement membrane; rather than forming fibrils, they organize into a planar meshwork that constitutes a major portion of the entire basement membrane (Section 14.2.11). The tissue arrangement of the remaining collagen types remains unclear.

Figure 14-33. Electron micrograph of a portion of a fibroblast surrounded by collagen fibrils in connective tissue. The extensively developed rough endoplasmic reticulum of the fibroblast reflects The Cell's high capacity for active Synthesis and Secretion of collagen and other extracellular matrix macromolecules. (Courtesy of Russell Ross.)

Many proteins with repeating Amino acid sequences have arisen through DNA segment duplications (Section 10.5.4), and fibrillar collagens appear to have evolved in this manner. Indeed, the genes encoding the a-chains of these collagens are very large (30–40 kb) and contain about 50 exons. Most exons consist of 54 NUCLEOTIDES or a multiple thereof, suggesting that these collagens originated through multiple duplications of an ancestral gene containing 54 nucleotides. This does not apply to Type IV collagen, which evidently evolved via a different pathway.

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14.2.7. Secreted Collagens Possess Nonhelical Segments at Both Ends [14, 15]

Individual polypeptide chains of collagen are synthesized on membrane-bound ribosomes and translocated into the lumen of the endoplasmic reticulum as longer precursors known as pro-a-chains. These precursors possess not only a short amino-terminal "signal peptide" required to guide the secreted protein across the ER membrane (Section 8.6.5), but also sets of additional amino acids called propeptides at both the amino and carboxy termini. Within the lumen of the endoplasmic reticulum, proline and Lysine residues are hydroxylated to form hydroxyproline and hydroxylysine, respectively. Each pro-a-chain then associates via Hydrogen Bonds with two other chains to form a triple-helical molecule known as procollagen (Fig. 14-34). In the extracellular space, the secreted forms of fibrillar collagens (though not Type IV collagen) are converted into collagen molecules by the Cleavage of their propeptides (see below).

Figure 14-34. In fibrillar collagen molecules, a-chains are initially synthesized as pro-a-chains containing extra peptides at both ends (highlighted in black), which are subsequently cleaved. The C-terminal propeptide appears to facilitate triple-helix formation during procollagen assembly. Note that the C-terminal propeptides in the procollagen molecule are covalently linked by Disulfide Bonds and frequently contain an oligosaccharide chain. The amino-terminal propeptides form a short triple-helical "minicollogen" region. The final collagen molecule comprises only the portion of the procollagen molecule highlighted in red; the remaining regions are cleaved away.

In other proteins, hydroxyproline and hydroxylysine residues (Fig. 14-35) are rare. Why are they present in collagen? Indirect evidence indicates that the hydroxyl groups of hydroxyproline residues form interchain hydrogen bonds that stabilize the triple helix. In particular, conditions that prevent proline hydroxylation (such as a deficiency in ascorbic acid, or Vitamin C) inhibit procollagen helix formation. Under normal conditions, collagens undergo continuous (albeit slow) degradation by specific extracellular Enzymes called collagenases. In scurvy—a human disease caused by dietary Vitamin C Deficiency—nonhydroxylated pro-a-chains are unable to form a triple helix and are rapidly degraded. Consequently, the gradual loss of previously established normal collagen in the matrix causes blood vessels to become extremely fragile and teeth to become loose. This indicates that collagen turnover and replacement occur relatively rapidly here. In contrast, in many other adult tissues, the renewal of collagen (and other extracellular matrix macromolecules) is normally very slow; an extreme example is bone, where collagen molecules persist for about 10 years before being degraded and replaced. By comparison, the half-lives of most cellular proteins are measured in hours or days.

Hydroxylation of lysine residues plays a different role: it is required for an unusual type of glycosylation (whose function remains unknown) and is crucial for cross-linking collagen molecules during their extracellular organization (Section 14.2.9).

14.2.8. Procollagen Molecules of Types I, II, and III Are Cleaved After Secretion to Form Collagen Molecules That Assemble into Fibrils [16]

Following secretion, the propeptides of type I, II, and III procollagen molecules are cleaved by specific enzymes outside the cell, converting procollagen into collagen (also referred to as tropocollagen). The resulting collagen molecules, 1.5 nm thick, assemble in the extracellular space into much larger collagen fibrils (10–300 nm thick). Fibril formation is driven in part by the intrinsic self-assembly tendency of collagen molecules. However, fibrillogenesis takes place near the cell surface, frequently within deep invaginations of the plasma membrane, and the underlying cortical Cytoskeleton can influence the site, rate, and orientation of fibril assembly (Section 14.2.18).

Figure 14-35. Structure of hydroxyproline and hydroxylysine residues—two modified amino acids commonly found in collagen.

Propeptides serve at least two functions: (1) they direct the intracellular assembly of triple-helical collagen molecules; and (2) because they are cleaved only after secretion, they prevent The formation of large collagen fibrils inside the cell, which would have catastrophic consequences for the cell. However, it is equally important to rid the tissue of propeptides once they have fulfilled their task. In certain hereditary disorders, such as Ehlers-Danlos syndrome, this process is defective, thereby impairing collagen fibril formation; As a result, affected individuals suffer from fragile skin and hypermobile joints.

Under the electron microscope, fixed and stained collagen fibrils exhibit a characteristic cross-striation with a 67 nm periodicity. This banding pattern reflects how individual molecules are packed within the fibril: as shown in Figure 14-36, they are arranged such that adjacent molecules are staggered relative to one another by nearly a quarter of their length (67 nm). This staggering arrangement presumably maximizes the tensile strength of the aggregate and produces the striations visible in negatively stained fibrils (Fig. 14-37). Nevertheless, the precise three-dimensional packing of molecules within these cylindrical fibrils remains incompletely understood.

Once collagen fibrils have formed in the extracellular space, their tensile strength is greatly enhanced by the formation of covalent cross-links both within and between individual collagen molecules (Fig. 14-38). Covalent bonds of this type occur exclusively in collagen and elastin. Blocking their formation renders collagen-containing tissues fragile, causing structures such as skin, tendons, and blood vessels to rupture easily. The quantity and type of cross-links vary among different tissues. For example, in the Achilles tendon, where tensile strength is paramount, collagen cross-linking is particularly abundant.

Fig. 14-36. Diagram of the staggered arrangement of collagen molecules (shown as arrows) within a collagen fibril. Molecules in adjacent rows are shifted by 67 nm relative to one another, and the gaps between molecules in a longitudinal row are 35 nm. With this spacing, the longitudinal arrangement of molecules repeats every five rows; for example, molecules in rows 1 and 6 lie directly opposite each other.

Fig. 14-37. This diagram illustrates how the staggered arrangement of collagen molecules results in the striated appearance of the fibril after negative staining. Because the contrasting agent fills only the gaps between molecules in each row, these gaps appear as dark bands. Bottom: an electron micrograph of a negatively stained fibril (courtesy of Robert Horne).

Fig. 14-38. Intramolecular and intermolecular cross-links between modified lysine side chains in a collagen fibril. Cross-linking occurs in several stages. First, certain lysine and hydroxylysine residues are deaminated by the extracellular enzyme lysyl oxidase, generating highly reactive aldehyde groups. These groups then spontaneously react to form covalent bonds with each other or with other lysine or hydroxylysine residues, so that more than two amino acid side chains may participate in a single cross-link. Some of the bonds formed are relatively unstable and eventually are modified into a variety of more stable cross-links. Note that most cross-links form between the short nonhelical segments at both ends of the collagen molecules (see Fig. 14-35).

14.2.9. The Organization of Collagen Fibrils in the Extracellular Matrix is Adapted to Tissue Needs [17]

Collagen fibrils vary in thickness and are organized differently in various tissues. For example, in mammalian skin they are interwoven like wickerwork, enabling them to resist mechanical stress from all directions. In tendons, they are gathered into parallel bundles aligned along the main axis, whereas in mature bone and cornea their arrangement resembles alternating layers of plywood, with the fibrils in each layer running parallel to one another and nearly at right angles to those in adjacent layers. A similar organization is found in tadpole skin (Fig. 14-39).

Connective tissue cells themselves determine the size and arrangement of collagen fibrils. Cells can express one or more genes for Different types of fibrillar procollagen molecules (including minor types not listed in Table 14-3), thereby regulating the distribution of molecules following secretion. By controlling the sequential Cleavage of the amino- and carboxy-terminal propeptides, secreting various types and amounts of noncollagenous matrix macromolecules alongside collagen, and directing collagen fibril formation in close association with the plasma membrane, cells can dictate the geometry and properties of the fibrils in their immediate environment. Finally, the formation of a greater or lesser number of cross-links in collagen depends on the required tensile strength. Figure 14-40 schematically outlines the steps of fibrillar collagen synthesis and higher-order structure assembly.

14.2.10. Cells Can Participate in the Organization of Secreted Collagen Fibrils by Altering Matrix Tension [18]

There is yet another mechanism by which collagen-secreting cells determine the Spatial Organization of the matrix they produce. Fibroblasts exert traction on the collagen they have synthesized by crawling over it and pulling on it, which promotes its compaction into layers and elongation into fibers. This mechanical role of fibroblasts in Structuring the collagen matrix has been clearly demonstrated in vitro. When fibroblasts are placed in a culture dish containing a gel of randomly oriented interlaced collagen fibrils, the cells begin to migrate and pull the surrounding fibrils along with them, causing the gel to shrink to a fraction of its original volume. In a similar manner, a group of fibroblasts can surround themselves with a capsule of densely packed collagen fibers oriented tangentially.

Fig. 14-39. Electron micrograph of a cross-section of tadpole skin. Layers of collagen fibrils are laid down like the plies of plywood, such that fibrils in adjacent layers intersect at right angles. This arrangement is also found in mature bone and the cornea. (Courtesy of Jerome Gross.)

Fig. 14-40. Schematic Overview of the various intracellular and extracellular events in collagen fibril formation. As an example of how fibrils can be arranged in an orderly fashion in the extracellular space, their subsequent assembly into a large collagen fiber visible by light Microscopy is shown. Covalent cross-links that stabilize the extracellular aggregates are omitted. Many hereditary disorders in humans are known in which collagen fibril formation is impaired, which is hardly surprising given the large number of enzymatic reactions involved in this process.

Fig. 14-41. Micrograph of the region between two explants of embryonic chick heart. These explants, containing numerous fibroblasts and Muscle cells, were grown on a collagen gel for 4 days. Note the dense band of parallel collagen fibers forming between the explants. (D. Stopak, A. K. Harris. Dev. Biol. 90: 383–398, 1982.)

When two pieces of embryonic tissue containing fibroblasts are placed a short distance apart on a collagen gel, the collagen organizes into a compact band of oriented fibers connecting the two explants (Fig. 14-41). Fibroblasts then migrate from both explants along these parallel fibers. Thus, fibroblasts influence the arrangement of collagen fibers, which in turn affect the distribution of fibroblasts. It is quite possible that fibroblasts play a similar role in organizing the extracellular matrix at the macroscopic level—for instance, in the formation of tendons and ligaments, as well as the tough, Dense connective tissue sheaths that surround and hold together most organs.

14.2.11. Type IV Collagen Molecules Are Organized into a Sheet-Like Network [19]

Type IV collagen molecules differ in several respects from fibrillar collagen. First, the regular repeating Amino Acid Sequence Gly-X-Y in the type IV a chain is interrupted in numerous places, locally disrupting the triple-helical STRUCTURE OF THE collagen molecule. Second, type IV "procollagen" molecules are not cleaved upon exiting the cell and thus retain their propeptides; interacting via these uncleaved propeptide domains, they organize into a sheet-like, multilaminar network rather than into fibrils. Electron microscopic studies indicate that these molecules appear to associate via their carboxy-terminal propeptides into dimers, which further associate to form an extensive network (Fig. 14-42). Disulfide and other covalent cross-links stabilize these associations. Type IV collagen sheets appear to form the foundation of all basement membranes; Other components of the basement membrane will be discussed later (Section 14.2.15).

Fig. 14-42. Hypothetical scheme of the assembly of type IV collagen molecules into the multilayered network that forms the foundation of every basement membrane. The model is based on ELECTRON MICROGRAPHS OF rotary-shadowed preparations of such molecules during in vitro assembly. (Based on P. D. Yurchenco, E. C. Tsilibary, A. S. Charonis, H. Furthmayr. J. Histochem. Cytochem. 34: 93–102, 1986.)

14.2.12. Elastin is a Random-Coil, Cross-Linked Protein That Confers Resilience to Tissues [20]

Certain tissues, such as skin, blood vessels, and lungs, must be not only strong but also elastic. An extensive network of elastic fibers in the extracellular matrix provides these tissues with The ability to recoil after temporary stretching. The major component of such fibers, elastin, is a highly hydrophobic, nonglycosylated protein (about 830 amino acids long) that, much like collagen, is exceptionally rich in proline and glycine, but unlike collagen contains very little hydroxyproline and no hydroxylysine at all. Elastin molecules are secreted into the extracellular space, where they form fibers and sheets in which the molecules are cross-linked into an extensively branched network (Fig. 14-43). Cross-links form between lysine residues via the same mechanism as in collagen (see Fig. 14-38). The function of elastin (unlike that of most other proteins) requires its peptide chains to remain extended and maintain a flexible, random-coil conformation (Fig. 14-44). It is precisely this structure—a network of cross-linked, randomly folded elastic fibers—that allows the entire network to stretch and snap back like a rubber band (Fig. 14-45). Elastin fibers can stretch to at least five times their resting length without breaking, while long, inextensible collagen fibrils woven into the elastic fiber network limit the extensibility of the whole matrix and thereby prevent tissue rupture.

Fig. 14-43. A dense network of elastic fibers in a cross-section of human skin connective tissue (dermis). Scanning electron micrograph. To remove collagen and glycosaminoglycans, the tissue was subjected to heat under pressure. (T. Tsuji, R. M. Lavker, A. M. Kligman. J. Microscop. 115: 165-173, 1978.)

Fig. 14-44. Diagram of various random conformations of an elastin molecule. Unlike most proteins, elastin does not adopt a unique structure, but constantly transitions from one partially unfolded random conformation to another, a third, and so on.

Fig. 14-45. Elastin molecules are linked by covalent cross-links (highlighted in color) into an extensive network. Because each molecule in such a network is capable of stretching and contracting, the entire network stretches and contracts like rubber.

Elastic fibers are composed of more than just elastin. They also contain a glycoprotein that is distributed mainly as microfibrils on the fiber surface. Elastic fibers form in close association with the plasma membrane of the cell that secretes elastin and the microfibrillar glycoprotein. Microfibrils appear earlier than elastin itself and presumably help the cell organize the elastin molecules it secretes into the fibers and sheets that form in the extracellular matrix.

14-14

14.2.13. Fibronectin is an extracellular glycoprotein that promotes cell-matrix adhesion [21]

The extracellular matrix contains several adhesive glycoproteins that bind to both cells and other matrix macromolecules, promoting cell attachment to the matrix. Of these, the best characterized is fibronectin—a large, fibril-forming glycoprotein found throughout the animal kingdom. It is a dimer composed of two identical subunits (each nearly 2,500 amino acid residues long); the subunits are joined by a pair of disulfide bonds near their carboxyl ends and folded into a series of globular domains separated by segments of a flexible polypeptide chain (Fig. 14-46). As sequencing shows, the fibronectin molecule consists primarily of three short, repeatedly occurring amino acid sequences, suggesting that the gene encoding fibronectin evolved through the repeated duplication of three small genes.

There are three forms of fibronectin: 1) a soluble dimeric form (plasma fibronectin), which circulates in blood and tissue fluids and is thought to promote blood clotting, wound healing, and phagocytosis; 2) fibronectin oligomers that can be temporarily attached to the cell surface (surface fibronectin); and 3) a poorly soluble fibrillar form of fibronectin in the extracellular matrix (matrix fibronectin). In surface and matrix aggregates, fibronectin dimers are linked to one another by additional disulfide cross-links.

Fig. 14-46. A. Schematic diagram of the structure of a fibronectin dimer. B. Electron micrographs of individual platinum-shadowed molecules. The two polypeptide chains are similar but not identical; they are joined near the carboxyl end by two disulfide bonds. Each chain is folded into a series of globular domains connected by flexible polypeptide segments. Individual domains are designed to bind a specific molecule or cell (as indicated for three domains). For simplicity, not all known binding sites are shown. [B-J. Engel et al. J. Mol. Biol. 150: 97-120, 1981. Copyright Academic Press Inc. (London) Ltd.]

Fibronectin is a multifunctional molecule in which different globular domains play distinct roles. For example, one domain binds to collagen, another to heparin, a third to specific receptors On the surface of various cell types, and so on (see Fig. 14-46). Thus, fibronectin participates in matrix organization and promotes cell attachment to it.

The Role of various domains, particularly the cell-binding domains, has been studied by cleaving the molecule into individual domains with Proteolytic Enzymes or by synthesizing specific protein fragments chemically or using recombinant DNA technology. For instance, the domain responsible for cell binding was isolated from proteolytic fragments, and its amino acid sequence was determined. Synthetic peptides corresponding to various segments of this domain were prepared, and it was discovered that a specific tripeptide sequence (Arg-Gly-Asp, or R-G-D) is responsible for the binding activity. Peptides containing this RGD sequence compete for cell-binding sites and thereby inhibit cell attachment to fibronectin; conversely, when these peptides are bound to a solid surface, they promote cell attachment to it. The RGD sequence is not unique to fibronectin—it is shared by numerous extracellular adhesive proteins and is recognized by a whole family of homologous cell-surface receptors that bind these proteins (Section 14.2.17). Although the molecules recognized by these receptors share a common tripeptide sequence, each receptor specifically recognizes its own small group of adhesive molecules; thus, receptor binding must also depend on other regions of the adhesive protein sequence.

Fibronectin is important not only for Cell Adhesion, but also for cell migration. In invertebrate and vertebrate embryos, it appears to guide migration in many cases. For example, large amounts of fibronectin are found along The pathway of prospective mesoderm cells during Gastrulation in amphibians (Section 16.1.4). The migration of these cells can be inhibited either by injecting Antibodies against fibronectin into the blastocoel or by introducing Polypeptides containing the cell-binding tripeptide but lacking the matrix-binding domains of fibronectin. It is believed that fibronectin promotes cell migration by assisting in their attachment to the matrix. This action must be delicately balanced so that cell-matrix adhesion occurs without resulting in their immobilization. We will return later to the question of how such a balance can be achieved by the numerous adhesive molecules involved in charting the pathways of morphogenetic movements.

14.2.14. Multiple Forms of fibronectin are synthesized via alternative RNA splicing [22]

As a member of the extensive family of RGD-containing adhesive molecules, fibronectin itself can exist in many forms, as already noted; even within a single dimer, The polypeptide chains may differ slightly. Yet all the various fibronectin polypeptide chains are encoded by a single large gene; in the rat, it is over 70 kb long and contains about 50 exons, making it one of the largest genes characterized to date. Transcription yields a single long RNA molecule that undergoes Alternative Splicing, generating one or more of approximately 20 different mRNAs depending on the cell type. It remains unclear what determines the choice of RNA splicing pathways and what the functional differences are between the resulting polypeptide chains. Some evidence suggests that one of the functions of alternative splicing of the human fibronectin RNA transcript is to add an extra cell-binding domain, distinct from the RGD-containing region, to certain fibronectin molecules.

Fibronectin is not the only secreted glycoprotein involved in cell-matrix adhesion. For example, tenascin is also an extracellular adhesive glycoprotein, although it is much less abundant than fibronectin and most common in embryonic tissues. In The Nervous system, it is secreted by glial cells, and some Neurons are thought to bind to it via a specific cell-surface proteoglycan. Tenascin is a large complex of six disulfide-linked polypeptide chains radiating from a center like the spokes of a wheel (see Fig. 14-51).

Some cells, particularly epithelial cells, secrete a different type of extracellular adhesive glycoprotein called laminin, which is a major protein of all basement membranes. It binds both to epithelial cells (as well as certain other cell types) and to type IV collagen, the principal collagen type in the basement membrane.

14.2.15. The basement membrane is a specialized form of extracellular matrix containing mainly type IV collagen, proteoglycans, and laminin [23]

The basement membrane is a thin layer of Specialized Extracellular Matrix underlying sheets of epithelial cells; it also surrounds individual muscle cells, fat cells, and Schwann cells (which wrap around peripheral nerve fibers to form myelin). Thus, the basement membrane separates these cells or cell layers from the surrounding or underlying connective tissue. In other locations, such as renal glomeruli or pulmonary alveoli, the basement membrane is situated between two different cell layers, where it acts as a highly efficient filter (Fig. 14-47). However, the role of basement membranes is not limited to structural support and filtration functions. They can determine cell polarity, influence cell metabolism, organize proteins in adjacent Plasma Membranes, induce Cell Differentiation, and, like fibronectin, serve as specific "highways" for cell Migrations.

Fig. 14-47. Three arrangements of the basement membrane (shown as a black line). The basement membrane may surround cells (e.g., muscle cells), underlie layers of epithelial cells, or be positioned between two cell layers (as in renal glomeruli). Note that in renal glomeruli, both cell layers contain gaps, so that the permeability barrier determining which molecules pass from the blood into the urine is the basement membrane. Because the membrane in glomeruli is the result of the fusion of two basement membranes formed by endothelial and epithelial cells, it is twice as thick as most such membranes.

Fig. 14-48. Basement membrane of the chick embryo cornea. Scanning electron micrograph. Part of the epithelial cells (Ep) has been removed to reveal the upper surface of the basement membrane (BM). Note that a dense network of collagen fibrils (C) interacts with the lower surface of the membrane. The macromolecules of the basement membrane are synthesized by the overlying epithelial cells. (Courtesy of Robert Trelstad.)

Fig. 14-49. A. Electron micrographs of laminin molecules (platinum-shadowed preparation). B. Diagram of the laminin structure. This multidomain glycoprotein consists of three polypeptide chains (A, B₁, and B₂) linked by disulfide bonds into the shape of an asymmetric cross. [A-J. Engel et al. J. Mol. Biol., 150, 97-120, 1981. Copyright Acad. Press Inc. (London) Ltd; B-after V. L. Hogan et al. In: Basement Membranes (S. Shibata, ed.), pp. 147-154. Amsterdam, Elsevier, 1985.]

The basement membrane is synthesized primarily by the cells resting upon it (Fig. 14-48). It is essentially a dense sheet of type IV collagen (see Fig. 14-42) with additional specific molecules on both of its surfaces that facilitate its attachment to neighboring cells or the matrix. Although the composition of basement membranes varies somewhat from tissue to tissue and even from region to region (Section 14.2.17), all these membranes contain type IV collagen along with proteoglycans (primarily heparan sulfates) and the glycoproteins laminin and entactin. Laminin is a large (molecular mass approx. 850,000) complex of three very long polypeptide chains arranged in a cross shape and held together by disulfide bonds (Fig. 14-49). Like fibronectin, it consists of several functional domains: one binds to type IV collagen, one to heparan sulfate, and one or more others to cell-surface protein receptors for laminin. It is also believed that a single dumbbell-shaped entactin molecule is tightly bound to each laminin molecule at the junction between the short arms of the "cross" and its long arm.

Fig. 14-50. Structure of the basement membrane underlying epithelial cells, as seen in cross-sections under an electron microscope.

Fig. 14-51. Comparison of the shape and size of some major extracellular matrix macromolecules.

As seen in electron micrographs following conventional fixation and contrasting, most basement membranes consist of two layers: an electron-lucent layer (lamina lucida, or rare layer) adjacent to the plasma membrane of the cells resting on the basement membrane (typically epithelial cells), and an underlying electron-dense layer (lamina densa). In some cases, a third layer containing collagen fibrils (lamina reticularis) is also present, anchoring the basement membrane to the underlying connective tissue. Some authors refer to the entire three-layer complex as the "basement membrane" (Fig. 14-50), which is typically thick enough to be resolved with a light microscope. The detailed Molecular organization of the basement membrane is yet to be fully elucidated, although immunoelectron microscopy indicates that the lamina densa appears to consist primarily of type IV collagen with proteoglycan molecules on both of its sides; laminin is thought to be present mainly on the side of the lamina densa facing the plasma membrane, where it participates in anchoring epithelial cells to the basement membrane, whereas fibronectin helps mediate the binding of matrix macromolecules and connective tissue cells on the opposite side.

Figure 14-51 compares the Sizes and Shapes of some of the most important basement membrane macromolecules.

14.2.16. Basement membranes perform diverse and complex functions [24]

The Functions of the basement membrane are extraordinarily diverse. In the renal glomeruli, an unusually thick basement membrane acts as a molecular filter, regulating the passage of molecules from the blood into the urine (see Fig. 14-47). Proteoglycans appear to be essential for this function, as their removal by specific enzymes leads to a loss of the membrane's filtration properties. The basement membrane can also serve as a selective barrier for cells. For example, the basement membrane underlying an epithelial layer generally prevents fibroblasts of the underlying connective tissue from contacting the epithelial cells, yet it does not obstruct the passage of macrophages, lymphocytes, and nerve fibers.

The basement membrane plays a crucial role in tissue regeneration following injury. When muscular, nervous, or Epithelial Tissue is damaged, the surviving basement membrane serves as a scaffold for the migration of regenerating cells. This allows the original tissue architecture to be readily restored. A striking example of the basement membrane's role in regeneration is found at the Neuromuscular Junction, where a nerve cell transmits a stimulus to a Skeletal Muscle fiber.

At the neuromuscular junction (synapse), the basal lamina possesses a specialized chemical architecture recognized, for instance, by antibodies that bind exclusively to this region. One of the functions of the basal lamina here appears to be the coordination of the spatial organization of components on both sides of the synapse. Evidence supporting The Central Role of the synaptic basal lamina in nerve or muscle regeneration will be discussed in Chapter 19 (Section 19.8.3). Such studies clearly demonstrate that we still have much to learn about the chemical and functional specialization of the basal lamina. They also suggest that minor (and as yet unidentified) extracellular matrix components may play a critical role in directing morphogenetic processes during embryonic development.

14.2.17. Integrins Mediate Cell Attachment to the Extracellular Matrix [25]

To understand how the extracellular matrix interacts with cells, we must examine both the cell-surface molecules that bind to matrix components and the matrix molecules themselves. As noted previously, certain proteoglycans are integral components of the plasma membrane; their core proteins either span the lipid bilayer or are covalently attached to it. By binding to most extracellular matrix components, these proteoglycans facilitate cell adhesion to the matrix. Conversely, matrix components also bind to the cell surface via specific receptor proteoglycans. Given such complex macromolecular interactions within the extracellular space, the question of where the plasma membrane ends and the extracellular matrix begins is largely semantic. For instance, the cell glycocalyx frequently comprises components of both structures (see Section 6.3.1).

Matrix receptors differ from cell-surface receptors for hormones and other soluble signaling molecules in that they bind their ligands with relatively low affinity (K_a = 10⁶-10⁸ l/mol) and are expressed at cell surfaces in concentrations roughly 10 to 100 times higher. Consequently, such receptors likely function cooperatively, enabling cells to respond to an organized array of matrix ligands rather than isolated molecules. This notion is supported by the observation that soluble matrix fragments attaching to cells generally fail to elicit the cellular response triggered by the same components immobilized within the matrix.

The fibronectin receptor of mammalian fibroblasts is one of the most thoroughly characterized matrix receptors. It was originally identified as a plasma membrane glycoprotein that binds to a fibronectin affinity Column and can be eluted with a small peptide containing the cell-attachment sequence RGD (Section 14.2.13). The receptor is a noncovalently linked complex of two distinct high-molecular-weight polypeptide chains, designated the a- and ß-chains. It functions as a transmembrane linker, mediating interactions between intracellular cytoskeletal Actin and extracellular fibronectin (Figure 14-52). As we will see later, such transmembrane interactions can polarize both the cell and the matrix. Numerous other matrix receptors—including those binding collagen and laminin—have been characterized and shown to be structurally related to the fibroblast fibronectin receptor. Collectively termed integrins, all are heterodimers featuring a- and ß-chains homologous to those of the fibronectin receptor. Most of these likely recognize RGD sequences within their respective matrix binding partners.

There are at least three distinct subfamilies within the vast integrin superfamily; members of a given subfamily share a common a-chain but differ in their ß-chains. One subfamily includes the fibroblast fibronectin receptor and at least five other members. A second subfamily comprises platelet receptors that bind various matrix components, including fibronectin and fibrinogen—a protein essential for platelet aggregation during blood clotting. In Glanzmann's thrombasthenia, an inherited deficiency of these receptors, hemostasis is severely impaired. The third integrin subfamily consists of receptors expressed predominantly on leukocyte surfaces; one is designated LFA-1 (lymphocyte function-associated antigen 1), and another is called Mac-1 because of its high Abundance on macrophages. These receptors participate in both cell-cell and cell-matrix interactions, playing a vital role in the defensive Immune Response against infection. Individuals with leukocyte adhesion deficiency lack the ability to synthesize the common ß-subunit; consequently, their leukocytes are devoid of an entire receptor family, rendering these patients susceptible to recurrent bacterial infections. Several cell-surface glycoproteins involved in position-specific cell adhesion in Drosophila larvae also belong to the integrin superfamily, although their phylogenetic relationship to the three mammalian subfamilies remains unclear.

Figure 14-52. The cell-surface fibronectin receptor and its subunits. Electron microscopy of isolated receptors indicates a molecular shape closely resembling this diagram. A globular "HEAD" protrudes more than 20 nm beyond the lipid bilayer. By binding to extracellular fibronectin on the outside and to the intracellular cytoskeleton (via the attachment protein talin) on the inside, the receptor acts as a transmembrane linker. Both the a- and ß-chains are glycosylated (not shown) and held together by noncovalent forces. The a-chain is initially synthesized as a single polypeptide chain with a Molecular Weight of 140,000, which is subsequently cleaved into a small transmembrane chain and a large extracellular chain linked by a disulfide bond. The extracellular domain of the ß-chain contains Cysteine-rich repeat motifs, implying a high density of interchain disulfide bonds (not shown). The fibronectin receptor belongs to an extensive superfamily of homologous matrix receptors known as integrins, most of which recognize RGD sequences in their target extracellular proteins.

However, not all matrix receptors belong to this superfamily. For example, certain cells appear to use an unrelated transmembrane glycoprotein to attach to collagen, and many cells, as mentioned earlier, possess integral membrane proteoglycans that anchor them to the extracellular matrix.

14.2.18. The Cytoskeleton and Extracellular Matrix Interact Across the Plasma Membrane [26]

Extracellular matrix macromolecules exert profound effects on the behavior of cultured cells, influencing not only their motility but also their shape, polarity, metabolism, and differentiation. For instance, corneal epithelial cells grown on artificial substrates synthesize very little collagen; however, when cultured on laminin, collagen, or fibronectin, they accumulate and secrete collagen in large quantities. Further Examples of matrix-mediated Regulation of cellular metabolism and differentiation will be discussed in Chapter 17 (Section 17.7.1).

The matrix can also direct the ORGANIZATION OF THE cellular cytoskeleton. Typically, the basal surfaces of epithelial cells grown on plastic or Glass assume an irregular Morphology, and the underlying intracellular cytoskeleton appears disorganized. Yet when identical cells are cultured on a substratum composed of appropriate extracellular matrix macromolecules, their basal surfaces flatten and smooth out, and the subjacent cytoskeleton becomes as highly ordered as in intact tissue. Comparable results have been obtained with cultures of oncogenically transformed fibroblasts. Transformed cells frequently produce less fibronectin than normal cultured cells and exhibit distinct behavioral abnormalities: for instance, they adhere poorly to substrates, fail to spread out properly, and are unable to form organized intracellular bundles of actin filaments known as stress fibers (Section 11.1.17). In some of these cells, the fibronectin deficiency is at least partially responsible for their aberrant phenotype; when such cells are cultured on a matrix of organized fibronectin fibrils, they flatten and assemble intracellular stress fibers aligned parallel to the extracellular fibronectin strands.

Interactions between the extracellular matrix and the cytoskeleton are reciprocal: intracellular actin filaments can dictate the spatial arrangement of secreted fibronectin molecules. For example, in culture, extracellular fibronectin fibrils align themselves along the axes of adjacent intracellular stress fibers in neighboring fibroblasts (Figure 14-53). Treating such cells with cytochalasin—an agent that disrupts intracellular actin filaments—causes the fibronectin fibrils to detach from the cell surface (much like the cell rounding observed during mitosis). Clearly, a physical linkage must exist between extracellular fibronectin and intracellular actin filaments across the fibroblast plasma membrane. This connection is mediated by the previously described fibronectin receptors, transmembrane proteins that couple fibronectin to actin filaments via intracellular attachment proteins such as talin (see Section 11.2.8 and Figure 14-52). The talin-binding domain of the receptor contains a Tyrosine residue whose phosphorylation by a tyrosine-specific protein kinase presumably inactivates this site, thereby severing The Link Between fibronectin and cortical actin filaments. It is thought that cell attachment to the matrix may be regulated in this manner by specific growth factors that stimulate tyrosine-specific Kinases (see Figure 13-37).

Fig. 14-53. Immunofluorescence micrographs showing extracellular fibronectin fibers (A) and intracellular actin filament bundles (B) in three cultured rat fibroblasts. Fibronectin was visualized using rhodamine-conjugated antifibronectin antibodies, and actin using fluorescein-conjugated antiactin antibodies. Note that the orientation of the fibronectin fibers closely parallels that of the actin filament bundles. (R. O. Hynes, A. T. Destree. Cell 15: 875-886, 1978. Copyright Cell Press.)

Because the cellular cytoskeleton can organize the matrix macromolecules it secretes, which in turn organize the cytoskeleton of contacting cells, the extracellular matrix can, in principle, propagate structural order from cell to cell (Fig. 14-54). Thus, it is thought that the extracellular matrix plays a central role in establishing and maintaining cell orientation within tissues and organs during development; for example, the parallel alignment of fibroblasts and collagen fibers in tendons may partly reflect such cell-matrix interactions. Transmembrane matrix receptors act as "mediators" in this ordering process.

Summary

Cells in connective tissues are embedded in a complex extracellular matrix that not only binds Cells and Tissues together but also influences the development, polarity, and behavior of contacting cells. The matrix contains various fiber-forming proteins interwoven within a hydrated gel composed of a network of glycosaminoglycan chains. Glycosaminoglycans are a diverse group of long, negatively charged polysaccharide chains that (with the exception of hyaluronic acid) are covalently linked to core proteins to form proteoglycan molecules.

Fig. 14-54. Hypothetical Scheme for the cell-to-cell propagation of structural order via the extracellular matrix. For simplicity, the diagram illustrates how a single cell influences the orientation of neighboring cells, but the same mechanism can also explain mutual cell-to-cell interactions.

Fiber-forming proteins fall into two functional classes: predominantly structural (collagen and elastin) and primarily adhesive (such as fibronectin and laminin). Fibrillar collagens (types I, II, and III) are ropelike triple-helical molecules that aggregate in the extracellular space into long fibrils, which in turn can assemble into a variety of highly ordered structures. Type IV collagen molecules assemble into sheetlike networks that form the structural backbone of all basal laminae. Elastin molecules, cross-linked extensively, form a network of fibers and sheets that can stretch and recoil, imparting resilience to the matrix. Fibronectin and laminin are prominent examples of large adhesive matrix glycoproteins; fibronectin is widely distributed in connective tissues, whereas laminin is found predominantly in the basal lamina. Through their multiple binding domains, these proteins promote cell adhesion and participate in the organizing Influence of the extracellular matrix on cells. Many of these adhesive glycoproteins contain a common tripeptide sequence (RGD) that is recognized by integrins—members of a superfamily of homologous transmembrane receptors for matrix components.

All matrix proteins and polysaccharides are locally secreted by the cells in contact with the matrix; in close association with the outer surface of the plasma membrane, these molecules can become organized. Because the structure and orientation of the matrix in turn influence the orientation of contacting cells, it is highly likely that structural order is propagated across the matrix from cell to cell.