Chapter 19:  Cell Junctions, Cell Adhesion, and the Extracellular Matrix

Occluding Junctions Form a Selective Permeability Barrier Across Epithelial Cell Sheets

All epithelia have at least one important function in common: they serve as selective permeability barriers, separating fluids on either side that have a different chemical composition. This function requires that the adjacent cells be sealed together by occluding junctions. Tight junctions have this barrier role in vertebrates, as we illustrate by considering the epithelium of the mammalian small intestine, or gut.

The epithelial cells lining the small intestine form a barrier that keeps the gut contents in the gut cavity, the lumen. At the same time, however, the cells must transport selected nutrients across the epithelium from the lumen into the extracellular fluid that permeates the connective tissue on the other side (see Figure 19-1). From there, these nutrients diffuse into small blood vessels to provide nourishment to the organism. This transcellular transport depends on two sets of membrane-bound membrane transport proteins. One set is confined to the apical surface of the epithelial cell (the surface facing the lumen) and actively transports selected molecules into the cell from the gut. The other set is confined to the basolateral (basal and lateral) surfaces of the cell, and it allows the same molecules to leave the cell by facilitated diffusion into the extracellular fluid on the other side of the epithelium. To maintain this directional transport, the apical set of transport proteins must not be allowed to migrate to the basolateral surface of the cell, and the basolateral set must not be allowed to migrate to the apical surface. Furthermore, the spaces between epithelial cells must be tightly sealed, so that the transported molecules cannot diffuse back into the gut lumen through these spaces (Figure 19-2).

The tight junctions between epithelial cells are thought to have both of these roles. First, they function as barriers to the diffusion of some membrane proteins (and lipids) between apical and basolateral domains of the plasma membrane (see Figure 19-2). Mixing of such proteins and lipids occurs if tight junctions are disrupted, for example, by removing the extracellular Ca²⁺ that is required for tight junction integrity. Second, tight junctions seal neighboring cells together so that, if a low-molecular-weight tracer is added to one side of an epithelium, it will generally not pass beyond the tight junction (Figure 19-3). This seal is not absolute, however. Although all tight junctions are impermeable to macromolecules, their permeability to small molecules varies greatly in different epithelia. Tight junctions in the epithelium lining the small intestine, for example, are 10,000 times more permeable to inorganic ions, such as Na⁺, than the tight junctions in the epithelium lining the urinary bladder. These differences reflect differences in tight junction proteins that form the junctions.

Epithelial cells can transiently alter their tight junctions to permit an increased flow of solutes and water through breaches in the junctional barriers. Such paracellular transport is especially important in the absorption of amino acids and monosaccharides from the lumen of the intestine, where their concentration can increase enough after a meal to drive passive transport in the desired direction.

When tight junctions are visualized by freeze-fracture electron microscopy, they seem to be composed of a branching network of sealing strands that completely encircles the apical end of each cell in the epithelial sheet (Figure 19-4A and B). In conventional electron micrographs, the outer leaflets of the two interacting plasma membranes are seen to be tightly apposed where sealing strands are present (Figure 19-4C). The ability of tight junctions to restrict the passage of ions through the spaces between cells is found to increase logarithmically with increasing numbers of strands in the network, suggesting that each strand acts as an independent barrier to ion flow.

Each tight junction sealing strand is composed of a long row of transmembrane adhesion proteins embedded in each of the two interacting plasma membranes. The extracellular domains of these proteins join directly to one another to occlude the intercellular space (Figure 19-5). The major transmembrane proteins in a tight junction are the claudins, which are essential for tight junction formation and function and differ in different tight junctions. A specific claudin found in kidney epithelial cells, for example, is required for Mg²⁺ to be resorbed from the urine into the blood. A mutation in the gene encoding this claudin results in excessive loss of Mg²⁺ in the urine. A second major transmembrane protein in tight junctions is occludin, the function of which is uncertain. Claudins and occludins associate with intracellular peripheral membrane proteins called ZO proteins (a tight junction is also known as a zonula occludens), which anchor the strands to the actin cytoskeleton.

In addition to claudins, occludins, and ZO proteins, several other proteins can be found associated with tight junctions. These include some that regulate epithelial cell polarity and others that help guide the delivery of components to the appropriate domain of the plasma membrane. Thus, the tight junction may serve as a regulatory center to help in coordinating multiple cell processes.

In invertebrates, septate junctions are the main occluding junction. More regular in structure than a tight junction, they likewise form a continuous band around each epithelial cell. But their morphology is distinct because the interacting plasma membranes are joined by proteins that are arranged in parallel rows with a regular periodicity (Figure 19-6). A protein called Discs-large, which is required for the formation of septate junctions in Drosophila, is structurally related to the ZO proteins found in vertebrate tight junctions. Mutant flies that are deficient in this protein not only lack septate junctions but also develop epithelial tumors. This observation suggests that the normal regulation of cell proliferation in epithelial tissues may depend, in part, on intracellular signals that emanate from occluding junctions.

Anchoring Junctions Connect the Cytoskeleton of a Cell Either to the Cytoskeleton of Its Neighbors or to the Extracellular Matrix

The lipid bilayer is flimsy and cannot by itself transmit large forces from cell to cell or from cell to extracellular matrix. Anchoring junctions solve the problem by forming a strong membrane-spanning structure that is tethered inside the cell to the tension-bearing filaments of the cytoskeleton (Figure 19-7).

Anchoring junctions are widely distributed in animal tissues and are most abundant in tissues that are subjected to severe mechanical stress, such as heart, muscle, and epidermis. They are composed of two main classes of proteins (Figure 19-8). Intracellular anchor proteins form a distinct plaque on the cytoplasmic face of the plasma membrane and connect the junctional complex to either actin filaments or intermediate filaments. Transmembrane adhesion proteins have a cytoplasmic tail that binds to one or more intracellular anchor proteins and an extracellular domain that interacts with either the extracellular matrix or the extracellular domains of specific transmembrane adhesion proteins on another cell. In addition to anchor proteins and adhesion proteins, many anchoring junctions contain intracellular signaling proteins that enable the junctions to signal to the cell interior.

Anchoring junctions occur in two functionally different forms:

• 1

  Adherens junctions and desmosomes hold cells together and are formed by transmembrane adhesion proteins that belong to the cadherin family.

• 2

  Focal adhesions and hemidesmosomes bind cells to the extracellular matrix and are formed by transmembrane adhesion proteins of the integrin family.

On the intracellular side of the membrane, adherens junctions and focal adhesions serve as connection sites for actin filaments, while desmosomes and hemidesmosomes serve as connection sites for intermediate filaments (see Table 19-1, p. 1067).

Adherens Junctions Connect Bundles of Actin Filaments from Cell to Cell

Adherens junctions occur in various forms. In many nonepithelial tissues, they take the form of small punctate or streaklike attachments that indirectly connect the cortical actin filaments beneath the plasma membranes of two interacting cells. But the prototypical examples of adherens junctions occur in epithelia, where they often form a continuous adhesion belt (or zonula adherens) just below the tight junctions, encircling each of the interacting cells in the sheet. The adhesion belts are directly apposed in adjacent epithelial cells, with the interacting plasma membranes held together by the cadherins that serve here as transmembrane adhesion proteins.

Within each cell, a contractile bundle of actin filaments lies adjacent to the adhesion belt, oriented parallel to the plasma membrane. The actin is attached to this membrane through a set of intracellular anchor proteins, including catenins, vinculin, and α-actinin, which we consider later. The actin bundles are thus linked, via the cadherins and anchor proteins, into an extensive transcellular network (Figure 19-9). This network can contract with the help of myosin motor proteins (discussed in Chapter 16), and it is thought to help in mediating a fundamental process in animal morphogenesis—the folding of epithelial cell sheets into tubes and other related structures (Figure 19-10).

The assembly of tight junctions between epithelial cells seems to require the prior formation of adherens junctions. Anti-cadherin antibodies that block the formation of adherens junctions, for example, also block the formation of tight junctions.

Desmosomes Connect Intermediate Filaments from Cell to Cell

Desmosomes are buttonlike points of intercellular contact that rivet cells together (Figure 19-11A). Inside the cell, they serve as anchoring sites for ropelike intermediate filaments, which form a structural framework of great tensile strength (Figure 19-11B). Through desmosomes, the intermediate filaments of adjacent cells are linked into a net that extends throughout the many cells of a tissue. The particular type of intermediate filaments attached to the desmosomes depends on the cell type: they are keratin filaments in most epithelial cells, for example, and desmin filaments in heart muscle cells.

The general structure of a desmosome is illustrated in Figure 19-11C, and some of the proteins that form it are shown in Figure 19-11D. The junction has a dense cytoplasmic plaque composed of a complex of intracellular anchor proteins (plakoglobin and desmoplakin) that are responsible for connecting the cytoskeleton to the transmembrane adhesion proteins. These adhesion proteins (desmoglein and desmocollin), like those at an adherens junction, belong to the cadherin family. They interact through their extracellular domains to hold the adjacent plasma membranes together.

The importance of desmosome junctions is demonstrated by some forms of the potentially fatal skin disease pemphigus. Affected individuals make antibodies against one of their own desmosomal cadherin proteins. These antibodies bind to and disrupt the desmosomes that hold their skin epithelial cells (keratinocytes) together. This results in a severe blistering of the skin, with leakage of body fluids into the loosened epithelium.

Anchoring Junctions Formed by Integrins Bind Cells to the Extracellular Matrix: Focal Adhesions and Hemidesmosomes

Some anchoring junctions bind cells to the extracellular matrix rather than to other cells. The transmembrane adhesion proteins in these cell-matrix junctions are integrins—a large family of proteins distinct from the cadherins. Focal adhesions enable cells to get a hold on the extracellular matrix through integrins that link intracellularly to actin filaments. In this way, muscle cells, for example, attach to their tendons at the myotendinous junction. Likewise, when cultured fibroblasts migrate on an artificial substratum coated with extracellular matrix molecules, they also grip the substratum at focal adhesions, where bundles of actin filaments terminate. At all such adhesions, the extracellular domains of transmembrane integrin proteins bind to a protein component of the extracellular matrix, while their intracellular domains bind indirectly to bundles of actin filaments via the intracellular anchor proteins talin, α-actinin, filamin, and vinculin (Figure 19-12B).

Hemidesmosomes, or half-desmosomes, resemble desmosomes morphologically and in connecting to intermediate filaments, and, like desmosomes, they act as rivets to distribute tensile or shearing forces through an epithelium. Instead of joining adjacent epithelial cells, however, hemidesmosomes connect the basal surface of an epithelial cell to the underlying basal lamina (Figure 19-13). The extracellular domains of the integrins that mediate the adhesion bind to a laminin protein (discussed later) in the basal lamina, while an intracellular domain binds via an anchor protein (plectin) to keratin intermediate filaments. Whereas the keratin filaments associated with desmosomes make lateral attachments to the desmosomal plaques (see Figure 19-11C and D), many keratin filaments associated with hemidesmosomes have their ends buried in the plaque (see Figure 19-13).

Although the terminology for the various anchoring junctions can be confusing, the molecular principles (for vertebrates, at least) are relatively simple (Table 19-2). Integrins in the plasma membrane anchor a cell to extracellular matrix molecules; cadherin family members in the plasma membrane anchor it to the plasma membrane of an adjacent cell. In both cases, there is an intracellular coupling to cytoskeletal filaments, either actin filaments or intermediate filaments, depending on the types of intracellular anchor proteins involved.

Gap Junctions Allow Small Molecules to Pass Directly from Cell to Cell

With the exception of a few terminally differentiated cells such as skeletal muscle cells and blood cells, most cells in animal tissues are in communication with their neighbors via gap junctions. Each gap junction appears in conventional electron micrographs as a patch where the membranes of two adjacent cells are separated by a uniform narrow gap of about 2–4 nm. The gap is spanned by channel-forming proteins (connexins). The channels they form (connexons) allow inorganic ions and other small water-soluble molecules to pass directly from the cytoplasm of one cell to the cytoplasm of the other, thereby coupling the cells both electrically and metabolically. Dye-injection experiments suggest a maximal functional pore size for the connecting channels of about 1.5 nm, implying that coupled cells share their small molecules (such as inorganic ions, sugars, amino acids, nucleotides, vitamins, and the intracellular mediators cyclic AMP and inositol trisphosphate) but not their macromolecules (proteins, nucleic acids, and polysaccharides) (Figure 19-14). This cell coupling has important functional implications, many of which are only beginning to be understood.

Evidence that gap junctions mediate electrical and chemical coupling has come from many experiments. When, for example, connexin mRNA is injected into either frog oocytes or gap-junction-deficient cultured cells, channels with the properties expected of gap-junction channels can be demonstrated electrophysiologically where pairs of injected cells make contact.

The mRNA injection approach has been useful for identifying new gap-junction proteins. Genetic studies in the fruit fly Drosophila identified the gene shaking B, which, when mutated, resulted in flies that failed to jump in response to a visual stimulus. Although these flies had defective gap junctions, the sequence of the Shaking B protein did not resemble a connexin, and the function of the protein was unclear. An injection of the shaking B mRNA into frog oocytes, however, led to the formation of functional gap-junction channels, just like those formed by connexins. Shaking B thus became the first member of a new family of invertebrate gap-junction proteins called innexins. There are more than 15 innexin genes in Drosophila and 25 in the nematode C. elegans.

A Gap-Junction Connexon Is Made Up of Six Transmembrane Connexin Subunits

Connexins are four-pass transmembrane proteins, six of which assemble to form a channel, a connexon. When the connexons in the plasma membranes of two cells in contact are aligned, they form a continuous aqueous channel that connects the two cell interiors (Figure 19-15A). The connexons hold the interacting plasma membranes at a fixed distance apart—hence the gap.

Gap junctions in different tissues can have different properties. The permeability of their individual channels can vary, reflecting differences in the connexins that form the junctions. In humans, for instance, there are 14 distinct connexins, each encoded by a separate gene and each having a distinctive, but sometimes overlapping, tissue distribution. Most cell types express more than one type of connexin, and two different connexin proteins can assemble into a heteromeric connexon, the properties of which differ from those of a homomeric connexon constructed from a single type of connexin. Moreover, adjacent cells expressing different connexins can form intercellular channels in which the two aligned half-channels are different (Figure 19-15B). Each gap junction can contain a cluster of a few to many thousands of connexons (Figure 19-16B).

Gap Junctions Have Diverse Functions

In tissues containing electrically excitable cells, coupling via gap junctions serves an obvious purpose. Some nerve cells, for example, are electrically coupled, allowing action potentials to spread rapidly from cell to cell, without the delay that occurs at chemical synapses. This is advantageous when speed and reliability are crucial, as in certain escape responses in fish and insects. Similarly, in vertebrates, electrical coupling through gap junctions synchronizes the contractions of both heart muscle cells and the smooth muscle cells responsible for the peristaltic movements of the intestine.

Gap junctions also occur in many tissues that do not contain electrically excitable cells. In principle, the sharing of small metabolites and ions provides a mechanism for coordinating the activities of individual cells in such tissues and for smoothing out random fluctuations in small molecule concentrations in different cells. In the liver, for example, the release of noradrenaline from sympathetic nerve endings in response to a fall in blood glucose levels stimulates hepatocytes to increase glycogen breakdown and release glucose into the blood. Not all the hepatocytes are innervated by sympathetic nerves, however. By means of the gap junctions that connect hepatocytes, the signal is transmitted from the innervated hepatocytes to the noninnervated ones. Thus, mice with a mutation in the major connexin gene expressed in the liver fail to mobilize glucose normally when blood glucose levels fall.

The normal development of ovarian follicles also depends on gap-junction-mediated communication—in this case, between the oocyte and the surrounding granulosa cells. A mutation in the gene that encodes the connexin that normally couples these two cell types causes infertility (Figure 19-17).

Cell coupling via gap junctions also seems to be important in embryogenesis. In early vertebrate embryos, beginning with the late eight-cell stage in mouse embryos, most cells are electrically coupled to one another. As specific groups of cells in the embryo develop their distinct identities and begin to differentiate, they commonly uncouple from surrounding tissue. As the neural plate folds up and pinches off to form the neural tube, for instance (see Figure 19-10), its cells uncouple from the overlying ectoderm. Meanwhile, the cells within each group remain coupled with one another and therefore tend to behave as a cooperative assembly, all following a similar developmental pathway in a coordinated fashion.

The Permeability of Gap Junctions Can Be Regulated

Like conventional ion channels (discussed in Chapter 11), individual gap-junction channels do not remain continuously open; instead, they flip between open and closed states. Moreover, the permeability of gap junctions is rapidly (within seconds) and reversibly reduced by experimental manipulations that decrease the cytosolic pH or increase the cytosolic concentration of free Ca²⁺ to very high levels. Thus, gap-junction channels are dynamic structures that can undergo a reversible conformational change that closes the channel in response to changes in the cell.

The purpose of the pH regulation of gap-junction permeability is unknown. In one case, however, the purpose of Ca²⁺ control seems clear. When a cell is damaged, its plasma membrane can become leaky. Ions present at high concentration in the extracellular fluid, such as Ca²⁺ and Na⁺, then move into the cell, and valuable metabolites leak out. If the cell were to remain coupled to its healthy neighbors, these too would suffer a dangerous disturbance of their internal chemistry. But the large influx of Ca²⁺ into the damaged cell causes its gap-junction channels to close immediately, effectively isolating the cell and preventing the damage from spreading to other cells.

Gap-junction communication can also be regulated by extracellular signals. The neurotransmitter dopamine, for example, reduces gap-junction communication between a class of neurons in the retina in response to an increase in light intensity (Figure 19-18). This reduction in gap-junction permeability helps the retina switch from using rod photoreceptors, which are good detectors of low light, to cone photoreceptors, which detect color and fine detail in bright light.

Figure 19-19 summarizes the various types of junctions formed by vertebrate cells in an epithelium. In the most apical portion of the cell, the relative positions of the junctions are the same in nearly all vertebrate epithelia. The tight junction occupies the most apical position, followed by the adherens junction (adhesion belt) and then by a special parallel row of desmosomes; together these form a structure called a junctional complex. Gap junctions and additional desmosomes are less regularly organized.

In Plants, Plasmodesmata Perform Many of the Same Functions as Gap Junctions

The tissues of a plant are organized on different principles from those of an animal. This is because plant cells are imprisoned within rigid cell walls composed of an extracellular matrix rich in cellulose and other polysacharides, as we discuss later. The cell walls of adjacent cells are firmly cemented to those of their neighbors, which eliminates the need for anchoring junctions to hold the cells in place. But a need for direct cell-cell communication remains. Thus, plant cells have only one class of intercellular junctions, plasmodesmata (singular, plasmodesma). Like gap junctions, they directly connect the cytoplasms of adjacent cells.

In plants, however, the cell wall between a typical pair of adjacent cells is at least 0.1 μm thick, and so a structure very different from a gap junction is required to mediate communication across it. Plasmodesmata solve the problem. With a few specialized exceptions, every living cell in a higher plant is connected to its living neighbors by these structures, which form fine cytoplasmic channels through the intervening cell walls. As shown in Figure 19-20A, the plasma membrane of one cell is continuous with that of its neighbor at each plasmodesma, and the cytoplasm of the two cells is connected by a roughly cylindrical channel with a diameter of 20–40 nm. Thus, the cells of a plant can be viewed as forming a syncytium, in which many cell nuclei share a common cytoplasm.

Running through the center of the channel in most plasmodesmata is a narrower cylindrical structure, the desmotubule, which is continuous with elements of the smooth endoplasmic reticulum in each of the connected cells (Figures 19-20B and 19-21A and B). Between the outside of the desmotubule and the inner face of the cylindrical channel formed by plasma membrane is an annulus of cytosol through which small molecules can pass from cell to cell. As each new cell wall is assembled during the cytokinesis phase of cell division, plasmadesmata are created within it. They form around elements of smooth ER that become trapped across the developing cell plate (discussed in Chapter 18). They can also be inserted de novo through pre-existing cell walls, where they are commonly found in dense clusters called pit fields (Figure 19-21C). When no longer required, plasmadesmata can be readily removed.

In spite of the radical difference in structure between plasmodesmata and gap junctions, they seem to function in remarkably similar ways. Evidence obtained by injecting tracer molecules of different sizes suggests that plasmo-desmata allow the passage of molecules with a molecular weight of less than about 800, which is similar to the molecular-weight cutoff for gap junctions. As with gap junctions, transport through plasmodesmata is regulated. Dye-injection experiments, for example, show that there can be barriers to the movement of even low-molecular-weight molecules between certain cells, or groups of cells, that are connected by apparently normal plasmodesmata; the mechanisms that restrict communication in these cases are not understood.

During plant development, groups of cells within the shoot and root meristems signal to one another in the process of defining their future fates (discussed in Chapter 21). Some gene regulatory proteins involved in this process of cell fate determination pass from cell to cell through plasmodesmata. They bind to components of the plasmodesmata and override the size exclusion mechanism that would otherwise prevent their passage. In some cases, the mRNA that encodes the protein can also pass through. Some plant viruses also exploit this route: infectious viral RNA, or even intact virus particles, can pass from cell to cell in this way. These viruses produce proteins that bind to components of the plasmodesmata to increase dramatically the effective pore size of the channel. As the functional components of plasmodesmata are unknown, it is unclear how endogenous or viral macromolecules regulate the transport properties of the channel to pass through it.

Summary

Many cells in tissues are linked to one another and to the extracellular matrix at specialized contact sites called cell junctions. Cell junctions fall into three functional classes: occluding junctions, anchoring junctions, and communicating junctions. Tight junctions are occluding junctions that are crucial in maintaining the concentration differences of small hydrophilic molecules across epithelial cell sheets. They do so in two ways. First, they seal the plasma membranes of adjacent cells together to create a continuous impermeable, or semipermeable, barrier to diffusion across the cell sheet. Second, they act as barriers in the lipid bilayer to restrict the diffusion of membrane transport proteins between the apical and the basolateral domains of the plasma membrane in each epithelial cell. Septate junctions serve as occluding junctions in invertebrate tissues.

The main types of anchoring junctions in vertebrate tissues are adherens junctions, desmosomes, focal adhesions, and hemidesmosomes. Adherens junctions and desmosomes connect cells together and are formed by cadherins, while focal adhesions and hemidesmosomes connect cells to the extracellular matrix and are formed by integrins. Adherens junctions and focal adhesions are connecting sites for bundles of actin filaments, whereas desmosomes and hemidesmosomes are connecting sites for intermediate filaments.

Gap junctions are communicating junctions composed of clusters of connexons that allow molecules smaller than about 1000 daltons to pass directly from the inside of one cell to the inside of the next. Cells connected by gap junctions share many of their inorganic ions and other small molecules and are therefore chemically and electrically coupled. Gap junctions are important in coordinating the activities of electrically active cells, and they have a coordinating role in other groups of cells as well. Plasmodesmata are the only intercellular junctions in plants. Although their structure is entirely different, and they can sometimes transport informational macromolecules, in general, they function like gap junctions.

Animal Cells Can Assemble into Tissues Either in Place or After They Migrate

Many simple tissues, including most epithelial tissues, derive from precursor cells whose progeny are prevented from wandering away by being attached to the extracellular matrix, to other cells, or to both (Figure 19-22). But the accumulating cells do not simply remain passively stuck together; instead, the tissue architecture is generated and actively maintained by selective adhesions that the cells make and progressively adjust.

Selective adhesion is even more essential for the development of tissues that have more complex origins involving cell migration. In these tissues, one population of cells invades another and assembles with it, and perhaps with other migrant cells, to form an orderly structure. In vertebrate embryos, for example, cells from the neural crest break away from the epithelial neural tube, of which they are initially a part, and migrate along specific paths to many other regions (discussed in Chapter 21). There they assemble with other cells and with one another to differentiate into a variety of tissues, including those of the peripheral nervous system (Figure 19-23).

Cell motility and cell adhesion combine to bring about these kinds of morphogenetic events. The process requires some mechanism for directing the cells to their final destination. This may involve chemotaxis or chemorepulsion, the secretion of a soluble chemical that attracts or repels migrating cells, respectively, or pathway guidance, the laying down of adhesive or repellent molecules in the extracellular matrix or on cell surfaces to guide the migrating cells along the right paths. Then, once a migrating cell has reached its destination, it must recognize and join other cells of the appropriate type to assemble into a tissue. How this latter process occurs can be studied if cells of different embryonic tissues are artificially mingled, after which they often spontaneously sort out to restore a more normal arrangement, as we discuss next.

Dissociated Vertebrate Cells Can Reassemble into Organized Tissues Through Selective Cell-Cell Adhesion

Unlike adult vertebrate tissues, which are difficult to dissociate, embryonic vertebrate tissues are easily dissociated. This is usually done by treating the tissue with low concentrations of a proteolytic enzyme such as trypsin, sometimes combined with the removal of extracellular Ca²⁺ and Mg²⁺ with a divalent-cation chelator (such as EDTA). These reagents disrupt the protein-protein interactions (many of which are divalent-cation-dependent) that hold cells together. Remarkably, the dissociated cells often reassemble in vitro into structures that resemble the original tissue. Such findings reveal that tissue structure is not just a product of history; it is actively maintained and stabilized by the system of affinities that cells have for one another and for the extracellular matrix.

A striking example of this phenomenon is seen when dissociated cells from two embryonic vertebrate organs, such as the liver and the retina, are mixed together and artificially formed into a pellet: the mixed aggregates gradually sort out according to their organ of origin. More generally, disaggregated cells are found to adhere more readily to aggregates of their own organ than to aggregates of other organs. Evidently there are cell-cell recognition systems that make cells of the same differentiated tissue preferentially adhere to one another; these adhesive preferences are presumably important in stabilizing tissue architecture.

Cells adhere to each other and to the extracellular matrix through cell-surface proteins called cell adhesion molecules (CAMs)—a category that includes the transmembrane adhesion proteins we have already discussed. CAMs can be cell-cell adhesion molecules or cell-matrix adhesion molecules. Some CAMs are Ca²⁺-dependent, whereas others are Ca²⁺-independent. The Ca²⁺-dependent CAMs seem to be primarily responsible for the tissue-specific cell-cell adhesion seen in early vertebrate embryos, explaining why these cells can be disaggregated with Ca²⁺-chelating agents.

CAMs were initially identified by making antibodies against cell-surface molecules and then testing the antibodies for their ability to inhibit cell-cell adhesion in a test tube. Those rare antibodies that inhibit the adhesion were then used to characterize and isolate the adhesion molecule recognized by the antibodies.

Cadherins Mediate Ca²⁺-dependent Cell-Cell Adhesion

The cadherins are the major CAMs responsible for Ca²⁺-dependent cell-cell adhesion in vertebrate tissues. The first three cadherins that were discovered were named according to the main tissues in which they were found: E-cadherin is present on many types of epithelial cells; N-cadherin on nerve, muscle, and lens cells; and P-cadherin on cells in the placenta and epidermis. All are also found in various other tissues; N-cadherin, for example, is expressed in fibroblasts, and E-cadherin is expressed in parts of the brain. These and other classical cadherins are related in sequence throughout their extracellular and intracellular domains. There are also a large number of nonclassical cadherins, with more than 50 expressed in the brain alone. The nonclassical cadherins include proteins with known adhesive function, such as the desmosomal cadherins discussed earlier and the diverse protocadherins found in the brain. They also include proteins that appear to have nonadhesive functions, such as T-cadherin, which lacks a transmembrane domain and is attached to the plasma membrane of nerve and muscle cells by a glycosylphosphatidylinositol (GPI) anchor, and the Fat protein, which was first identified as the product of a tumor-suppressor gene in Drosophila. Together, the classical and nonclassical cadherin proteins constitute the cadherin superfamily (Table 19-3).

Cadherins are expressed in both invertebrates and vertebrates. Virtually all vertebrate cells seem to express one or more cadherins, according to the cell type. They are the main adhesion molecules holding cells together in early embryonic tissues. In culture, the removal of extracellular Ca²⁺ or treatment with anti-cadherin antibodies disrupts embryonic tissues, and, if cadherin-mediated adhesion is left intact, antibodies against other adhesion molecules have little effect. Mutations that inactivate the function of E-cadherin cause mouse embryos to fall apart and die early in development.

Most cadherins are single-pass transmembrane glycoproteins about 700–750 amino acids long. Structural studies suggest that they associate in the plasma membrane to form dimers or larger oligomers. The large extracellular part of the polypeptide chain is usually folded into five or six cadherin repeats, which are structurally related to immunoglobulin (Ig) domains (Figure 19-24A and B). The crystal structures of E- and N-cadherin have helped to explain the importance of Ca²⁺ binding for cadherin function. The Ca²⁺ ions are positioned between each pair of cadherin repeats, locking the repeats together to form a stiff, rodlike structure: the more Ca²⁺ ions that are bound, the more rigid the structure is. If Ca²⁺ is removed, the extracellular part of the protein becomes floppy and is rapidly degraded by proteolytic enzymes (Figure 19-24C).

Cadherins Have Crucial Roles in Development

E-cadherin is the best-characterized cadherin. It is usually concentrated in adherens junctions in mature epithelial cells, where it helps connect the cortical actin cytoskeletons of the cells it holds together (see Figure 19-9B). E-cadherin is also the first cadherin expressed during mammalian development. It helps cause compaction, an important morphological change that occurs at the eight-cell stage of mouse embryo development. During compaction, the loosely attached cells, called blastomeres, become tightly packed together and joined by intercellular junctions. Antibodies against E-cadherin block blastomere compaction, whereas antibodies that react with various other cell-surface molecules on these cells do not.

It seems likely that cadherins are also crucial in later stages of vertebrate development, since their appearance and disappearance correlate with major morphogenetic events in which tissues segregate from one another. As the neural tube forms and pinches off from the overlying ectoderm, for example, neural tube cells lose E-cadherin and acquire other cadherins, including N-cadherin, while the cells in the overlying ectoderm continue to express E-cadherin (Figure 19-25). Then, when the neural crest cells migrate away from the neural tube, these cadherins become scarcely detectable, and another cadherin (cadherin-7) appears that helps hold the migrating cells together as loosely associated cell groups. Finally, when the cells aggregate to form a ganglion, they re-express N-cadherin (see Figure 19-23).

If N-cadherin is overexpressed in the emerging neural crest cells, the cells fail to escape from the neural tube. Thus, not only do cell groups that originate from one cell layer exhibit distinct patterns of cadherin expression when separating from one another, but these switches in cadherin expression seem to be intimately involved in the separation process.

Cadherins Mediate Cell-Cell Adhesion by a Homophilic Mechanism

How do cell-cell adhesion molecules such as the cadherins bind cells together? Three possibilities are illustrated in Figure 19-26: (1) in homophilic binding, molecules on one cell bind to other molecules of the same kind on adjacent cells; (2) in heterophilic binding, the molecules on one cell bind to molecules of a different kind on adjacent cells; (3) in linker-dependent binding, cell-surface receptors on adjacent cells are linked to one another by secreted multivalent linker molecules. Although all three mechanisms have been found to operate in animals, cadherins usually link cells by the homophilic mechanism. In a line of cultured fibroblasts called L cells, for example, the cells neither express cadherins nor adhere to one another. When L cells are transfected with DNA encoding E-cadherin, the transfected cells become adherent to one another by a Ca²⁺-dependent mechanism, and the adhesion is inhibited by anti-E-cadherin antibodies. Since cadherin proteins can bind directly to one another and the transfected cells do not bind to untransfected L cells, one can conclude that E-cadherin binds cells together through the interaction of two E-cadherin molecules on different cells.

If L cells expressing different cadherins are mixed together, they sort out and aggregate separately, indicating that different cadherins preferentially bind to their own type (Figure 19-27A), mimicking what happens when cells derived from tissues that express different cadherins are mixed together. A similar segregation of cells occurs if L cells expressing different amounts of the same cadherin are mixed together (Figure 19-27B). It therefore seems likely that both qualitative and quantitative differences in the expression of cadherins have a role in organizing tissues.

In the nervous system especially, there are many different cadherins, each with a distinct but overlapping pattern of expression (Figure 19-28A). As they are concentrated at synapses, they are thought to have a role in synapse formation and stabilization. Some of the nonclassical cadherins, such as the protocadherins, are strong candidates for helping to determine the specificity of synaptic connections. Like antibodies, they differ in their N-terminal (variable) regions but are identical in their C-terminal (constant) regions. The extracellular variable region and intracellular constant region are encoded by separate exons, with the variable-region exons arranged in tandem arrays upstream of the constant-region exons (Figure 19-28B). The diversity of protocadherins is generated by a combination of differential promoter usage and alternative RNA splicing, rather than by site-specific recombination as occurs in antibody diversification (discussed in Chapter 24).

Cadherins Are Linked to the Actin Cytoskeleton by Catenins

Most cadherins, including all classical and some nonclassical ones, function as transmembrane adhesion proteins that indirectly link the actin cytoskeletons of the cells they join together. This arrangement occurs in adherens junctions (see Figure 19-9B). The highly conserved cytoplasmic tail of these cadherins interacts indirectly with actin filaments by means of a group of intracellular anchor proteins called catenins (Figure 19-29). This interaction is essential for efficient cell-cell adhesion, as classical cadherins that lack their cytoplasmic domain cannot hold cells strongly together.

As discussed earlier, the nonclassical cadherins that form desmosomes interact with intermediate filaments, rather than with actin filaments. Their cytoplasmic domain binds to a different set of intracellular anchor proteins, which in turn bind to intermediate filaments (see Figure 19-11D).

Some cells can regulate the adhesive activity of their cadherins. This regulation may be important for the cellular rearrangements that occur within epithelia when these cell sheets change their shape and organization during animal development (see Figure 19-10). The molecular basis of this regulation is uncertain but may involve the phosphorylation of anchor proteins attached to the cytoplasmic tail of the cadherins.

Some cadherins can help transmit signals to the cell interior. Vascular endothelial cadherin (VE-cadherin), for example, not only mediates adhesion between endothelial cells but also is required for endothelial cell survival. Although endothelial cells that do not express VE-cadherin still adhere to one another via N-cadherin, they do not survive (see Table 19-3, p. 1082). Their survival depends on an extracellular signal protein called vascular endothelial growth factor (VEGF), which binds to a receptor tyrosine kinase (discussed in Chapter 15) that uses VE-cadherin as a co-receptor.

Selectins Mediate Transient Cell-Cell Adhesions in the Bloodstream

White blood cells lead a nomadic life, moving to and fro between the bloodstream and the tissues, and this necessitates special adhesive properties. These properties depend on selectins. Selectins are cell-surface carbohydrate-binding proteins (lectins) that mediate a variety of transient, Ca²⁺-dependent, cell-cell adhesion interactions in the bloodstream. There are at least three types: L-selectin on white blood cells, P-selectin on blood platelets and on endothelial cells that have been locally activated by an inflammatory response, and E-selectin on activated endothelial cells. Each selectin is a transmembrane protein with a highly conserved lectin domain that binds to a specific oligosaccharide on another cell (Figure 19-30A).

Selectins have an important role in binding white blood cells to endothelial cells lining blood vessels, thereby enabling the blood cells to migrate out of the bloodstream into a tissue. In a lymphoid organ, the endothelial cells express oligosaccharides that are recognized by L-selectin on lymphocytes, causing the lymphocytes to loiter and become trapped. Conversely, at sites of inflammation, the endothelial cells switch on expression of selectins, which recognize the oligosaccharides on white blood cells and platelets, flagging the cells down to help deal with the local emergency. Selectins do not act alone, however; they collaborate with integrins, which strengthen the binding of the blood cells to the endothelium. The cell-cell adhesions mediated by both selectins and integrins are heterophilic (see Figure 19-26): selectins bind to specific oligosaccharides on glycoproteins and glycolipids, while integrins bind to specific proteins.

Selectins and integrins act in sequence to let white blood cells leave the bloodstream and enter tissues. The selectins mediate a weak adhesion because the binding of the lectin domain of the selectin to its carbohydrate ligand is of low affinity. This allows the white blood cell to adhere weakly and reversibly to the endothelium, rolling along the surface of the blood vessel propelled by the flow of blood. The rolling continues until the blood cell activates its integrins (discussed later), now causing the cell to bind strongly to the endothelial cell surface and to crawl out of the blood vessel between adjacent endothelial cells (Figure 19-30B).

Members of the Immunoglobulin Superfamily of Proteins Mediate Ca²⁺-independent Cell-Cell Adhesion

Cadherins, selectins, and integrins all depend on extracellular Ca²⁺ (or Mg²⁺ for some integrins) to function in cell adhesion. The molecules responsible for Ca²⁺-independent cell-cell adhesion belong mainly to the large and ancient immunoglobulin (Ig) superfamily of proteins. These proteins contain one or more Ig-like domains that are characteristic of antibody molecules (discussed in Chapter 24). One of the best-studied examples is the neural cell adhesion molecule (N-CAM), which is expressed by a variety of cell types, including most nerve cells. N-CAM is the most prevalent of the Ca²⁺-independent cell-cell adhesion molecules in vertebrates, and, like cadherins, it is thought to bind cells together by a homophilic mechanism (between N-CAM molecules on adjacent cells). Some Ig-like cell-cell adhesion proteins, however, use a heterophilic mechanism. Intercellular adhesion molecules (ICAMs) on endothelial cells, for example, bind to integrins on blood cells when blood cells migrate out of the bloodstream, as just discussed.

There are at least 20 forms of N-CAM, all generated by alternative splicing of an RNA transcript produced from a single gene. In all forms, the large extracellular part of the polypeptide chain is folded into five Ig-like domains (Figure 19-31). Some forms of N-CAM carry an unusually large quantity of sialic acid (with chains containing hundreds of repeating sialic acid units). By virtue of their negative charge, these long polysialic acid chains hinder cell adhesion, and there is increasing evidence that N-CAM heavily loaded with sialic acid serves to prevent adhesion, rather than cause it.

Although cadherins and Ig family members are frequently expressed on the same cells, the adhesions mediated by cadherins are much stronger, and they are largely responsible for holding cells together, segregating cell collectives into discrete tissues, and maintaining tissue integrity. N-CAM and other members of the Ig family seem to contribute more to the fine-tuning of these adhesive interactions during development and regeneration. In the developing rodent pancreas, for example, the formation of the islets of Langerhans requires cell aggregation, followed by cell sorting. Whereas inhibition of cadherin function prevents cell aggregation and islet formation, loss of N-CAM only impairs the cell sorting process, so that disorganized islets form.

Similarly, whereas mutant mice that lack N-cadherin die early in development, mutant mice that lack N-CAM develop relatively normally, although they do have some defects in neural development. Mutations in other genes that encode Ig-like cell adhesion proteins, however, can cause more severe neural defects. L1 gene mutations in humans, for example, cause mental retardation and other neurological defects resulting from abnormalities in the migration of nerve cells and their axons.

The importance of Ig-like cell adhesion proteins in connecting the neurons of the developing nervous system has been demonstrated dramatically in Drosophila. An N-CAM-like protein called fasciclin III (FAS3) is expressed transiently on some motor neurons, as well as on the muscle cells they normally innervate. If FAS3 is genetically removed from these neurons, they fail to recognize their muscle targets and do not make synapses with them. Conversely, if motor neurons that normally do not express FAS3 are made to express this protein, they now synapse with FAS3-expressing muscle cells to which they normally do not connect. It seems that FAS3 mediates these synaptic connections by a homophilic “matchmaking” mechanism.

Like the cadherins, some Ig-like proteins do more than just bind cells together. They can also transmit signals to the cell interior. Some forms of N-CAM in nerve cells, for example, associate with Src family cytoplasmic tyrosine kinases (discussed in Chapter 15), which relay signals onward by phosphorylating intracellular proteins on tyrosines. Other Ig family members are transmembrane tyrosine phosphatases (discussed in Chapter 15) that help guide growing axons to their target cells, presumably by dephosphorylating specific intracellular proteins.

Multiple Types of Cell-Surface Molecules Act in Parallel to Mediate Selective Cell-Cell Adhesion

A single type of cell utilizes multiple molecular mechanisms in adhering to other cells. Some of these mechanisms involve organized cell junctions, while others do not (Figure 19-32). Each cell in a multicellular animal contains an assortment of cell-surface receptors that enables the cell to respond specifically to a complementary set of soluble extracellular signal molecules, such as hormones and growth factors. Likewise, each cell in a tissue has a particular combination (and concentration) of cell-surface adhesion molecules that enables it to bind in its own characteristic way to other cells and to the extracellular matrix. And just as receptors for soluble extracellular signal molecules generate intracellular signals that alter the cell's behavior, so too can cell adhesion molecules, although the signaling mechanisms they use are generally not as well understood.

Unlike receptors for soluble signal molecules, which bind their specific ligand with high affinity, the receptors that bind to molecules on cell surfaces or in the extracellular matrix usually do so with relatively low affinity. These low-affinity receptors rely on the enormous increase in binding strength gained through the simultaneous binding of multiple receptors to multiple ligands on an opposing cell or in the adjacent matrix. One could call this the “Velcro principle.”

We have seen, however, that the interaction of the extracellular binding domains of these cell-surface molecules is not enough to ensure cell adhesion. At least in the case of cadherins and, as we shall see, integrins, the adhesion molecules must also attach (via anchor proteins) to the cytoskeleton inside the cell. The cytoskeleton is thought to assist and stabilize the lateral clustering of the adhesion molecules to facilitate multipoint binding. The cytoskeleton is also required to enable the adhering cell to exert traction on the adjacent cell or matrix (and vice versa). Thus, the mixture of specific types of cell-cell adhesion molecules present on any two cells, as well as their concentration, cytoskeletal linkages, and distribution on the cell surface, determine the total affinity with which the two cells bind to each other.

Nonjunctional Contacts May Initiate Cell-Cell Adhesions That Junctional Contacts Then Orient and Stabilize

We have seen that adhesive contacts between cells play a crucial part in organizing the formation of tissues and organs in developing embryos or in adult tissues undergoing repair after injury. Most often, these contacts do not involve the formation of organized intercellular junctions that show up as specialized structures in the electron microscope. The interacting plasma membranes are simply seen to come close together and run parallel, separated by a space of 10–20 nm. This type of “nonjunctional” contact may be optimal for cell locomotion—close enough to provide traction and to allow transmembrane adhesion proteins to interact, but not so tight, or so solidly anchored to the cytoskeleton, as to immobilize the cell.

A reasonable hypothesis is that nonjunctional cell-cell adhesion proteins initiate cell-cell adhesions, which are then oriented and stabilized by the assembly of full-blown intercellular junctions. Many of the transmembrane proteins involved can diffuse in the plane of the plasma membrane and, in this or other ways, can be recruited to sites of cell-cell (and cell-matrix) contact, enabling nonjunctional adhesions to enlarge and mature into junctional adhesions. This has been demonstrated for some integrins and cadherins, which help initiate cell adhesion and then later become integral parts of cell junctions. The migrating tip of an axon, for example, has an even distribution of cadherins on its surface, which helps it adhere to other cells along the migration pathway. It also has an intracellular pool of cadherins in vesicles just under the plasma membrane. When the axon reaches its target cell, it is thought to release the intracellular cadherin molecules onto the cell surface, where they help form a stable contact, which matures into a chemical synapse.

As discussed earlier, antibodies against adherens junction proteins block the formation of tight junctions, as well as adherens junctions, suggesting that the assembly of one type of junction can be a prerequisite for the formation of another. An increasing number of monoclonal antibodies and peptide fragments have been produced that can block a single type of cell adhesion molecule. Moreover, an increasing number of genes encoding these cell-surface proteins have been identified, creating new opportunities for manipulating the adhesive machinery of cells in culture and in experimental animals. It is now possible, therefore, to inactivate the various cell-cell adhesion proteins in combinations—a requirement for deciphering the rules of cell-cell recognition and binding used to build complex tissues.

Summary

Cells dissociated from various tissues of vertebrate embryos preferentially reassociate with cells from the same tissue when they are mixed together. This tissue-specific recognition process in vertebrates is mediated mainly by a family of Ca²⁺-dependent cell-cell adhesion proteins called cadherins, which hold cells together by a homophilic interaction between these transmembrane proteins on adjacent cells. For this interaction to be effective, the cytoplasmic part of the cadherins must be linked to the cytoskeleton by cytoplasmic anchor proteins called catenins.

Two other families of transmembrane adhesion proteins have major roles in cell-cell adhesion. Selectins function in transient Ca²⁺-dependent cell-cell adhesions in the bloodstream by binding to specific oligosaccharides on the surface of another cell. Members of the immunoglobulin superfamily, including N-CAM, mediate Ca²⁺-independent cell-cell adhesion processes that are especially important during neural development.

Even a single cell type uses multiple molecular mechanisms in adhering to other cells (and to the extracellular matrix). Thus, the specificity of cell-cell (and cell-matrix) adhesion seen in embryonic development must result from the integration of several different adhesion systems, of which some are associated with specialized cell junctions, while others are not.

The Extracellular Matrix Is Made and Oriented by the Cells Within It

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.

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.

Glycosaminoglycan (GAG) Chains Occupy Large Amounts of Space and Form Hydrated Gels

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.

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 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 Is Thought to Facilitate Cell Migration During Tissue Morphogenesis and Repair

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.

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.

Proteoglycans Are Composed of GAG Chains Covalently Linked to a Core Protein

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.

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).

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.

Proteoglycans Can Regulate the Activities of Secreted Proteins

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.

GAG Chains May Be Highly Organized in the Extracellular Matrix

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).

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).

Cell-Surface Proteoglycans Act as Co-receptors

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.

Collagens Are the Major Proteins of the Extracellular Matrix

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).

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.

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.

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).

Collagens Are Secreted with a Nonhelical Extension at Each End

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.

After Secretion, Fibrillar Procollagen Molecules Are Cleaved to Collagen Molecules, Which Assemble into Fibrils

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.

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.

Fibril-associated Collagens Help Organize the Fibrils

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).

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.

• 1

  Their triple-stranded helical structure is interrupted by one or two short nonhelical domains, which makes the molecules more flexible than fibrillar collagen molecules.

• 2

  They are not cleaved after secretion and therefore retain their propeptides.

• 3
  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.

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 Help Organize the Collagen Fibrils They Secrete by Exerting Tension on 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.

Elastin Gives Tissues Their Elasticity

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.

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).

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.

Fibronectin Is an Extracellular Protein That Helps Cells Attach to the Matrix

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.

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.

Fibronectin Exists in Both Soluble and Fibrillar Forms

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.

Intracellular Actin Filaments Regulate the Assembly of Extracellular Fibronectin Fibrils

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).

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.

Glycoproteins in the Matrix Help Guide Cell Migration

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.

Basal Laminae Are Composed Mainly of Type IV Collagen, Laminin, Nidogen, and a Heparan Sulfate Proteoglycan

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.

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.

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.

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.

The shapes and sizes of some of the extracellular matrix molecules discussed in this chapter are compared in Figure 19-59.

Basal Laminae Perform Diverse Functions

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.

The Extracellular Matrix Can Influence Cell Shape, Cell Survival, and Cell Proliferation

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.

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.

The Controlled Degradation of Matrix Components Helps Cells Migrate

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).

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.

Summary

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.

Integrins Are Transmembrane Heterodimers

Integrins are crucially important because they are the main receptor proteins that cells use to both bind to and respond to the extracellular matrix. An integrin molecule is composed of two noncovalently associated transmembrane glycoprotein subunits called α and β (Figure 19-64; see also Figure 19-12B). Because the same integrin molecule in different cell types can have different ligand-binding specificities, it seems that additional cell-type-specific factors can interact with integrins to modulate their binding activity.

The binding of integrins to their ligands depends on extracellular divalent cations (Ca²⁺ or Mg²⁺, depending on the integrin), reflecting the presence of divalent-cation-binding domains in the extracellular part of the α and β subunits. The type of divalent cation can influence both the affinity and the specificity of the binding of an integrin to its ligands.

Many matrix proteins in vertebrates are recognized by multiple integrins. At least 8 integrins bind fibronectin, for example, and at least 5 bind laminin. A variety of human integrin heterodimers are formed from 9 types of β subunits and 24 types of α subunits. This diversity is further increased by alternative splicing of some integrin RNAs. Some of the best-studied integrins are listed in Table 19-6.

β₁ subunits form dimers with at least 12 distinct α subunits. They are found on almost all vertebrate cells: α₅β₁, for example, is a fibronectin receptor and α₆β₁ a laminin receptor on many types of cells. Mutant mice that cannot make any β₁ integrins die at implantation, whereas mice that are only unable to make the α₇ subunit (the partner for β₁ in muscle) survive but develop muscular dystrophy (as do mice that cannot make the laminin ligand for the α₇β₁ integrin).

The β₂ subunits form dimers with at least four types of α subunit. They are expressed exclusively on the surface of white blood cells, where they have an essential role in enabling these cells to fight infection. The β₂ integrins mainly mediate cell-cell rather than cell-matrix interactions, binding to specific ligands on another cell, such as an endothelial cell. The ligands, sometimes referred to as counterreceptors, are members of the Ig superfamily of cell-cell adhesion molecules discussed earlier. The β₂ integrins enable white blood cells, for example, to attach firmly to endothelial cells at sites of infection and migrate out of the bloodstream into the infected site (see Figure 19-30B). Humans with the genetic disease called leucocyte adhesion deficiency are unable to synthesize β₂ subunits. As a consequence, their white blood cells lack the entire family of β₂ receptors, and they suffer repeated bacterial infections.

The β₃ integrins are found on a variety of cells, including blood platelets. They bind several matrix proteins, including fibrinogen. Platelets interact with fibrinogen during blood clotting, and humans with Glanzmann's disease, who are genetically deficient in β₃ integrins, bleed excessively.

Integrins Must Interact with the Cytoskeleton to Bind Cells to the Extracellular Matrix

Integrins function as transmembrane linkers (or “integrators”), mediating the interactions between the cytoskeleton and the extracellular matrix that are required for cells to grip the matrix. Most integrins are connected to bundles of actin filaments. The α₆β₄ integrin found in hemidesmosomes is an exception: it is connected to intermediate filaments. After the binding of a typical integrin to its ligand in the matrix, the cytoplasmic tail of the β subunit binds to several intracellular anchor proteins, including talin, α-actinin, and filamin. These anchor proteins can bind directly to actin or to other anchor proteins such as vinculin, thereby linking the integrin to actin filaments in the cell cortex. Given the right conditions, this linkage leads to a clustering of the integrins and the formation of focal adhesions between the cell and the extracellular matrix, as discussed earlier.

If the cytoplasmic domain of the β subunit is deleted using recombinant DNA techniques, the shortened integrins still bind to their ligands, but they no longer mediate robust adhesion, and they fail to cluster at focal adhesions. It seems that integrins must interact with the cytoskeleton to bind cells strongly to the matrix, just as cadherins must interact with the cytoskeleton to hold cells together efficiently. The cytoskeletal attachment may help cluster the integrins, providing a stronger aggregate bond; it may also lock the integrin in a conformation that allows the integrin to bind its ligand more tightly.

Just as cadherins can promote cell-cell adhesion without forming mature adherens junctions, integrins can mediate cell-matrix adhesion without forming mature focal adhesions. In both cases, however, the transmembrane adhesion proteins may still bind to the cytoskeleton. For integrins, this kind of adhesion occurs when cells are spreading or migrating, and it results in the formation of focal complexes. For such focal complexes to mature into the focal adhesions that are typical of many well-spread cells, the activation of the small GTPase Rho is required. The activation of Rho leads to the recruitment of more actin filaments and integrins to the contact site (discussed in Chapter 16).

Cells Can Regulate the Activity of Their Integrins

We discuss below how integrin clustering activates intracellular signaling pathways. But signaling also operates in the opposite direction: signals generated inside the cell can either enhance or inhibit the ability of integrins to bind to their ligand outside the cell (Figure 19-65). This regulation is poorly understood, but it may involve the phosphorylation of the cytoplasmic tails of the integrins, the association of the tails with activating cytoplasmic proteins, or both.

The ability of a cell to control integrin-ligand interactions from within is termed inside-out signaling. It is particularly important in platelets and white blood cells, where integrins usually have to be activated before they can mediate adhesion. In most other cells, integrins are usually maintained in an adhesion-competent state. Regulated adhesion allows white blood cells to circulate unimpeded until they are activated by an appropriate stimulus. Because the integrins do not need to be synthesized de novo, the signaled adhesion response can be rapid. Platelets, for example, are activated either by contact with a damaged blood vessel or by various soluble signal molecules. In either case, the stimulus triggers intracellular signaling pathways that rapidly activate a β₃ integrin in the platelet membrane. This induces a conformational change in the extracellular domain of the integrin that enables the protein to bind the blood-clotting protein fibrinogen with high affinity. The fibrinogen links platelets together to form a platelet plug, which helps stop bleeding.

Similarly, the weak binding of a T lymphocyte to its specific antigen on the surface of an antigen-presenting cell (discussed in Chapter 24) triggers intracellular signaling pathways in the T cell that activate its β₂ integrins. The activated integrins then enable the T cell to adhere strongly to the antigen-presenting cell so that it remains in contact long enough to become stimulated fully. The integrins may then return to an inactive state, allowing the T cell to disengage.

There are occasions, especially during development, when cells other than blood cells also regulate the activity of their integrins. If a constitutively active integrin (made by deleting the cytoplasmic tail of the α subunit) is expressed in a developing Drosophila embryo, for example, it disrupts normal muscle development. The muscle precursor cells expressing the activated integrin cannot disengage from the extracellular matrix and therefore cannot migrate normally.

Integrins Activate Intracellular Signaling Pathways

We have already discussed how integrins function as transmembrane linkers that connect extracellular matrix molecules to actin filaments in the cell cortex and thereby regulate the shape, orientation, and movement of cells. But the clustering of integrins at the sites of contact with the matrix (or with another cell) can also activate intracellular signaling pathways. Signaling is initiated by the assembly of signaling complexes at the cytoplasmic face of the plasma membrane, much as in signaling by conventional signaling receptors (discussed in Chapter 15).

Whereas activated integrins, like activated conventional signaling receptors, can induce global cell responses, often including changes in gene expression, activated integrins are especially adept at stimulating localized changes in the cytoplasm close to the cell-matrix contact. This may be a fundamental feature of signaling by transmembrane cell adhesion proteins in general. In the developing nervous system, for example, the growing tip of an axon is guided mainly by its responses to local adhesive (and repellent) cues in the environment that are recognized by transmembrane cell adhesion proteins. The primary effects of the adhesion proteins are thought to result from the activation of intracellular signaling pathways that act locally in the axon tip, rather than through cell-cell adhesion itself or signals conveyed to the cell body.

Many of the signaling functions of integrins depend on a cytoplasmic protein tyrosine kinase called focal adhesion kinase (FAK). Focal adhesions are often the most prominent sites of tyrosine phosphorylation in cells in culture (see Figure 17-50), and FAK is one of the major tyrosine-phosphorylated proteins found in focal adhesions (although it can also associate with conventional signaling receptors). When integrins cluster at sites of cell-matrix contact, FAK is recruited to focal adhesions by intracellular anchor proteins such as talin, which binds to the integrin β subunit, or paxillin, which binds to one type of integrin α subunit. The clustered FAK molecules cross-phosphorylate each other on a specific tyrosine, creating a phosphotyrosine docking site for members of the Src family of cytoplasmic tyrosine kinases. These kinases then phosphorylate FAK on additional tyrosines, creating docking sites for a variety of intracellular signaling proteins; they also phosphorylate other proteins at focal adhesions. In this way, the signal is relayed into the cell (as discussed in Chapter 15).

One way to analyze the function of FAK is to examine focal adhesions in cells from mutant mice that lack the protein. FAK-deficient fibroblasts still adhere to fibronectin and form focal adhesions. Suprisingly, they form too many focal adhesions rather than too few; as a result, cell spreading and migration are slowed (Figure 19-66). This unexpected finding suggests that FAK normally helps disassemble focal adhesions and that this loss of adhesions is required for normal cell migration. By interacting with both conventional signaling receptors and focal adhesions, FAK can couple migratory signals to changes in cell adhesion. Many cancer cells have elevated levels of FAK, which may help explain why they are often more motile than their normal counterparts.

Integrins and conventional signaling receptors can work together in several ways. The signaling pathways activated by conventional signaling receptors can increase the expression of integrins or extracellular matrix molecules, while those activated by integrins can increase the expression of conventional signaling receptors or the ligands that bind to them. The intracellular signaling pathways themselves can also interact and reinforce one another. While some conventional signaling receptors and integrins activate the Ras/MAP kinase pathway (see Figure 15-56) independently, for example, they often act together to sustain the activation of this pathway long enough to induce cell proliferation. Integrins and conventional signaling receptors cooperate to stimulate many types of cell response. Many cells in culture, for example, will not grow or proliferate in response to extracellular growth factors unless the cells are attached via integrins to extracellular matrix molecules. For some cell types, including epithelial, endothelial, and muscle cells, even cell survival depends on signaling through integrins. When these cells lose contact with the extracellular matrix, they undergo programmed cell death, or apoptosis. This dependence on attachment to the extracellular matrix for survival and proliferation may help ensure that the cells survive and proliferate only when they are in their appropriate location, which may protect animals against the spread of cancer cells. Attachment-dependent cell survival is exploited for special purposes in embryonic development, as shown in Figure 19-67. The signaling pathways that integrins activate to promote cell survival are similar to those activated by conventional signaling receptors, as discussed in Chapters 15 and 17.

The cell adhesion molecules discussed in this chapter are summarized in Table 19-7.

Summary

Integrins are the principal receptors used by animal cells to bind to the extracellular matrix. They are heterodimers and function as transmembrane linkers between the extracellular matrix and the actin cytoskeleton. A cell can regulate the adhesive activity of its integrins from within. Integrins also function as signal transducers, activating various intracellular signaling pathways when activated by matrix binding. Integrins and conventional signaling receptors often cooperate to promote cell growth, cell survival, and cell proliferation.

The Composition of the Cell Wall Depends on the Cell Type

All cell walls in plants have their origin in dividing cells, as the cell plate forms during cytokinesis to create a new partition wall between the daughter cells (discussed in Chapter 18). The new cells are usually produced in special regions called meristems (discussed in Chapter 21), and they are generally small in comparison with their final size. To accommodate subsequent cell growth, their walls, called primary cell walls, are thin and extensible, although tough. Once growth stops, the wall no longer needs to be extensible: sometimes the primary wall is retained without major modification, but, more commonly, a rigid, secondary cell wall is produced by depositing new layers inside the old ones. These may either have a composition similar to that of the primary wall or be markedly different. The most common additional polymer in secondary walls is lignin, a complex network of phenolic compounds found in the walls of the xylem vessels and fiber cells of woody tissues.The plant cell wall thus has a “skeletal” role in supporting the structure of the plant as a whole, a protective role as an enclosure for each cell individually, and a transport role, helping to form channels for the movement of fluid in the plant. When plant cells become specialized, they generally adopt a specific shape and produce specially adapted types of walls, according to which the different types of cells in a plant can be recognized and classified (Figure 19-69; see also Panel 21-3).

Although the cell walls of higher plants vary in both composition and organization, they are all constructed, like animal extracellular matrices, using a structural principle common to all fiber-composites, including fibreglass and reinforced concrete. One component provides tensile strength, while another, in which the first is embedded, provides resistance to compression. While the principle is the same in plants and animals, the chemistry is different. Unlike the animal extracellular matrix, which is rich in protein and other nitrogen-containing polymers, the plant cell wall is made almost entirely of polymers that contain no nitrogen, including cellulose and lignin. Trees make a huge investment in the cellulose and lignin that comprise the bulk of their biomass. For a sedentary organism that depends on CO₂, H₂O and sunlight, these two abundant biopolymers represent “cheap,” carbon-based, structural materials, helping to conserve the scarce fixed nitrogen available in the soil that generally limits plant growth.

In the cell walls of higher plants, the tensile fibers are made from the polysaccharide cellulose, the most abundant organic macromolecule on Earth, tightly linked into a network by cross-linking glycans. In primary cell walls, the matrix in which the cellulose network is embedded is composed of pectin, a highly hydrated network of polysaccharides rich in galacturonic acid. Secondary cell walls contain additional components, such as lignin, which is hard and occupies the interstices between the other components, making the walls rigid and permanent. All of these molecules are held together by a combination of covalent and noncovalent bonds to form a highly complex structure, whose composition, thickness and architecture depends on the cell type.

We focus here on the primary cell wall and the molecular architecture that underlies its remarkable combination of strength, resilience, and plasticity, as seen in the growing parts of a plant.

The Tensile Strength of the Cell Wall Allows Plant Cells to Develop Turgor Pressure

The aqueous extracellular environment of a plant cell consists of the fluid contained in the walls that surround the cell. Although the fluid in the plant cell wall contains more solutes than does the water in the plant's external milieu (for example, soil), it is still hypotonic in comparison with the cell interior. This osmotic imbalance causes the cell to develop a large internal hydrostatic pressure, or turgor pressure, that pushes outward on the cell wall, just as an inner tube pushes outward on a tire. The turgor pressure increases just to the point where the cell is in osmotic equilibrium, with no net influx of water despite the salt imbalance (see Panel 11-1, pp. 628–629). This pressure is vital to plants because it is the main driving force for cell expansion during growth, and it provides much of the mechanical rigidity of living plant tissues. Compare the wilted leaf of a dehydrated plant, for example, with the turgid leaf of a well-watered one. It is the mechanical strength of the cell wall that allows plant cells to sustain this internal pressure.

The Primary Cell Wall Is Built from Cellulose Microfibrils Interwoven with a Network of Pectic Polysaccharides

The cellulose molecules provide tensile strength to the primary cell wall. Each molecule consists of a linear chain of at least 500 glucose residues that are covalently linked to one another to form a ribbonlike structure, which is stabilized by hydrogen bonds within the chain (Figure 19-70). In addition, intermolecular hydrogen bonds between adjacent cellulose molecules cause them to adhere strongly to one another in overlapping parallel arrays, forming a bundle of about 40 cellulose chains, all of which have the same polarity. These highly ordered crystalline aggregates, many micrometers long, are called cellulose microfibrils, and they have a tensile strength comparable to steel. Sets of microfibrils are arranged in layers, or lamellae, with each microfibril about 20–40 nm from its neighbors and connected to them by long cross-linking glycan molecules that are bound by hydrogen bonds to the surface of the microfibrils. The primary cell wall consists of several such lamellae arranged in a plywoodlike network (Figure 19-71).

The cross-linking glycans are a heterogeneous group of branched polysaccharides that bind tightly to the surface of each cellulose microfibril and thereby help to cross-link microfibrils into a complex network. Their function is analogous to that of the fibril-associated collagens discussed earlier (see Figure 19-49). There are many classes of cross-linking glycans, but they all have a long linear backbone composed of one type of sugar (glucose, xylose, or mannose) from which short side chains of other sugars protrude. It is the backbone sugar molecules that form hydrogen bonds with the surface of cellulose microfibrils, cross-linking them in the process. Both the backbone and the side-chain sugars vary according to the plant species and its stage of development.

Coextensive with this network of cellulose microfibrils and cross-linking glycans is another cross-linked polysaccharide network based on pectins (see Figure 19-71). Pectins are a heterogeneous group of branched polysaccharides that contain many negatively charged galacturonic acid units. Because of their negative charge, pectins are highly hydrated and associated with a cloud of cations, resembling the glycosaminoglycans of animal cells in the large amount of space they occupy (see Figure 19-37). When Ca²⁺ is added to a solution of pectin molecules, it cross-links them to produce a semirigid gel (it is pectin that is added to fruit juice to make jelly). Certain pectins are particularly abundant in the middle lamella, the specialized region that cements together the walls of adjacent cells (see Figure 19-71); here, Ca²⁺ cross-links are thought to help hold cell-wall components together. Although covalent bonds also play a part in linking the components together, very little is known about their nature. Regulated separation of cells at the middle lamella underlies such processes as the ripening of tomatoes and the abscission (detachment) of leaves in the fall.

In addition to the two polysaccharide-based networks that are present in all plant primary cell walls, proteins can contribute up to about 5% of the wall's dry mass. Many of these proteins are enzymes, responsible for wall turnover and remodelling, particularly during growth. Another class of wall proteins contains high levels of hydroxyproline, as in collagen. These proteins are thought to strengthen the wall, and they are produced in greatly increased amounts as a local response to attack by pathogens. From the genome sequence of Arabidopsis, it has been estimated that more than 700 genes are required to synthesize, assemble, and remodel the plant cell wall. Some of the main polymers found in the primary and secondary cell wall are listed in Table 19-8.

For a plant cell to grow or change its shape, the cell wall has to stretch or deform. Because of their crystalline structure, however, individual cellulose microfibrils are unable to stretch. Thus, stretching or deformation of the cell wall must involve either the sliding of microfibrils past one another, the separation of adjacent microfibrils, or both. As we discuss next, the direction in which the growing cell enlarges depends in part on the orientation of the cellulose microfibrils in the primary wall, which in turn depends on the orientation of microtubules in the underlying cell cortex at the time the wall was deposited.

Microtubules Orient Cell-Wall Deposition

The final shape of a growing plant cell, and hence the final form of the plant, is determined by controlled cell expansion. Expansion occurs in response to turgor pressure in a direction that depends in part on the arrangement of the cellulose microfibrils in the wall. Cells, therefore, anticipate their future morphology by controlling the orientation of microfibrils that they deposit in the wall. Unlike most other matrix macromolecules, which are made in the endoplasmic reticulum and Golgi apparatus and are secreted, cellulose, like hyaluronan, is spun out from the surface of the cell by a plasma-membrane-bound enzyme complex (cellulose synthase), which uses as its substrate the sugar nucleotide UDP-glucose supplied from the cytosol. As they are being synthesized, the nascent cellulose chains assemble spontaneously into microfibrils that form on the extracellular surface of the plasma membrane—forming a layer, or lamella, in which all the microfibrils have more or less the same alignment (see Figure 19-71). Each new lamella forms internally to the previous one, so that the wall consists of concentrically arranged lamellae, with the oldest on the outside. The most recently deposited microfibrils in elongating cells commonly lie perpendicular to the axis of cell elongation (Figure 19-72). Although the orientation of the microfibrils in the outer lamellae that were laid down earlier may be different, it is the orientation of these inner lamellae that is thought to have a dominant influence on the direction of cell expansion (Figure 19-73).

An important clue to the mechanism that dictates this orientation came from observations of the microtubules in plant cells. These are arranged in the cortical cytoplasm with the same orientation as the cellulose microfibrils that are currently being deposited in the cell wall in that region. These cortical microtubules form a cortical array close to the cytosolic face of the plasma membrane, held there by poorly characterized proteins (Figure 19-74). The congruent orientation of the cortical array of microtubules (lying just inside the plasma membrane) and cellulose microfibrils (lying just outside) is seen in many types and shapes of plant cells and is present during both primary and secondary cell-wall deposition, suggesting a causal relationship.

If the entire system of cortical microtubules is disassembled by treating a plant tissue with a microtubule-depolymerizing drug, the consequences for subsequent cellulose deposition are not as straightforward as might be expected. The drug treatment has no effect on the production of new cellulose microfibrils, and in some cases cells can continue to deposit new microfibrils in the preexisting orientation. Any developmental change in the microfibril pattern that would normally occur between successive lamellae, however, is invariably blocked. It seems that a preexisting orientation of microfibrils can be propagated even in the absence of microtubules, but any change in the deposition of cellulose microfibrils requires that intact microtubules be present to determine the new orientation.

These observations are consistent with the following model. The cellulose-synthesizing complexes embedded in the plasma membrane are thought to spin out long cellulose molecules. As the synthesis of cellulose molecules and their self-assembly into microfibrils proceeds, the distal end of each microfibril presumably forms indirect cross-links to the previous layer of wall material as it becomes integrated into the texture of the wall. At the growing, proximal end of each microfibril, the synthesizing complexes would therefore need to move through the membrane in the direction of synthesis. Since the growing cellulose microfibrils are stiff, each layer of microfibrils would tend to be spun out from the membrane in the same orientation as the previously laid down layer, with the cellulose synthase complex following along the preexisting tracks of oriented microfibrils outside the cell. Oriented microtubules inside the cell, however, can change this predetermined direction in which the synthase complexes move: they can create boundaries in the plasma membrane that act like the banks of a canal to constrain movement of the synthase complexes (Figure 19-75). In this view, cellulose synthesis can occur independently of microtubules but is constrained spatially when cortical microtubules are present to define membrane domains within which the enzyme complex can move.

Plant cells can change their direction of expansion by a sudden change in the orientation of their cortical array of microtubules. Because plant cells cannot move (being constrained by their walls), the entire morphology of a multicellular plant depends on the coordinated, highly patterned control of cortical microtubule orientations during plant development. It is not known how the organization of these microtubules is controlled, although it has been shown that they can reorient rapidly in response to extracellular stimuli, including low-molecular-weight plant growth regulators such as ethylene and gibberellic acid (see Figure 21-113).

Summary

Plant cells are surrounded by a tough extracellular matrix in the form of a cell wall, which is responsible for many of the unique features of a plant's life style. The cell wall is composed of a network of cellulose microfibrils and cross-linking glycans embedded in a highly cross-linked matrix of pectin polysaccharides. In secondary cell walls, lignin may be deposited. A cortical array of microtubules can determine the orientation of newly deposited cellulose microfibrils, which in turn determines directional cell expansion and therefore the final shape of the cell and, ultimately, of the plant as a whole.