CHAPTER 20

Integrating Cells into Tissues

Antibody staining of mouse small intestinal villi, fingerlike projections of cell layers (see Figures 20-13 through 20-15). NPC1L1, a protein located primarily on
the plasma membrane involved with cholesterol metabolism, is green. Villin, which binds to actin bundles in microvilli (very small membrane projections on
the apical surfaces of absorptive cells that take up digested nutrients), is red. Nuclei (DNA) are blue. The image shows all colors merged together
(colocalization of green and red appears yellow). The yellow lines show how the apical surfaces of the cells face the lumen of the small intestine (black) from
which nutrients are absorbed.

OUTLINE

20.1 Cell-Cell and Cell–Extracellular Matrix Adhesion: An Overview

20.2 Cell-Cell and Cell–Extracellular Matrix Junctions and Their Adhesion Molecules
20.3 The Extracellular Matrix I: The Basal Lamina
20.4 The Extracellular Matrix II: Connective Tissue
20.5 Adhesive Interactions in Motile and Nonmotile Cells
20.6 Plant Tissues

Vertebrates have many hundreds of different cell types, including

In the development of complex multicellular organisms such as plants
and animals, progenitor cells differentiate into distinct types that have
characteristic compositions, structures, and functions. Cells of a given
type often aggregate into a tissue to cooperatively perform a common
function — muscle contracts, neural tissue conducts electric impulses,
xylem tissue in plants transports water. Different tissues can be
organized into an organ, again to perform one or more specific
functions. For instance, the muscles, valves, and blood vessels of a
heart work together to pump blood. The coordinated functioning of
many types of cells and tissues permits the organism to move,
metabolize, reproduce, and carry out other essential activities. Indeed,
the complex and diverse morphologies of plants and animals are
examples of the whole being greater than the sum of the individual
parts.
leukocytes (white blood cells) and erythrocytes (red blood cells),
photoreceptors in the retina, fat-storing adipocytes, fibroblasts in
connective tissue, and the hundreds of different subtypes of neurons in
the human brain. A large-scale international effort, the Human Cell
Atlas Project, is using single-cell RNA sequencing and other methods to
generate a comprehensive catalogue of all human cell types and
subtypes.
Multicellular animals — also called metazoans — usually comprise
differentiated cells organized into distinct tissues, although there are
some less complex exceptions (e.g., sponges). Even rather simple
animals exhibit distinctive tissue organization. The adult form of the
roundworm Caenorhabditis elegans contains a mere 959 cells, yet these
cells fall into 12 different general cell types and many distinct subtypes.
Despite their diverse forms and functions, animal cells typically can be
classified as components of just five main classes of tissue: epithelial
tissue, connective tissue, muscular tissue, neural tissue, and blood. Various
cell types are arranged in precise patterns of staggering complexity to
generate tissues and organs. The physiological costs of such
complexity include increased requirements for information, material,
energy, and time during the development of an individual organism.
Although these costs of generating and maintaining complex tissues
and organs are high, they confer the ability to thrive in varied and
variable environments — a major evolutionary advantage.
One of the defining characteristics of animals with complex tissues and
organs (most metazoans) is that the external and internal surfaces of
most of their tissues and organs — and indeed, the exterior of the
entire organism — are built from tightly packed sheet-like layers of
cells known as epithelia. The formation of an epithelium and its
subsequent remodeling into more complex collections of epithelial and
nonepithelial tissues is a hallmark of the development of most
metazoans. Sheets of tightly attached epithelial cells act as regulatable,
selectively permeable barriers, which permit the generation of
chemically and functionally distinct compartments in an organism,
such as the stomach and bloodstream. As a result, distinct and
sometimes opposite functions (e.g., digestion and synthesis) can
efficiently proceed simultaneously within an organism. Such
compartmentalization also permits more sophisticated regulation of
diverse biological functions. In many ways, the roles of complex
tissues and organs in an organism are analogous to those of organelles
and membranes in individual cells.
The assembly of distinct tissues and their organization into organs are
determined by molecular interactions at the cellular level (Figure 20-1).
These interactions would not be possible without the temporally,
spatially, and functionally regulated expression of a wide array of
adhesion molecules. Cell-surface molecules engaged in adhesion are
called adhesion receptors. Cells in tissues can adhere directly to one
another (cell-cell adhesion) through specialized membrane proteins
called cell-adhesion molecules (CAMs) or cell-cell adhesion
receptors, which often cluster on the plasma membrane, sometimes
into specialized structures called cell junctions. In the fruit fly
Drosophila melanogaster, at least 500 genes (∼4 percent of the total) are
estimated to be involved in cell adhesion, and in mammals there are
over 1000 such genes. Cells in animal tissues also adhere indirectly
(cell-matrix adhesion) through the binding of cell-matrix adhesion
receptors in the plasma membrane to components of the surrounding
extracellular matrix (ECM), a complex interdigitating meshwork of
proteins and polysaccharides secreted by cells into the spaces between
them. Some classes of adhesion receptors (e.g., integrins) can mediate
both cell-cell adhesion as well as cell-matrix adhesion.

FIGURE 20-1 Overview of major cell-cell and cell-matrix adhesive interactions. Schematic cutaway drawing of a typical epithelial tissue, such as in the inner
surface of the intestines. The apical (upper) surface of each cell is packed with fingerlike microvilli (1) that project into the intestinal lumen; the basal (lower)
surface (2) rests on extracellular matrix (ECM). The ECM (3) associated with epithelial cells is usually organized into various interconnected layers — such as
the basal lamina (3a), connecting fibers (not shown), and connective tissue (3b) — in which large, interdigitating ECM macromolecules bind to one another
and to the cells (3). Cell-adhesion molecules (CAMs, also called cell-cell adhesion receptors) bind to CAMs on other cells, mediating cell-cell adhesion (4), and
cell-matrix adhesion receptors bind to various components of the ECM, mediating cell-matrix adhesion (5). Both types of adhesion receptors are usually
integral membrane proteins whose cytosolic domains often bind to multiple intracellular adapter proteins. These adapters, directly or indirectly, link the
CAM to the cytoskeleton (actin or intermediate filaments) and to intracellular signaling pathways (see Figure 20-8). As a consequence, information can be
transferred by adhesion receptors and the macromolecules to which they bind from the cell exterior to the intracellular environment and vice versa. In some
cases, a complex aggregate of adhesion receptors, adapters, and associated proteins is assembled. Specific, localized aggregates of adhesion receptors form
various types of cell junctions, which play important roles in holding tissues together and facilitating communication between cells and their environment.
Tight junctions (6), lying just under the apical surface, prevent the diffusion of many substances through the extracellular spaces between the cells. Through
connexon channels, gap junctions (7) allow the movement of small molecules and ions between the cytosols of adjacent cells. The remaining three types of
junctions, adherens junctions (8 and 4), desmosomes (9), hemidesmosomes (10 and 5), and focal adhesions (also called focal contacts; 11) link the
cytoskeleton of a cell to other cells or to the ECM. See V. Vasioukhin and E. Fuchs, 2001, Curr. Opin. Cell Biol. 13:76–84.

The illustration on the left shows two ovals, the top one is labeled tight junction and shows a vertical line of cells close together with pieces coming off to the
right. The oval below is labeled gap junction and shows two yellow vertical lines with two orange tubes connecting them at the center. The top tube is closed
and the bottom one is open and is labeled connexon. In the center of the illustration is a schematic of a sample of the intestinal wall. At the top is a wavy line
labeled apical surface, representing the villi. Between two villi are examples of the chain of cells going down to the basal lamia at the bottom of the diagram.
The ovals from the left side are indicated as enlargements of two areas on the left example. The right example has a green oval half way batten villi and lamia
and labeled adherens junction, and a blue oval further down labeled desmosome. The lamia shows root-like lines. At the right of the illustration, an oval is at
the top, labeled cell-cell adhesions. It shows chains of cells labeled, from left to right, Adapters, cell adhesion molecules (C A M's), then actin. The lower oval
is labeled cell-matrix adhesions. It shows adapters attached to actin at the top, attached below to adhesion receptors, which are then lowered into a blue
area labeled E C M.

Cell-cell and cell-matrix adhesions not only allow cells to aggregate 20.1 Cell-Cell and Cell–Extracellular Matrix Adhesion: An Overview
into distinct tissues, but also provide a means for the bidirectional
transfer of information between the exterior and the interior of cells.
There are many different types of cells in the body that dynamically
As we will see, both types of adhesions are intrinsically associated with
interact with each other in a myriad of ways. These interactions,
the cytoskeleton and cellular signaling pathways. As a result, a cell’s
achieved via adhesion molecules, must be precisely and carefully
surroundings influence its shape and functional properties (“outside-
controlled in time and space and in their physical characteristics (e.g.,
in” effects); likewise, cellular shape and function influence a cell’s
strength of adhesion) to correctly determine the structures and
surroundings (“inside-out” effects). Thus connectivity and
functions of tissues in a complex organism. It is not surprising,
communication are intimately related properties of cells in tissues.
therefore, that cell-cell and cell-matrix adhesion molecules exhibit
Information transfer is important to many biological processes,
diverse structures, or that their expression levels vary in different cells
including cell survival, proliferation, differentiation, and migration.
and tissues. As a consequence, they mediate the very specific and
Therefore, it is not surprising that defects that interfere with adhesive
distinctive cell-cell and cell-matrix interactions that hold tissues
interactions and the associated flow of information can cause or
together as well as permit essential communication between cells and
contribute to diseases, including a wide variety of neuromuscular and
their environment. We begin this overview with a brief orientation to
skeletal disorders, heart and vascular defects, clotting abnormalities,
some of the various types of adhesion molecules present on cells and
cancer, and others.
within the extracellular matrix, their major functions in organisms,
and their evolutionary origin. In subsequent sections, we will examine
In this chapter, we examine various types of adhesion molecules found in detail the unique structures and properties of various participants in
on the surfaces of cells and in the surrounding extracellular matrix. cell-cell and cell-matrix interactions.
Interactions between these molecules allow the organization of cells
into tissues and have profound effects on tissue development, function,
and pathology. Many adhesion molecules are members of families or
superfamilies of related proteins. While each type of adhesion
molecule performs a distinct role, we will focus on the common
features shared by members of some of these families to illustrate the
general principles underlying their structures and functions. Because
of the particularly well-understood nature of the adhesion molecules in
tissues that form tight epithelia, as well as their very early evolutionary Cell-Adhesion Molecules Bind to One Another and to Intracellular Proteins
development, we will initially focus on epithelial tissues, such as the
walls of the intestinal tract and the skin. Epithelial cells are normally
Cell-cell adhesion is mediated through membrane proteins called cell-
nonmotile (immobile), also called sessile; however, during
adhesion molecules (CAMs, also called cell-cell adhesion receptors). Many
development, wound healing, and in certain pathological states (e.g.,
CAMs fall into one of four major families: the cadherins, the
cancer), epithelial cells can transform into motile cells. Changes in the
immunoglobulin (Ig) superfamily, the integrins, and sugar-binding
expression and function of adhesion molecules play a key role in this
proteins called lectins, one subset of which are called selectins. As the
transformation, as they do in normal biological processes involving
schematic structures in Figure 20-2 illustrate, CAMs often comprise
cell movement, such as the crawling of white blood cells into sites of
multiple, distinct domains, many of which can be found in more than
infection. We therefore follow the discussion of epithelial tissues with
one kind of protein. The functions of these domains vary. Some confer
a discussion of adhesion in nonepithelial, developing, and motile
the ability to bind specifically to their partner CAMs on neighboring
tissues.
cells or even to CAMs on the same cell. Some of these domains are
present in multiple copies and contribute to the length of the CAMs
The evolutionary lineages of plants and animals diverged before and thus help define the distance between the plasma membranes of
multicellular organisms arose (see Figure 1-1). Thus multicellularity cells bound together by the CAMs. Other membrane proteins, whose
and the molecular means for assembling tissues and organs structures do not belong to any of the major classes of CAMs in Figure
presumably arose independently in animal and plant lineages. Not 20-2, are also adhesion receptors and participate in cell-cell and cell-
surprisingly, then, animals and plants exhibit many differences in the matrix adhesion in various tissues. One example is a set of adhesion G
organization and development of tissues. For this reason, we first protein–coupled receptors (GPCRs, see Chapter 15). As we will see
consider the organization of tissues in animals and then deal later, integrins can function both as cell-cell and cell-matrix adhesion
separately with plants. receptors. Figure 20-2 depicts an integrin serving as a cell-matrix
adhesion receptor that binds to ECM components. Some Ig-
superfamily CAMs can play this dual role as well.
Through their extracellular domains, CAMs mediate adhesive
interactions between cells of the same type (homotypic adhesion) or
between cells of different types (heterotypic adhesion). A CAM on one
cell can directly bind to the same kind of CAM on an adjacent cell
(homophilic binding) or to a different class of CAM (heterophilic
binding) (see Figure 20-2). CAMs can be broadly distributed along the
regions of plasma membranes that contact other cells or clustered in
discrete patches or spots that are sometimes called cell junctions. Cell-
cell adhesions can be tight and long lasting or relatively weak and
transient. For example, the associations between neurons in the spinal
cord or the metabolic cells in the liver exhibit tight adhesion. In
contrast, immune-system cells in the blood often exhibit only brief,
weak interactions that allow them to roll along and pass through a
blood vessel wall on their way to fight an infection within a tissue.
The cytosolic domains of adhesion receptors recruit sets of
multifunctional adapter proteins (see Figure 20-1). These adapters act
as linkers that directly or indirectly connect adhesion receptors to
elements of the cytoskeleton (see Chapters 17 and 18); they can also
recruit intracellular molecules that function in signaling pathways (see
Chapters 15 and 16) to modify cellular behavior, including gene
expression, and the activities of a variety of intracellular proteins
including the adhesion receptors themselves. In many cases, a
complex aggregate of adhesion receptors, adapter proteins, and other
associated proteins is assembled at the inner surface of the plasma
membrane. These complexes facilitate two-way, outside-in and inside-
out, communication between cells and their surroundings.
The formation of many cell-cell adhesions entails two types of
molecular interactions, called trans and cis binding interactions (Figure
20-3). Trans interactions are also called intercellular or adhesive
interactions, and cis interactions are also called lateral (in the same
cell) interactions. In trans interactions, CAMs on one cell bind to the
CAMs on an adjacent cell. In cis interactions, monomeric CAMs on one
cell bind to one or more CAMs in the same cell’s plasma membrane.
For some adhesion molecules the trans interactions between adjacent
cells can increase the probability of stable cis interaction formation
and thus determine the order in which these interactions occur (trans
prior to cis is shown in Figure 20-3). It appears that trans and cis
interactions are mutually reinforcing. Furthermore, the association of
intracellular molecules with the cytosolic domains of CAMs can
dramatically influence the intermolecular interactions of CAMs by
promoting their clustering together (e.g., cis interactions) or by
altering their conformation in a way that increases the affinity of trans
interactions. Just like Velcro, CAMs can generate very tight adhesion
when many weak interactions are combined, and this is especially the
case when CAMs are concentrated in small, well-defined areas such as
cell junctions.

FIGURE 20-2 Major families of cell-adhesion molecules (CAMs) and adhesion receptors. Adhesion receptors can mediate binding, either by homophilic
interactions (binding to other molecules of the same type; left) or heterophilic interactions (binding to different types of molecules; right). E-cadherins on one
cell (light blue, cell 1) commonly mediate cell-cell adhesion by forming homophilic cross-bridges with identical E-cadherins on adjacent cells (dark blue, cell
2). E-cadherins also can bind to neighboring E-cadherin molecules on the same cells (see Figures 20-3 and 20-14). Members of the immunoglobulin (Ig)
superfamily of adhesion receptors form homophilic linkages (as shown here for NCAM) or heterophilic linkages (to other types of CAMs, not shown).
Heterodimeric integrins (e.g., αv and β3 chains) can function as cell-matrix adhesion receptors that bind to very large, multi-adhesive extracellular proteins
such as fibronectin, only a small part of which is shown here. Selectins, shown as dimers, contain a carbohydrate-binding lectin domain that recognizes
specialized sugar structures on glycoproteins (as shown here) or glycolipids on adjacent cells (heterophilic interactions). Note that CAMs such as E-cadherins,
often form higher order oligomers within the plane of the plasma membrane. Many adhesion molecules contain multiple, distinct domains, some of which
are found in more than one kind of CAM. The cytoplasmic domains of adhesion receptors are often associated with adapter proteins that link them to the
cytoskeleton or to signaling pathways. See R. O. Hynes, 1999, Trends Cell Biol. 15(12):M33–M37; R. O. Hynes, 2002, Cell 110:673–687; and J. Brasch et al., 2012,
Trends Cell Biol. 22:299–310.
The illustration on the left shows two types of homophilic interactions. The diagram has a blue bar at the top labeled cell 1 and a blue bar at the bottom
labeled cell 2. These interactions are connected between these cells. One is labeled Cadherins (E-Cadherin) and is represented as a set of 5 light blue beads
hanging down and attached to a set of 5 darker blue beads. The connections between the light blue beads are labeled as calcium-binding sites. Next to this at
the right is an interaction labeled Ig-superfamily C A M's (N C A M). The chain of bead-like structures here are two orange ovals followed by 10 green circles
with the middle 4 joining each other, then two orange ovals at the bottom. None are labeled. On the heterophilic side, the first diagram is labeled integrins
(alpha V Beta 3) A blue chain of beads labeled alpha hangs straight down. To the right, the beta chain hangs down at an angle to join with the alpha chain and
join to a light orange chain that is moving sideways and is labeled fibronectin. The bottom cell is not shown here. The second type is labeled selectins (P-
selectin). Two pink chains hang down parallel to each other and join to black branched lines labeled sugars. The sugars go to cell 2, which has the label
glycoprotein.

FIGURE 20-3 Model for the generation of cell-cell adhesions. Trans interactions between CAMs on adjacent cells can hold the plasma membranes closely
together and permit the subsequent formation of cis (lateral) interactions that collect the CAMs into clusters. The parts of the molecules that participate in
these trans and cis interactions vary among the different CAMs. Multiple trans interactions concentrated in space by cis interactions generate a strong,
Velcro-like adhesion between the cells. The models shown here are based on CAMs called classical cadherins (described in greater detail later in this chapter).
See M. S. Steinberg and P. M. McNutt, 1999, Curr. Opin. Cell Biol. 11:554–560; and J. Brasch et al., 2012, Trends Cell Biol. 22:299–310.

The illustration shows the surfaces of two neighboring cells. Cell 1, at the top of the diagram, has several chains of cadherins embedded in the surface. Cell 2
also contains these chains. The tips of the chains are interacting with the tips of chains embedded in the same molecule laterally; this interaction is labeled
cis. The chains of one cell, on encountering the cadherin chains of another cell, form trans interactions, the tips of the chains interacting with the cis chains
on the other cell.

Adhesive interactions between cells and the consequences of those
interactions on cell structure and function vary considerably,
depending on the tissue and the particular CAMs participating. For
example, some CAMs require calcium ions to form effective adhesions
while others do not. Among the many variables that determine the
nature of adhesion between two cells are: the binding affinity of the
interacting molecules (thermodynamic properties); the overall “on”
and “off” rates of association and dissociation for each interacting
molecule (kinetic properties); the spatial distribution or density of
adhesion molecules (ensemble properties); the active versus inactive
states of CAMs with respect to adhesion (biochemical properties); and
external forces such as stretching and pulling, for example, in muscle
or the laminar and turbulent flow of cells and surrounding fluids in the
circulatory system (mechanical properties).
The Extracellular Matrix Participates in Adhesion, Signaling, and Other
Functions
The ECM is a complex combination of proteins and polysaccharides
that is secreted and assembled by cells into a network in which the
components bind to one another. The ECM is often involved in holding
cells and tissues together. The composition, physical properties, and
functions of the ECM are carefully controlled and can vary depending
on the tissue type, its location, its physiological state, and chemical
modifications of its components, including changes that can occur as a
consequence of pathology. These modifications include enzymatic
phosphorylation, sulfation and desulfation, cross-linking, cleavage by
proteases and glycosidases, and oxidation, as well as nonenzymatic
addition of glucose (glycation).
The ECM is usually sensed by cells as a consequence of binding to cell-
matrix adhesion receptors on their plasma membranes, which then
instruct the cells to behave appropriately in response to their
environments or modulate the structure and function of the ECM
based on the state of the cells. Different cells can bind to the same
patch of ECM via their adhesion receptors and thus be indirectly bound
together. ECM components include proteoglycans, a unique type of
glycoprotein (a protein with covalently attached sugars); collagens and
other proteins that often form fibers; soluble multi-adhesive matrix
proteins; and others (Table 20-1). A common feature of many ECM
proteins is that they comprise repeating structural domains
(sometimes called repeats) that form very large proteins. Within one
molecule, these repeats can be either very similar (homologous) to
other repeats in the protein, distinct from other repeats, or both. The
presence of nonidentical repeats that can exhibit distinct binding
properties contributes to the multiligand and multifunctional
characteristics of many adhesive proteins. For example, multi-
adhesive matrix proteins, such as fibronectin and laminin, are long,
flexible molecules that contain multiple repeats. They are responsible
for binding various types of collagen, other matrix proteins,
polysaccharides, and extracellular signaling molecules as well as cell-
matrix adhesion receptors. These proteins are important organizers of
the extracellular matrix. Through their interactions with adhesion
receptors, they also regulate cell-matrix adhesion — and thus cell
shape and behavior.

TABLE 20-1 • Extracellular Matrix Proteins

Proteoglycans Collagens Multi-adhesive matrix proteins

The illustration shows a

purple stack of
flattened ovals with
three tails at the top, The illustration shows two types of collagens. A blue line
labeled Perlecan. with an oval head on the right is labeled sheet forming; for
example, type 4. An enlarged section shows a braid of 3
lines, with the lines stretched at the center. A blue rod-
shaped structure is labeled fibrillar collagens; for example,
types 1, 2, and 3. An enlarged section shows a braid of
three lines evenly braided.
 The illustration shows three diagrams. The diagram on
the top labeled Laminin shows a cross-shaped row of
beads with a group of 5 orange circles grouped at the
left. The diagram in the center labeled fibronectin shows
an orange rod-shaped structure. An enlarged area shows
a double row of orange ovals. The diagram at the bottom
diagram is labeled nidogen slash entactin shows a
curved line that is enlarged to show that the line is made
of a set of circles.

Cells contribute to the assembly of the ECM not only by secreting its
components, but also by participating directly in the assembly of those
components into complex structures containing large fibrils and
amorphous macromolecules. Once assembled, the ECM often is not
static, but rather highly dynamic in that its chemical, physical, and
biological properties can be altered quantitatively or qualitatively as a
consequence of cells secreting enzymes, such as proteases, and other
molecules into the extracellular space. These alterations in the ECM,
which are usually referred to as remodeling, can involve covalent
chemical modifications (including chemical cross-linking of ECM
molecules), partial or essentially complete proteolytic cleavage of ECM
components, and addition of newly synthesized ECM molecules. An
example we have already encountered, and will discuss in more detail
in Section 20.4, is the role of ECM components in triggering activation
of TGF-β by releasing it from its inhibitor (see Figure 16-22).
The relative volumes occupied by cells and their surrounding matrix
vary greatly among different animal tissues. Some connective tissue,
for instance, is mostly matrix with relatively few cells (Figure 20-4a),
whereas many other tissues, such as epithelia, are composed of very
densely packed cells with relatively little matrix (Figure 20-4b). The
density of packing of the molecules within the ECM itself can also vary
greatly.

FIGURE 20-4 Variation in the relative density of cells and ECM in different tissues. (a) Dense connective tissue contains mostly extracellular matrix consisting
of tightly packed ECM fibers (pink) interspersed with rows of relatively sparse fibroblasts, the cells that synthesized this ECM (purple). (b) Squamous
epithelium viewed from the top, showing epithelial cells tightly packed into a quilt-like pattern with the plasma membranes of adjacent cells close to one
another and little ECM between the cells (see also Figure 20-10b).

The micrograph labeled (a) shows two rows of three narrow oval blue shapes labeled fibroblasts. The pink area between the fibroblasts is labeled E C M. The
micrograph labeled (b) shows several irregular shaped cells with adjacent cell membranes. The blue dots in the center of the cells are labeled nuclei.

H. V. Wilson’s classic studies of adhesion in marine sponge cells dissociated and individual cells from two sponge species are mixed, the
showed conclusively that one primary function of the ECM is to cells of one species will adhere to one another, but not to cells from the
literally hold tissue together. Figures 20-5a and 20-5b, which re-create other species. This specificity is due in part to species-specific adhesive
Wilson’s classic work, show that when sponges are mechanically proteins in the ECM that bind to the cells via adhesion receptors. These
adhesive proteins can be purified and used to coat colored beads,
which, when mixed, aggregate with one another with a specificity
similar to that of intact sponge cells (Figure 20-5c, d).
The ECM plays a multitude of other roles in addition to facilitating cell
adhesion (Table 20-2). Different combinations of components tailor the
ECM for specific purposes at different anatomic sites, for example,
strength in a tendon, strength and rigidity in teeth and bones,
cushioning in cartilage, and transparency in the vitreous humor in the
eyeball. The composition of the ECM also provides positional and
signaling information for cells, letting a cell know where it is and what
it should do. ECM remodeling can modulate the interactions of a cell
with its environment. Furthermore, the ECM serves as a reservoir for
many extracellular signaling molecules that control cell growth and
differentiation. In addition, it provides a lattice through or on which
cells either can move or are prevented from moving, particularly in the
early stages of tissue assembly. Morphogenesis — the stage of
embryonic development in which tissues, organs, and body parts are
formed by cell movements and rearrangements — is critically
dependent on cell-matrix adhesion as well as cell-cell adhesion. For
example, cell-matrix interactions are required for branching
morphogenesis (formation of branching structures) to form blood
vessels, the air sacs in the lung, mammary and salivary glands, and
other structures (Figure 20-6).

aggregation factor (AF) from the ECM of either M. prolifera (MAF) or H.

panicea (HAF). (c) When beads of both colors were coated with MAF, they all
aggregated together, forming yellow aggregates (combination of red and
green). (d) MAF (red) and HAF (green) coated beads do not readily form
mixed aggregates, but rather assemble into distinct clumps held together by
homotypic adhesion. (Magnification 40×.)

[Parts (a) and (b) republished with permission from Springer, from X.
Fernández-Busquets and M. M. Burger, 2003, “Circular Proteoglycans from
Sponges: First Members of the Spongican Family,” Cell. Mol. Life Sci.
60(1):88–112; permission conveyed through Copyright Clearance Center, Inc.
Parts (c) and (d) from J. Jarchow and M. M. Burger, 1998, “Species-Specific
Association of the Cell-Aggregation Molecule Mediates Recognition in Marine
Sponges,” Cell Adhes. Commun. 6(5):405–414; © Taylor and Francis,
www.tandfonline.com.]

EXPERIMENTAL FIGURE 20-5 Mechanically separated marine sponges
reassemble through species-specific homotypic cell adhesion. (a) Two intact
sponges, Microciona prolifera (orange) and Halichondria panicea (yellow),
growing in the wild. (b) After mechanical disruption and mixing of the
individual cells from the two sponge species, their individual cells were
allowed to reassociate for about 30 minutes with gentle stirring. The cells
aggregated with species-specific homotypic adhesion, forming clumps of M.
prolifera cells (orange) and H. panicea cells (yellow). In parts (c) and (d) red
or green fluorescently labeled beads were coated with the proteoglycan
The image labeled (a) shows sponges in their natural habitat. The
micrograph labeled (b) shows agglomerated sponge cells after dispersion.
The fluorescence micrographs labeled (c) and (d) show clumps of cells after
the treatment with M A F - MA F and M A F - H A F, respectively. The
micrograph labeled (c) shows more orange spots with a few green spots
interspersed. The micrograph labeled (d) shows the orange and green spots
clumped together away from each other.

TABLE 20-2 • Functions of the Extracellular Matrix

1. Anchoring and surrounding cells to maintain solid-tissue three-dimensional architecture and define tissue boundaries

2. Determining the biomechanical properties (stiffness/elasticity, porosity, shape) of the extracellular environment

3. Controlling cellular polarity, survival, proliferation, differentiation, and fate (e.g., asymmetric division of stem cells; see Chapter 22),
and thus embryonic and neonatal development and adult function and responses to the environment and to disease

4. Inhibiting or facilitating cell migration (e.g., serving as either a barrier to movement or, conversely, as a “track” along which cells — or
portions of cells — can move)

5. Binding to and acting as a reservoir of growth factors; in some cases, the ECM (a) helps generate an extracellular concentration
gradient of the growth factor, (b) serves as a co-receptor for the growth factor, or (c) aids in proper binding of the growth factor to its
receptor (ECM component and growth factor jointly serve as a receptor’s combined ligand)

6. Activating cell surface signaling receptors

 fibronectin. Anti-fibronectin antibody (Anti-FN) treatment blocked branch
formation (arrowheads). Inhibition of fibronectin’s adhesion receptor (an
integrin) also blocks branch formation (not shown). Scale bar, 100 μm.

[Republished with permission of Nature, from T. Sakai, M. Larsen, and K. M.

Yamada, 2003, “Fibronectin Requirement in Branching Morphogenesis,”
Nature 423(6942):876–881; permission conveyed through Copyright
Clearance Center, Inc.]

EXPERIMENTAL FIGURE 20-6 Antibodies to fibronectin block branching
morphogenesis in developing mouse tissues. Immature salivary glands were
isolated from mouse embryos and allowed to undergo branching
morphogenesis in vitro for 10 hours in the absence (a) or presence (b) of an
antibody that binds to and blocks the activity of the ECM molecule
The micrograph labeled (a) shows a control mouse salivary gland. The gland
features branched lobes with pits in-between. The pits are marked with
arrows.
The micrograph labeled (b) shows a mouse salivary gland treated with an
antibody. The gland is smooth without branching.

Disruptions in cell-matrix and cell-cell interactions can have
devastating consequences for the development of tissues. Figure 20-7
shows the dramatic changes in the skeletal system of embryonic mice
when the genes for either of two key ECM molecules, collagen II and
perlecan, are inactivated. Disruptions in adhesion and ECM functions
are also characteristic of various pathologies, including cardiovascular,
musculoskeletal, kidney, skin, eye, and bone diseases as well as
metastatic cancer, in which cancer cells leave their normal locations
and spread throughout the body.

short stature (e.g., dwarfism), with many skeletal elements shortened and
disfigured.

[Republished with permission from John Wiley & Sons, Inc., from E.
Gustafsson et al., 2003, “Role of Collagen Type II and Perlecan in Skeletal
Development,” Ann. NY Acad. Sci. 995:140–150; permission conveyed
through Copyright Clearance Center, Inc.]

The illustration on the left shows a wild type mouse skeleton stained to
reveal areas of bone and cartilage.

The illustration in the center shows a mouse skeleton with collagen 2

EXPERIMENTAL FIGURE 20-7 Inactivating the genes for some ECM proteins
results in defective skeletal development in mice. These photographs show
skeletons of normal (left), collagen II–deficient (center), and perlecan-
deficient (right) murine embryos that were isolated and stained to visualize
the cartilage (blue) and bone (red). Absence of these key ECM components
leads to forms of
deficiency. The skeleton is stained to reveal areas of bone and cartilage. The
skeleton is smaller than the wild type mouse skeleton and has shortened
forelegs and almost no back legs.
The illustration on the right shows a mouse skeleton with a perlecan
deficiency. The skeleton is stained to reveal areas of bone and cartilage. The
skeleton is smaller than the wild type mouse skeleton and has less bone,
more cartilage, and small stubs.

Although many CAMs and adhesion receptors were initially identified and characterized because of their adhesive properties, they also play major
roles in signaling, using many of the pathways discussed in Chapters 15 and 16. Figure 20-8 illustrates how integrin adhesion receptors physically
and functionally interact, via adapters and signaling molecules, with a broad array of intracellular signaling pathways to influence cell survival,
gene transcription, cytoskeletal organization, cell motility, and cell proliferation. Conversely, changes in the activities of signaling pathways
inside cells can influence the structures of CAMs and adhesion receptors — for example, by altering adapter binding to the cytosolic portions of
the CAMs — and so modulate their ability to interact with other cells and with the ECM. Thus outside-in and inside-out signaling involve numerous
interconnected pathways.

FIGURE 20-8 Integrin adhesion receptor–mediated signaling pathways
control diverse cell functions. Binding of integrins to their ligands induces
The Evolution of Multifaceted Adhesion Molecules Enabled the Evolution of Diverse Animal Tissues
Cell-cell and cell-matrix adhesions are responsible for the formation,
composition, architecture, and function of animal tissues. Not
surprisingly, some adhesion molecules are evolutionarily ancient and
are among the most highly conserved proteins in multicellular
organisms. Some sponges, the most primitive multicellular organisms,
express certain CAMs and multi-adhesive ECM molecules whose
structures are strikingly similar to those of the corresponding human
proteins. The evolution of metazoans has depended on the evolution of
diverse adhesion molecules with novel properties and functions whose
levels of expression differ in different types of cells. Some CAMs and
adhesion receptors (e.g., cadherins, integrins, and Ig-superfamily
CAMs such as L1CAM) and some ECM components (e.g., type IV
collagen, laminin, nidogen/entactin, and perlecan-like proteoglycans)
are highly conserved because they play crucial roles in many different
organisms, whereas other adhesion molecules are less well conserved.
Fruit flies, for example, do not have certain types of collagen or the
ECM protein fibronectin, which play important roles in mammals.
The diversity of adhesion molecules arises in large part from two
phenomena that can generate the numerous closely related proteins,
called isoforms, that constitute a protein family. In some cases, the
different members of a protein family are encoded by multiple genes
conformational changes in their cytoplasmic domains, directly or indirectly
altering their interactions with cytoplasmic proteins (outside-in signaling).
These cytoplasmic proteins include adapter proteins (e.g., talins, kindlins,
paxillin, vinculin) and signaling kinases [Src-family kinases, focal adhesion
kinase (FAK), integrin-linked kinase (ILK)] that transmit signals via diverse
signaling pathways, thereby influencing cell proliferation, cell survival,
cytoskeletal organization, cell migration, and gene transcription.
Components of several signaling pathways, some of which are associated
directly with the plasma membrane, are shown in green boxes. Many of the
components of the pathways shown here are shared with other cell-surface-
activated signaling pathways (e.g., receptor tyrosine kinases shown on the
right) and are discussed in Chapters 15 and 16. In turn, intracellular signaling
pathways can, via adapter proteins, modify the ability of integrins to bind to
their extracellular ligands (inside-out signaling). See W. Guo and F. G.
Giancotti, 2004, Nat. Rev. Mol. Cell Biol. 5:816–826; and R. O. Hynes, 2002, Cell
110:673–687.
The illustration shows a plasma membrane as a gray bar with exterior
labeled above it and cytosol below it. Above the membrane is the label E C M
with a blue and pink chain of beads labeled integrin (adhesion receptor).
The two chains are joined at the top with a ligand. The integrin moves
through the membrane to a rectangle with the label: various adaptors and
signaling kinases. Using downward arrows, a series of these adaptors are
represented as green rectangles with labels inside. The first column of
rectangles, at left, is labeled, top to bottom: P I 3 K, P I (3, 4, 5) P subscript 3,
and A k t slash P K B. The center set of green rectangles is labeled: R a c slash
R h o slash C d c 42 (small G T Pases), N F-k B, J U N. The right-hand set is
labeled: G R B 2, S o s, R a s, R a f, M E K, E R K slash M A P K. Below all of these
is a box labeled cellular responses to adhesion receptor signaling. The
following responses are listed in the box: Cell proliferation (cycle), cell
survival, cytoskeletal organization, cell migration, gene transcription. At the
right end of the plasma membrane is a receptor Tyrosine Kinase. It is a blue-
colored four connected circles above the membrane with two triangle
structures labeled bound ligands and blue irregular structures below the
membrane.
that arose from a common ancestor by gene duplication and divergent
evolution (see the human β-like globin gene cluster in Chapter 7). In
other cases, a single gene produces an RNA transcript that can undergo
alternative splicing to yield multiple mRNAs, each encoding a distinct
protein isoform (see Figure 7-3 and Section 9.2). Both phenomena
contribute to the diversity of some protein families, such as the
cadherins. Particular isoforms of an adhesive protein are often
expressed in some cell types and tissues, but not others.
Cell-Adhesion Molecules Mediate Mechanotransduction
Mechanotransduction is the reciprocal interconversion of a
mechanical force — or stimulus — and biochemical processes. These
interconversions underlie a variety of biological activities, such as
signaling, regulated gene expression, cell proliferation, cell migration,
and interactions among cells and between cells and the ECM. Evolution
has produced a wide variety of mechanosensors that respond to a
mechanical stimulus by changing their shape and activity. Examples
include at least one G protein–coupled receptor that appears to
respond directly to extracellular shear stress — a force parallel to the
surface of the cell, such as that resulting from the flow of blood in a
blood vessel — and a variety of ion channel mechanosensors whose
opening is sensitive to mechanical stretch of the plasma membrane.
These include certain transient receptor potential (TRP) channels as
well as Piezo nonselective cation channels (see Chapter 23), which are
found in neurons and non-neuronal cells and that respond to a variety
of mechanical stimuli, including touch, airway stretch, blood pressure,
and shear and osmotic forces.
Mechanotransduction in the context of cell-cell and cell-matrix
interactions usually involves a cell-surface adhesion receptor that
transmits mechanical force or biochemical information across the
plasma membrane and one or more intracellular or extracellular
mechanosensors that respond to the mechanical stimulus by changing
shape and activity. For example, tension applied across the length of a
multidomain mechanosensor protein, such as the ECM protein
fibronectin or the integrin adapter protein talin, can separate
otherwise tightly packed domains, or even literally pull apart
(disassemble) individual domains. As a consequence, one or more
binding sites that were otherwise inaccessible (cryptic) are exposed.
Figure 20-9 shows examples of domains that are unfolded by
mechanical force, exposing cryptic binding sites. The newly accessible
binding sites can then recruit binding partners — in some cases after
phosphorylation — and alter cellular or extracellular functions. For
example, the stretching of fibronectins by integrins induces domain
separation and consequently their assembly into fibrils, which is an
early step in the assembly of collagen and other molecules into ECM.
The mechanical forces in mechanotransduction can be forces
generated within a cell, such as myosin-driven movement of actin
filaments (Chapter 17), or outside a cell, such as blood flow, movement
of adjacent cells, or contraction or expansion of ECM. As we will see
later in this chapter, cell-cell and cell-matrix adhesion sites play
particularly important roles in mechanotransduction.

FIGURE 20-9 Models of domains in mechanosensor proteins responding to mechanical forces. (a) Hypothetical model of the partial unfolding of a fibronectin
type III domain in the ECM molecule fibronectin when that protein is subjected to mechanical force. Mechanical force generated within the cell by actin
movement and mechanotransduced via multiple integrin adhesion receptors bound to the extracellular dimeric fibronectin can partially unfold the
fibronectin. The unfolding is thought to expose a putative, previously hidden binding site on fibronectin (blue segment) that has the potential to form β
sheets with other fibronectin molecules, recruiting them to form fibronectin fibrils, and helping assemble the ECM. (b) Hypothetical model of the partial
unfolding of a domain (the R1 five-helix bundle) in the intracellular integrin adapter protein talin when it is subjected to mechanical stretching force. This
force is generated by actin, which can bind to and pull on the C-terminus of talin while talin’s N-terminus is bound to the cytoplasmic tail of integrin’s β
subunit. The unfolding is thought to expose this domain’s otherwise cryptic α-helical vinculin binding site (blue). Vinculin, an actin-binding protein, can then
bind to the integrin-talin complex via the exposed site and in turn bind to actin, thus promoting the assembly of multiple actin fibers. The assembly of actin
fibers indirectly linked by adapters to integrins strengthens integrin-mediated adhesion and helps to build focal adhesions (see Figures 20-14e and 20-40).

[Part (a) Data from E. P. Gee et al., 2013, J. Biol. Chem. 288:21329–21340; and M. A. Schumacher et al., 2013, J. Biol. Chem. 288:33738–33744. Part (b) Data from
M. Yao et al., 2014, Sci. Rep. 4:4610; and E. Papagrigoriou et al., 2004, EMBO J. 23:2942–2951.]

The illustration labeled (a) titled Fibronectin type 3 domain shows gray ribbons with one green, one red, and one blue area highlighted. The blue area is
labeled inaccessible binding site. A sideward arrow from the ends of the domain is labeled mechanical force (stretching) and goes to a diagram where the red
and blue parts are stretched into a single ribbon off to the right and the green highlight is still in the same place. The stretched blue ribbon is labeled
accessible binding site. The illustration labeled (b) titled Talin five-helix bundle domain shows gray ribbons with one blue area highlighted. The blue area is
labeled inaccessible binding site. Then the sideward arrow with the stretching shows a blue ribbon off to the right of the bundle and labeled accessible
binding site.

KEY CONCEPTS OF SECTION 20.1

Cell-Cell and Cell–Extracellular Matrix Adhesion: An Overview

 Cell-cell and cell–extracellular matrix (ECM) interactions are critical for assembling cells into tissues, controlling cell shape and function,
and determining the developmental fate of cells and tissues. Diseases may result from abnormalities in the structures or expression of
adhesion molecules.
 Cell-cell adhesion molecules (CAMs, also called cell-cell adhesion receptors) mediate direct intercellular cell-cell adhesions (homotypic and
heterotypic), and cell-matrix receptors mediate cell attachment to the ECM (see Figure 20-1). These interactions bind cells into tissues and
facilitate communication between cells and their environments.
 The cytosolic domains of cell adhesion receptors bind adapter proteins that mediate interaction with cytoskeletal fibers and intracellular
signaling proteins.
 The major families of CAMs are the cadherins, lectins, Ig-superfamily CAMs, and integrins (see Figure 20-2). Members of the integrin and
IgCAM superfamilies can also function as cell-matrix adhesion receptors.
 Tight cell-cell adhesions entail both trans (adhesive or intercellular) interactions of like (homophilic) or different (heterophilic) CAMs and cis
(lateral) oligomerization of CAMs (see Figure 20-3). The combination of trans and cis interactions produces a Velcro-like adhesion between
cells.
 The ECM is a dynamic, complex meshwork of proteins and polysaccharides that contributes to the structure and function of a tissue (see
Table 20-2). The major classes of ECM molecules are proteoglycans, collagens, and multi-adhesive matrix proteins, such as fibronectin and
laminin.
 CAMs and adhesion receptors, together with their cytoplasmic adapter proteins, play major roles in “outside-in” and “inside-out” signaling,
facilitating critically important communication between cells and their surroundings.
 The evolution of adhesion molecules with specialized structures and functions permits cells to assemble into diverse classes of tissues with
varying functions.
 Mechanotransduction, the interconversion of a mechanical stimulus or force and biochemical processes, is mediated by adhesion receptors
and mechanosensors. Adhesion receptors themselves can also be mechanosensors. Mechanotransduction permits cells to respond to
mechanical forces from their environments and to exert mechanical forces on their surroundings.
20.2 Cell-Cell and Cell–Extracellular Matrix Junctions and Their Adhesion
Molecules
Cells in epithelial and in nonepithelial tissues use many, but not all, of
the same cell-cell and cell-matrix adhesion molecules. We begin our
detailed discussion of adhesion with epithelia because of their
relatively simple organization, as well as their fundamental role in
evolution and development. In this section, we focus on regions of the
cell surface that contain clusters of adhesion molecules in discrete
patches or spots, called anchoring junctions, tight junctions, and gap
junctions. Anchoring and tight junctions play critical roles in mediating
cell-cell and cell-matrix adhesion, and all three types of junctions
mediate intercellular or cell-matrix communication.
Epithelial Cells Have Distinct Apical, Lateral, and Basal Surfaces
Cells that form epithelial tissues are said to be polarized because their
plasma membranes are organized into discrete regions. Typically, the
distinct surfaces of a polarized epithelial cell are called the apical (top),
lateral (side), and basal (base or bottom) surfaces (Figure 20-10; see
also Figure 20-1). The apical, lateral, and basal surfaces of epithelial
cells can exhibit distinctive characteristics. The area of the apical
surface is often greatly expanded by the formation of microvilli —
small, fingerlike projections (see Chapter 22). A complex set of
evolutionarily conserved molecules, called polarity regulators, is
required to generate the functional and structural asymmetry of
epithelial and other cells (see Chapter 22). Adhesion molecules play
essential roles in generating and maintaining these distinct surfaces.

Epithelia in different body locations have characteristic morphologies
and functions (see Figure 20-10; see also Figure 1-4). Stratified
(multilayered) epithelia commonly serve as barriers and protective
surfaces (e.g., the skin), whereas simple (single-layered) epithelia often
selectively move ions and small molecules from one side of the
epithelium to the other. For instance, the simple columnar epithelium
lining the stomach secretes hydrochloric acid into the lumen; a similar
epithelium lining the small intestine transports products of digestion
from the lumen of the intestine across the cells into the blood (see
Figure 11-30).
In simple columnar epithelia, adhesive interactions between the lateral
surfaces hold the cells together in a two-dimensional sheet, whereas
those at the basal surface connect the cells to a specialized underlying
extracellular matrix called the basal lamina. Often the basal and lateral
surfaces are similar in composition and are collectively called the
basolateral surface. The basolateral surfaces of most simple epithelia
are usually on the side of the cell closest to the blood vessels, whereas
FIGURE 20-10 Principal types of epithelia. (a) Simple columnar epithelia
consist of elongated cells, including mucus-secreting cells (in the lining of
the stomach and cervical tract) and absorptive cells (in the lining of the
small intestine). The thin protrusions at the apical surface are microvilli (see
Figure 20-11). (b) Simple squamous epithelia, composed of thin cells, line
the blood vessels (endothelial cells/endothelium) and many body cavities.
(c) Transitional epithelia, composed of several layers of cells with different
shapes, line certain cavities subject to expansion and contraction (e.g., the
urinary bladder). (d) Stratified squamous (nonkeratinized) epithelia line
surfaces such as the mouth and vagina; these linings resist abrasion and
generally do not participate in the absorption or secretion of materials into
or out of the cavity. The basal lamina, a thin fibrous network of collagen and
other ECM components, supports all epithelia and connects them to the
underlying connective tissue.
The illustration labeled (a) titled simple columnar shows tall thick rounded
rectangular cells grouped above connective tissue, which is represented as a
brown area of cells at the bottom. The top is labeled apical surface; the side
of the rectangular cells has the label: lateral surface. At the bottom of the
rectangles, the label reads basal surface, and the top of the connective
tissue is labeled basal lamina. The illustration labeled (b) titled simple
squamous shows the same connective tissue at the bottom, but the
rectangles now look like a flat layer on top. The illustration labeled (c) titled
transitional shows four layers of cells, and they are different sizes, thick on
top, small ones in the middle, and taller ones at the bottom above the
connective tissue. The illustration labeled (d) titled stratified squamous
(non-keratinized) shows the cells flattened in the top layers.
the apical surface is not in stable, direct contact with other cells or the
ECM. In animals with closed circulatory systems, blood flows through
vessels whose inner lining is composed of flattened epithelial cells
called endothelial cells. In general, epithelial cells are sessile, in that
adhesion molecules firmly and stably attach them to one another and
their associated ECM. One especially important mechanism that
generates strong, stable adhesions is the concentration of subsets of
these molecules into clusters called cell junctions.
Three Types of Junctions Mediate Many Cell-Cell and Cell-Matrix Interactions
All epithelial cells in a sheet are connected to one another and to the
ECM by specialized junctions. Although hundreds of individual
dispersed adhesion molecule–mediated interactions are sufficient to
cause cells to adhere, the clustered groups of adhesion molecules at
cell junctions play special roles in imparting strength and rigidity to a
tissue, transmitting information between the extracellular and the
intracellular space, controlling the passage of ions and molecules
across cell layers, and serving as conduits for the movement of ions
and molecules from the cytoplasm of one cell to that of its immediate
neighbor. Particularly important to epithelia is the formation of
junctions that help form tight seals between the cells, allowing the
epithelial sheet to serve as a barrier to the flow of molecules from one
side of the sheet to the other.
Three major classes of animal-cell junctions are prominent features of
simple columnar epithelia (Figure 20-11 and Table 20-3): anchoring
junctions, tight junctions, and gap junctions. Anchoring junctions
and tight junctions perform the key task of holding the tissue together.
As we shall see, tight junctions also control the flow of solutes through
the extracellular spaces between the cells forming an epithelial sheet.
Tight junctions are found primarily in epithelial cells, whereas
anchoring junctions can be seen in both epithelial and nonepithelial
cells. Anchoring junctions and tight junctions in epithelia are
organized into three parts: (1) adhesive proteins (CAMs, adhesion
receptors) in the plasma membrane that connect one cell to another
cell on the lateral surfaces or to the ECM on the basal surfaces; (2)
adapter proteins, which connect the CAMs or adhesion receptors to
cytoskeletal filaments and signaling molecules; and (3) the cytoskeletal
filaments themselves.

FIGURE 20-11 Principal types of cell junctions connecting the columnar epithelial cells lining the small intestine. (a) Schematic cutaway drawing of intestinal
epithelial cells. The basal surface of the cells rests on a basal lamina, and the apical surface is packed with microvilli that project into the intestinal lumen.
Tight junctions, lying just under the microvilli, prevent the diffusion of many substances between the intestinal lumen and internal body fluids (such as the
blood) via the extracellular space between cells. Gap junctions allow the movement of small molecules and ions between the cytosols of adjacent cells. The
remaining three types of junctions — adherens junctions, desmosomes, and hemidesmosomes — are critical to cell-cell and cell-matrix adhesion and
signaling. (b) Electron micrograph of a thin section of epithelial cells in the rat intestine, showing the relative locations of the different junctions.

[Part (b) M. G. Farquhar and G. E. Palade, 1963, J. Cell Biol. 17(2):375–412, Fig. 1; https://doi.org/10.1083/jcb.17.2.375.]

The illustration labeled (a) shows a cube-shaped section of epithelial cells with microvilli as tan tubes on the top. A cutaway shows how various junctions are
repeated and connected. The different types of junctions are labeled. At the top, below the microvilli is a tight junction with three layers of skinny ovals.
Below this is a long red tube labeled adherens junction, with brown dots inside labeled actin and myosin filaments. Further down is an orange oval-shaped
labeled gap junction. Next is a blue circle labeled desmosome. Connected to this, inside the cube are blue lines labeled intermediate filaments. At the
bottom, but above the connective tissue layer is a pink oval labeled hemidesmosome. The micrograph labeled (b) shows the same area as that of the
illustration. The tight junction, gap junction, adherens junction, and desmosome are labeled.

TABLE 20-3 • Cell Junctions

Junction Adhesion Principal CAMs Cytoskeletal Intracellular Function

Type or Adhesion Attachment Adapters
Receptors

Anchoring junctions

1. Adherens junctions Cell-cell Cadherins Actin filaments Catenins, vinculin Shape, tension,
signaling, force
transmission
 2. Desmosomes Cell-cell Desmosomal Intermediate Plakoglobin, Strength, durability,
cadherins filaments plakophilins, signaling
desmoplakins

3. Hemidesmosomes Cell- Integrin (α6β4) Intermediate Plectin, Shape, rigidity,

matrix filaments dystonin/BPAG1 signaling

4. Focal contacts, Cell- Integrins Actin filaments Talin, kindlin, Shape, signaling,
fibrillar, and 3-D matrix paxillin, vinculin force transmission,
adhesions cell movement

Tight junctions Cell-cell Occludin, Actin filaments ZO-1,2,3, PAR3, Controlling solute
claudins, JAMs cingulin flow, cell polarity,
signaling

Gap junctions Cell-cell Connexins, Via adapters to ZO-1,2,3 Communication,

innexins, other junctions small-molecule
pannexins transport between
cells

Plasmodesmata (plants only) Cell-cell Undefined Actin filaments NET1A Communication,

molecule transport
between cells

Gap junctions permit the rapid diffusion of small, water-soluble
molecules between the cytoplasms of adjacent cells. Along with
anchoring and tight junctions, gap junctions help a cell communicate
with its environment. However, they are structurally very different
from anchoring junctions and tight junctions and do not play a key role
in strengthening cell-cell and cell-matrix adhesions. Found in both
epithelial and nonepithelial cells, gap junctions resemble the distinct
cell junctions in plants called plasmodesmata, which we discuss in
Section 20.6.
Four types of anchoring junctions are present in cells. Two participate
in cell-cell adhesion and two participate in cell-matrix adhesion.
Adherens junctions connect the lateral membranes of adjacent
epithelial cells and are usually located near the apical surface, just
below the tight junctions (see Figure 20-11). A circumferential belt of
actin and myosin filaments in a complex with the adherens junctions
functions as a tension cable that can internally brace the cell and
thereby control its shape. Epithelial and some other types of cells, such
as smooth muscle and heart cells, are also bound tightly together by
desmosomes, snap-like points of contact sometimes called spot
desmosomes. Hemidesmosomes, found mainly on the basal surface of
epithelial cells, and focal contacts, also called focal adhesions, anchor
an epithelium to components of the underlying ECM, much like nails
holding down a carpet. Adherens junctions, desmosomes, and focal
adhesions are found in many different types of cells; hemidesmosomes
appear to be restricted to epithelial cells.
Cadherins Mediate Cell-Cell Adhesions in Adherens Junctions and
Desmosomes
The primary CAMs in adherens junctions and desmosomes belong to
the cadherin family of glycoproteins that can contain oligosaccharides
that are either N-linked (see Chapters 13 and 14) or O-linked (described
in Figure 20-31 in Section 20.4). In vertebrates, this protein superfamily
of more than a hundred members can be grouped into at least six
subfamilies, including classical cadherins, clustered protocadherins,
and desmosomal cadherins, which we will describe below. The
diversity of cadherins arises from the presence of multiple cadherin
genes and alternative RNA splicing. It is not surprising that there are
many different types of cadherins in vertebrates; many different types
of cells in the widely diverse tissues of these animals use cadherins to
mediate adhesion and communication, the detailed requirements for
which may differ for different types of cells and tissues. Members of
the cadherin superfamily can also control cell morphology, such as the
assembly and tight packing of microvilli on the apical surfaces of some
epithelial cells (see Figures 20-10a and 20-11a). The brain expresses the
largest number of different cadherins, including protocadherins,
presumably owing to the necessity of forming many specific cell-cell
contacts to help establish its complex wiring pattern. Invertebrates,
however, are able to function with fewer than 20 cadherins.
Classical Cadherins

Bundles of intermediate filaments running parallel to the cell surface
or through the cell connect desmosomes and hemidesmosomes,
imparting shape and rigidity to the cell, as do actin filaments that
connect the cytoskeleton with focal contacts and adherens junctions.
The close interaction between these junctions and the cytoskeleton
helps transmit shear forces from one region of a cell layer to the
epithelium as a whole, providing strength and rigidity to the entire
epithelial cell layer. Desmosomes and hemidesmosomes are especially
important in maintaining the integrity of skin epithelia. As a
consequence, mutations that interfere with hemidesmosomal
anchoring in the skin can lead to a condition in which the epithelium
becomes detached from its underlying matrix and extracellular fluid
accumulates at the basolateral surface, forcing the skin to balloon
outward, forming a blister.
The classical cadherins include E-, N-, and P-cadherins, named for the
type of tissue in which they were initially identified (epithelial, neural,
and placental, respectively). E- and N-cadherins are the most widely
expressed, particularly during early differentiation. Sheets of polarized
epithelial cells, such as those that line the small intestine or kidney
tubules, contain abundant E-cadherin along their lateral surfaces.
Although E-cadherin is concentrated in adherens junctions, it is
present throughout the lateral surfaces, where it is thought to link
adjacent cell membranes.
The results of experiments with L cells, a line of cultured mouse
fibroblasts, demonstrated that E-cadherins preferentially mediate
homophilic interactions. L cells express no cadherins and adhere
poorly to each other and to other types of cells. When the E-cadherin
gene was introduced into L cells, the cells were found to adhere
preferentially to other cells expressing E-cadherin (Figure 20-12).
These engineered cadherin-expressing L cells formed epithelium-like
aggregates with one another and with epithelial cells isolated from
EXPERIMENTAL FIGURE 20-12 E-cadherin mediates Ca2+ -dependent
adhesion of L cells. Under standard cell-culture conditions, in the presence
of
The role of E-cadherin in adhesion can also be demonstrated by
experiments with cultured epithelial cells called Madin-Darby canine
kidney (MDCK) cells (see Figure 4-3). A green fluorescent protein–
labeled form of E-cadherin has been used in these cells to show that
clusters of E-cadherin mediate the initial attachment of the cells and
EXPERIMENTAL FIGURE 20-13 E-cadherin mediates adhesive connections in
cultured MDCK epithelial cells. An E-cadherin gene fused to green
fluorescent
Each classical cadherin molecule contains a single transmembrane
domain, a relatively short C-terminal cytosolic domain, and five
extracellular cadherin domains (called EC1–EC5) (see Figure 20-2). The
extracellular domains are necessary for Ca2+ binding and
cadherin-mediated cell-cell adhesion. Classical cadherin–mediated
adhesion entails both cis lateral (on the same cell) clustering and trans
adhesive (intercellular) molecular interactions (see Figures 20-3 and
20-14a–c). The binding of three Ca2+ ions at each of the sites
located between the cadherin repeats (see Figures 20-2 and 20-14a)
stabilizes the elongated and curved structure of the extracellular
domain. As we shall see shortly, the curved structure of cadherin’s
extracellular domain is necessary for the proper molecular
complementarity that stabilizes cis and trans binding between
lungs. Although most E-cadherins exhibit primarily homophilic
binding, some mediate heterophilic interactions.
calcium in the extracellular fluid, L cells do not aggregate into sheets (left).
Introduction of a gene that causes the expression of E-cadherin in these cells
results in their aggregation into epithelium-like clumps in the presence of
calcium (center), but not in its absence (right). Bar, 60 μm.
[© C. L. Adams et al., 1998, J. Cell Biol. 142(4):1105–1119, Fig. 1E;
https://doi.org/10.1083/jcb.142.4.1105.]
The adhesiveness of cadherins depends on the presence of
extracellular Ca2+ ; it is this property (calcium adhering) that gave
rise to their name. For example, the adhesion of L cells expressing E-
cadherin is prevented when the cells are bathed in a solution that is
low in Ca2+ (see Figure 20-12). Some adhesion molecules require
some minimal amount of Ca2+ in the extracellular fluid to
function properly, whereas others, such as IgCAMs, are Ca2+
independent.
the subsequent zippering of the cells into sheets (Figure 20-13). In this
experimental system, the addition of an antibody that binds to E-
cadherin, preventing its homophilic interactions, blocks the Ca2+
- dependent attachment of MDCK cells to one another and the
subsequent formation of intercellular adherens junctions.
protein (GFP) was introduced into cultured MDCK cells. The cells were then
mixed together in a calcium-containing medium, and the distribution of
fluorescent E-cadherin was visualized over time (shown in hours). Clusters of
E-cadherin mediate the initial attachment and subsequent zippering up of
the epithelial cells and the formation of junctions (bicellular junctions are
where two cells join and appear as lines; tricellular junctions are the sites of
intersection of three cells).
[© C. L. Adams et al., 1998, J. Cell Biol. 142(4):1105–1119, Fig. 2B;
https://doi.org/10.1083/jcb.142.4.1105.]
The label at the top of the photos reads, Time after mixing cells in hours (0,
2, 4, 6, 8 hours). The photo at 0 hours shows three dim and separate cells. In
2 hours, the cells are brighter, a connection is forming, and arrows point to
the developing connection. In 4 hours, the connections are bigger. In 6
hours, the connections are bigger and longer. In 8 hours, the connections
have extended three ways between the three cells.
cadherin molecules. The cis and trans interactions of cadherins,
together with their interactions with cytoplasmic adapter and
cytoskeletal molecules, permit the zippering up of cadherins into
adhesive arrays. Binding of the EC1 domain of one cadherin molecule
to the EC1 domain of another on the adjacent cell is responsible for
trans binding (Figure 20-14a; see also Figure 20-3). Although the
dissociation constant (Kd) for EC1–EC1 homophilic binding
measured using isolated domains in solution is on the order of
10−5−10−4 mol/L (relatively weak, or low-affinity,
binding), the multiple low-affinity interactions in arrays of intact
cadherin molecules on adjacent cells sum to produce a very tight
intercellular adhesion.
FIGURE 20-14 Trans (intercellular) and cis (on the same cell) interactions of classical cadherins in typical adherens junctions and of protocadherins. (a) The
exoplasmic cadherin domains [EC1-EC5, see ovals in (b)] of E-cadherins at adherens junctions on adjacent cells are clustered by homophilic cis and trans
interactions (see Figure 20-3). The Ca2+ -dependent elongated and curved structure of cadherin’s extracellular domains is necessary for stable cis and
trans interactions (examples highlighted by dashed circles). (b) EC1-EC2 cis interaction. Cis interactions are defined by the binding of an EC1 domain of one
cadherin to an EC2 domain of an adjacent cadherin on the same cell. In (b) and (c) the structure of each extracellular cadherin domain is represented using a
ribbon diagram and highlighted by an oval. (c) EC1-EC1 trans interaction: Two views rotated by 90° of the trans binding of an EC1 domain of one cadherin to
an EC1 domain of a cadherin on the adjacent cell. Only the EC1 and a portion of the EC2 domains are shown. Note that a small segment of polypeptide at the
N-terminus of each of the two EC1 domains [highlighted in yellow (cell 1) and blue (cell 2)] swings out and replaces the equivalent segment from its binding
partner (strand swap, dashed oval on the right). The strand swap places the side chain of a tryptophan residue on each of the segments into a binding pocket
on the adjacent EC1 domain — an interaction that substantially stabilizes the trans binding. (d) The clustered protocadherins at neuron-to-neuron interfaces
form a lattice of cis and trans interactions with their exoplasmic cadherin domains (ovals, EC1-EC6) that differs from that in classical cadherins (part a).
Different protocadherin isoforms are represented by different colors (lighter colors expressed by Cell 1, darker colors expressed by Cell 2). The dimeric,
homo- (same color) and heterophilic (different colors) cis interactions form between the EC6 domain of one molecule and EC5-EC6 domains of its partner (cis
dashed circle). The dimeric, homophilic trans interactions form between the EC1-EC4 domains of one molecule with the EC1-EC4 domains of the same
isoform (same color) on the adjacent cell. (e) The cytosolic domains of the classical E-cadherins bind directly or indirectly to multiple adapter proteins (e.g.,
β-catenin), which both connect the junctions to actin filaments (F-actin) of the cytoskeleton and participate in intracellular signaling pathways. Somewhat
different sets of adapter proteins are illustrated in the two cells to emphasize the variety of adapters in adherens junctions. Some of these adapters, such as
vinculin and ZO-1, can interact with several different CAMs. See V. Vasioukhin and E. Fuchs, 2001, Curr. Opin. Cell Biol. 13:76–84; and J. Brasch, et al., 2012,
Trends Cell Biol. 22:299–310.

[Parts (a–c) Data from O. J. Harrison et al., 2011, Structure 19: 244–256, PDB ID 3q2w. Part (d) Data from J. Brasch et al., 2019, Nature 569: 280–283, PDB ID
6e6b.]

The illustration labeled (a) shows cell 1 at left and cell 2 at right, each with 5 rows of blue ovals moving toward the center between the cells. At the joint,
where two of cell one ovals join, is the labeled cis, and a white arrow points to illustration (b). Also in illustration (a), where an oval from cell 1 joins an oval
from cell 2 is labeled trans and a white arrow is pointing to illustration (c). The illustration labeled (b) shows a three-dimensional ribbon of the inside of each
oval. Each oval is labeled from the cell 1 side E C 1 through E C 5. The E C 1 from the top row joins to the E C 1 from the next row down. The illustration labeled
(c) shows the E C 1 from cell 1 joining to the E C 1 from cell 2, using three-dimensional ribbon diagrams. A small area of ribbon at the center is highlighted with
yellow (cell 1) and blue (cell 2) colors showing how they interact. Next to the first ribbon diagram is another one from a 90-degree turn of the same ribbon
diagram. The illustration labeled (d) titled protocadherin shows a schematic with cell 1 membrane at left and cell 2 membrane at right. From each cell, 3 sets
of 6 ovals in chains extend toward the other cell. In this diagram, each pair of chains cross each other near the cell membrane, then the top chain from cell 1
pair joins four ovals to the bottom chain from cell 2. The illustration labeled (e) titled E-cadherin shows oval chains between the cell membranes of cell 1 at
left and cell 2 at right. In the cytosol of cell 1, the ends of 4 chains have a protein attached, labeled from top to bottom: beta-catenin, alpha-catenin, F-actin, Z
O-1. In the cytosol of cell 2, four different proteins are diagramed and labeled, from top to bottom: V A S P, vinculin, alpha-actinin, and p120-catenin.

Determination of the structures of the extracellular domains of
cadherins, together with analyses of the structures and binding
properties of many mutants of the key binding domains, have provided
a clear picture of the cis and trans interactions that underlie classical
cadherin–mediated cell adhesion. The key features of cadherin cis and
trans binding interactions are (1) the calcium-dependent curvature of
the five extracellular cadherin domains that permits proper relative
orientations of the EC1 and EC2 domains (see Figures 20-2 and 20-14a);
(2) for cis interactions, the binding of one side of an EC1 domain to a
complementary surface on the EC2 domain of an adjacent molecule on
the same membrane (see Figures 20-2 and 20-14a and b); and (3) for
trans interactions, the binding of a different surface of the EC1 domain
to an EC1 domain from a cadherin molecule on the adjacent cell
(Figure 20-14c). The trans EC1–EC1 binding is stabilized when a small
segment of the protein at the N-terminus of each of the two EC1
domains swings out and replaces the equivalent segment from its
binding partner (strand swap; see Figure 20-14c).
Another group of CAMs, called clustered protocadherins, provides an
excellent example of how evolution can employ common molecular
building blocks — in this case, cadherin domains — for distinct
structural and functional purposes. Clustered protocadherins play a
role in establishing the complex wiring diagram of neurons in
mammalian brains. Multiple protocadherin genes encode 52 different
clustered protocadherin proteins (isoforms) in humans (58 in mice).
The random combinations of 10–15 different isoforms that are
expressed in any individual neuron provide a bar code that helps a
neuron distinguish itself from other neurons and thus helps generate
neuronal wiring diagrams. The domain architecture of these
protocadherins with six extracellular cadherin domains (EC1–EC6)
differs from that of classical cadherins with five domains, and the
organization of the cis and trans binding (see Figure 20-14d) differs
markedly from that of classical cadherins (see Figure 20-14a–c). In
clustered protocadherins, cis interactions are mediated by the EC6
domain of one molecule binding to the EC5 and EC6 domains of an
adjacent molecule (Figure 20-14d). These cis interactions can be both
homophilic and heterophilic, allowing the same or different isoforms
to associate with each other into cis dimers [different colors represent
different isoforms in part (d)]. In contrast, trans interactions are
mediated by strictly homophilic, antiparallel binding of the EC1-EC4-
domains (see Figure 20-14d). Trans binding only occurs when the
sequences of all four of these EC domains perfectly match those of a
corresponding protocadherin isoform on the adjacent cell. The lattices
of intermixed protocadherin isoforms that form an interface between
the membranes of neurons are responsible for their influence on
neuronal wiring.
The cytosolic domains of CAMs have profound effects on the binding
properties and functions of these adhesive molecules. For example, the
C-terminal cytosolic domain of classical cadherins is linked to the actin
cytoskeleton by adapter proteins (Figure 20-14e). These linkages are
essential for strong adhesion. Indeed, cadherins together with their
adaptor proteins in adherens junctions participate in
mechanotransduction; a moderate increase in the mechanical tension
at the junctions generated either intracellularly by actin and myosin, or
by extracellular forces such as those transferred from adjacent cells or
shear force due to blood flow, induces the formation of larger clusters
of cadherins and stronger intercellular adhesion. An important
mechanosensor is the adapter protein α-catenin, which is attached to
the cytoplasmic domain of cadherin via the protein β-catenin and also
binds directly to F-actin (see Figure 20-14e). When the cadherin/β-
catenin/α-catenin/F-actin complex is subjected to mechanical stress,
the C-terminal actin-binding domain of α-catenin undergoes a
conformational change to bind more tightly to the actin. In addition, a
tension-induced conformational change in α-catenin’s adhesion
modulation domain uncovers otherwise cryptic binding sites for
additional proteins, including the actin-binding adaptor vinculin. As a
consequence, additional F-actin filaments can be recruited (see Figure
20-14e) and these further enlarge and strengthen the adherens
junction. We will see vinculin playing a similar role in integrin-
mediated adhesion later in this chapter.
Disruption of the interactions between classical cadherins and α-
catenin or β-catenin dramatically reduces cadherin-mediated cell-cell
adhesion. This disruption occurs spontaneously in tumor cells, which
sometimes fail to express α-catenin, and can be induced
experimentally by depleting the cytosolic pool of accessible β-catenin.
The cytosolic domains of cadherins also interact with other adaptor
molecules including p120-catenin, which can influence the stability of
the cadherin. Interestingly, β-catenin plays a dual role: it not only
mediates cytoskeletal attachment, but also serves as a signaling
molecule, translocating to the nucleus and altering gene transcription
in the Wnt signaling pathway (see Figure 16-26).
Classical cadherins play a critical role during tissue differentiation.
Each classical cadherin has a characteristic tissue distribution. In the
course of differentiation, the amounts or types of cell-surface
cadherins change, affecting many aspects of cell-cell adhesion, cell
migration, and cell division. For instance, the normal reorganization of
tissues during morphogenesis is often accompanied by the conversion
of nonmotile epithelial cells into motile cells, called mesenchymal cells,
that are precursors for other tissues. This epithelial-to-mesenchymal
transition (EMT) is associated with a reduction in the expression of E-
cadherin (Figure 20-15a, b). The EMT is also associated with pathology,
as in the conversion of epithelial cells into malignant carcinoma cells.
For example, certain ductal breast tumors and hereditary diffuse
Infection with rhinoviruses (RV) is the most frequent cause of the
common cold, and infection with virulent class C rhinoviruses (RV-C)
can cause more severe illnesses, including exacerbation of asthma. To
enter cells and replicate, RV-C must bind to cell-surface receptors. A
cadherin family member called CDHR3, which is highly expressed in
epithelial cells in the human airway, is a receptor for RV-C. Pathogens
such as RV-C often evolve to co-opt proteins that have normal
functions in their target (host) tissues. Genetic studies have shown that
a naturally occurring mutation in humans that changes a cysteine to
tyrosine (C → Y) in the EC5 domain of CDHR3 is associated with
increased wheezing illnesses and hospitalizations for childhood
asthma. In cultured cells, this C → Y mutation increases the cell-surface
expression of CDHR3 and the binding and replication of RV-C.
gastric cancer (Figure 20-15c) characteristically involve a loss of E-
cadherin activity. It is well known that animal cell-cell contact can
inhibit cell proliferation. During tissue development, once dividing
epithelial cells have formed a well-defined, tightly bound epithelium,
they have no need for further cell division unless they are damaged or
receive a signal to undergo the EMT. It is now clear that one
mechanism used to inhibit proliferation of epithelial cells in epithelia
is E-cadherin- and catenin-mediated regulation of the Hippo pathway
that controls cell proliferation (see Chapter 21).
EXPERIMENTAL FIGURE 20-15 E-cadherin activity is lost during the epithelial-
to-mesenchymal transition and during cancer progression. A protein called
Snail that suppresses the expression of E-cadherin is associated with the
epithelial-to-mesenchymal transition (EMT). (a) Normal epithelial MDCK
cells grown in culture. (b) Expression of the snail gene in MDCK cells causes
them to undergo an EMT. (c) Distribution of E-cadherin detected by
immunohistochemical staining (dark brown) in a thin section of tissue from
a patient with hereditary diffuse gastric cancer. E-Cadherin is seen at the
intercellular borders of normal stomach gastric gland epithelial cells; no E-
cadherin is seen at the borders of underlying invasive carcinoma cells.
[Parts (a) and (b) republished with permission from Elsevier, from A.
Martinez Arias, 2001, “Epithelial Mesenchymal Interactions in Cancer and
Development,” Cell 105(4):425–431; permission conveyed through Copyright
Clearance Center, Inc. Part (c) republished with permission from John Wiley
& Sons, Inc., from F. Carneiro et al., 2004, “Model of the Early Development
of Diffuse Gastric Cancer in E-Cadherin Mutation Carriers and Its
Implications for Patient Screening,” J. Pathol. 203(2):681–687; permission
conveyed through Copyright Clearance Center, Inc.]
The firm epithelial cell-cell adhesions mediated by cadherins in
adherens junctions permit the formation of a second class of
intercellular junctions in epithelia — tight junctions, to which we will
turn shortly.
Treatments that disrupt the RV-C/cadherin (CDHR3) interaction have
the potential to prevent or treat respiratory diseases caused by RV-C.
Desmosomal Cadherins
Desmosomes (Figure 20-16) contain two specialized cadherins,
desmoglein and desmocollin, whose cytosolic domains are distinct from
those in the classical cadherins. The cytosolic domains of desmosomal
cadherins bind to adapter proteins such as plakoglobin (similar in
structure to β-catenin) and plakophilins, and these bind to a member
of the plakin family of adapters, called desmoplakin. These adapters
form the thick cytoplasmic plaques that are characteristic of
desmosomes. The desmoplakins directly mediate plaque binding to
intermediate filaments.

The cadherin desmoglein was identified through studies of an
unusual but revealing skin disease called pemphigus vulgaris, an
autoimmune disease. Patients with autoimmune disorders synthesize
self-attacking (or “auto”) antibodies that bind to a normal body protein.
In pemphigus vulgaris, the auto-antibodies disrupt adhesion between
epithelial cells, causing blisters of the skin and mucous membranes.
The predominant auto-antibodies in patients were shown to be specific
for desmoglein; indeed, the addition of such antibodies to normal skin
induces the formation of blisters and disruption of cell adhesion.
Integrins Mediate Cell-Matrix Adhesions, Including Those in Epithelial-Cell
Hemidesmosomes
To be stably anchored to solid tissues and organs, simple columnar
epithelial sheets must be firmly attached via their basal surfaces to the
underlying ECM (basal lamina). This attachment occurs via adhesion
receptors called integrins (see Figure 20-2), which are located both
FIGURE 20-16 Desmosomes. (a) Model of a desmosome between epithelial
cells with attachments to the sides of intermediate filaments. The key CAMs
in desmosomes are the desmosomal cadherins desmoglein and
desmocollin. Adapter proteins bound to the cytoplasmic domains of these
cadherins include plakoglobin, desmoplakins, and plakophilins. See B. M.
Gumbiner, 1993, Neuron 11:551–564; L. A. Staehelin and B. E. Hull, 1978, Sci.
Am. 238:140; and D. R. Garrod, 1993, Curr. Opin. Cell Biol. 5:30–40. (b)
Electron micrograph of a thin section of a desmosome connecting two
cultured differentiated human keratinocytes. Bundles of intermediate
filaments radiate from the two darkly staining cytoplasmic plaques that line
the inner surface of the adjacent plasma membranes. Inset: Electron
microscopic tomograph of a desmosome linking two human epidermal cells
(plasma membranes, pink; desmosomal cadherins, blue; bar, 35 nm).
[Part (b) republished with permission from Nature, from A. Al-Amoudi et al.,
2007, “The Molecular Architecture of Cadherins in Native Epidermal
Desmosomes,” Nature 450(7171): 832–837; permission conveyed through
Copyright Clearance Center, Inc.]
The illustration labeled (a) shows a schematic of a desmosome in blue. Two
cell membranes are with the blue connections of rows of blue ovals between
them in the intercellular space. In the cytosol of each cell, intermediate
filaments are labeled. The blue ovals are labeled desmoglein and
desmocollin (cadherins). Inside the cell membrane on each side of the ovals
is a large blue elongated oval labeled cytoplasmic plaque (plakoglobin,
desmoplakins, plakophilins). The micrograph labeled (d) shows a
desmosome with labels intermediate filaments, cytoplasmic plaques, and
plasma membranes.
within and outside of anchoring junctions called hemidesmosomes (see
Figure 20-11a). Hemidesmosomes comprise integral membrane
proteins linked via cytoplasmic adapter proteins (e.g., plakins) to
keratin-based intermediate filaments. The principal ECM adhesion
receptor in epithelial hemidesmosomes is integrin α6β4.
Integrins function as cell-cell and cell-ECM adhesion receptors in a
wide variety of epithelial and nonepithelial cells, mediating many cell-
matrix and cell-cell interactions (Table 20-4). In vertebrates, at least 24
integrin heterodimers, composed of 18 types of α subunits and 8 types
of β subunits in various αβ heterodimeric combinations, are known. A
single type of β chain can interact with any one of several different
types of α chains, forming distinct integrins that bind different ligands.
This phenomenon of combinatorial diversity allows a relatively small
number of components to serve a large number of distinct functions.
Although most cells express several distinct integrins that bind the
same or different ligands, many integrins are expressed predominantly
in certain types of cells. Not only do many integrins bind more than
one ligand, but there are ligands that can bind to any one of several
different integrins.
TABLE 20-4 • Selected Vertebrate Integrins

Subunit Composition Primary Cellular Distribution Ligands

α1β1 Many types Mainly collagens

α2β1 Many types Mainly collagens; also laminins

α3β1 Many types Laminins

α4β1 Hematopoietic cells Fibronectin; VCAM-1

α5β1 Fibroblasts Fibronectin

α6β1 Many types Laminins

αLβ2 T lymphocytes ICAM-1, ICAM-2

αMβ2 Monocytes Serum proteins (e.g., C3b, fibrinogen, factor X); ICAM-1

αIIbβ3 Platelets Serum proteins (e.g., fibrinogen, von Willebrand factor, vitronectin); fibronectin

α6β4 Epithelial cells Laminin

NOTE:
The integrins are grouped into subfamilies having a common β subunit. Ligands shown in red are CAMs; all others are ECM or serum proteins.
Some subunits can have multiple spliced isoforms with different cytosolic domains.

SOURCE: Data from R. O. Hynes, 1992, Cell 69(1):11.

All integrins appear to have evolved from two ancient general
subgroups: those that bind proteins containing the tripeptide sequence
Arg-Gly-Asp, usually called the RGD motif (fibronectin is one such
protein), and those that bind laminin. Several integrin α subunits
contain a distinctive, inserted domain, the I-domain, which can
mediate binding of certain integrins to various collagens in the ECM.
Some integrins with I-domains are expressed exclusively on leukocytes
(white blood cells) and red and white blood cell precursor
(hematopoietic) cells. I-domains also recognize CAMs on other cells,
including members of the Ig superfamily (e.g., ICAMs, VCAMs), and
thus participate in cell-cell adhesion.
Integrins typically exhibit low affinities for their ligands, with
dissociation constants (Kd) between 10−6 and 10−7 mol/L
. However, the multiple weak interactions generated by the
binding of hundreds or thousands of integrin molecules to their
ligands on cells or in the ECM allow a cell to remain firmly anchored to
its ligand-expressing target.
Parts of both the α subunit and the β subunit of an integrin molecule
contribute to the primary extracellular ligand-binding site (see Figure
20-2). Ligand binding to integrins also requires the simultaneous
binding of divalent cations. Like that of other adhesion molecules, the
cytosolic region of integrins interacts with adapter proteins, which in
turn bind to the cytoskeleton and to intracellular signaling molecules
(see Figure 20-8). Most integrins are linked via adapters to the actin
cytoskeleton, including two of the integrins that connect the basal
surface of epithelial cells to the basal lamina via the ECM molecule
laminin. Some integrins, however, interact with intermediate
filaments. The cytosolic domain of the β4 chain in the α6β4 integrin in
hemidesmosomes (see Figure 20-1), which is much longer than the
cytosolic domains of other integrin β chains, binds to specialized
adapter proteins, which in turn interact with keratin-based
intermediate filaments (see Table 20-4). Other integrins (e.g., α3β1) are
the adhesion receptors in the focal contacts linking the epithelial basal
lamina with the actin cytoskeleton (see Figure 20-1).
As we will see, the diversity of integrins and their ECM ligands allows
integrins to participate in a wide array of key biological processes,
including the inflammatory response and the migration of cells to their
correct locations during morphogenesis. The importance of integrins
in diverse processes is highlighted by the defects exhibited by
knockout mice engineered to have mutations in various integrin
subunit genes. These defects include major abnormalities in
development, blood vessel formation, leukocyte function,
inflammation, bone remodeling, and blood clotting. Despite their
differences, all these processes depend on integrin-mediated
interactions between the cytoskeleton and either the ECM or CAMs on
other cells.
In addition to their adhesion function, integrins can mediate outside-in
and inside-out signaling (see Figure 20-8). The engagement of integrins
by their extracellular ligands can, through adapter proteins bound to
the integrin’s cytosolic region, influence the cytoskeleton and
intracellular signaling pathways (outside-in signaling). Conversely,
intracellular signaling pathways can alter the structure of integrins and
consequently their abilities to adhere to their extracellular ligands and
mediate cell-cell and cell-matrix interactions (inside-out signaling). We
will see a detailed example of integrin-mediated signaling later in this
chapter. Integrin- mediated signaling pathways influence processes as
diverse as cell survival, cell proliferation, and programmed cell death
(see Chapter 21).
Tight Junctions Seal Off Body Cavities and Restrict Diffusion of Membrane
Components
 For polarized epithelial cells to function as barriers and mediators of
selective transport, extracellular fluids surrounding their apical and
basolateral membranes must be kept separate. Tight junctions
between adjacent epithelial cells are usually located in a band
surrounding the cell just below the apical surface (Figure 20-17; see
Tight junctions prevent the diffusion of macromolecules and, to
varying degrees, small water-soluble molecules and ions across an
epithelium via the spaces between cells. They also help establish and
maintain the polarity of epithelial cells by preventing the diffusion of
membrane proteins and glycolipids between the apical and the
also Figure 20-11). These specialized junctions form a barrier that seals
off body cavities such as the intestinal lumen and separates the blood
from the cerebral spinal fluid of the central nervous system (i.e., the
blood-brain barrier).
FIGURE 20-17 Tight junctions. (a) Freeze-fracture preparation of tight-
junction zone between two intestinal epithelial cells. The fracture plane
passes through the plasma membrane of one of the two adjacent cells (see
also Figure 20-11). A honeycomb-like network of ridges and grooves below
the microvilli constitutes the tight-junction zone. (b) Schematic drawing
shows how a tight junction might be formed by the linkage of rows of
protein particles in adjacent cells. In the inset micrograph of an ultrathin
sectional view of a tight junction, the adjacent cells can be seen in close
contact where the rows of proteins interact. See L. A. Staehelin and B. E.
Hull, 1978, Sci. Am. 238:140.
[Part (b) republished with permission from Nature, from S. Tsukita, M.
Furuse, and M. Itoh, 2001, “Multifunctional Strands in Tight Junctions,” Nat.
Rev. Mol. Cell Biol. 2(4):285–293; permission conveyed through Copyright
Clearance Center, Inc.]
The micrograph labeled (a) shows gray rows of cells labeled tight junction,
which is below an area labeled villi. The illustration labeled (b) shows the
microvilli at the top, and the below is the tight junction labeled below. The
tight junction has sections labeled intercellular spaces and linkage of
protein particles in adjacent cells. Also labeled are rows of protein particles
that stick out from the linkages. Next to this is a small micrograph of the
linkage-intercellular space chain.
basolateral regions of the plasma membrane, ensuring that these
regions contain different membrane components. Indeed, the lipid
compositions of the apical and basolateral regions of the plasma
membrane’s exoplasmic leaflet (see Chapter 10) are distinct.
Essentially all cell surface glycolipids are restricted to the exoplasmic
face of the apical membrane, as are all proteins linked to the
membrane by a glycosylphosphatidylinositol (GPI) anchor (see Figure
10-19). In contrast, the apical and basolateral regions of the plasma
membrane’s cytosolic leaflet have uniform membrane composition in
epithelial cells; their lipids and proteins can apparently diffuse
laterally from one region of the membrane to the other.
Tight junctions are composed of thin bands of plasma-membrane
proteins that completely encircle the cell and are in contact with
similar thin bands on adjacent cells. When thin sections of the tight
junctions in cells are viewed in an electron microscope, the lateral
surfaces of adjacent cells appear to touch each other at intervals and
even to fuse in the zone just below the apical surface (see Figure 20-
11b). In freeze-fracture preparations, tight junctions appear as an
interlocking network of ridges and grooves in the plasma membrane
(Figure 20-17a). Very high magnification reveals that rows of protein
particles 3–4 nm in diameter form the ridges seen in freeze-fracture
micrographs of tight junctions. In the model shown in Figure 20-17b,
the tight junction is formed by a double row of these particles, one row
donated by each cell. Treatment of an epithelium with the protease
At the intersection of three cells connected to one another by tight
junctions (see Figure 20-13 and Figure 20-18a), two additional
transmembrane proteins are incorporated into the tight junctions:
tricellulin, which has four membrane-spanning helices; and angulins,
which have a single transmembrane helix and one extracellular
immunoglobulin domain and appear to be required for the assembly of
tricellulin where the cells intersect. As with tight junctions, there
appear to be tension-sensitive proteins that localize at tricellular
vertices in at least some adherens junctions.
As is the case for adherens junctions and desmosomes, cytosolic
adapter proteins and their connections to the cytoskeleton are critical
trypsin destroys the tight junctions, supporting the proposal that
proteins are essential structural components of these junctions.
There are a number of integral membrane proteins found in tight
junctions; two of the best studied are occludin and claudin (from the
Latin claudere, “to close”). Each of these proteins has four membrane-
spanning α helices (Figure 20-18) and are representative of a group of
such cell-surface proteins called tetraspanins. The mammalian claudin
gene family encodes as many as 27 homologous proteins that exhibit
distinct tissue-specific patterns of expression and, as described below,
exhibit distinct properties. A group of junction adhesion molecules
(JAMs) have also been found to contribute to homophilic adhesion and
other functions of tight junctions. JAMs and another junctional
protein, the coxsackievirus and adenovirus receptor (CAR), contain a
single transmembrane α helix and belong to the Ig superfamily of
CAMs. The extracellular domains of rows of occludin, claudin, and
JAMs in the plasma membrane of one cell apparently form extremely
tight links with similar rows of the same proteins in an adjacent cell,
creating a tight seal. Ca2+ -dependent cadherin-mediated adhesion
also plays an important role in tight-junction formation, stability, and
function.
FIGURE 20-18 Proteins that compose tight junctions. (a)
Immunofluorescence localization of occludin (green) and tricellulin (red) in
mouse intestinal epithelium. Note that tricellulin is predominantly
concentrated in tricellular junctions. (b) The junction adhesion molecule
(JAM) has a single transmembrane domain and an extracellular region with
two immunoglobulin domains, whereas occludin and claudins contain four
transmembrane helices. The transmembrane helices of claudin-15 form a
four-helix bundle, and the extracellular loops contain a five-stranded β sheet
(seen edgewise in this view) and α helices. These extracellular secondary
structures participate in cis interactions that form a single-file row of
claudins, which in turn interact via other cis contacts with an adjacent row
of claudins in the same membrane of each cell. There are also trans
interactions between the rows of claudins in adjacent cells (see also Figure
20-20).
[Part (a) J. Ikenouchi et al., 2005, J. Cell Biol. 171(6):939–945, Fig. 3A;
https://doi.org/10.1083/jcb.200510043. Part (b) structure of claudin-15 data
from H. Suzuki et al., 2014, Science 344:304–307, PDB ID 4p79.]
The micrograph labeled (a) shows the fluorescence staining of occludin with
a net of green threads attaching to green bead-like areas and the staining of
tricellulin with a dim net of threads and enlargements. The illustration
labeled (b) shows the schematic of J A M looking like a spring shape moving
to a red square in the cell membrane. The N terminus and C terminus are
labeled. The schematic of occludin looks like a pair of headphones above
the cell membrane with long coiled wires leading into the cytosol. The
claudin schematic looks like a pair of headphones with short wires into the
cytosol. This schematic includes a three-dimensional ribbon diagram with
four highlighted ribbons represents the areas that are in the cell membrane.
components of tight junctions and can mediate signaling. For example,
when one specific claudin, claudin-6, is engaged in intercellular tight
junctions, its cytoplasmic domain can initiate a kinase signaling
cascade that results in the activation of nuclear receptors (e.g., the
retinoic acid receptor γ and the estrogen receptor α) to control cell
behavior (see Chapter 8). Also, the long C-terminal cytosolic segment
of occludin binds to PDZ domains in some large, multidomain adapter
proteins. PDZ domains are about 80 to 90 amino acids long and are
found in various cytosolic proteins; they mediate binding to other
cytosolic proteins or to the C-termini of particular plasma-membrane
proteins. Cytosolic proteins containing a PDZ domain often have more
than one of them. In the human genome, there are more than 250 PDZ
domains in hundreds of proteins. Proteins with multiple PDZ domains
can serve as scaffolds on which to assemble proteins into larger
functional complexes. Several multiple-PDZ-domain–containing
adapter proteins are associated with tight junctions, including the
zonula occludens (ZO) proteins ZO-1, ZO-2, and ZO-3, which not only
interact with occludin, claudin, and other adapter and signaling
proteins but also mediate association with actin fibers. These
interactions appear to stabilize the linkage between occludin and
claudin molecules that is essential for maintaining the integrity of tight
junctions. ZO proteins, which may form phase-separated biomolecular
condensates at tight junctions, can also function as adapters in
adherens junctions (see Figure 20-14) and gap junctions.
As a consequence of tight junctions, many nutrients cannot move
across an epithelium between cells; instead, their transport is achieved
in large part through the transcellular pathway via specific
membrane-bound transport proteins (Figure 20-20a; see also Figure 11-
30). The barrier to diffusion provided by tight junctions, however, is
not absolute; some can exhibit size- and ion-selective permeability,
depending on the type of claudins incorporated into the tight junction.
For some, but not all, claudins the intercellular trans association of
their closely packed extracellular domains results in the formation of
paracellular channels (pores) (Figure 20-20b). Thus certain small
molecules and ions can move from one side of the epithelium to the
other
The role of tight junctions as permeability barriers has been
demonstrated in many experiments. For example, in one simple
experiment, lanthanum hydroxide (an electron-dense colloid of high
molecular weight) is injected into the pancreatic blood vessel of an
experimental animal; a few minutes later, the pancreatic epithelial
acinar cells are fixed and prepared for microscopy. As shown in Figure
20-19, the lanthanum hydroxide diffuses from the blood into the space
that separates the lateral surfaces of adjacent acinar cells, but it cannot
penetrate past the tight junction.
EXPERIMENTAL FIGURE 20-19 Tight junctions prevent passage of large
molecules through extracellular spaces between epithelial cells. Tight
junctions in the pancreas are impermeable to the large water-soluble colloid
lanthanum hydroxide (dark stain) administered from the basolateral side of
the epithelium.
[From D. S. Friend and N. B. Gilula, 1972, J. Cell Biol. 53(3):758–776.]
The micrograph has the following labels: at the top, the apical surface of the
left cell and the right cell are labeled. Below them, a narrow area is labeled
tight junction. Just below the tight junction is a black thick line labeled
lanthanum hydroxide (between cells). The lateral surface of the left cell and
right cell are labeled next to the black thick line.
through the paracellular pathway (see Figure 20-20). The importance
of selective permeability is highlighted by the evolutionary
conservation of the molecules that establish it and the diseases that
arise when it is disrupted. For example, murine embryos cannot
develop properly if selective permeability is disrupted because proper
fluid balance on the two sides of epithelia cannot be maintained.
Similarly, the kidneys depend on proper tight-junction permeability to
establish the ion gradients necessary for normal regulation of body
fluids and waste removal. Owing at least in part to the varying
properties of the different types of claudin molecules located in
different tight junctions, the permeability of the tight junctions to ions,
small molecules, and water varies enormously among different
epithelial tissues.

FIGURE 20-20 Transcellular and paracellular pathways of transepithelial
transport. (a) Transcellular transport requires the cellular uptake of
molecules on one side of the cell and subsequent release on the opposite
side by mechanisms for crossing membranes (see Chapter 11). In
paracellular transport
The permeability of tight junctions can be altered by intracellular
signaling pathways, especially G protein and cyclic AMP–coupled
pathways (see Chapter 15). The regulation of tight-junction
permeability is often studied by measuring ion flux (electrical
resistance, called transepithelial resistance) or the movement of
radioactive or fluorescent molecules across monolayers of MDCK or
other epithelial cells.
The importance of paracellular transport is apparent in several
human diseases. In hereditary hypomagnesemia, a defect in the
claudin16 gene prevents the normal paracellular flow of magnesium in
the kidney. This defect results in an abnormally low blood level of
magnesium, which can lead to convulsions. Furthermore, a mutation
in the claudin14 gene causes hereditary deafness, apparently by
altering transport around hair-cell epithelia in the cochlea of the inner
ear.
Some pathogens have evolved to exploit the molecules in tight
junctions. Some use junctional proteins as co-receptors to attach to
cells prior to infecting them (e.g., hepatitis C virus uses claudin-1 and
occludin, together with two other co-receptors, to enter liver cells).
Others break down the tight-junction barrier and cross epithelia via
paracellular movement, and still others produce toxins that alter
barrier function. For example, toxins produced by Vibrio cholerae, the
enteric bacterium that causes cholera, alter the permeability barrier of
the intestinal epithelium by altering the composition or activity of tight
junctions. Vibrio cholerae also releases a protease that disrupts tight
junctions by degrading the extracellular domain of occludin. Other
bacterial toxins can affect the ion-pumping activity of membrane
transport proteins in intestinal epithelial cells. Toxin-induced changes
molecules move extracellularly through channels in the tight junctions,
whose permeability to small molecules and ions depends on the
composition of the junctional components (mainly claudins) and the
physiological state of the epithelial cells. A cross-sectional plane through the
tight junction linking the right-most two cells is indicated by dashed lines.
Part (b) shows a perpendicular view of this plane (90 ° rotation). (b) A
computational model of claudin-15 in a tight junction. No other tight-
junctional proteins are included in this model. This perpendicular view of
the cross-sectional plane illustrated in part (a) shows the transmembrane
helices of claudin-15 molecules spanning the tight junctional membranes. In
each of the two cells, there are adjacent, single-file rows of claudins (orange
and blue) that form trans intercellular contacts with the rows of claudins in
the adjacent cell. Extracellular channels that are oriented perpendicular to
this cross-sectional plane (outlined by gray circles) are formed by the
extracellular domains of claudin-15. These channels permit the paracellular
movement of small molecules and ions across the epithelium. There are
negatively charged side chains of aspartic acid residues that line the
channels and appear to contribute to the cation specificity of claudin-15-
dependent channels.
[Part (a) Data from S. Tsukita et al., 2001, Nat. Rev. Mol. Cell Biol. 2:285. Part
(b) Information from P. Samanta et al., 2018, J. Gen. Physiol. 150:949–968.]
The illustration labeled (a) shows a schematic of three pathways between
cells. 4 rectangular cells are with nuclei. Between the first two cells is a
pathway labeled paracellular pathway. Between the second and third cells
is a transcellular pathway, and between the third and fourth cells is an
extracellular channel. The illustration labeled (b) shows a three-dimensional
ribbon diagram of the extracellular channel. Cell 1 is at the left and the
cytosol of cell 1 is labeled. The cell membranes of cells 1 and 2 are wide and
have the ribbons inside them. Between cell 1 and cell 2, the connections
between the ribbons are displayed. The area inside the membrane is also
labeled claudins in the tight junction.
in tight-junction permeability (increased paracellular transport) and in
protein-mediated ion pumping (increased transcellular transport) can
result in massive losses of internal body ions and water into the
gastrointestinal tract, which in turn leads to diarrhea and potentially
lethal dehydration (see Chapter 11).
It is clear that tight junctions are important for maintaining tissue
integrity and function. Thus these junctions should and can respond to
dynamic changes in epithelia, for example, when they are subjected to
either external forces (such as stretching of the bladder by filling with
urine) or internal forces (actin-myosin-induced changes in tension). It
is clear that when there are changes in the mechanical forces at the
tight junction there are coordinated changes in the composition of
junctional adaptor proteins and paracellular permeability. The
molecular mechanisms underlying mechanotransduction at tight
junctions are not as well understood as those at adherens junctions,
with some studies suggesting that ZO-1 may serve as a mechanosensor.
Gap Junctions Composed of Connexins Allow Small Molecules to Pass
Directly Between the Cytosols of Adjacent Cells
Early electron micrographs of tissues revealed sites of cell-cell contact
with a characteristic intercellular gap (Figure 20-21a). This feature,
which was found in virtually all animal cells that contact other cells,
prompted early morphologists to call these regions gap junctions. In
retrospect, the most important feature of these junctions is not the 2–4
nm gap itself, but a well-defined set of cylindrical particles that cross
the gap and compose pores connecting the cytosols of adjacent cells
(Figure 20-21b, c). As we will see in Section 20.6, plant cells also
assemble pores that connect the cytosols of adjacent cells, but those channels, called plasmodesmata, differ considerably in structure from
gap junctions.

FIGURE 20-21 Gap junctions. (a) In this thin section through a gap junction connecting two mouse liver cells, the two plasma membranes are closely
associated for a distance of several hundred nanometers, separated by a gap of 2–3 nm. (b) Numerous roughly hexagonal particles are visible in this
perpendicular view of the cytosolic face of a region of plasma membrane enriched in gap junctions. Each hexagonal particle aligns with a similar particle on
an adjacent cell, forming a channel connecting the two cells. (c) Schematic model of a gap junction connecting two plasma membranes. Both membranes
contain connexon hemichannels, cylinders of six dumbbell-shaped connexin molecules. Two connexons join in the gap between the cells to form a gap-
junction channel, 1.4–2.0 nm in diameter, that connects the cytosols of the two cells. (d) Structure of recombinant human Cx26 gap junction as determined
by x-ray crystallography (3.5-Å resolution). Left: Space-filling model of a side view of the complete structure of two attached connexons oriented as in part (c).
Each of the six connexins that comprise a connexon has four transmembrane helices and is shown in a distinct color. The structures of the loops connecting
the transmembrane helices are not well defined and not shown. Right: View from the cytosol perpendicular to the membrane bilayers, looking down on the
connexon with its central pore. The diameter of the pore’s channel is ∼14 Å, and it is lined by many polar/charged amino acids. See S. Nakagawa, S. Maeda,
and T. Tsukihara, 2010, Curr. Opin. Struct. Biol. 20(4):423–430.

[Part (a) courtesy of Daniel Goodenough. Part (b) D. L. Caspar et al., 1977, 74:605–628, Fig. 2b; https://doi.org/10:1083/jcb.74.2.605. Part (c) Data from L. A.
Staehelin and B. E. Hull, 1978, Sci. Am. 238:140. Part (d) Data from S. Maeda et al., 2009, Nature 458:597–602, PDB ID 2zw3.]

The micrograph labeled (a) shows the gap junction as two long lines running vertically close together but not twisted. The micrograph labeled (b) shows
many ends of tubes gathered. The illustration labeled (c) shows a schematic of a gap junction. The two cell membranes are vertical yellow lines with a pair of
orange tubes connecting them at the center. The top tube looks closed and is labeled connexon hemichannel. The bottom tube looks open and is labeled
gap junction channel. The area between the membranes is labeled intercellular area. A white arrow from the bottom tube points to illustration (d). The
illustration labeled (d) shows two three-dimensional space-filling models of the gap junction, one side view, and one top view. In the side view, the cytosol
and membrane of cell 1 and cell 2 are labeled at the top, with the intercellular area labeled in the middle. At the bottom are width measurements for each
part of the gap junction. From left to right: the area in cell 1 cytosol is 19 angstroms, the area inside the membrane is 38 angstroms, the area of the
intercellular area is 40 angstroms, cell 2 membrane, 38 angstroms, and cell 2 cytosol, 19 angstroms.

In many animal tissues, anywhere from a few to thousands of gap-
junction particles cluster together in patches (e.g., along the lateral
surfaces of epithelial cells; see Figure 20-11). When the plasma
membrane is purified and then sheared into small fragments, some
pieces mainly containing patches of gap junctions are generated.
Owing to their relatively high protein content, these fragments have a
higher density than the bulk of the plasma membrane and can be
purified by equilibrium density-gradient centrifugation (see Figure 4-
37). When these preparations are viewed perpendicular to the
membrane, the gap junctions appear as arrays of hexagonal particles
that enclose water-filled channels (see Figure 20-21b).
The effective pore size of gap junctions can be measured by injecting a
cell with a fluorescent dye covalently linked to membrane bilayer–
impermeable molecules of various sizes and observing with a
fluorescence microscope whether the dye passes into neighboring
cells. Gap junctions between mammalian cells permit the passage of
molecules as large as 1.2 nm in diameter. In insects, these junctions
are permeable to molecules as large as 2 nm in diameter. Generally
speaking, molecules smaller than 1200 Da pass freely and those larger
than 2000 Da do not pass; the passage of intermediate-sized molecules
is variable and limited. Thus ions, many low-molecular-weight
precursors of cellular macromolecules, products of intermediary
metabolism, and small intracellular signaling molecules can pass from
cell to cell through gap junctions.
In neural tissue, some neurons are connected by gap junctions through
which ions pass rapidly, thereby allowing very rapid transmission of
electrical signals. Impulse transmission through these connections,
called electrical synapses, is almost a thousand times as rapid as at
chemical synapses (see Chapter 23). Gap junctions are also present in
many non-neural tissues, where they help to integrate the electrical
and metabolic activities of many cells. In the heart, for instance, gap
junctions rapidly pass ionic signals among cardiac muscle cells, which
are tightly bound together via desmosomes. Thus gap junctions
contribute to the electrically stimulated coordinate contraction of
cardiac muscle cells during a heartbeat. As discussed in Chapter 15,
some extracellular hormonal signals induce the production or release
of small intracellular signaling molecules called second messengers (e.g.,
cyclic AMP, IP3 , and Ca2+ ) that regulate cellular metabolism.
Because many second messengers can be transferred between cells
through gap junctions, hormonal stimulation of one cell has the
potential of triggering a coordinated response by that cell as well as
many of its neighbors. Such gap-junction-mediated signaling plays an
important role, for example, in the secretion of digestive enzymes by
the pancreas and in the coordinated muscular contractile waves
(peristalsis) in the intestine. Another vivid example of gap-junction-
mediated transport is the phenomenon of metabolic coupling, or
metabolic cooperation, in which a cell transfers nutrients or
intermediary metabolites to a neighboring cell that is itself unable to
synthesize them. Gap junctions play critical roles in the development
of egg precursors (oocytes) in the ovaries by mediating the movement
of both metabolites and signaling molecules, such as cyclic GMP,
between an oocyte and its surrounding granulosa cells, as well as
between neighboring granulosa cells.
A current model of the structure of the gap junction is shown in Figure
20-21c–d. Vertebrate gap junctions are composed of connexins, a
family of structurally related transmembrane proteins with molecular
weights between 26,000 and 60,000. Each vertebrate hexagonal particle
consists of 12 noncovalently associated connexin molecules: six form a
cylindrical hemichannel, called a connexon, in one plasma membrane
that is joined to a connexon in the adjacent cell membrane, forming a
continuous aqueous channel (diameter ∼14 Å) between the cells. Each
individual connexin molecule has four membrane-spanning α helices
with a topology similar to that of claudin (see Figure 20-18), resulting in
24 transmembrane α helices in each connexon hemichannel.
A completely different family of proteins, the innexins, forms the gap
junctions in invertebrates. A third family of innexin-like proteins,
called pannexins, has been found in both vertebrates and invertebrates.
Pannexins form hexamer hemichannels (pannexons) whose opening
can be regulated by changes in membrane potential or mechanical
stress. When open, pannexons permit direct exchange of small
molecules (such as ATP) and ions between the intracellular and
extracellular spaces. Pannexons are thought to play key roles in release
of ATP from cells into the extracellular space. Extracellular ATP (as
well as ADP and AMP) can function as an intercellular messenger or
transmitter by binding to and activating the cell-surface purinergic
receptors P1, P2X, or P2Y on target cells.
There are 21 different connexin genes in humans, and different sets of
connexins are expressed in different cell types. The existence of this
diversity, together with the generation of mutant mice with
inactivating mutations in connexin genes, has highlighted the
importance of connexins in a wide variety of cellular systems. Some
cells express a single connexin that forms homotypic connexons. Most
cells, however, express at least two connexins; these different proteins
can assemble into heteromeric connexons, which in turn form
heterotypic gap-junction channels. Diversity in channel composition
leads to differences in channel permeability. For example, channels
made from a 43-kDa connexin isoform, Cx43 — the most ubiquitously
expressed connexin — are more than a hundred times as permeable to
ADP and ATP as those made from Cx32 (32 kDa).
The permeability of gap junctions is regulated by post-translational
modification of connexins (e.g., phosphorylation) and is sensitive to
changes in environmental conditions such as intracellular pH and
Ca2+ concentration, membrane potential, and the intercellular
potential between adjacent interconnected cells (“voltage gating”). The
N-termini of connexins appear to be especially important in the gating
mechanism. Thus, as is the case for many ion channels (see Chapter
11), the channel in some gap junctions can be either opened or closed.
One example of the physiological regulation of gap junctions occurs
during mammalian childbirth. The smooth muscle cells in the
mammalian uterus must contract strongly and synchronously during
labor to expel the fetus. To facilitate this coordinated activity,
immediately before and during labor there is an approximately five- to
tenfold increase in the amount of the major connexin in these cells,
Cx43, and an increase in the number and size of gap junctions, which is
reversed rapidly postpartum (following childbirth).
The assembly of connexins, their trafficking within cells, and the
formation of functional gap junctions apparently depend on N-
cadherin and its associated adapter proteins (e.g., α- and β-catenins,
ZO-1, and ZO-2) as well as desmosomal proteins (plakoglobin,
desmoplakin, and plakophilin-2). PDZ domains in ZO-1 and ZO-2 bind
to the C-terminus of Cx43 and mediate its interaction with catenins and
N-cadherin. The relevance of these relationships is particularly evident
in the heart, which depends on gap junctions for rapid coordinated
electrical coupling and on adjacent adherens junctions and
desmosomes for mechanical coupling between cardiomyocytes to
achieve the intercellular integration of electrical activity and
movement required for normal cardiac function. It is noteworthy that
ZO-1 serves as an adapter for adherens (see Figure 20-14), tight, and
gap junctions, suggesting that this and other adapters can help
integrate the formation and functions of these diverse junctions.
Mutations in connexin genes cause at least eight human diseases,
including neurosensory deafness (Cx26 and Cx31), cataracts or heart
malformations (Cx43, Cx46, and Cx50), and the X-linked form of
Charcot-Marie-Tooth disease (Cx32), which is marked by progressive
degeneration of peripheral nerves.

Tunneling Nanotubes Can Mediate Metabolic Coupling and Transfer
Organelles Between Animal Cells
Tunneling nanotubes can serve as an alternative to gap junctions for
moving molecules between cells. Tunneling nanotubes are tubelike
projections of the plasma membrane that form a continuous channel
connecting the cytosols of animal cells (Figure 20-22) and can transfer
chemical and electrical signals between cells in a manner analogous to
structures in plants called plasmodesmata (described in Section 20.6).
Tunneling nanotubes are typically unbranched, straight tubes and can
have a wide variety of diameters (50–300 nm) and lengths (extending
between cells from <10 μm to>100 μm , they can be
longer than several cell diameters). All tunneling nanotubes have actin
filaments passing through the central channel, and in some types of
cells they also contain microtubules. Remarkably, functional
mitochondria can travel between cells by passing through tunneling
nanotubes in cell culture (see Figure 20-22) and in vivo, thereby
rescuing receiving cells that have mitochondrial defects or
deficiencies. Thus the concept of metabolic coupling described for gap
junctions can be extended to include the movement of small molecules
and organelles through tunneling nanotubes. Pathogens may also use
tunneling nanotubes to spread between cells.

EXPERIMENTAL FIGURE 20-22 Microscopic visualization of a tunneling

nanotube and mitochondria in cultured human cells. (a) Cultured human
retinal pigment epithelial cells (ARPE-19 cell line) were incubated with a
fluorescent dye (JC-1) that specifically stains mitochondria and then
examined by a combination of conventional bright-field microscopy (see
Chapter 4) to visualize the cells and fluorescence microscopy to visualize
mitochondria (green intracellular fluorescence). A typical tunneling
nanotube can be seen connecting cells 1 and 2. Inset (b) shows a higher
magnification of the bright-field-only image with two bulges in the tunneling
nanotube highlighted by dashed circles. Inset (c) shows a higher
magnification of the same region of the combination image indicating two
likely mitochondria within the tunneling nanotube at the positions of those
bulges.

[D. Wittig et al., 2012, “Multi-Level Communication of Human Retinal

Pigment Epithelial Cells via Tunneling Nanotubes.” PLoS One 7(3):e33195;
https://doi.org/10.1371/journal.pone.0033195.]

KEY CONCEPTS OF SECTION 20.2

Cell-Cell and Cell–Extracellular Matrix Junctions and Their Adhesion Molecules

 Epithelial cells have distinct apical, basal, and lateral surfaces. Microvilli projecting from the apical surfaces of many epithelial cells
considerably expand the cells’ surface areas.
 Three major classes of cell junctions — anchoring junctions, tight junctions, and gap junctions — assemble epithelial cells into sheets and
mediate communication between them (see Figures 20-1 and 20-11). Anchoring junctions can be further subdivided into adherens
junctions, focal contacts, desmosomes, and hemidesmosomes.
 Adherens junctions and desmosomes are cadherin-containing anchoring junctions that bind the membranes of adjacent cells, giving
strength and rigidity to the entire tissue.
 Cadherins are cell-adhesion molecules (CAMs) responsible for Ca2+ -dependent interactions among cells in epithelial and other tissues.
They promote strong cell-cell adhesion by mediating both lateral (cis) and adhesive intercellular (trans) interactions.
 Adapter proteins that bind to the cytosolic domain of cadherins and other cell-cell and cell matrix adhesion receptors mediate the
association of cytoskeletal and signaling molecules with the plasma membrane (see Figures 20-8 and 20-14). Strong cell-cell adhesion
depends on the linkage of the interacting adhesion receptors to the cytoskeleton.
 Hemidesmosomes are integrin-containing anchoring junctions that attach cells to elements of the underlying extracellular matrix.
 Integrins are a large family of αβ heterodimeric cell-surface proteins that mediate both cell-cell and cell-matrix adhesions and inside-out
and outside-in signaling in numerous tissues.
 Tight junctions block the diffusion of proteins and some lipids in the plane of the plasma membrane, contributing to the polarity of
epithelial cells. They also limit and regulate the extracellular (paracellular) flow of water and solutes from one side of the epithelium to the
other (see Figure 20-20). Two key integral membrane proteins found in tight junctions are occludin and claudin.
 Gap junctions are constructed of multiple copies of connexin proteins, which are assembled into a transmembrane channel that connects
the cytosols of two adjacent cells (see Figure 20-21). Small molecules and ions can pass through gap junctions, permitting metabolic and
electrical coupling of adjacent cells.
 Tunneling nanotubes are tubelike projections of plasma membranes that form a continuous channel connecting the cytosols of nearby
animal cells (see Figure 20-22).
20.3 The Extracellular Matrix I: The Basal Lamina

In animals, the extracellular matrix (ECM) has multiple functions (see Table 20-2). The ECM helps organize cells into tissues and coordinates their
cellular functions by activating intracellular signaling pathways that control cell growth, proliferation, and gene expression. The ECM can directly
influence cell and tissue structure and function. In addition, it can serve as a repository for inactive or inaccessible signaling molecules (e.g.,
growth factors) that are released to function when the ECM is disassembled or remodeled by hydrolyases, such as proteases. Indeed, hydrolyzed
fragments of ECM macromolecules can themselves have independent biological activity. The ensemble of proteins that compose the ECM itself
and associated proteins that covalently modify (e.g., chemically cross-link, phosphorylate, cleave), bind to, or otherwise regulate the composition
and structure of the ECM is called the matrisome. Proteomic (Chapter 3) and genomic analyses suggest that there are approximately 1030 and 1110
genes that encode the human and mouse matrisomes, respectively. Dysfunction of matrisome components can cause a wide variety of diseases
that affect many different tissues and organs. It is noteworthy that there are ECM components, as well as extracellular domains of plasma-
membrane proteins, that are phosphorylated on serine, threonine, or tyrosine side chains. Kinases that are present in the luminal compartments
of the secretory pathway and some that are apparently secreted into the extracellular space catalyze these phosphorylations.

Many functions of the ECM and, indeed, some features of the assembly of the ECM require transmembrane adhesion receptors, including the
integrins, that bind directly to ECM components and that also interact, through adapter proteins, with the cytoskeleton. Adhesion receptors bind
to three types of molecules abundant in the ECM of all tissues (see Table 20-1):
 Proteoglycans, a group of glycoproteins that cushion cells and bind a wide variety of extracellular and cell-surface molecules
 Collagen fibers, which provide structural integrity, mechanical strength, and resilience
 Soluble multi-adhesive matrix proteins, such as laminin and fibronectin, which bind to and cross-link adhesion receptors and
other ECM components

We begin our description of the structures and functions of these major ECM components in the context of the basal lamina: the specialized sheet
of ECM that plays a particularly important role in determining the overall architecture and function of epithelial tissues. In the following section,
we discuss the ECM molecules commonly found in nonepithelial tissues, including connective tissue.

The Basal Lamina Provides a Foundation for Assembly of Cells into Tissues

In animals, most organized groups of cells in epithelial and nonepithelial tissues are underlain or surrounded by the basal lamina, a sheet-like
meshwork of ECM components usually no more than 60–120 nm thick (Figure 20-23). The basal lamina is structured differently in different tissues.
In columnar and other epithelia such as intestinal lining and skin, it is a foundation on which only one surface of the cells rests. In other tissues,
such as muscle or fat, the basal lamina surrounds each cell. Basal laminae play important roles in regeneration after tissue damage and in
embryonic development. For instance, the basal lamina helps four- and eight-celled embryos adhere together in a ball (see Figures 22-2 and 22-3).
In the development of the nervous system, neurons migrate along ECM pathways that contain basal laminal components. In higher animals, two
distinct basal laminae are employed to form a tight barrier that limits diffusion of molecules between the blood and the brain (blood-brain
barrier), and in the kidney, a specialized basal lamina serves as a selectively permeable blood filter. In muscle, the basal lamina helps protect the
cell membranes from damage during contraction and relaxation. Thus the basal lamina is important for organizing cells into tissues and distinct
compartments, repairing tissues, forming permeability barriers, and guiding migrating cells during development. It is therefore not surprising
that its components have been highly conserved throughout evolution.

Most of the ECM components in the basal lamina are synthesized by
the cells that rest on it. Four ubiquitous protein components, each of
which comprises multiple distinct repeating domains, are found in
basal laminae (Figure 20-24):
 Type IV collagen, trimeric molecules with both rodlike
and globular domains that form a two-dimensional
network
 Laminins, a family of multi-adhesive, cross-shaped
proteins that form a fibrous two-dimensional network
with type IV collagen and that also bind to integrins
and other adhesion receptors
 Perlecan, a large proteoglycan whose protein contains
multiple copies of seven different structural domains
(total of 48 domains) and which binds to and cross-
links many ECM components and cell-surface
molecules
Nidogen (also called entactin), a rodlike molecule that cross-links type
IV collagen, perlecan, and laminin, which helps incorporate other
components into the ECM and also stabilizes basal laminae

FIGURE 20-23 A basal lamina separates epithelial cells and some other cells
from connective tissue. (a) Transmission electron micrograph of a thin
section of cells (top) and underlying connective tissue (bottom). The
electron-dense layer of the basal lamina can be seen to follow the
undulations of the basal surfaces of the cells. (b) Electron micrograph of a
quick-freeze deep-etch preparation of skeletal muscle, showing the plasma
membrane, basal lamina, and surrounding connective-tissue collagen fibers.
In this preparation, the basal lamina is revealed as a meshwork of
filamentous proteins that associates with the plasma membrane and the
thicker collagen fibers of the connective tissue.
In the micrograph labeled (a) the bottom area is light in color and labeled
connective tissue. At the top of the light area is a tube-like line going across
it and labeled basal lamina. A dark line on top of this is labeled basal surface,
and the area above this is labeled cytosol. The micrograph labeled (b) shows
a micrograph of bone tissue. There is a thin area that separates the plasma
membrane and basal lamina and moves in a crooked line from bottom left
to top right of the micrograph. This area is labeled adhesion receptors. An
area below and to the right of the basal lamina is labeled collagen fibers.
Other ECM molecules, such as members of the evolutionarily ancient
family of glycoproteins called fibulins, are incorporated into various
basal laminae, depending on the tissue and the particular functional
requirements of the basal lamina.
As depicted in Figure 20-1, one side of the basal lamina is linked to cells
by adhesion receptors, including integrins in hemidesmosomes, which
bind to laminin in the basal lamina. The other side of the basal lamina
is anchored to the adjacent connective tissue by a layer of collagen
fibers embedded in a proteoglycan-rich matrix. In stratified squamous
epithelia (e.g., skin; see Figure 20-10d), this linkage between the
dermis and epidermis is mediated by anchoring fibrils of type VII
collagen. Loss-of-function mutations in the gene encoding type VII
collagen results in the rare and debilitating blistering disease called
dystrophic epidermolysis bullosa. Together, the basal lamina and the
anchoring collagen fibrils form the structure called the basement
membrane.
FIGURE 20-24 Major protein components of the basal lamina. Type IV collagen and laminin each form two-dimensional networks (see Figures 20-25 and 20-
27), which are cross-linked by nidogen/entactin, perlecan, and laminin molecules, and which interact via laminins with the plasma membranes of adjacent
cells.

The basal lamina is composed of a layer of meshed collagen 4 and a layer composed of a mixture of laminin, which itself is composed of perlecan and
laminin. Beneath the laminin layer, the plasma membrane is located. In this layer are small short strings of yellow circles, which are in a legend outside the
diagram as nidogen slash entactin.

Laminin, a Multi-Adhesive Matrix Protein, Helps Cross-Link Components of the Basal Lamina

Laminin, the principal multi-adhesive matrix protein in basal laminae, is a heterotrimeric protein comprising α, β, and γ chains. At least 16
laminin isoforms in vertebrates are assembled from 5 α, 3 β, and 3 γ chains, with each chain numbered to reflect the chain composition: laminin-
111 (α1β1γ1) or laminin-511 (α5β1γ1). Each laminin isoform exhibits a distinctive pattern of tissue- and developmental stage–specific expression.
As shown in Figure 20-25, many laminins are large, cross-shaped proteins (molecular weight of about 820,000), although some are Y- or rod-
shaped. Globular domains at the N-terminus of each subunit bind to one another and thus mediate the self-assembly of laminins into mesh-like
networks. Five globular LG domains at the C-terminus of the laminin α subunit mediate Ca2+ -dependent binding to cell-surface laminin
receptors, including certain integrins (see Table 20-4) as well as sulfated glycolipids, syndecan, and dystroglycan, which will be described further
in Section 20.4. Some of these interactions are via negatively charged carbohydrates on the receptors. LG domains are found in a wide variety of
other proteins and can mediate binding to steroids and proteins as well as carbohydrates. Laminin is the principal basal laminal ligand of
integrins.
 globular domains, and coiled-coil region in which
laminin’s three chains are covalently linked by
several disulfide

bonds. Different regions of laminin bind to

adhesion receptors and various matrix
components (indicated by arrows and brackets).
Right: Laminins assemble into a lattice via
interactions between their N-terminal globular
domains. See G. R. Martin and R. Timpl, 1987,
Annu. Rev. Cell Biol. 3:57–85; M. Durbeej, 2010,
Cell Tissue Res. 339:259–268; and S. Meinen et al.,
2007, J. Cell Biol. 176:979–993. (b) Electron
micrographs of an intact laminin molecule,
showing its characteristic cross shape (left), and
the carbohydrate-binding LG domains near the C-
terminus (right).

[Left: Republished with permission from Elsevier,

from J. Engel et al., 1981, “Shapes, Domain
Organizations and Flexibility of Laminin and
Fibronectin, Two Multifunctional Proteins of the
Extracellular Matrix,” J. Mol. Biol. 150(1):97–120;
permission conveyed through Copyright
Clearance Center, Inc. Right: Republished with
permission from Elsevier, from R. Timpl et al.,
2000, “Structure and Function of Laminin LG
Modules,” Matrix Biol. 19(4):309–317; permission
conveyed through the Copyright Clearance
Center, Inc.]

The illustration labeled (a) shows laminin with

three chains, alpha, beta, and gamma. The alpha
FIGURE 20-25 Laminin is a heterotrimeric multi-adhesive matrix protein found in all basal laminae. chain is at the top of a cross-shaped diagram and
(a) Schematic model of cross-shaped laminin molecule showing the general shape, location of is labeled: laminins (self-assembly) integrins. The
beta chain
goes off to the left and is labeled: laminins (self-assembly) collagens. The gamma chain goes off to the right and is also labeled laminins (self-assembly)
collagens, but adds a nidogen near the centerline of the cross shape. At the bottom of the cross shape are 5 pink circles labeled: LG domains, bind cellular
receptors (integrins, syndecan, sulfated glycolipids, dystroglycan. A white arrow goes from the end of the gamma chain to a diagram that shows the cross
shape binding to several cross shapes
.

Sheet-Forming Type IV Collagen Is a Major Structural Component of the proteins made from three polypeptides, each encoded by one of at
Basal Lamina least 43 genes in humans, usually called collagen α chains. The three α
chains in a collagen molecule can be identical (forming a homotrimer)
or different (forming a heterotrimer). All or parts of the three-stranded
Type IV collagen is, together with laminin, a principal structural
collagen molecule can twist together into a special triple helix called a
component of all basal laminae and can bind to adhesion receptors,
collagenous triple helix. When there is more than one triple-helical
including some integrins. Collagen IV is one of at least 28 types of
segment, these segments are joined by nonhelical regions of the
collagen in humans that participate in the formation of distinct ECMs
protein, as we will see shortly for type IV collagen. Within a helical
in various tissues (Table 20-5). There are also at least 20 additional
segment, each of the three α chains twists into a left-handed helix, and
collagen-like proteins (such as host defense collagens) in the human
the three chains then wrap around one another to form a right-handed
proteome. Although collagen isoforms differ in certain structural
triple helix (Figure 20-26).
features and in their tissue distribution, all collagens are trimeric

TABLE 20-5 • Selected Collagens

Type Molecule Composition Structural Features Representative Tissues

Fibrillar Collagens

I [α1(I)]2[α2(I)] 300-nm-long fibrils Skin, tendon, bone, ligaments,

dentin, interstitial tissues

II [α1(II)]3 300-nm-long fibrils Cartilage, vitreous humor

III [α1(III)]3 300-nm-long fibrils; often with type I Skin, muscle, blood vessels
 V [α1(V)]2[α2(V)],[α1(V)]3
390-nm-long fibrils with globular N- Cornea, teeth, bone, placenta, skin,
terminal extension; often with type I smooth muscle

Fibril-Associated Collagens

VI [α1(VI)][α2(VI)][α3(VI)]
Lateral association with type I; periodic Most interstitial tissues
globular domains

IX [α1(IX)][α2(IX)][α3(IX)]
Lateral association with type II; N-terminal Cartilage, vitreous humor
globular domain; bound GAG

Sheet-Forming and Anchoring Collagens

IV [α1(IV)]2[α2(IV)] Two-dimensional network All basal laminae

VII [α1(VII)]3 Long fibrils Below basal lamina of the skin

XV [α1(XV)]3 Core protein of chondroitin sulfate Widespread; near basal lamina in

proteoglycan muscle

Transmembrane Collagens

XIII [α1(XIII)]3 Integral membrane protein Hemidesmosomes in skin

XVII [α1(XVII)]3 Integral membrane protein Hemidesmosomes in skin

Host Defense Collagens

Collectins Oligomers of triple helix; lectin domains Blood, alveolar space

C1q Oligomers of triple helix Blood (complement)

Class A scavenger Homotrimeric membrane proteins Macrophages

receptors

SOURCE: Data from K. Kühn, 1987, in R. Mayne and R. E. Burgeson, eds., Structure and Function of Collagen Types, Academic Press, p. 2; M. Van Der Rest
and R. Garrone, 1991, FASEB J. 5:2814.

The collagen triple helix can form because of an unusual abundance of
three amino acids in the α chains: glycine, proline, and a modified
form of proline called hydroxyproline (see Figure 2-15). They make up
the characteristic repeating sequence motif Gly-X-Y, where X and Y can
be any amino acid but are often proline in position X and
hydroxyproline in position Y, and less often lysine and hydroxylysine.
Glycine is essential because its small side chain, a hydrogen
atom, is the only one that can fit into the crowded center of the three-
stranded helix (see Figure 20-26b). Hydrogen bonds help hold the three
chains together. Although the rigid peptidyl-proline and peptidyl-
hydroxyproline linkages are not compatible with formation of a classic
single-stranded α helix, they stabilize the distinctive collagenous triple
helix. The hydroxyl group in hydroxyproline in the Y position helps
hold its ring in a conformation that stabilizes the three-stranded helix.

The unique properties of each collagen isoform are due mainly to
differences in (1) the number and lengths of the collagenous triple-
helical segments; (2) the segments that flank or interrupt the triple-
helical segments and that fold into other kinds of three-dimensional
structures; and (3) covalent modification of the α chains (e.g.,
hydroxylation, glycosylation, oxidation, cross-linking). For example,
the chains in type IV collagen are designated IVα chains. Mammals
express six homologous IVα chains, which assemble into three
different heterotrimeric type IV collagens with distinct properties. All
subtypes of type IV collagen, however, form a 400-nm-long triple helix
(Figure 20-27) that is interrupted about 24 times by nonhelical
segments and flanked by a large globular domain at the C-terminus of
FIGURE 20-26 The collagen triple helix. (a) Left: Side view of the crystal
structure of a polypeptide fragment whose sequence is based on repeating
sets of three amino acids, Gly-X-Y, characteristic of collagen α chains. Center:
Each chain is twisted into a left-handed helix, and three chains wrap around
one another to form a right-handed triple helix. Right: A schematic model
illustrates the triple-helical nature of the structure and shows the left-
handed twist of the individual collagen α chains (red line). (b) View down the
axis of the triple helix. The proton side chains of the glycine residues
(orange) point into the very narrow space between the polypeptide chains in
the center of the triple helix. In collagen mutations in which other amino
acids replace glycine, the proton in glycine is replaced by larger groups that
disrupt the packing of the chains and destabilize the triple-helical structure.
[Data from R. Z. Kramer et al., 2001, J. Mol. Biol. 311:131, PDB ID 1bkv.]
There are several distinct cell-surface receptors for collagen IV and
other types of collagen (other collagens are discussed in the next
section). These cell-surface receptors include certain integrins,
discoidin domain receptors 1 and 2 (which are tyrosine kinase
receptors), glycoprotein VI (on platelets), leukocyte-associated Ig-like
receptor-1, members of the mannose receptor family, and a modified
form of the protein CD44. They can play critical roles in helping to
assemble the ECM and in integrating cellular activity with the ECM.
the chain and a smaller globular domain at the N-terminus. The
nonhelical regions introduce flexibility into the molecule. Through
both lateral associations and interactions entailing the globular N- and
C-termini, type IV collagen molecules assemble into a branching and
covalently cross-linked, irregular two-dimensional fibrous network
that forms a lattice on which, together with the laminin lattice, the
basal lamina is built (see Figures 20-24 and 20-27). Multiple, unusual
sulfilimine (―S═N―) or thioether bonds between
hydroxylysine (or lysine) and methionine residues covalently cross-
link some adjacent C-terminal domains of type IV collagen molecules,
and thus contribute to the stability of the network.
 FIGURE 20-27 Structure and assembly of type IV collagen. (a) Schematic
representation of type IV collagen. This 400-nm-long molecule has a small
noncollagenous globular domain at the N-terminus and a large globular
domain at the C-terminus. The collagenous triple helix is interrupted by
nonhelical segments that introduce flexible kinks into the molecule. Lateral
interactions between triple-helical segments, as well as head-to-head and
tail-to-tail interactions between the globular domains, form dimers,
tetramers, and higher order complexes, yielding a sheet-like network. See A.
Boutaud et al., 2000, J. Biol. Chem. 275:30716. (b) Electron micrograph of
type IV collagen network formed in vitro. The lacy appearance results from
the flexibility of the molecule, the side-to-side binding between triple-helical
segments (white arrows), and the interactions between C-terminal globular
domains (yellow arrows).

[Part (b) P. D. Yurchenco and G. C. Ruben, 1987, J. Cell Biol. 105(6):2559–

2568, Fig. 1c; https://doi.org/10.1083/jcb.105.6.2559.]

The illustration labeled (a) shows a collagen triple helix depicted

schematically with a blue line. A region of the non-helical protein is above
this. The N-terminal and C-terminal globular domains are highlighted as
circles at each end. Downward arrows indicate that association can occur by
the association of 2 C-terminal head groups (two blue lines joined), forming
dimers, or association of four of the non-helical domains, forming tetramers
(four blue lines joined at the center). The association of different collagen
structures leads to a network structure.

Perlecan, a Proteoglycan, Cross-Links Components of the Basal Lamina and

In the kidney, a double basal lamina called the glomerular Cell-Surface Receptors
basement membrane separates the epithelium that lines the urinary
space from the endothelium that lines the surrounding blood-filled
Perlecan, the major secreted proteoglycan in basal laminae, consists
capillaries. Defects in this structure, which is responsible for
of a large multidomain core protein (∼470 kDa) to which
ultrafiltration of the blood and initial urine formation, can lead to renal
polysaccharides are covalently attached. The core protein is made up
failure. For instance, mutations that alter the C-terminal globular
of multiple repeats of five distinct domains, including laminin-like LG
domain of certain IVα chains are associated with progressive renal
domains (3 copies), EGF-like domains (12 copies), and Ig domains (22
failure as well as sensorineural hearing loss and ocular abnormalities,
copies). The many globular repeats give it the appearance of an
a condition known as Alport’s syndrome. In Goodpasture’s syndrome, a
approximately 200-nm-long string of pearls when visualized by
relatively rare autoimmune disease, antibodies bind to the α3 chains of
electron microscopy; hence the name perlecan. Perlecan contains three
type IV collagen found in the glomerular basement membrane and
types of covalent polysaccharide chains: N-linked chains (see Chapter
lungs. This binding sets off an immune response that causes cellular
14), O-linked chains, and glycosaminoglycans (GAGs) (O-linked sugars
damage, resulting in progressive renal failure and pulmonary
and GAGs are discussed further in Section 20.4). GAGs are long, linear
hemorrhage.
polymers of repeating disaccharides. Glycoproteins containing
covalently attached GAG chains are called proteoglycans. Both the
protein and the GAG components of perlecan contribute to its ability to
incorporate into and define the structure and function of basal
laminae. Because its multiple domains and its polysaccharide chains
have distinct binding properties, perlecan binds to dozens of other
molecules, including other ECM components (e.g., laminin,
nidogen/entactin), cell-surface receptors, and polypeptide growth
factors. Simultaneous binding to these molecules results in perlecan-
mediated cross-linking. Perlecan can be found in basal laminae and in
non–basal laminal ECM. The adhesion receptor dystroglycan can bind
perlecan directly, via perlecan’s LG domains, and indirectly, via its
binding to laminin. In humans, mutations in the perlecan gene can
lead either to dwarfism or to muscle abnormalities, apparently due to
dysfunction of the neuromuscular junction that controls muscle firing.

KEY CONCEPTS OF SECTION 20.3

The Extracellular Matrix I: The Basal Lamina

 The matrisome is the ensemble of proteins that compose the ECM itself and associated proteins that covalently modify (e.g., chemically
cross-link, phosphorylate, cleave) the ECM.
 The basal lamina, a thin meshwork of ECM molecules, separates most epithelia and other organized groups of cells from adjacent
connective tissue. Together, the basal lamina and the immediately adjacent collagen network form a structure called the basement
membrane.
 Four ECM proteins are found in all basal laminae (see Figure 20-24): laminin (a multi-adhesive matrix protein), type IV collagen, perlecan (a
proteoglycan), and nidogen/entactin.
 Adhesion receptors such as integrin anchor cells to the basal lamina, which in turn is connected to other ECM components (see Figure 20-1).
Laminin in the basal lamina is the principal ligand of α6β4 integrin (see Table 20-4).
 Laminin and other multi-adhesive matrix proteins are multidomain molecules that bind multiple adhesion receptors and ECM components.
 The large, flexible molecules of type IV collagen interact end to end and laterally to form a mesh-like scaffold to which other ECM
components and adhesion receptors can bind (see Figures 20-24 and 20-27).
 Type IV collagen is a member of the collagen family of proteins, which is distinguished by the presence of repeating tripeptide sequences of
Gly-X-Y that give rise to the collagen triple-helical structure (see Figure 20-26). Different collagens are distinguished by the length and
chemical modifications of their α chains and by the presence or absence of segments that interrupt or flank their triple-helical regions.
 Perlecan, a large, multidomain, secreted proteoglycan that is present primarily in basal laminae, binds many ECM components and
adhesion receptors. Proteoglycans consist of membrane-associated or secreted core proteins covalently linked to one or more specialized
polysaccharide chains called glycosaminoglycans (GAGs).

20.4 The Extracellular Matrix II: Connective Tissue
Connective tissue, such as tendon and cartilage, differs from other
solid tissues in that, rather than being filled primarily with cells, most
of its volume is made up of extracellular matrix packed with insoluble
protein fibers (see Figure 20-4). ECM in connective tissue has several
key components, some of which are found in other types of tissues as
well:
 Collagens, trimeric molecules that are often bundled
together into fibers (fibrillar collagens)
 Glycosaminoglycans (GAGs), specialized linear
polysaccharide chains of specific repeating
disaccharides that can be highly hydrated and confer
diverse binding and physical properties (e.g.,
resistance to compression)
 Proteoglycans, glycoproteins containing one or more
covalently bound GAG chains
 Multi-adhesive proteins, large multidomain proteins
often comprising many copies (“repeats”) of a few
distinctive domains that bind to and cross-link a
variety of adhesion receptors and ECM components
 Elastin, a protein that forms the amorphous core of
elastic fibers
Collagen is the most abundant fibrous protein in connective tissue.
Rubberlike elastin fibers, which can be stretched and relaxed, are also
present in deformable sites (e.g., skin, tendons, heart). The
fibronectins, a family of multi-adhesive matrix proteins, form their
own distinct fibrils in the ECM of most connective tissues. Although
several types of cells are found in connective tissues, the various ECM
components are produced largely by cells called fibroblasts. In this
section, we explore the structure and function of the various ECM
components in connective tissue, and we see how the ECM is degraded
and remodeled by a variety of specialized proteases.
Fibrillar Collagens Are the Major Fibrous Proteins in the ECM of Connective
Tissues
About 80–90 percent of the collagen in the body consists of fibrillar
collagens (types I, II, and III), located primarily in connective tissues
(see Table 20-5). Because of its abundance in tendon-rich tissue such as
rat tail, type I collagen is easy to isolate and was the first collagen to be
characterized. Its fundamental structural unit is a long (300 nm), thin
(1.5 nm diameter) triple helix (see Figure 20-26) consisting of two α1(I)
chains and one α2(I) chain, each 1050 amino acids in length. The triple-
stranded molecules pack tightly together and wrap around one
another, forming microfibrils that associate into higher order polymers
called collagen fibrils, which in turn often aggregate into larger bundles
called collagen fibers (Figure 20-28).
FIGURE 20-28 Biosynthesis of fibrillar collagens. Step 1: Procollagen α chains are synthesized on ribosomes associated with the endoplasmic reticulum
(rough ER), and in the ER, asparagine-linked oligosaccharides are added to the C-terminal propeptide. Step 2: Propeptides associate to form trimers and are
covalently linked by disulfide bonds, and selected residues in the Gly-X-Y triplet repeats are covalently modified [certain prolines and lysines are
hydroxylated; galactose or galactose-glucose (hexagons) are attached to some hydroxylysines; prolines are cis → trans isomerized]. Step 3: The modifications
facilitate zipper-like formation and stabilization of triple helices, and binding by the ER chaperone protein Hsp47, which may stabilize the helices or prevent
premature aggregation of the trimers, or both. Steps 4 and 5: The folded procollagens are packaged into large transport vesicles with the help of specialized
membrane proteins in the ER (not shown) and then transported to and through the Golgi complex, where some lateral association into small bundles takes
place. The chains are then secreted (step 6), the N- and C-terminal propeptides are removed (step 7), and the trimers assemble into fibrils and are covalently
cross-linked (step 8). The 67 nm staggering of the trimers gives the fibrils a striated appearance in electron micrographs ( inset). Step 9: The fibrils can
assemble into larger and larger bundles, some of which form the tendons that attach muscle to bone. See A. V. Persikov and B. Brodsky, 2002, Proc. Nat’l.
Acad. Sci. USA 99:1101–1103.

[Electron micrograph republished with permission from John Wiley & Sons, Inc., from J. Gross, 1953, “Evaluation of Structural and Chemical Changes in
Connective Tissue,” Ann. NY Acad. Sci. 56(4):674–683; permission conveyed through Copyright Clearance Center, Inc.]

In this schematic, the top left shows a cell inside the cytosol. Inside the cell,
steps 1-5 a blue circle represents the ribosome associated with the rough
endoplasmic reticulum. At step 3, a braid of 3 blue lines is with circles at
each end and a green circle labeled H s p 47 attached toward the right end.
The Golgi complex is at step 5 represented as light blue lines with blue
circles on each end. Step 6 is the packages from the Golgi that enter the
extracellular space and is arranged in rows to build collagen molecules. The
molecules make fibrils, which look like blue wires that are gathered into
blue cables in the last step to the right of the cell diagram. A micrograph is
added at the bottom left that shows the cables of collagen as light gray lines.
Classes of collagen that are less abundant, but nevertheless important,
include fibril-associated collagens, which link the fibrillar collagens to
one another or to other ECM components; sheet-forming and anchoring
collagens, which form two-dimensional networks in basal laminae (type
IV) and connect the basal lamina in skin to the underlying connective
tissue (type VII); transmembrane collagens, which function as adhesion
receptors; and host defense collagens, which help the body recognize and
eliminate pathogens. Interestingly, several collagens (e.g., types IX,
XVIII, and XV) are also proteoglycans with covalently attached GAGs
(see Table 20-5).
Fibrillar Collagen Is Secreted and Assembled into Fibrils Outside the Cell
Fibrillar collagens are secreted proteins, produced primarily by
fibroblasts in the ECM. Collagen biosynthesis and secretion follow the
normal pathway for a secreted protein, described in detail in Chapters
13 and 14. The collagen α chains are synthesized as longer precursors,
called pro-α chains, by ribosomes attached to the endoplasmic
reticulum (ER). The pro-α chains undergo a series of covalent
modifications and fold into triple-helical procollagen molecules before
their transport from the ER to the Golgi complex and subsequent
release from cells (see Figure 20-28). Specialized membrane proteins in
the ER allow the packaging of the very large collagen molecules into
large transport vesicles for transfer to the Golgi complex.
After the secretion of procollagen from the cell, extracellular
peptidases remove the N-terminal and C-terminal propeptides. The
resulting molecules, which consist almost entirely of a triple-stranded
helix because of long stretches of the characteristic collagen repeating
sequence motif Gly-X-Y, associate laterally to generate fibrils with a
diameter of 50–200 nm. In fibrils, adjacent collagen molecules are
displaced from one another by 67 nm, about one-quarter of their
length. This staggered array produces a striated effect that can be seen
in both light and electron microscopic images of collagen fibrils (see
Figure 20-28, inset). The unique properties of the fibrillar collagens are
mainly due to the formation of fibrils.
Short segments at either end of the fibrillar collagen α chains that are
not composed of the repeating sequence motif Gly-X-Y, and thus are
not triple-helical, are of particular importance in the formation of
collagen fibrils. Lysine and hydroxylysine side chains in these
segments are covalently modified by extracellular lysyl oxidases to
form aldehydes in place of the amine group at the end of the side
chain. These reactive aldehyde groups form covalent cross-links with
lysine, hydroxylysine, and histidine residues in adjacent molecules.
The cross-links stabilize the side-by-side packing of collagen molecules
and generate a very strong fibril. The removal of the terminal
propeptides and covalent cross-linking take place in the extracellular
space to prevent the potentially catastrophic assembly of large fibrils
within the cell.
The post-translational modifications of pro-α chains are crucial
for the formation of mature collagen molecules and their assembly
into fibrils. Defects in these modifications have serious consequences,
which ancient mariners frequently experienced. For example, ascorbic
acid (vitamin C) is an essential cofactor for the hydroxylases
responsible for adding hydroxyl groups to proline and lysine residues
in pro-α chains. In cells deprived of ascorbate, as in the disease scurvy,
the pro-α chains are not hydroxylated sufficiently to form stable triple-
helical procollagen at normal body temperature, and the procollagen
that forms cannot assemble into normal fibrils. Without the structural
support of collagen, blood vessels, tendons, and skin become fragile.
Fresh fruit in the diet can supply sufficient vitamin C to support the
formation of normal collagen. Historically, British sailors were
provided with limes to prevent scurvy, leading to their being called
“limeys.” Mutations in lysyl hydroxylase genes can also cause
connective-tissue defects.
Type I and II Collagens Associate with Nonfibrillar Collagens to Form Diverse
Structures
Collagens differ in the structures of the fibers they form and in how
these fibers are organized into networks. Of the predominant types of
collagen found in connective tissues, type I collagen forms long fibers,
whereas networks of type II collagen are more mesh-like. In tendons,
for instance, the long type I collagen fibers connect muscles to bones
and must withstand enormous forces. Because type I collagen fibers
have great tensile strength, tendons usually can be stretched without
being broken. Indeed, gram for gram, type I collagen is stronger than
steel. Two quantitatively minor fibrillar collagens, type V and type XI,
co-assemble into fibers with type I collagen, thereby regulating the
structures and properties of the fibers. Incorporation of type V
collagen, for example, results in smaller-diameter fibers.
Type I collagen fibrils are also used as the reinforcing rods in the
construction of bone. Bones and teeth are hard and strong because
they contain large amounts of dahllite, a crystalline calcium- and
phosphate-containing mineral. Most bones are about 70 percent
mineral and 30 percent protein, the vast majority of which is type I
collagen. Bones form when certain cells (chondrocytes and
osteoblasts) secrete collagen fibrils that are then mineralized by
deposition of small dahllite crystals.
In many connective tissues, particularly skeletal muscle, proteoglycans
and a fibril-associated collagen called type VI collagen are noncovalently
bound to the sides of type I fibrils and may bind the fibrils together to
The fibrils of type II collagen, the major collagen in cartilage, are
smaller in diameter than type I fibrils and are oriented randomly in a
viscous proteoglycan matrix. The rigid collagen fibrils impart strength
to the matrix and allow it to resist large deformations. Type II fibrils
are cross-linked to matrix proteoglycans by type IX collagen, another
fibril-associated collagen. Type IX collagen and several related types
have two or three triple-helical segments connected by flexible kinks
and a globular N-terminal segment (Figure 20-29b). The globular N-
terminal segment of type IX collagen extends from the type II fibril at
the end of one of its helical segments, as does a chondroitin sulfate
GAG chain (chondroitin sulfate is described below) that is sometimes
linked to one of the type IX chains. These protruding nonhelical
structures are thought to anchor the type II fibril to proteoglycans and
other components of the matrix. The interrupted triple-helical
structure of type IX and related collagens prevents them from
assembling into fibrils, although they can associate with fibrils formed
from other collagen types and form covalent cross-links to them.
Mutations affecting type I collagen and its associated proteins
cause a variety of human diseases. Certain mutations in the genes
encoding the type I collagen α1(I) or α2(I) chains lead to osteogenesis
imperfecta, or brittle-bone disease. Because every third position in a
collagen α chain must be a glycine for the triple helix to form (see
Figure 20-26), mutations of glycine to almost any other amino acid are
deleterious, resulting in poorly formed and unstable helices. A defect
in only one of the three α chains in a collagen molecule can disrupt the
whole molecule’s triple-helical structure and function. A mutation in a
single copy (allele) of either the α1(I) gene or the α2(I) gene, both
located on autosomes, can cause this disorder. Thus it normally shows
autosomal dominant inheritance (see Chapter 6).
form thicker collagen fibers (Figure 20-29a). Type VI collagen is
unusual in that the molecule consists of a relatively short triple helix
with globular domains at both ends. The lateral association of two type
VI monomers generates an antiparallel dimer. The end-to-end
association of these dimers through their globular domains forms type
VI microfibrils. These microfibrils have a beads-on-a-string appearance,
with about 60-nm-long triple-helical regions separated by 40-nm-long
globular domains.
FIGURE 20-29 Interactions of fibrillar collagens with fibril-associated
collagens. (a) In tendons, type I fibrils are all oriented in the direction of the
stress applied to the tendon. Proteoglycans and type VI collagen bind
noncovalently to type I fibrils, coating the surface. The microfibrils of type VI
collagen, which contain globular and triple-helical segments, bind to type I
fibrils and link them together into thicker fibers. See R. R. Bruns et al., 1986,
J. Cell Biol. 103:393. (b) In cartilage, type IX collagen molecules are
covalently bound at regular intervals along type II fibrils. A chondroitin
sulfate chain, covalently linked to the α2(IX) chain at the flexible kink,
projects outward from the fibril, as does the globular N-terminal region. See
L. M. Shaw and B. R. Olsen, 1991, Trends Biochem. Sci. 16:191.
The illustration labeled (a) shows two stacks of blue disks representing the
type 1 collagen fibrils with orange short tubes going along between the
stacks and labeled Type 6 collagen. Short black line segments are in the
stacks and labeled proteoglycan. The illustration labeled (b) shows one
stack of blue disks labeled Type 2 collagen fibril. On the front of this stake
are two strings of orange cylinders with black lines between labeled kink. A
red thread comes from between two orange cylinders and is labeled
chondroitin sulfate. The orange cylinders are labeled Type 9 collagen.
Absence or malfunctioning of fibril-associated collagen in muscle
tissue due to mutations in the type VI collagen genes cause dominant
or recessive congenital muscular dystrophies with generalized muscle
weakness, respiratory insufficiency, muscle wasting, and muscle-
related joint abnormalities. Skin abnormalities have also been reported
with type VI collagen disease.
Proteoglycans and Their Constituent GAGs Play Diverse Roles in the ECM
As we saw with perlecan in the basal lamina, proteoglycans play an
important role in cell-matrix adhesion. Proteoglycans are a subset of
secreted or cell-surface glycoproteins containing covalently linked,
specialized polysaccharide chains called glycosaminoglycans (GAGs).
GAGs are long linear polymers of specific repeating disaccharides.
Usually one sugar is either a uronic acid (D-glucuronic acid or L-
iduronic acid) or D-galactose; the other sugar is N-acetylglucosamine or
N-acetylgalactosamine (Figure 20-30). One or both of the sugars
contain at least one anionic group (carboxylate or sulfate). Thus each
GAG chain bears many negative charges. GAGs are classified into
several major types based on the nature of the repeating disaccharide
unit: heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan
sulfate, and hyaluronan (see Figure 20-30). A hypersulfated form of
heparan sulfate called heparin, produced mostly by mast cells, plays a
key role in allergic reactions. It is also used medically as an anticlotting
drug because of its ability to activate a natural clotting inhibitor called
antithrombin III.

With the exception of hyaluronan, which is discussed below, all the
major GAGs occur naturally as components of proteoglycans. Like
other secreted and transmembrane glycoproteins, proteoglycan core
proteins are synthesized in the endoplasmic reticulum, and the GAG
chains are assembled on and
FIGURE 20-30 The repeating disaccharides of glycosaminoglycans (GAGs).
Each of the four classes of GAGs is formed by polymerization of monomeric
units into repeats of a particular disaccharide and subsequent
modifications, including addition of sulfate groups and inversion
(epimerization) of the carboxyl group on carbon 5 of D-glucuronic acid to
yield L-iduronic acid. The squiggly lines represent covalent bonds that are
oriented either above (D-glucuronic acid) or below ( L-iduronic acid) the ring.
Heparin is generated by hypersulfation of heparan sulfate, whereas
hyaluronan is not sulfated.
The illustration labeled (a) shows the chemical structure of hyaluronan, with
twenty-five thousand dimers composed of D-glucuronic acid and N-acetyl-D-
glucosamine.
The illustration labeled (b) shows the chemical structure of chondroitin
sulfate, with 250 dimers composed of D-glucuronic acid and N-acetyl D-
galactosamine or L-iduronic acid and N-acetyl-D-galactosamine.
The illustration labeled (c) shows the chemical structure of heparin sulfate,
with two hundred dimers composed of D-glucuronic or L-iduronic acid and
N-acetyl- or N-sulfo-D-glucosamine.
The illustration labeled (d) shows the chemical structure of keratan sulfate,
with 20 to 40 dimers composed of D-galactose and N-acetyl-d-glucosamine.
covalently attached to these cores in the Golgi complex. To generate
heparan or chondroitin sulfate chains, a three-sugar linker is first
attached to the hydroxyl side chains of certain serine residues in a core
protein (Figure 20-31a); thus these GAGs are O-linked
oligosaccharides, several examples of which are shown in Figure 20-
O-linked oligosaccharides are attached to the hydroxyl side chains of
specific serine and theonine residues in a variety of secreted proteins
and the exoplasmic (extracellular) domains of some membrane
proteins. For example, mucin-like O-linked chains are covalently
bound to glycoproteins via an N-acetylgalactosamine (GalNAc)
monosaccharide to which are covalently attached a variety of other
sugars, often including sialic acid (SA; Figure 20-31b). The high
concentration of long O-linked chains on mucin glycoproteins on the
apical plasma membranes of epithelial cells apparently contributes to
the bending of the membranes and the formation of microvilli (see
Figures 20-11, 17-1, and 17-2). The only O-linked glycoproteins in
31.
FIGURE 20-31 Hydroxyl O-linked oligosaccharides.
(a) Synthesis of a GAG, in this case chondroitin
sulfate, is initiated by transfer of a xylose (Xyl)
residue to a serine residue in the core protein,
most likely in the Golgi complex, followed by
sequential addition of two galactose (Gal)
residues. Glucuronic acid (GlcUA) and N-
acetylgalactosamine (GalNAc) residues are then
added sequentially to these linking sugars and
some of the GalNAc monomers are sulfated,
forming the chondroitin sulfate chain. Heparan
sulfate chains are connected to core proteins by
the same three-sugar linker. Keratan sulfate GAGs
are covalently attached to proteins via N-linked
rather than O-linked connections. (b) Mucin-like
O-linked chains are covalently bound to
glycoproteins via an N-acetylgalactosamine
(GalNAc). (c) In the integral membrane protein α-
dystroglycan on cell surfaces, a complex
polysaccharide is attached to hydroxyl side
chains via an O-mannose (O-Man). A polymer of
the GlcUA-Xyl disaccharide ([-Xyl-GlcUA]n)
called matriglycan is attached
to a phosphorylated O-linked mannose via an
oligosaccharide containing N-acetylglucosamine
(GlcNAc), ribitol 5-phosphate (Ribito5P), Xyl, and
GlcUA. Matriglycan provides a binding site for
ECM molecules, such as laminin and perlecan
(described in detail in Section 20.5).
baker’s yeast are mannose-linked (O-Man). In other eukaryotes (not in
nematode worms or plants), certain specialized O-linked sugars are
bound to proteins via mannose monosaccharides. Some proteins,
including classical cadherins and protocadherins (Section 20.2), have
relatively short O-mannose linked oligosaccharides. Others have
longer oligosaccharides attached via the O-mannose, such as that
attached to the cell-surface protein dystroglycan (Figure 20-31c;
described in detail in Section 20.5).
In contrast to protein-linked heparan or chondroitin sulfate chains, the
linkers for the addition of keratan sulfate chains are oligosaccharide
chains attached to asparagine residues; such N-linked
oligosaccharides are present in many glycoproteins (see Chapters 13
and 14), although only a subset carry GAG chains. All GAG chains are
elongated by the alternating addition of sugar monomers to form the
disaccharide repeats characteristic of a particular GAG; the chains are
often modified subsequently by the covalent linkage of small molecules
such as sulfate. The mechanisms responsible for determining which
proteins are modified with GAGs, the sequence of disaccharides to be
added, the sites to be sulfated, and the lengths of the GAG chains are
unknown. The ratio of polysaccharide to protein in all proteoglycans is
much higher than that in most other glycoproteins.
Function of GAG Chain Modifications
Similar to the sequence of amino acids in proteins, the arrangement of
the sugar residues in GAG chains and the modification of specific
sugars in those chains can determine their function and that of the
proteoglycans that contain them. For example, groupings of certain
modified sugars in the GAG chains of heparin sulfate proteoglycans
Diversity of Proteoglycans
The proteoglycans constitute a remarkably diverse group of molecules
that are abundant in the ECM of all animal tissues and are also
expressed on the cell surface. For example, of the five major classes of
heparan sulfate proteoglycans, three are located in the ECM (perlecan,
agrin, and type XVIII collagen) and two are cell-surface proteins. The
latter include integral membrane proteins (syndecans) and GPI-
anchored proteins (glypicans); the GAG chains in both types of cell-
surface proteoglycans extend into the extracellular space. The
sequences and lengths of proteoglycan core proteins vary
considerably, and the number of attached GAG chains ranges from just
a few to more than 100. Moreover, a core protein is often linked to two
different types of GAG chains, generating a hybrid proteoglycan. The
basal laminal proteoglycan perlecan is primarily a heparan sulfate
proteoglycan with three to four GAG chains, although it can sometimes
have a bound chondroitin sulfate chain. Additional diversity in
proteoglycans occurs because the numbers, compositions, and
sequences of the GAG chains attached to otherwise identical core
proteins can differ considerably. Laboratory generation and analysis of
mutants with defects in proteoglycan production in Drosophila, C.
can control the binding of growth factors to certain receptors or the
activities of proteins in the blood-clotting cascade.
In the past, the chemical and structural complexity of proteoglycans
posed a daunting barrier to analyzing and understanding their
structures and their many diverse functions. In recent years,
investigators employing classic and state-of-the-art biochemical
techniques, mass spectrometry, and genetics have begun to elucidate
the detailed structures and functions of these ubiquitous ECM
molecules. The results of ongoing studies suggest that sets of sugar-
residue sequences containing common modifications, rather than
single unique sequences, are responsible for specifying distinct GAG
functions. A case in point is a set of five-residue (pentasaccharide)
sequences found in a subset of heparin GAGs that controls the activity
of antithrombin III (ATIII), an inhibitor of the key blood-clotting
protease thrombin. When these pentasaccharide sequences in heparin
are sulfated at two specific positions (Figure 20-32), heparin can
activate ATIII, thereby inhibiting clot formation. Several other sulfates
can be present in the active pentasaccharide in various combinations,
but they are not essential for the anticlotting activity of heparin. The
rationale for generating sets of similar active sequences rather than a
single, unique sequence is not well understood.
FIGURE 20-32 The pentasaccharide GAG sequence
that regulates the activity of antithrombin III
(ATIII). Sets of modified five-residue sequences in
the much longer GAG called heparin with the
composition shown here bind to ATIII and
activate it, thereby inhibiting blood clotting. The
sulfate groups in red type are essential for this
heparin function; the modifications in blue type
may be present but are not essential. Other sets
of modified GAG sequences are thought to
regulate the activity of other target proteins.
elegans, and mice have clearly shown that proteoglycans play critical
roles in development; for example, as participants in various signaling
pathways (see Chapter 16 for examples in the TGF-β and Wnt
pathways).
Syndecans are cell-surface proteoglycans expressed by epithelial and
nonepithelial cells that bind to collagens and multi-adhesive matrix
proteins such as fibronectin, anchoring cells to the ECM. Like that of
many integral membrane proteins, the cytosolic domain of syndecan
interacts with the actin cytoskeleton and in some cases with
intracellular regulatory molecules. In addition, cell-surface
proteoglycans such as syndecan bind many protein growth factors and
other external signaling molecules, thereby helping to regulate cellular
metabolism and function. For instance, syndecans in the hypothalamic
region of the brain modulate feeding behavior in response to food
deprivation. They do so by participating in the binding to cell-surface
receptors of antisatiety peptides that help control feeding behavior. In
the fed state, the syndecan extracellular domain decorated with
heparan sulfate GAG chains is released from the cell surface by
proteolysis, thus suppressing the activity of the antisatiety peptides and
feeding behavior. In mice engineered to overexpress the syndecan-1
gene in the hypothalamic region of the brain and other tissues, normal
control of feeding by antisatiety peptides is disrupted, and the animals
overeat and become obese.
Hyaluronan Resists Compression, Facilitates Cell Migration, and Gives
Cartilage Its Gel-Like Properties
Hyaluronan, also called hyaluronic acid (HA) or hyaluronate, is a
nonsulfated GAG (see Figure 20-30a) made by a plasma-membrane-
bound enzyme called HA synthase and is secreted directly into the
extracellular space as it is synthesized. (A similar approach is used by
plant cells to make the ECM component cellulose.) Hyaluronan is a
major component of the ECM that surrounds migrating and
proliferating cells, particularly in embryonic tissues. In addition, it
forms the backbone of complex proteoglycan aggregates found in
many ECMs, particularly cartilage. Because of its remarkable physical
properties, hyaluronan imparts stiffness and resilience as well as a
lubricating quality to many types of connective tissue such as joints.
Hyaluronan molecules range in length from a few disaccharide repeats
to about 25,000. The typical hyaluronan in joints such as the elbow has
10,000 repeats for a total mass of 4×106 Da and a length of 10 μm
(about the diameter of a small cell). Individual segments of a
hyaluronan molecule fold into a rodlike conformation because of the β
glycosidic linkages between the sugars and extensive intrachain
hydrogen bonding. Mutual repulsion between negatively charged
carboxylate groups that protrude outward at regular intervals also
contributes to these locally rigid structures. Overall, however,
hyaluronan is not a long, rigid rod like fibrillar collagen; rather, it is
very flexible in solution, bending and twisting into many
conformations, forming a random coil.
Because of the large number of anionic residues on its surface, the
typical hyaluronan molecule binds a large amount of water and
behaves as if it were a large hydrated sphere with a diameter of about
500 nm. As the concentration of hyaluronan increases, the long chains
begin to entangle, forming a viscous gel. Even at low concentrations,
hyaluronan forms a hydrated gel; when placed in a confining space,
such as that between two cells, the long hyaluronan molecules tend to
push outward. This outward pushing creates a swelling, or turgor
pressure, within the extracellular space. In addition, the binding of
cations by carboxylate (COO−) groups on the surface of
hyaluronan increases the concentration of ions and thus the osmotic
pressure in the gel. As a result, large amounts of water are taken up,
contributing to the turgor pressure. These swelling forces give
connective tissues their ability to resist compression forces, in contrast
to collagen fibers, which are best able to resist stretching forces. Other
highly charged GAG chains are similarly hydrated.
Hyaluronan is bound to the surface of many migrating cells by a
number of adhesion receptors, such as the receptor called CD44, which
contains hyaluronan-binding domains, each with a similar three-
dimensional conformation. Because of its loose, hydrated, porous
nature, the hyaluronan coating bound to cells appears to keep them
apart from one another, giving them the freedom to move about and
proliferate. The cessation of cell movement and the initiation of cell-
cell attachments are frequently correlated with a decrease in
hyaluronan, a decrease in hyaluronan receptors, and an increase in the
extracellular enzyme hyaluronidase, which degrades hyaluronan in the
matrix. These alterations of hyaluronan are particularly important
during the many cell migrations that facilitate differentiation and in
the release of a mammalian egg cell from its surrounding cells after
ovulation.
The predominant proteoglycan in cartilage, called aggrecan, assembles
with hyaluronan into very large aggregates, illustrative of the complex
structures that proteoglycans sometimes form. The backbone of this
proteoglycan aggregate is a long molecule of hyaluronan to which
multiple aggrecan molecules are bound tightly but noncovalently
(Figure 20-33). A single hyaluronan-aggrecan aggregate, one of the
largest macromolecular complexes known, can be more than 4 μm
long and have a volume larger than that of a bacterial cell. These
aggregates give cartilage its unique gel-like properties and its
resistance to deformation, essential for distributing the load in weight-
bearing joints.

The aggrecan core protein (∼250,000 MW) has one N-terminal globular
domain that binds with high affinity to a specific decasaccharide
sequence within hyaluronan. This specific sequence is generated by
covalent modification of some of the repeating disaccharides in the
hyaluronan chain. The interaction between aggrecan and hyaluronan
is facilitated by a link protein that binds to both the aggrecan core
protein and hyaluronan (Figure 20-33b). Aggrecan and the link protein
have in common a link domain, about 100 amino acids long, that is
found in numerous ECM and cell-surface hyaluronan-binding proteins
in both cartilaginous and noncartilaginous tissues. These proteins
almost certainly arose in the course of evolution from a single
ancestral gene that encoded just this domain.
Fibronectins Connect Cells and ECM, Influencing Cell Shape, Differentiation,
and Movement
Many different cell types synthesize fibronectin, an abundant multi-
adhesive matrix protein found in all vertebrates. The discovery that
fibronectin functions as an adhesion molecule stemmed from
observations that it is present on the surfaces of normal fibroblasts,
which adhere tightly to petri dishes in laboratory experiments, but is
absent from the surfaces of tumorigenic (i.e., cancerous) cells, which
adhere weakly. The 20 or so isoforms of fibronectin are generated by
alternative splicing of the RNA transcript produced from a single gene
FIGURE 20-33 Structure of proteoglycan aggregate from cartilage. (a)
Electron micrograph of an aggrecan aggregate from fetal bovine epiphyseal
cartilage (artificially colored). Aggrecan core proteins are bound at ∼40-nm
intervals to a molecule of hyaluronan. (b) Schematic representation of an
aggrecan monomer bound to hyaluronan. In aggrecan, both keratan sulfate
and chondroitin sulfate chains are attached to the core protein. The N-
terminal domain of the core protein binds noncovalently to a hyaluronan
molecule. Binding is facilitated by a link protein, which binds to both the
hyaluronan molecule and the aggrecan core protein. Each aggrecan core
protein has 127 Ser-Gly sequences at which GAG chains can be added. The
molecular weight of an aggrecan monomer averages 2×106 . The
entire aggregate, which may contain upward of 100 aggrecan monomers,
has a molecular weight in excess of 2×108 and is about as large as
the bacterium Escherichia coli.
[Part (a) republished with permission from Elsevier, from J. A. Buckwalter
and L. Rosenberg, 1983, “Structural Changes During Development in Bovine
Fetal Epiphyseal Cartilage,” Collagen Rel. Res. 3(6):489–504; permission
conveyed through Copyright Clearance Center, Inc.]
The micrograph labeled (a) shows a horizontal squiggly yellow line labeled
hyaluronan molecule. From this molecule, all along both sides and around
the end are threads that start green and end red. The green part is labeled
keratan sulfate and the red part is labeled chondroitin sulfate. The
illustration labeled (b) takes one of the green and red threads and makes a
schematic diagram of it using lines to represent the parts. There is a yellow
tube at the top to represent the hyaluronan molecule. A gray circle attached
to it below is labeled link protein. There is a central line attached to the
hyaluronan. One end of the central line near hyaluronan is labeled N-
terminal hyaluronan-binding domain and the other end is labeled aggrecan
core protein. The green and red lines are attached along the sides with
linking sugars.
(see Figure 5-28). Fibronectins are essential for the migration and
differentiation of many cell types in embryogenesis. These proteins are
also important for wound healing because they promote blood clotting
and facilitate the migration of macrophages and other immune-system
cells into the affected area.
Fibronectins help attach cells to the ECM by binding to other ECM
components, particularly fibrillar collagens and heparan sulfate
proteoglycans, and to adhesion receptors such as integrins (see Figure
20-2). Through their interactions with adhesion receptors, fibronectins
influence the shape and movement of cells and the organization of the
cytoskeleton. Conversely, by regulating their receptor-mediated
attachments to fibronectin and other ECM components, cells can
sculpt the immediate ECM environment to suit their needs.
Fibronectins are dimers of two similar polypeptides linked at their C-
termini by two disulfide bonds; each chain is about 60–70 nm long and
2–3 nm thick. Partial digestion of fibronectin with small amounts of
proteases and analysis of the fragments showed that each chain
comprises several functional regions with different ligand-binding
specificities (Figure 20-34a). Each region, in turn, contains multiple
copies of certain domain-encoding sequences that can be classified
into one of three types. These domains are designated fibronectin type
I, II, and III repeats, on the basis of similarities in amino acid
sequence, although the sequences of any two repeats of a given type
are not identical. These linked repeats give the molecule the repeats composing the regions confers on fibronectin its ability to bind
appearance of beads on a string. The combination of the different multiple ligands.

FIGURE 20-34 Organization of fibronectin and its binding to integrin. (a) Scale model of fibronectin is shown docked by two type III repeats to the extracellular
domains of integrin. Only one of the two similar chains, which are linked by disulfide bonds near their C-termini, in the dimeric fibronectin molecule is shown.
Each chain contains about 2446 amino acids and is composed of three types of repeating amino acid sequences (type I, II, or III repeats) or domains. The EIIIA,
EIIIB — both type III repeats — and IIICS domain are variably spliced into the structure at locations indicated by arrows. Circulating fibronectin lacks EIIIA,
EIIIB, or both. At least five different sequences may be present in the IIICS region as a result of alternative splicing (see Figure 5-28). Each chain contains
several multi-repeat-containing regions, some of which contain specific binding sites for heparan sulfate, fibrin (a major constituent of blood clots), collagen,
and cell-surface integrins. The integrin-binding domain is also known as the cell-binding domain. Structures of fibronectin’s domains were determined from
fragments of the molecule. (b) A high-resolution structure shows that the RGD sequence motif extends outward in a loop from its compact type III domain on
the same side of fibronectin as the synergy region, which also contributes to high-affinity binding to integrins.

[Data from D. J. Leahy, I. Aukhil, and H. P. Erickson, 1996, Cell 84:155, PDB ID 1fnf.]

The illustration labeled (a) shows fibronectin which comprises type 1 repeats at the N-terminal end, where fibrin and heparan sulfate can bind, followed by
type 2 repeats, where collagen can bind, followed by type three repeats. In the long region composed of type three repeats, there are several cell-binding
regions. E 3 B, R G D, where integrin binds, and E 3 A. This section is followed by further binding sites for heparan sulfate and three C S. At the c-terminal,
there are further type 1 repeats followed by disulfide bonds to other fibronectin fibers.

One of the type III repeats in the cell-binding region of fibronectin
mediates binding to certain integrins. The results of studies with
synthetic peptides corresponding to parts of this repeat identified the
tripeptide sequence Arg-Gly-Asp, called the RGD motif, as the minimal
sequence within this repeat
required for recognition by those integrins. In one study,
heptapeptides with and without the RGD motif were tested for their
ability to mediate the adhesion of rat kidney cells to a culture dish. The
results showed that heptapeptides containing the RGD motif mimicked
intact fibronectin’s ability to stimulate integrin-mediated adhesion,
whereas variant heptapeptides lacking this sequence were ineffective
(Figure 20-35).

A three-dimensional model of fibronectin binding to integrin, based on
partial structures of both fibronectin and integrin, has been assembled.
In a high-resolution structure of the integrin-binding fibronectin type
III repeat and its neighboring type III domain, the RGD motif is at the
apex of a loop that protrudes outward from the molecule, in a position
facilitating binding to integrins (Figure 20-34b). Although the RGD
motif is required for binding to several different integrins, its affinity
for integrins is substantially less than that of intact fibronectin or of the
entire cell-binding region in fibronectin. Thus structural features near
the RGD motif in fibronectins (e.g., parts of adjacent repeats, such as
the synergy region; see Figure 20-34b) and in other RGD-containing
proteins must enhance their binding to certain integrins. Moreover,
the simple, soluble dimeric forms of fibronectin produced by the liver
or by fibroblasts are initially in a nonfunctional conformation that
binds poorly to integrins because the RGD motif is not readily
accessible. The adsorption of fibronectin onto a collagen matrix or
basal lamina — or, experimentally, to a plastic tissue culture dish —
results in a conformational change that enhances the ability of
fibronectin to bind to cells. Possibly, this conformational change
increases the accessibility of the RGD motif for integrin binding.
Microscopy and other experimental approaches (e.g., biochemical-
binding experiments) have demonstrated the role of integrins in cross-
linking fibronectin and other ECM components to the cytoskeleton. For
example, the colocalization of cytoskeletal actin filaments and
integrins within cells can be visualized by fluorescence microscopy
(Figure 20-36a). The binding of cell-surface integrins to fibronectin in
the ECM induces the actin cytoskeleton–dependent movement of some
integrin molecules in the plane of the plasma membrane. The ensuing
mechanical tension due to the relative movement of different integrins
bound to a single fibronectin dimer stretches the fibronectin (see
.
EXPERIMENTAL FIGURE 20-35 A specific tripeptide sequence (RGD) in the
cell-binding region of fibronectin is required for cell adhesion. The cell-
binding region of fibronectin contains an integrin-binding hexapeptide
sequence, GRGDSP in the single-letter amino acid code. Together with an
additional C-terminal cysteine (C) residue, this heptapeptide and several
variants were synthesized chemically. Different concentrations of each
synthetic peptide were added to polystyrene dishes that had the protein
immunoglobulin G (IgG) firmly attached to their surfaces; the peptides were
then chemically cross-linked to the IgG. Subsequently, cultured normal rat
kidney cells were added to the dishes and incubated for 30 minutes to allow
adhesion. After the unbound cells were washed away, the relative amounts
of cells that had adhered firmly were determined by staining the bound cells
with a dye and measuring the intensity of the staining with a
spectrophotometer. The results shown here indicate that cell adhesion
increased above the background level with increasing peptide
concentration for those peptides containing the RGD motif, but not for the
variants lacking this sequence (modification underlined).
[Data from M. D. Pierschbacher and E. Ruoslahti, 1984, Proc. Nat’l. Acad. Sci.
USA 81:5985.]
In the graph, the vertical axis plots the relative amounts of bound cells by
stain intensity ranging from 0 to 1.4 in increments of 0.2. The horizontal axis
plots peptide concentration in nanomoles per milliliter ranging from 0 to ten
thousand in increments of 9, 90, and 900. Curves for four proteins containing
R G D sequences are sigmoidal. Curves of proteins without R G D sequence
show no cell binding.
Figure 20-9), a mechanosensor, and promotes self-association of
fibronectins into multimeric fibrils
The force needed to unfold and expose functional self-association sites
in fibronectin is much less than that needed to disrupt fibronectin-
integrin binding. Thus fibronectin molecules remain bound to integrin
while cell-generated mechanical forces induce fibril formation. In
effect, the integrins, through adapter proteins, transmit the
intracellular forces generated by the actin cytoskeleton to extracellular
fibronectin (inside-out signaling via mechanotransduction). Gradually,
the initially formed fibronectin fibrils mature into highly stable matrix
components by covalent cross-linking. In some electron micrographs,
exterior fibronectin fibrils appear to be aligned in a seemingly
continuous line with bundles of actin fibers within the cell (Figure 20-
36b). These observations and the results from other studies provided
the first example of a molecularly well-defined adhesion receptor
forming a bridge between the intracellular cytoskeleton and the ECM
components — a phenomenon now known to be widespread.
Elastic Fibers Permit Many Tissues to Undergo Repeated Stretching and
Recoiling
Elastic fibers are found in the ECM of a wide variety of tissues that are
subject to mechanical strain or deformation, such as the lungs, which
expand and contract during breathing (Figure 20-37a); the blood
vessels, through which blood pulses due to the heartbeat; and the skin
and many other tissues that stretch and contract. Elastic fibers permit
the rubberlike reversible elastic stretching and recoiling of these
tissues.

The major component of an elastic fiber, which can be several hundred
to several thousand nanometers in diameter, is an insoluble,
amorphous core composed of the protein elastin. Elastin consists of
aggregates of monomeric tropoelastin molecules that are covalently
cross-linked via a lysyl oxidase–mediated process similar to that seen
in collagen. Repetitive proline- and glycine-enriched hydrophobic
sequence motifs contribute to the ability of tropoelastins to self-
EXPERIMENTAL FIGURE 20-36 Integrins mediate linkage between fibronectin
in the ECM and the cytoskeleton. (a) Immunofluorescent micrograph of a
fixed cultured fibroblast showing colocalization of the α5β1 integrin (green)
and actin-containing stress fibers (red). The cell was incubated with two
types of monoclonal antibodies: an integrin-specific antibody linked to a
green-fluorescing dye and an actin-specific antibody linked to a red-
fluorescing dye. Stress fibers are long bundles of actin microfilaments that
radiate inward from points where the cell contacts a substratum. At the
distal ends of these fibers, near the plasma membrane, the coincidence of
actin and fibronectin-binding integrin produces a yellow fluorescence. (b)
Electron micrograph of the junction of fibronectin and actin fibers in a
cultured fibroblast. Individual actin-containing 7 nm microfilaments,
components of a stress fiber, end at the obliquely sectioned cell membrane.
The microfilaments appear aligned with and in close proximity to the
thicker, densely stained fibronectin fibrils on the outside of the cell.
[Part (a) J. L. Duband et al., 1988, J. Cell Biol. 107:1385–1396, cover;
https://doi.org/10.1083/jcb.107.4.1385. Part (b) republished with permission
from Elsevier, from I. I. Singer, 1979, “The Fibronexus: A Transmembrane
Association of Fibronectin-Containing Fibers and Bundles of 5 nm
Microfilaments in Hamster and Human Fibroblasts,” Cell 16(3):675–685;
permission conveyed through Copyright Clearance Center, Inc.]
The micrograph labeled (a) shows bright red streaks coming from upper left
to lower right, with tiny green spots along the ends. The micrograph labeled
(b) shows long gray streaks at the left labeled fibronectin fibrils. A light area
above these is labeled cell exterior. Thick gray areas to the right are labeled
plasma membrane, and tiny thin gray lines streaking left to right and on the
right side of the micrograph are labeled actin-containing microfilaments.
The area below these is labeled cell interior.
FIGURE 20-37 Elastic and collagen fibers in connective tissue. (a) Light-
microscopic image of loose connective tissue from the lung. Collagen fibers
(bundles of collagen fibrils) are stained pink; elastic fibers and nuclei of cells
are stained purple. (b) Longitudinal and (c) cross-sectional electron
microscopic images of elastic fibers and collagen fibrils (coll) in the skin of a
mouse. The elastic fibers have a solid core of elastin (e) integrated into and
surrounded by a bundle of microfibrils (mf). Scale bars, 0.25 μm.
[Parts (b) and (c) republished with permission from Elsevier, from J. Choi et
al., 2009, “Analysis of Dermal Elastic Fibers in the Absence of Fibulin-5
Reveals Potential Roles for Fibulin-5 in Elastic Fiber Assembly,” Matrix Biol.
28(4):211–220; permission conveyed through Copyright Clearance Center,
Inc.]
The micrograph labeled (a) titled connective tissue shows a pink web of
fibers in and around cells with dark nuclei. The collagen fibers and elastic
fibers are labeled. The micrograph labeled (b) shows a lower darkest area
labeled collagen fibrils, a layer of dark and white splotches above it labeled
elastic fiber, and an area at the top looking like many black circles clustered
along and labeled collagen fibrils. The micrograph labeled (c) shows the
cross-sectional view of the micrograph (b), which looks like a dark spot in
the center surrounded by the black circles from the collagen fibrils.
associate, extend under stress, and recoil efficiently after stretching.
The elastin core is surrounded by a collection of 10–12-nm-diameter
microfibrils (Figure 20-37b) made up of the proteins fibrillin, fibulin,
and associated proteins such as LTBPs (latent TGF-β binding proteins).
The microfibrils serve as scaffolds for the assembly of the elastic fiber’s
core. Elastin-free microfibrils are found in the eye, where they
transmit muscular force to reshape the lens for focusing and may weapons testing in the 1950s and 1960s, 14C was introduced into the
provide structural support to the cornea. atmosphere and hence the food chain. This environmental 14C has
been used as the radioactive pulse in what is essentially a pulse-chase
experiment to determine the stability of proteins of interest.
Similar to other components of the ECM, microfibrils participate in cell
signaling. In the secretory pathway, LTBPs bind the inactive form of
the secreted signaling protein transforming growth factor β (TGF-β; a
cytokine, see Figure 16-22) prior to their co-secretion and
Metalloproteases Remodel and Degrade the Extracellular Matrix
incorporation into microfibrils. Biomechanical stress mediated by cell-
surface integrins binding to and pulling on the LTBP/TGF-β complex or
proteolytic cleavage are thought to be the direct cause of active TGF-β Many key physiological processes, including morphogenesis during
release from the ECM and subsequent signaling (see Figure 16-22). development, control of cellular proliferation and motility, response to
Similar integrin-mediated activation of the LTBP/TGF-β complex injury, and even survival, require not only the production of ECM, but
bound to other ECM proteins provides active TGF-β for regulation of a also its remodeling or degradation. Because of its enormous
wide variety of biological processes. importance as a key element in the extracellular environment of
multicellular organisms, remodeling and degradation of the ECM must
be carefully controlled. Degradation of the ECM is often mediated by
zinc-dependent ECM metalloproteases. Given the wide array of ECM
A variety of diseases, many involving skeletal and cardiovascular
components, it is not surprising that there are many such
abnormalities, are consequences of mutations in the genes encoding
metalloproteases with varying substrate specificities and sites of
the structural proteins of elastic fibers or the proteins that contribute
expression. In many cases, their names incorporate the names of their
to their proper assembly. For example, mutations in the fibrillin-1 gene
substrates, as for the metalloproteases called collagenases, gelatinases,
cause Marfan syndrome, whose varied symptoms can include bone
elastases, and aggrecanases. Some are secreted into the extracellular
overgrowth, loose joints, abnormally long extremities and face, and
fluid, and others are closely associated with the plasma membranes of
cardiovascular defects due to weakness in the walls of the aorta and
cells, either tightly bound in a noncovalent association with the
other blood vessels. There has been considerable speculation that
membrane or as integral membrane proteins. Many are initially
President Abraham Lincoln’s unusually tall, elongated body may have
synthesized as inactive precursors that must be specifically activated to
been a consequence of Marfan syndrome.
function.

In mammals, most tropoelastin synthesis occurs immediately before

ECM metalloproteases are divided into three major subgroups based
and after birth during the late fetal and neonatal periods. Thus most of
on the enzymes’ structures: matrix metalloproteases (MMPs) (of
the body’s elastin needs to be very durable, lasting an entire lifetime.
which there are 23 in humans), a disintegrin and metalloproteinases
The extraordinary stability of elastin has been measured in a variety of
(ADAMs), and ADAMs with thrombospondin motifs (ADAMTSs). These
ways. Pulse-chase experiments (see Chapter 3) using radioactive amino
proteases can degrade ECM components as well as non-ECM
acid administration can be used to measure the life span of elastin in
components such as adhesion receptors. Indeed, a key function of
animals. In humans, two other methods employed to study the
ADAMs is cleaving extracellular domains from integral membrane
longevity of elastin have revealed that the mean lifetime of an elastin
proteins. In Chapter 16 we learned how ADAMs play key roles in
molecule in human lungs is about 70 years! The first method takes
signaling by Notch and EGF receptors. One mechanism used to control
advantage of a naturally occurring phenomenon: the slow, natural rate
the activities of these proteases is the production of protein inhibitors
of conversion of L-aspartic acid — incorporated into proteins during
called TIMPs (tissue inhibitors of metalloproteinases) and RECK
their synthesis — to D-aspartic acid. Thus the age of a long-lived protein
(reversion-inducing–cysteine-rich protein with kazal motifs). Some of
can be estimated using chemical analysis to determine what fraction of
these inhibitors have their own cell-surface receptors and functions
its L-aspartic acid has been converted over time to the D isomer,
independent of their ability to inhibit metalloproteinases. ECM-
together with knowledge of the age of the tissue from which it was
degrading proteases are associated with a variety of diseases, the best
isolated. The second method is a variation on the classic pulse-chase
known of which is metastatic (spreading) cancer (see Chapter 25).
experiments used in the laboratory. As a consequence of nuclear

KEY CONCEPTS OF SECTION 20.4

The Extracellular Matrix II: Connective Tissue

 Connective tissue, such as tendon and cartilage, differs from other solid tissues in that most of its volume is made up of extracellular matrix
(ECM) rather than cells (see Figure 20-4).
 The synthesis of fibrillar collagen (e.g., types I, II, and III) begins inside the cell with the chemical modification of newly made α chains and
their assembly into triple-helical procollagen within the endoplasmic reticulum. After secretion, procollagen molecules are cleaved,
associate laterally, and are covalently cross-linked into bundles called fibrils, which can form larger assemblies called fibers (see Figure 20-
28).
 The various collagens are distinguished by the ability of their helical and nonhelical regions to associate into fibrils, to form sheets, or to
cross-link other collagen types (see Table 20-5).
 Proteoglycans consist of membrane-associated or secreted core proteins covalently linked to one or more glycosaminoglycan (GAG) chains,
which are linear polymers of disaccharides that are often modified by sulfation.
 Cell-surface proteoglycans such as the syndecans facilitate cell-matrix interactions and help present certain external signaling molecules to
their cell-surface receptors.
 Hyaluronan, a highly hydrated GAG, is a major component of the ECM of migrating and proliferating cells. Certain adhesion receptors bind
hyaluronan to cells.
 Large proteoglycan aggregates containing a central hyaluronan molecule noncovalently bound to the core proteins of proteoglycan
molecules (e.g., aggrecan) contribute to the ability of the matrix to resist compression forces (see Figure 20-33).
 Fibronectins are abundant multi-adhesive matrix proteins that play a key role in migration and cellular differentiation. They contain binding
sites for integrins and ECM components (collagens, proteoglycans) and thus can attach cells to the ECM (see Figure 20-34).
 The tripeptide RGD motif Arg-Gly-Asp, found in fibronectins and some other matrix proteins, is recognized by several integrins.
 Elastic fibers permit repeated stretching and recoiling of tissues because of their highly elastic core of cross-linked, amorphous elastin,
which is surrounded by a network of microfibrils that help assemble the fibers and regulate signaling mediated by TGF-β.
 The remodeling or degradation of ECM is mediated by a large number of secreted and cell-membrane-associated zinc metalloproteases
that fall into several families (MMPs, ADAMs, ADAMTSs) and whose activities are regulated by protein inhibitors (TIMPs and RECK).
20.5 Adhesive Interactions in Motile and Nonmotile Cells
After adhesive interactions in epithelia form during differentiation,
they are often very stable and can last throughout the life span of the
cells or until the epithelium undergoes further differentiation.
Although such long-lasting nonmotile adhesion also exists in
nonepithelial tissues, some nonepithelial cells must be able to crawl
across or through a layer of ECM or other cells. Moreover, during
development or wound healing and in certain pathological states (e.g.,
cancer), epithelial cells can transform into more motile cells (the
epithelial-to-mesenchymal transition). Changes in expression of
adhesion molecules play a key role in this transformation, as they do in
other biological processes involving cell movement, such as the
crawling of white blood cells into tissue sites of infection. In this
section, we describe various cell-surface structures that mediate
transient adhesive interactions that are especially adapted for the
movement of cells as well as those that mediate long-lasting adhesion.
The intracellular mechanisms used to generate the mechanical forces
that propel cells and modify their shapes are covered in Chapters 17
and 18.
Integrin-containing adhesive structures are dynamic due to ongoing
import, export, or covalent modification of their components, and each
contains dozens of intracellular adapter and associated proteins. These
adhesive structures may in some cases assemble due to the formation
of phase-separated biomolecular condensates (see Chapter 3) in which
each component has the potential of binding directly to many other
components. The hundreds of such proteins identified to date have the
potential to engage in many hundreds of distinct protein-protein
interactions that may be subject to regulation. For example, binding
sites generated by phosphorylation of integrin and its associated
proteins, as well as by generation of phosphorylated derivatives of
phosphatidylinositol in the adjacent membrane, recruit additional
proteins into and can also cause release of some proteins from these
multiprotein complexes. A tightly controlled choreography of internal
signals, contributions of other signaling pathways such as those
Integrins Mediate Adhesion and Relay Signals Between Cells and Their
Three-Dimensional Environment
As already discussed, integrins can connect epithelial cells to the basal
lamina and, through adapter proteins, to intermediate filaments of the
cytoskeleton (see Figure 20-1). In both nonepithelial and epithelial
cells, integrins form a bridge between the ECM and the cytoskeleton.
In epithelial and nonepithelial cells, integrins in the plasma membrane
are also clustered with other molecules in various focal contacts (focal
adhesions) and focal contact–like adhesive structures called focal
complexes, 3-D adhesions, and fibrillar adhesions, as well as in circular
adhesions called podosomes. These structures are multiprotein
complexes that mediate (1) cell adhesion to the ECM — for example, via
integrin binding to fibronectin (see Figure 20-36), collagen or laminin,
(2) integrin association with the actin cytoskeleton, (3) adhesion-
dependent outside-in and inside-out signaling (see Figure 20-8), and (4)
mechanosensory coupling between cells and their environments.
These complexes are readily observed by fluorescence microscopy
with the use of antibodies that recognize integrins or other molecules
clustered with them (Figure 20-38).
EXPERIMENTAL FIGURE 20-38 Integrins cluster into adhesive structures with
various morphologies in nonepithelial cells. Immunofluorescence methods
were used to detect integrin-containing adhesive structures (green) on
cultured cells: (a) focal adhesions and (b) 3-D adhesions on the surfaces of
human fibroblasts. Cells were grown (a) directly on the flat surface of a
culture dish or (b) on a three-dimensional matrix of ECM components. The
shape, distribution, and composition of the integrin-based adhesions
formed by cells vary depending on the cells’ environment.
[Part (a) republished with permission from Nature, from B. Geiger et al.,
2001, “Transmembrane Crosstalk Between the Extracellular Matrix and the
Cytoskeleton,” Nat. Rev. Mol. Cell Biol. 2(11):793–805; permission conveyed
through Copyright Clearance Center, Inc. Part (b) Kenneth Yamada and Edna
Cukierman.]
The micrograph labeled (a) is titled focal adhesion. The cell is fluorescing at
points where integrins are expressed in bright green spots that look like a
butterfly pattern. Points of fluorescence are visible at points along the cell
membrane where the cell is in contact with a surface. The micrograph
labeled (b) is titled three-dimensional adhesion and shows green
fluorescence in three elongated clumps leaning from top left to bottom
right. Fluorescence image shows focal adhesion over the surface of the cells.
involving receptor tyrosine kinases (see Figure 20-8), and external
signals (such as the composition and rigidity of the ECM) regulates
these complexes. Together, they help define the precise composition
and activity of the integrin multiprotein complex and the consequent
influence that it has on cellular structure and activity (outside-in effect)
as well as the influence of the cellular actin cytoskeleton on the ECM
(inside-out effect). The adhesive structures can be stable and
substantially contribute to the architecture of a tissue. They can also be
transient. For example, the dynamic formation and dissolution of
adhesive structures can result in cells grabbing onto the ECM for
traction, exerting force to move relative to the adhesion site, and then
disassembling to release the cell, permitting net movement as cells
crawl through adjacent tissue.
Although found in many nonepithelial cells, integrin-containing
adhesive structures have been studied most frequently in fibroblasts
grown in cell culture on flat glass or plastic surfaces called substrata.
These conditions only poorly approximate the three-dimensional ECM
environment that normally surrounds such cells in vivo. When
fibroblasts are cultured in three-dimensional ECMs derived from cells
or tissues, they form adhesions to the three-dimensional ECM
substratum, called 3-D adhesions. These structures differ somewhat in
composition, shape, distribution, and activity from the focal or fibrillar
adhesions seen in cells growing on the flat substrata typically used in
cell culture experiments (see Figure 20-38). Cultured fibroblasts in
three-dimensional ECMs have anchoring junctions that display greater
adhesion and mobility, increased rates of cell proliferation, and
spindle-shaped morphologies more like those of fibroblasts in tissues
than do cells cultured on hard, flat surfaces. These and other
observations indicate that the topological, compositional, and
mechanical properties of the ECM all play a role in controlling the
shape and activity of a cell, due at least in part to integrin-mediated
bridging of the ECM to intracellular molecules. Tissue-specific
differences in these ECM characteristics probably contribute to the
tissue-specific properties of cells.
The importance of the three-dimensional environment of cells has
been highlighted by cell-culture studies of the morphogenesis,
functioning, and stability of specialized milk-producing mammary
epithelial cells and their cancerous transformed counterparts. For
example, the three-dimensional ECM-dependent outside-in signaling
mediated by integrins influences the epidermal growth factor–tyrosine
kinase receptor signaling system, and vice versa. The three-
dimensional ECM also permits the mammary epithelial cells to
generate in vivo–like circular epithelial structures, called acini, which
secrete the major protein constituents of milk. The use of such three-
dimensional ECM cell culture systems permits more realistic
comparisons of the responses of normal and cancer cells to potential
chemotherapeutic agents. Analogous systems employing both natural
and synthetic three-dimensional ECMs are being developed to provide
more in vivo–like conditions to study other complex tissues and
organs, such as the liver. In the presence of the appropriate ECM,
many types of 3-D, organ-like tissues called organoids, similar to those
in intact organisms, can now be generated from self-organizing,
pluripotent or adult stem cells (see Chapters 4 and 22).
Regulation of Integrin-Mediated Adhesion and Signaling Controls Cell
Function and Movement
Cells can exquisitely control the strength of integrin-mediated cell-
matrix interactions by regulating integrin’s expression levels, ligand-
binding activities, or both. Such regulation is critical to the role of
these interactions in cell migration and other functions involving cell
movement.
Integrin Binding
Integrin binding to ECM and neighboring cells can influence the
developmental fate of cells, determining the type of cell into which a
progenitor cell will differentiate (see Chapter 22). Integrins participate
in cells sensing the mechanical characteristics of their environments,
such as the rigidity of the ECM, and responding appropriately.
Most integrins can exist in at least three conformations. In the first,
inactive state (bent closed conformation, Figure 20-39a, left), the closely
apposed extracellular domains of the αβ heterodimer are bent over,
placing the ligand-binding site — located at the interface of the
propeller (α chain) and βA (β chain) domains — near the plasma
membrane and distant from the ECM. In the bent closed conformation,
there is only low-affinity ligand binding and the transmembrane
domains and cytoplasmic C-terminal tails of the two subunits are
closely bound together. In the second, partially active state, the
transmembrane and cytoplasmic domains of the α and β chains at least
partially separate and the extracellular domains partially straighten
into an extended closed conformation in which the ligand-binding site is
projected farther away from the cell surface and assumes an
intermediate affinity for ligands, such as a variety of ECM proteins
(Figure 20-39a, center). In the third, fully active, extended open
conformation, tension on the protein fully extends the extracellular
domain of the β chain and confers high affinity ligand binding that can
be as much as 4000-fold tighter than binding to the bent closed
conformation (Figure 20-39a, right). The changes in the conformations
of integrin’s cytoplasmic tails can influence the binding of intracellular
adaptor proteins. For example, two of integrin’s cytoplasmic adaptor
proteins, talin and kindlin, can bind to short sequence motifs in the
cytoplasmic tail of the β chain when it is separated from the tail of the
α chain in the extended closed and open forms, but not when these
sites are blocked in the bent closed form (see Figure 20-39a).
EXPERIMENTAL FIGURE 20-39 The three conformations of integrins. (a) Most integrins can be in one of three interconvertible conformations: inactive, low
affinity for extracellular ligands (bent closed, left); partially active, intermediate affinity (extended closed, center); and fully active, high affinity (extended
open, right). Tension across the integrin due to pulling by either its extracellular ligand or intracellular binding partners drives the integrin into the extended
open conformation. The extracellular ligand binding site is at the interface of the propeller domain on the α chain and the βA domain on the β chain. The
cytoplasmic domain of the β chain has binding sites for intracellular adaptor proteins, e.g., talin and kindlin (green and yellow ovals, respectively). These
sites are inaccessible (cryptic) in the bent closed form, but accessible in the extended forms due to separation of the transmembrane and cytoplasmic
domains of the α and β chains (indicated by double-headed arrow). (b) Single inactive (bent closed) integrin αIIbβ3 molecules ( left) were incorporated into
phospholipid nanodiscs (small bilayers in which the extracellular and cytoplasmic domains of the integrin are exposed to a buffer), and the integrin-binding
and activating head domain of the adapter protein talin was added to some of these preparations (right). Multiple electron microscopic images of individual
nanodiscs were collected and averaged. Phospholipid nanodiscs are indicated by dashed white circles, and the heights of the integrin extracellular regions
that extend above the nanodiscs are indicated by brackets. Binding of talin’s head domain to the cytoplasmic domain of the β chain traps the integrins in the
extended closed conformation.

[Part (a) Information from J. Wang et al., 2019, Nat. Commun. 10:5481. Part (b) F. Ye et al., 2010, J. Cell Biol. 188(1):157–173, Fig. 7;
https://doi.org/10:1083/jcb.200908045.]

The illustration labeled (a) shows three schematics. The first one, at left, is labeled inactive low affinity (bent closed). There is a cell membrane with a ligand
represented as a blue thin line hanging from it and the integrin is bent down and not connected to the ligand. In the center schematic, the integrin has
straightened upward and the blue side has connected to the membrane while the pink side top looks like an open circle starting to surround the ligand. This
is labeled partially active, intermediate affinity extended closed. The last diagram shows the pink side has closed around the ligand and stretched straight. A
dotted line is labeled tension along that pink line. The label read fully active, high affinity (extended open). The micrographs labeled (b) show the closed
integrin and labeled inactive integrin, bent closed. A dotted line outlines the membrane nanodisc. Another micrograph shows the open integrin connected to
the nanodisc labeled partially active integrin, extended closed.

One helpful way to think about these three forms of integrins is that
there is an equilibrium distribution of integrin molecules in all three
conformations (they can readily interconvert). When the integrin is
bound to neither extracellular ligands (e.g., ECM) nor intracellular
partners (e.g., talin), the large majority of unbound integrin molecules
is in the bent, inactive conformation. Binding of extracellular ligands
to the extended conformations traps more of the integrins in the
extended conformations than in the absence of the ligands (shifts the
equilibrium distribution) and thus facilitates the binding of
intracellular adaptor proteins such as talin or kindlin to the now
accessible sites in the β chain’s cytoplasmic domain. Similarly, binding
of intracellular adaptors to the extended conformations increases the
fraction of integrin molecules in extended conformations (again, a
shift in the equilibrium distribution) and facilitates extracellular ligand
binding to the intermediate and high-affinity ligand-binding sites. Thus
binding of ligands to integrins on either side of the membrane can
increase binding on the other side.
The structural features of integrin’s α and β subunits that permit them
to adopt the bent and extended conformations have been preserved
through metazoan evolution, indicating their importance for integrin
function. Indeed, these three conformations provide an attractive
explanation for the ability of integrins to mediate outside-in and inside-
out signaling. The binding to the integrin’s extracellular ligand-binding
site of certain ECM molecules or CAMs on other cells would shift the
distribution of conformations and thus result in more integrins in the
extended closed or extended open forms with at least partially
separated cytoplasmic tails. Intracellular adapter proteins could
recognize the separation of the tails and, as a result, bind to the tails.
Integrin-binding-dependent changes in these adapters can then alter
the cytoskeleton and activate or inhibit intracellular signaling
pathways. Conversely, changes in the metabolic or signaling state of
the cells could cause intracellular adapters to bind to or dissociate from
the cytoplasmic tails of the integrins and thus alter the distribution of
integrin conformations. Such binding can force the tails either to
separate or to associate (see Figure 20-39a). As a consequence, a
greater fraction of the integrins would be either in the bent closed
(inactive), extended closed (partially active), or extended open (fully
active) conformations, thereby altering their interactions with the ECM
or with other cells. Indeed, in vitro EM studies of purified integrins
reconstituted individually into lipid bilayer nanodiscs show that
binding of the head domain of the adapter/mechanosensor protein
talin (described in detail below; see Figure 20-40) to the cytoplasmic tail
of integrin’s β chain is sufficient to partially activate integrin, inducing
a straightening of the bent closed conformation into the extended
closed form (see Figure 20-39b).
We can see an example of integrins mediating intracellular-
extracellular communication in the initial formation of focal adhesions
in Figure 20-40. Although there are many intracellular proteins
involved, this figure focuses on talin, actin, and vinculin. Figure 20-40a
shows a model of an active conformation of talin and Figures 20-40b
and c show some of the key steps associated with integrin/talin-
mediated inside-out signaling. In the absence of stimulation, most of
the talin in the cytoplasm is condensed into an inactive, autoinhibited,
compact structure in which interdomain binding sterically blocks talin
binding to some of its many partners, including integrins and actin (see
Figure 20-40b, lower left).
FIGURE 20-40 Talin- and integrin-mediated intracellular-extracellular communication. (a) Top: A hypothetical model of an elongated, active conformation of
a talin monomer based on structures of fragments of the intact protein with secondary structures illustrated as tubes (α helices) and ribbons with
arrowheads (β sheets). The N-terminal “head” domain (four globular subdomains) is connected by an apparently unstructured domain to a rod domain that
comprises 13 α-helical bundles (4 or 5 helices per bundle), 9 of which contain either 1 or 2 helices (red, total of 11) that are cryptic binding sites for the protein
vinculin. Some bundles also contain cryptic actin-binding sites. These helices can only bind vinculin when exposed by unfolding (see Figure 20-9b). There is a
single α helix at the extreme C-terminus that is a dimerization domain. Bottom: A cartoon model. Head subdomains = gray ovals; rod domain bundles =
colored capsules; and dimerization domain = narrow rectangle. (b) Intracellular signaling induces the translocation of inactive talin in the cytoplasm to the
plasma membrane and its unfolding/activation (steps 1 and 2). The binding of activated talin to integrin’s β chain cytoplasmic domain stabilizes integrin’s
extended closed conformation, promoting binding to the ECM (step 3). The unfurled talin homodimerizes and binds several proteins, including actin
filaments that bind via talin’s C-terminal dimerization domain (shown) and several other sites (not shown) (step 4). The actin filaments exert force (step 5),
generating tension across the ECM/integrin/talin complex. This tension converts the integrin into the fully active, extended open form (step 6). (c)
Subsequent steps illustrated for the region outlined in the light gray dashed box in part (b) are shown. Additional force unfolds one or more of talin’s helical
bundles (step 7), exposing cryptic binding site(s) (red helix) that recruit vinculin and other proteins, including more actin (steps 8 and 9), resulting in growth
of the focal adhesion.

[Part (a) left information from B. T. Goult, J. Yan, and M. A. Schwartz, 2018, J. Cell Biol. 217(11):3776–3784; and M. Yao et al., 2014, Sci. Rep. 4:4610. Parts (b)
and (c) information from J. Wang et al., 2019, Nat. Commun. 10:5481; D. Dedden et al., 2019, Cell 179(1):120–131; S. Chakraborty et al., 2019, Biochemistry
58(47):4677–4695; Y. A. Kadry and D. A. Calderwood, 2020, Biochim. Biophys. Acta, Biomembr. 1862(5):183206.]

The illustration labeled (a) shows a horizontal three-dimensional ribbon model of talin. At left, a gray group of ribbons labeled talin (extended conformation),
connected to a center set of red and white ribbons labeled alpha-helical bundles, which are in turn connected to bundles labeled cryptic vinculin binding
alpha helices (red). Below this is the same structure drawn in schematic and labeled, from left to right, talin "head" (F E R M domain), unstructured (an area of
thin gray lines), rod domain (13 alpha-helical bundles, and dimerization. The illustration labeled (b) shows a schematic that starts with membranes as gray
bars with ligands, and an inactive integrin, with an inactive talin looking like a tangled web of rectangular beads. Next, to the right, the integrin is extended
and attaches to the partially active integrin. Then the pink part of the integrin separates from the blue part and dimerization is labeled at the other end of the
integrin. Next, the integrin is fully active, and now two parts are with the talin, each one stretched down with tension to meet together at an actin protein
represented as red circles. In the illustration, labeled (c) the helical part of the talin bends and reveals vinculin-binding areas represented as light blue ovals,
which now attach to the actin.

Intracellular signaling can induce the translocation of talin to the
inner leaflet of the plasma membrane, for example, by generating
polyphosphoinositides to which globular subdomains in talin’s head
domain can bind and by small GTP-binding proteins (Figure 20-40b,
steps 1 and 2). In steps 2 and 3, the compact autoinhibited rod domain
unfolds into the active conformation (see Figure 20-40a) and a portion
of the head domain binds to the cytoplasmic domain of integrin’s β
chain. Talin binds to integrin’s β chain cytoplasmic domain when the
transmembrane and cytoplasmic domains of integrin’s α and β chains
at least partly separate in the extended closed conformation. In this
conformation integrin binds to the ECM with intermediate affinity (see
Figure 20-39). The order of these events is not well established. The C-
terminal dimerization domain in the unfurled, extended, and active
talin mediates homodimerization, and actin filaments can bind to
several sites in the rod domains in the dimeric talin, including the C-
terminal dimerization domain (step 4, also see Figure 20-40a). Other
binding partners can also bind to the unfurled, active talin. Additional
signaling steps mediated by the partially active, extended closed
integrin (e.g., activation of kinases, such as focal adhesion kinase and
scr-like kinases) induce formin-mediated actin filament formation and
myosin engagement with actin filaments to exert force on the talin
(step 5, also see Chapter 17). As a consequence, tension develops in the
ECM-bound integrin/talin complex, converting the integrin into the
fully active, extended open form (step 6).
mediated signaling, such as recruitment of branched actin networks in
lamellipodia (see Chapter 17).
Platelet function, discussed in more detail below, provides a good
example of how cell-matrix interactions are modulated by control of
integrin-binding activity. Platelets are cell fragments that circulate in
the blood and clump together with ECM molecules to form a blood clot.
In its basal state, the αIIbβ3 integrin present on the plasma membranes
of platelets cannot bind tightly to its protein ligands (including
fibrinogen and fibronectin), all of which participate in the formation of
a blood clot, because it is in the inactive (bent closed) conformation.
During clot formation, platelets are activated by binding to ECM
proteins such as collagen and a large protein called von Willabrand
factor that, through binding to receptors, generate intracellular signals.
Platelets may also be activated by ADP or the clotting enzyme
thrombin. These signals induce changes in signaling pathways within
the platelet that result in an activating conformational change in the
platelet’s αIIbβ3 integrin. As a consequence, this integrin can bind
tightly to extracellular clotting proteins and participate in clot
formation. People with genetic defects in the β3 integrin subunit are
prone to excessive bleeding, attesting to the role of the αIIbβ3 integrin
in the formation of blood clots (see Table 20-4).

Additional force applied to the integrin/talin complex, exerted either

from the cytoplasm (e.g., actin filaments and myosin) or the ECM, can
pull apart the α-helical bundles in talin (Figure 20-40c, step 7), exposing
cryptic vinculin binding sites (red helix). The greater the force, the
more α-helical bundles in talin are opened and the more vinculin,
actin, and other proteins are recruited to the integrin/talin complex.
Vinculin binding to exposed sites on talin can then recruit other
proteins, such as actin filaments (steps 8 and 9). The unfolded bundles
can readily refold when the force is reduced. Strikingly, the more rigid
the structure of the ECM, the greater the tension across the
integrin/talin complex and the larger and stronger the adhesion.
During steps 3–9, integrins begin to cluster and the focal adhesion can
continue to grow and form a mature focal adhesion that firmly attaches
the cell to the ECM. Alternatively, the multiprotein adhesion complex
can dissociate with inactivation of the integrins and other components,
in part due to recruitment of inhibitory proteins.
Studies suggest that the efficient activation of integrins in intact cells
may also require the participation of another class of adapter proteins
called kindlins, which bind to a distinct site on the cytoplasmic tail of
integrin’s β chain (see Figure 20-39a). Kindlin plays a key role in the
integrin- and microfibril-mediated activation of TGF-β (inside-out
signaling involving elastic fibers and their microfibril-associated
protein LTBP, described earlier) and other pathways of integrin-
Integrin Expression
The attachment of cells to ECM components can also be modulated by
altering the number of integrin molecules exposed on the cell surface.
The α4β1 integrin, which is found on many hematopoietic cells, offers
an example of this regulatory mechanism. For these hematopoietic
cells to proliferate and differentiate, they must be attached to
fibronectin synthesized by supportive (stromal) cells in the bone
marrow. The α4β1 integrin on hematopoietic cells binds to a Glu-Ile-
Leu-Asp-Val (EILDV) sequence in fibronectin in the ECM, thereby
anchoring the cells to the matrix. This integrin also binds to a sequence
in a CAM called vascular CAM-1 (VCAM-1), which is present on stromal
cells of the bone marrow. Thus hematopoietic cells directly contact the
stromal cells as well as the ECM. Late in their differentiation,
hematopoietic cells decrease their expression of this integrin; the
resulting reduction in the number of α4β1 integrin molecules on the
cell surface is thought to allow mature blood cells to detach from the
ECM and stromal cells in the bone marrow and enter the circulation.
Connections Between the ECM and Cytoskeleton Are Defective in Muscular
Dystrophy

The importance of the adhesion receptor–mediated linkage
between ECM components and the cytoskeleton is highlighted by a set
of hereditary muscle-wasting diseases, collectively called muscular
dystrophies. Duchenne muscular dystrophy (DMD), the most common
type, is a sex-linked disorder, affecting 1 in 3300 boys, that results in
cardiac or respiratory failure, usually in the late teens or early
twenties. The first clue to understanding the molecular basis of this
disease came from the discovery that people with DMD carry
mutations in the gene encoding a protein named dystrophin. This very
FIGURE 20-41 The dystrophin glycoprotein complex (DGC) in skeletal muscle
cells. This schematic model shows that the DGC comprises three
Dystroglycan is a heterodimeric (α and β subunits) cell-surface
glycoprotein (see Figure 20-41). As noted in Section 20.4, about three of
the more than 20 O-linked oligosaccharide chains in dystroglycan’s α
subunit are directly linked to the hydroxyl group via a mannose sugar
that is phosphorylated (see Figure 20-31c). A phosphorylated
hexasaccharide connects the phosphorylated mannose to a GAG-like
polymer of xylose–glucuronic acid disaccharides called matriglycan.
One key enzyme in the synthesis of the matriglycan oligosaccharide
that is located in the Golgi complex is called LARGE.
The O-linked matriglycan binds to various components of the basal
lamina, including the multi-adhesive matrix protein laminin (see
Figures 20-25 and 20-41) and the proteoglycans perlecan and agrin. The
neurexins, a family of cell-surface adhesion molecules expressed by
neurons, also bind matriglycan. All of these bind to matriglycan via LG
domains (see Figure 20-41).
Dystroglycan’s transmembrane β subunit associates with a group of
integral membrane proteins called the sarcoglycan complex and
sarcospan, while its cytosolic domain binds directly to dystrophin and
directly or indirectly to other adapter and signaling proteins (see
Figure 20-41). Sarcospan is a tetraspanin, as are claudin and occludin
in tight junctions (see Figure 20-18b). The resulting large, heteromeric
assemblage, the dystrophin glycoprotein complex (DGC), links the ECM to
the actin cytoskeleton. Binding to laminin in the basal lamina leads to a
large protein was found to be a cytosolic adapter protein that contains
a long central rodlike region with 24 repeats of a three-α-helix bundle,
flanked by an N-terminal F-actin-binding domain and C-terminal
domains that bind to several plasma membrane and cytoplasmic
proteins (Figure 20-41). Several of the central repeats also bind to F-
actin and dystrophin links the actin filaments to an adhesion receptor
called dystroglycan (see Figure 20-41). It is noteworthy that when
dystrophin is subjected to tension some of its α-helical repeats unfold
and the protein substantially lengthens, a feature somewhat
reminiscent of the force-induced unfolding of talin (see Figure 20-40).
However, a role for dystrophin in mechanotransduction has not been
established.
subcomplexes: the α, β-Dystroglycan subcomplex; the integral membrane
protein sarcospan and the sarcoglycan complex; and the cytosolic adapter
subcomplex comprising dystrophin, other adapter proteins, and signaling
molecules. Through its O-linked matriglycan sugars (see Figure 20-31c), α-
dystroglycan binds to LG-domain-containing components of the basal
lamina, such as laminin and the GAG-containing proteoglycans perlecan and
agrin. Dystrophin — the protein that is defective in DMD — links β-
dystroglycan to the actin cytoskeleton, and α-dystrobrevin links dystrophin
to the sarcoglycan complex and sarcospan. Neuronal nitric oxide synthase
(nNOS), which produces the gaseous signaling molecule nitric oxide, binds
to dystrophin via the adaptors called syntrophins. See S. J. Winder, 2001,
Trends Biochem. Sci. 26:118–124; D. E. Michele and K. P. Campbell, 2003, J.
Biol. Chem. 278(18):15457–15460; T. Yoshida-Moriguchi and K. P. Campbell,
2015, Glycobiology 25(7):702–713; and Y. M. Kobayashi and K. P. Campbell,
2012, Chapter 66, “Skeletal Muscle Dystrophin-Glycoprotein Complex and
Muscular Dystrophy,” in J. A. Hill and E. N. Olson, eds., Muscle, vol. 2,
Elsevier/Academic Press.
The illustration shows agrin, laminin, and perlecan in the basal lamina.
These can interact with the O-linked sugars of the alpha-beta-dystroglycan
complex. This complex is attached to the sarcoglycan complex in the cell
membrane, which is also connected to dystrophin and various other
cytosolic proteins such as syntrophins, nitric oxide synthase, and alpha
dystrobrevin. Dystrophin is connected to actin.
cluster of DGCs on the cell surface. The DGC is associated with
signaling pathways within muscle and other types of cells. For
instance, the signaling enzyme neuronal nitric oxide synthase (nNOS)
is associated through syntrophin with the DGC in skeletal muscle. The
rise in intracellular Ca2+ during muscle contraction activates
nNOS to produce nitric oxide (NO), a signaling molecule that diffuses
into smooth muscle cells surrounding nearby blood vessels. NO
promotes smooth muscle relaxation, leading to a local rise in the flow
of blood supplying nutrients and oxygen to the active skeletal muscle.
Heart (cardiac) muscle contraction may be influenced by similar NOS-
syntrophin interactions.
Mutations in the genes encoding dystrophin, other DGC components,
laminin, or the multiple enzymes that mediate the addition of
matriglycan to dystroglycan can all disrupt the DGC-mediated link
between the exterior and the interior of muscle cells and cause
muscular dystrophies. In addition, dystroglycan mutations have been
shown to greatly reduce the clustering of acetylcholine receptors on
muscle cells at the neuromuscular junctions, which is also dependent
on the basal lamina proteins laminin and agrin. These and possibly
other effects of DGC defects apparently lead to a cumulative loss of the
mechanical stability of muscle cells as they undergo contraction and
relaxation, resulting in deterioration of the cells and muscular
dystrophy.
Dystroglycan is an elegant and medically relevant example of the
intricate networks of connectivity in cell biology. While originally
discovered in the context of studying muscular dystrophy, it was later
shown to be expressed in nonmuscle cells and, through its binding to
laminin, to play a key role in the assembly and stability of at least some
basement membranes. The extent of glycosylation of dystroglycan,
such as the length of the matriglycan repeats, varies in different
tissues, suggesting that alterations in its glycosylation regulate
function. Dystroglycan is essential for normal development, including
the proper functioning of some neuronal synapses (see Chapter 23).
Additional studies led to its identification as a cell-surface receptor for
the virus that causes the frequently fatal human disease Lassa fever
and other related viruses, all of which bind via matriglycan.
Furthermore, dystroglycan is the receptor on specialized cells in the
nervous system — Schwann cells — to which binds the pathogenic
bacterium Mycobacterium leprae, the organism that causes leprosy.
IgCAMs Mediate Cell-Cell Adhesion in Neural and Other Tissues
Numerous transmembrane proteins characterized by the presence of
multiple immunoglobulin domains in their extracellular regions
constitute the immunoglobulin (Ig) superfamily of CAMs, or IgCAMs
(e.g., see NCAM in Figure 20-2). The Ig domain is a common protein
domain, containing 70–110 residues. It was first identified in
antibodies, the antigen-binding immunoglobulins (see Chapter 24), but
has a much older evolutionary origin in CAMs. The human, Drosophila,
and C. elegans genomes include about 765, 150, and 64 genes,
respectively, that encode proteins containing Ig domains.
Immunoglobulin domains are found in a wide variety of cell-surface
proteins, including the T-cell receptors produced by lymphocytes and
many proteins that take part in adhesive interactions. Among the
IgCAMs are neural CAMs; intercellular CAMs (ICAMs), which function
in the movement of leukocytes into tissues; and junction adhesion
molecules (JAMs), which are present in tight junctions (see Figure 20-
18b).
As their name implies, neural CAMs are of particular importance in
neural tissues. One type, the NCAMs, primarily mediate homophilic
interactions. First expressed during morphogenesis, NCAMs play an
important role in the differentiation of muscle cells, glial cells, and
neurons. Their role in cell adhesion has been directly demonstrated by
the inhibition of adhesion with anti-NCAM antibodies. Numerous
NCAM isoforms, encoded by a single gene, are generated by alternative
mRNA splicing and by differences in glycosylation. Other neural CAMs
(e.g., L1-CAM) are encoded by different genes. In humans, mutations
in different parts of the L1-CAM gene cause various neuropathologies
(e.g., mental retardation, congenital hydrocephalus, and spasticity).
An NCAM comprises an extracellular region with five Ig domains and
two fibronectin type III domains, a single membrane-spanning
segment, and a cytosolic segment that interacts with the cytoskeleton
(see Figure 20-2). In contrast, the extracellular region of L1-CAM has
six Ig domains and four fibronectin type III domains. As with
cadherins, cis (on the same cell) interactions and trans (intercellular)
interactions probably play key roles in IgCAM-mediated adhesion (see
Figure 20-3); however, adhesion mediated by IgCAMs is Ca2+
independent.
Leukocyte Movement into Tissues Is Orchestrated by a Precisely Timed
Sequence of Adhesive Interactions
In adult organisms, several types of white blood cells (leukocytes)
participate in defense against infection caused by bacteria and viruses
and respond to tissue damage due to trauma or inflammation. To fight
infection and clear away damaged tissue, these cells must move rapidly
from the blood, where they circulate as unattached, relatively
quiescent cells, into the underlying tissue at sites of infection,
inflammation, or damage. We know a great deal about the movement
into tissue, termed extravasation, of four types of leukocytes:
neutrophils, which release several antibacterial proteins; monocytes,
the precursors of macrophages, which can engulf and destroy foreign
particles; and T and B lymphocytes, the antigen-recognizing cells of the
immune system (see Chapter 24).
Extravasation requires the successive formation and breakage of cell-
cell contacts between leukocytes in the blood and endothelial cells
lining the vessels. Some of these contacts are mediated by selectins, a
family of CAMs that mediate leukocyte–vascular endothelium
interactions. Endothelial cells express P- and E-selectins on their blood-
facing surfaces, activated platelets express P-selectin, and leukocytes
express L-selectin. All selectins contain a Ca2+ -dependent lectin
domain, which is located at the distal end of the extracellular region of
the molecule and recognizes particular sugars in glycoproteins or
glycolipids (see Figure 20-2). For example, the primary ligand for P-
and E-selectins is an oligosaccharide called the sialyl Lewis-x antigen, a
part of longer oligosaccharides present in abundance on leukocyte
glycoproteins and glycolipids.
Figure 20-42 illustrates the basic sequence of cell-cell interactions
leading to the extravasation of leukocytes. Various inflammatory
signals released in areas of infection or inflammation first cause
activation of the vascular endothelium. P-selectin exposed on the
surfaces of activated endothelial cells mediates the weak adhesion of
passing leukocytes. Because of the force of the blood flow and the rapid
“on” and “off” rates of P-selectin binding to its ligands, these bound
leukocytes are slowed, but not stopped, and literally roll along the
surface of the endothelium. Among the signals that promote activation
of the endothelium are chemokines, a group of small, secreted
proteins (8–12 kDa) produced by a wide variety of cells, including
endothelial cells and leukocytes.
FIGURE 20-42 Endothelium-leukocyte interactions: activation, binding, rolling, and extravasation. Step 1: In the absence of inflammation or infection,
leukocytes and endothelial cells lining blood vessels are in a resting state and not interacting. Step 2: Inflammatory signals released only in areas of
inflammation, infection, or both activate resting endothelial cells, resulting in the movement of vesicle-sequestered selectins to the cell surface. The exposed
selectins mediate weak binding of leukocytes by interacting with carbohydrate ligands on leukocytes. Blood flow forces the loosely bound leukocytes to roll
along the endothelial surface of the blood vessel (curved arrow). Activation of the endothelium also causes synthesis of platelet-activating factor (PAF) and
ICAM-1, both expressed on the endothelial cell surface. PAF and other, usually secreted, activators, including chemokines, then induce changes in the shapes
of the leukocytes and activation of leukocyte integrins such as αLβ2, which is expressed by T lymphocytes (step 3). The subsequent tight binding between
activated integrins on leukocytes and CAMs on the endothelium (e.g., ICAM-2 and ICAM-1) results in firm adhesion (step 4) and subsequent movement
(extravasation) into the underlying tissue (step 5). See R. O. Hynes and A. D. Lander, 1992, Cell 68:303.

The steps involved in the process are as follows. Step 1. Leukocyte in resting state (represented as a blue circle with a nucleus). The leukocyte has alpha L
beta 2 integrin on its surface, two yellow selectin ligands, and a P A F receptor. Endothelial cells below it contain I C A M - 2 receptors on the cell surface and
contain vesicles containing P-selectin. Step 2. Endothelial activation by the release of P selectin and leukocyte attachment via the selectin ligand and I C A M -
2. Step 3. Leukocyte activation, by docking of P A F on the endothelial cell with the P A F receptor. Step 4. Firm adhesion via integrin slash I C A M binding.
Step 5. Extravasation, where the leukocyte squeezes between endothelial cells as it moves from the blood into the tissue.

For tight adhesion to occur between activated endothelial cells and
leukocytes, β2-containing integrins on the surfaces of the leukocytes
must be activated
between endothelial cells that are primarily mediated by the CAM VE-
cadherin. There is general agreement that the leukocyte interactions
with endothelial cells mediated by CAMs initiate outside-in signaling in
the endothelial cells that involves phosphorylation, activation of small
GTPases, and an increase in cytosolic calcium concentration. These
signals weaken or disrupt VE-cadherin-mediated inter-endothelial-cell
adherens junctions and increase actin-myosin contraction, which pulls
the endothelial cells apart, thus permitting the paracellular, amoeboid
movement of the leukocyte between adjacent endothelial cells that is
responsible for most extravasation.

indirectly by chemokines or by other local activation signals such as

platelet-activating factor (PAF). Platelet-activating factor is unusual in
that it is a phospholipid rather than a protein; it is exposed on the
surfaces of activated endothelial cells at the same time that P-selectin is
exposed. The binding of PAF or other activators to their G protein–
coupled receptors on leukocytes leads to activation of the leukocyte
integrins (see Figure 20-39). These activated integrins then bind to
distinct IgCAMs on the surfaces of endothelial cells. These IgCAMs
include ICAM-2, which is expressed constitutively, and ICAM-1, whose
synthesis is induced by activation. ICAM-1 does not usually contribute
substantially to leukocyte adhesion to endothelial cells immediately
after activation, but rather participates at later times in cases of
chronic inflammation. The tight adhesion mediated by these Ca2+
-independent integrin-ICAM interactions leads to the cessation of
rolling and to the spreading of leukocytes on the surface of the
endothelium; soon the adhered cells move between adjacent
endothelial cells and into the underlying tissue. The extravasation step
itself (also called transmigration or diapedesis; step 5 in Figure 20-42)
requires the dissociation of otherwise stable adhesive interactions
The selective adhesion of leukocytes to the endothelium near sites of
infection or inflammation thus depends on the sequential appearance
and activation of several different CAMs on the surfaces of the
interacting cells. Different types of leukocytes express different
integrins, though all contain the β2 subunit. Nonetheless, all
leukocytes move into tissues by the general mechanism depicted in
Figure 20-42.
Many of the CAMs used to direct leukocyte adhesion are shared among
different types of leukocytes and target tissues, yet often only a
particular type of leukocyte is directed to a particular tissue. How is
this specificity achieved? A three-step model has been proposed to
account for the cell-type specificity of such leukocyte-endothelium
interactions. First, endothelial activation promotes initial relatively
weak, transient, and reversible binding (e.g., the interaction of
selectins and their carbohydrate ligands). Without additional local
activation signals, the leukocyte will quickly move on. Second, cells in
the immediate vicinity of the site of infection or inflammation release
or express chemical signals such as chemokines and PAFs that activate
only special subsets of the transiently attached leukocytes, depending
on the types of chemokine receptors those leukocytes express. Third,
additional activation-dependent CAMs (e.g., integrins) engage their
binding partners, leading to strong, sustained adhesion. Only if the
proper combination of CAMs, binding partners, and activation signals
are engaged together with the appropriate timing at a specific site will
a given leukocyte adhere strongly. Such combinatorial diversity and
cross talk allows a small set of CAMs to serve diverse functions
throughout the body — a good example of biological efficiency.
Leukocyte-adhesion deficiency is caused by a genetic defect in the
synthesis of the integrin β2 subunit. People with this disorder are
susceptible to repeated bacterial infections because their leukocytes
cannot extravasate properly and thus cannot effectively fight infection
within a tissue.
Some pathogenic viruses have evolved mechanisms to exploit cell-
surface proteins that participate in the normal response to
inflammation. For example, many of the RNA viruses that cause the
common cold (rhinoviruses) bind to and enter cells through ICAM-1,
and chemokine receptors can be important entry sites for human
immunodeficiency virus (HIV), the cause of AIDS. Integrins appear to
participate in the binding or internalization of a wide variety of viruses,
including reoviruses (which cause fever and gastroenteritis, especially
in infants), adenoviruses (which cause conjunctivitis and acute
respiratory disease), and foot-and-mouth disease virus (which causes
fever in cattle and pigs).

KEY CONCEPTS OF SECTION 20.5

Adhesive Interactions in Motile and Nonmotile Cells

 Many cells have integrin-containing clusters of proteins (e.g., focal adhesions, 3-D adhesions, podosomes) that physically and functionally
connect cells to the ECM and facilitate inside-out and outside-in signaling.
 Via interaction with integrins, the three-dimensional structure of the ECM surrounding a cell can profoundly influence the behavior of the
cell.
 Integrins exist in at least three conformations (bent closed, extended closed, extended open) that differ in their affinity for ligands and in
their interactions with cytosolic adapter proteins and extracellular ligands (see Figure 20-39). Switching between these conformations
depends, in part, on the application of mechanical force and allows regulation of integrin activity, which is important for control of cell
adhesion and movements. Talin, one of many cytoplasmic adaptors that interact with integrins, can function as a mechanosensor. Talin can
mediate integrin’s interactions with the cytoskeleton and thus its switching between conformational and functional states.
 Dystroglycan, an adhesion receptor, forms a large complex with dystrophin, other adapter proteins, and signaling molecules (see Figure 20-
41). This complex links the actin cytoskeleton to the surrounding ECM, providing mechanical stability to muscle. Mutations in various
components of this complex cause different types of muscular dystrophy.
 Neural cell-adhesion molecules, which belong to the immunoglobulin (Ig) family of CAMs, mediate Ca2+ - independent cell-cell adhesion
in neural and other tissues.
 The combinatorial and sequential interaction of several types of CAMs (e.g., selectins, integrins, and ICAMs) is critical for the specific
adhesion of different types of leukocytes to endothelial cells in response to local signals induced by infection or inflammation (see Figure
20-42).
 Thus at the cell, tissue, and organ levels, plants are generally less
complex than most animals.
20.6 Plant Tissues

Moreover, unlike animals, plants do not replace or repair old or

We turn now to the assembly of plant cells into tissues. The overall
structural organization of plants is generally simpler than that of
animals. For instance, plants have only four broad types of cells, which
in mature plants form four basic classes of tissue: dermal tissue
interacts with the environment, vascular tissue transports water and
dissolved substances such as sugars and ions, space-filling ground
tissue constitutes the major sites of metabolism, and sporogenous tissue
forms the reproductive organs. Plant tissues are organized into just
four main organ systems: stems have support and transport functions,
roots provide anchorage and absorb and store nutrients, leaves are the
sites of photosynthesis, and flowers enclose the reproductive structures.
damaged cells or tissues; they simply grow new organs. Indeed, the
developmental fate of any given plant cell is primarily based on its
position in the organism rather than on its lineage, whereas both are
important in animals (see Chapter 22). In both plants and animals, a
cell’s direct communication with its neighbors is important. Most
importantly for this chapter, and in contrast with animals, few cells in
plants contact one another directly through molecules incorporated
into their plasma membranes. Instead, plant cells are typically
surrounded by a rigid cell wall that contacts the cell walls of adjacent
cells (Figure 20-43a). Also in contrast with animal cells, a plant cell
rarely changes its position in the organism relative to other cells. These
features of plants and their organization have determined the
distinctive molecular mechanisms by which plant cells are
incorporated into tissues and communicate with one another.

FIGURE 20-43 Structure of the plant cell wall. (a) Overview of the organization of a typical plant cell, in which the organelle-filled cell with its plasma
membrane is surrounded by a well-defined extracellular matrix called the cell wall. (b) Schematic representation of the cell wall of an onion. Cellulose (highly
ordered glucose polymers) and hemicellulose (amorphous polysaccharide) are arranged into at least three layers in a matrix of the polysaccharide pectin and
glycoproteins. The sizes of the polymers and their separations are drawn to scale. To simplify the diagram, most of the hemicellulose cross-links and other
matrix constituents (e.g., glycoproteins such as extensin, complex organic polymers called lignins) are not shown. (c) Quick-freeze deep-etch electron
micrograph of the cell wall of a garden pea in which some of the pectin molecules were removed by chemical treatment. The abundant thicker fibers are
cellulose microfibrils, and the thinner fibers are hemicellulose cross-links (red arrowheads).

[Part (c) republished with permission from Oxford University Press, from T. Fujino et al., 2000, “Characterization of Cross-Links Between Cellulose Microfibrils,
and Their Occurrence During Elongation Growth in Pea Epicotyl,” Plant Cell Physiol. 2000, 41(4):486–494; permission conveyed through Copyright Clearance
Center, Inc.]

The illustration labeled (a) shows a typical plant cell drawing with labels nucleus, vacuole, Golgi, chloroplast, cell wall, and plasmodesmata. The illustration
labeled (b) shows a white arrow from the cell wall area of illustration (a) to the three-dimensional ribbon model. A dotted line cube is filled with cellulose
microfibril as orange ribbons above the plasma membrane. The dotted line is labeled primary wall. Pectin is represented in green lines and hemicellulose is
represented in black lines. The electron micrograph labeled (c) shows a plant cell wall with cellulose and red arrows mark the hemicellulose areas.

The Plant Cell Wall, a Plant’s ECM, Is a Laminate of Cellulose Fibrils in a
Matrix of Polysaccharides and Glycoproteins
The plant cell wall, an extracellular matrix that is mainly composed of
polysaccharides and is about 0.2 μm thick, completely coats the outside
of the plant cell’s plasma membrane. This structure serves some of the
same functions as the ECM produced by animal cells, even though the
two structures are composed of entirely different macromolecules and
have a different organization. About 1000 genes in the plant
Arabidopsis, a small flowering plant (also called “thale cress”; see
Chapters 1 and 7), are devoted to the synthesis and functioning of its
cell wall, including approximately 414 glycosyltransferase genes,
which encode enzymes that transfer specific sugar residues to proteins
or polysaccharides, and more than 316 glycosyl hydrolase genes, which
encode enzymes that degrade sugar containing polymers. Similar to
animal ECMs, the plant cell wall organizes cells into tissues, signals a
plant cell to grow and divide, and controls the shapes of plant organs
(morphogenesis). It is a dynamic structure that plays important roles in
controlling the differentiation of plant cells during embryogenesis and
growth, and it provides a barrier to protect against pathogen infection.
Just as the ECM helps define the shapes of animal cells, the cell wall
defines the shapes of plant cells. When the cell wall is digested away
from plant cells by hydrolytic enzymes, spherical cells enclosed by a
plasma membrane are left.
Because a major function of the plant cell wall is to withstand the
turgor pressure of the cell (between 14.5 and 435 pounds per square
inch; as much as three orders of magnitude greater than in animal cells
and about 13 times greater than in an automobile tire; see Chapter 11),
the cell wall is built for lateral strength. It is arranged into layers of
cellulose microfibrils: bundles of 18–36 parallel chains of extensively
hydrogen-bonded, long (as much as 4 μm or greater), linear polymers
of glucose in β glycosidic linkages. The cellulose microfibrils are
embedded in a matrix composed of pectin, a negatively charged
polymer of D-galacturonic acid and other monosaccharides, and
hemicellulose, a short, highly branched polymer of several five- and six-
carbon monosaccharides. The mechanical strength of the cell wall
depends on cross-linking of the microfibrils by hemicellulose chains
(Figure 20-43b, c). The layers of microfibrils prevent the cell wall from
stretching laterally. Cellulose microfibrils are synthesized on the
exoplasmic face of the plasma membrane from UDP-glucose and ADP-
glucose formed in the cytosol. The polymerizing enzyme, called
cellulose synthase, moves within the plane of the plasma membrane
along tracks of intracellular microtubules as cellulose is formed,
providing a distinctive mechanism for intracellular-extracellular
communication and ensuring that the cellulose microfibrils are
oriented properly to permit cell wall, and thus whole-cell, growth.
Unlike cellulose, pectin and hemicellulose are synthesized in the Golgi
complex and transported to the cell surface, where they form an
interlinked network that helps bind the walls of adjacent cells to one
another and cushions them. When purified, pectin binds water and
forms a gel in the presence of Ca2+ and borate ions — hence the
use of pectins in many processed foods. As much as 15 percent of the
cell wall may be composed of extensin, a glycoprotein that contains
abundant hydroxyproline and serine. Most of the hydroxyproline
residues are linked to short chains of arabinose (a five-carbon
monosaccharide), and the serine residues are linked to galactose.
Carbohydrate accounts for about 65 percent of extensin by weight, and
its protein backbone forms an extended rodlike helix with the hydroxyl
or O-linked carbohydrates protruding outward. Lignin — a complex,
insoluble polymer of phenolic residues — associates with cellulose and
is a strengthening material. Like cartilage proteoglycans, lignin resists
compression forces.
The cell wall is a selective filter whose permeability is controlled
largely by pectins. Whereas water and ions diffuse freely across and
through cell walls, the diffusion of large molecules, including proteins
larger than 20 kDa, is limited. This limitation may explain why many
plant hormones are small, water-soluble molecules, which can diffuse
across the cell wall and interact with receptors in the plasma
membrane of plant cells.
Ç
Loosening of the Cell Wall Permits Plant Cell Growth
Because the cell wall surrounding a plant cell prevents it from
expanding, the wall’s structure must be loosened when the cell grows.
The amount, type, and direction of plant-cell growth are regulated by
small-molecule hormones called auxins. The auxin-induced weakening
of the cell wall permits the expansion of the intracellular vacuole (see
Figure 20-42a) by uptake of water, leading to elongation of the cell. We
can grasp the magnitude of this phenomenon by considering that, if all
cells in a redwood tree were reduced to the size of a typical liver cell,
the tree would have a maximum height of only 1 meter, about a
hundredfold less than normal.
The cell wall undergoes its greatest changes at the meristem in a root or
shoot tip. Meristems are where cells divide and grow, as described in
Chapter 22. Young meristematic cells are connected by thin, flexible
primary cell walls, which can be loosened and stretched to allow
subsequent cell elongation. Their major components are cellulose,
pectin, and polysaccharides called xyloglucans. After cell elongation
ceases, the cell wall is generally thickened, either by the secretion of
additional macromolecules into the primary wall or, more usually, by
the formation of a secondary cell wall composed of several layers.
Cellulose, lignin, and polysaccharides called xylans and glucomannans
are the major components in secondary cell walls. In mature tissues
such as the xylem — the tubes that conduct salts and water from the
roots through the stems to the leaves — most of the cell eventually
degenerates, leaving only the cell wall. The unique properties of wood
and of plant fibers such as cotton are due to the molecular properties
of the cell walls in the tissues of origin.
Plasmodesmata Directly Connect the Cytosols of Adjacent Cells
The presence of a cell wall separating cells in plants imposes barriers
to cell-cell communication not faced by animals. One distinctive
mechanism used by plant cells to communicate directly is specialized
cell junctions called plasmodesmata, which are large tubes that extend
through the cell wall (Figure 20-44) and were discovered in 1885. Like
gap junctions, plasmodesmata can function as channels that directly
connect the cytosol of a cell to that of an adjacent cell without a
membrane barrier. Plasmodesmata appear to play an especially
important role in protection from pathogens and in regulating the
development of plant cells and tissues, as is suggested by their ability
to mediate intracellular movement of transcription factors and
ribonuclear protein complexes.
 filled with cytosol that interconnects the cytosols
of adjacent cells. The regulated deposition of a
glucose polymer called callose in the extracellular
spaces in the cell wall adjacent to the entrances
of the channels has the potential to block
intercellular transport through the
plasmodesmata, apparently by forcing the
closing of the channels by narrowing the annulus.
(b) Electron micrographs of thin sections of a
sugarcane leaf (brackets indicate individual
plasmodesmata). Left: Longitudinal view,
showing the ER and desmotubule running
through each annulus. Right: Perpendicular cross-
sectional views of plasmodesmata, each with its
annulus, desmotubule, and cytoplasmic sleeve.
(c) Cells from the plant Arabidopsis were grown in
culture, frozen, and, while cold, fixed with
glutaraldehyde and stained with osmium
tetroxide and uranyl acetate. Multiple views of
one plasmodesma were visualized by tilting the
sample in an electron microscope and a three-
dimensional tomographic model of the structure
was computed. Shown is the electron micrograph
of one view (black and white) with the colored
model of its plasmodesma; plasmamembrane =
yellow; ER and desmotubule membrane = blue;
and spoke-like tethers connecting these
membranes = red. The gap between the two
membranes is the cytoplasmic sleeve..

[Part (b) republished with permission from

Springer, from K. Robinson-Beers and R. F. Evert,
1991, “Fine Structure of Plasmodesmata in
Mature Leaves of Sugarcane,” Planta 184(3):307–
318; permission conveyed through Copyright
Clearance Center, Inc. Part (c) republished with
permission from Springer Nature, from W. J.
Nicolas et al., 2017, “Architecture and
Permeability of Post-Cytokinesis Plasmodesmata
Lacking Cytoplasmic Sleeves.” Nature Plants
3:17082; permission conveyed through Copyright
Clearance Center, Inc.]

The illustration labeled (a) shows a plasma

membrane and rough endoplasmic reticulum on
both sides of the cell membrane, having moved
through an annule. The E R is pink with red dots
and the cell wall looks like Swiss cheese, with the
holes being desmata. A desmotubule is labeled
coming through one of the desmata. The
fluorescence micrograph labeled (b) shows two
views of the desmata. The first view shows cell 1
and cell 2 with a nanotube between two cells
containing two endoplasmic reticulums. The
second view shows little black circles with black
dots in the centers. The dots are labeled
desmotubules and the circles are labeled
cytoplasmic sleeve. Together, both parts are
labeled annulus. The electron micrograph labeled
(c) shows the plasma membranes of cell 1 and
cell 2 highlighted in bright yellow, a bright blue
line between them labeled desmotubule with E R
in it moving from cell 1 to cell 2. Three-spoke like
tethers are labeled attached to the desmotubule
and the plasma membrane.

FIGURE 20-44 Plasmodesmata. (a) Schematic model of plasmodesmata, showing the desmotubule,
an extension of the endoplasmic reticulum (ER), and the annulus, a plasma-membrane-lined channel
Although plasmodesmata and gap junctions appear to resemble each
other functionally with respect to forming channels for direct diffusion
of molecules from one cell’s cytosol to that of another, their structures
differ dramatically in two significant ways. First, in animal-cell gap
junctions, the membranes of the connected cells are distinct, and the
cytosols are connected by a protein-lined tube of connexon
hemichannels (see Figure 20-21). In plant plasmodesmata (see Figure
20-44), the plasma membranes of the adjacent plant cells merge to
form a continuous single membrane that lines the wall of the channel,
called the annulus, that connects the two cells. The diameter of the
annulus varies from about 30–60 nm; its length can vary and may be
greater than 1 μm. The lipid and protein composition of the specialized
plasma membrane lining the annulus differs from that of the rest of
the plasma membrane that surrounds the cell. Second, passing though
the center of the plasmodesmata’s annulus is a narrow tube, called a
desmotubule, that is an extension of endoplasmic reticulum (ER) from
each of the connected cells. Between the desmotubule and the
plasmodesmata’s specialized plasma membrane is a gap whose size is
regulated and varies between 2–10 nm. This gap is called the
cytoplasmic sleeve, through which molecules diffuse from the cytosol of
one cell to that of the other. Filamentous actin runs throughout the
length of the cytoplasmic sleeve. The close apposition of the plasma
membrane and ER/desmotubule’s membrane in plasmodesmata is an
example of a specialized membrane contact site (MCS), such as that
described for mitochondria and ER in Chapter 12. A set of
transmembrane proteins in the desmotubule called MCTPs can span
the gap of the cytoplasmic sleeve and bind to anionic phospholipids on
the plasma membrane, thus tethering these two membranes. The
MCTP proteins appear to compose spoke-like projections observed by
electron microscopy that tether the two membranes (see Figure 20-
44c), help determine the size and properties of the cytoplasmic sleeve,
and thus control the movement of molecules through plasmodesmata.
In animal cells, tunneling nanotubes resemble plasmodesmata in that
they are membrane-lined channels that permit molecules to flow
between cells (see Section 20.2). However, unlike plasmodesmata,
there is no evidence for ER passing through tunneling nanotubes.
There are simple plasmodesmata (with a single pore, like those in
Figure 20-44) and complex plasmodesmata that branch into multiple
channels. The density of plasmodesmata varies depending on the plant
and cell type, and even the smallest meristematic cells have more than
a thousand connections with their neighbors. Proteomic analysis
suggests that there are more than 115 different proteins that are
concentrated in plasmodesmata and play important roles in
determining their structures and functions. These include the MCTPs,
actin-binding proteins (e.g., formin and the adaptor protein NET1A), a
variety of receptor-like proteins (e.g., kinases), and enzymes.
Many types of molecules travel from cell to cell through
plasmodesmata. Molecules smaller than about 1000 Da, including a
variety of metabolic and signaling compounds (ions, sugars, amino
acids), can generally diffuse freely through the cytoplasmic sleeve,
whose size and properties are highly regulated. In some
circumstances, the channel is clamped shut; in others, it is dilated
sufficiently to permit the passage of molecules larger than 10,000 Da,
including some transcription factors, nucleic acid/protein complexes,
metabolic products, and even plant viruses. Some of these require
special chaperones to facilitate transport. Soluble molecules pass
through the cytosolic sleeve, whereas membrane-bound molecules or
certain proteins within the ER lumen may pass from cell to cell via the
desmotubule. The deposition and breakdown of a glucose polymer
called callose in the extracellular spaces adjacent to the entrances of the
channels (see Figure 20-44a) is thought to regulate the closing and
opening of the channels, respectively. Specialized kinases may also
phosphorylate plasmodesmal components to regulate their activities
(e.g., opening of the channels). Among the factors that affect the
permeability of plasmodesmata is the cytosolic Ca2+
concentration: an increase in cytosolic Ca2+ reversibly inhibits
movement of molecules through these structures.
The Molecules That Plants Depend on for Adhesion and
Mechanotransduction Differ from Those in Animals
As noted earlier in this section, there are dramatic differences in the
organization of cells into tissues in plants and animals. These
differences have determined the distinctive molecular mechanisms by
which plant cells are incorporated into tissues and communicate with
one another, including their cell-cell and cell-matrix interactions.
Plant Adhesion Molecules
Systematic analyses of the Arabidopsis genome and biochemical
analyses of other plant species have provided no evidence for the
existence of plant homologs of most animal CAMs, adhesion receptors,
and ECM components; plants use other adhesive molecules for their
cell-cell and cell-matrix interactions. Currently, our understanding of
cell-matrix adhesion in plants is less advanced than our understanding
of cell-matrix adhesion in animals.
Because plant cells are surrounded by thick cell walls, their assembly
into tissues is primarily determined by cell-matrix interactions. Most of
the polysaccharides in the cell wall (cellulose, hemicellulose, pectins,
glycoproteins) can cross-link to one another. The adhesive properties
of pectins are thought to be especially important for determining both
the physical characteristics of cell walls and the interactions of the
walls with the plasma membranes of the underlying cells. Disruption
of the gene encoding glucuronyltransferase 1, a key enzyme in pectin
biosynthesis, dramatically interferes with the synthesis of specialized
pectins that help hold the cells in meristems tightly together. As a
consequence, the mutation prevents normal cell adhesion and
differentiation into photosynthetic cells. In vitro binding assays,
combined with in vivo studies and analyses of plant mutants, have
identified other macromolecules in the ECM that are important for
specialized types of adhesion. For example, a cysteine-rich protein
called stigma/stylar cysteine-rich adhesin (SCA) and a specialized pectin
that binds to SCA mediate normal adhesion of pollen, which contains
sperm cells, to the stigma or style in the female reproductive organ of
the Easter lily.
A number of plasma membrane proteins exhibit the potential to bind
to cell wall polysaccharides, particularly pectins, and thus mediate cell-
matrix adhesion. These include receptor-like proteins, such as Wall-
Associated Kinases (WAKs), GPI-anchored proteins, Hydroxyproline-
Rich Glycoproteins (HRGPs), and Glycosyl Inositol Phosphoceramides
(GIPCs). For example, five WAKs and WAK-like proteins are expressed
in the plasma membrane of Arabidopsis cells. These transmembrane
proteins have a cytoplasmic serine/threonine kinase domain, and their
extracellular regions contain multiple epidermal growth factor (EGF)
repeats, frequently found in animal cell-surface receptors. Some WAKs
have an extracellular pectin-binding domain that can recognize and
bind full-length pectin and pectin degradation fragments. Such binding
has been proposed to help cells monitor and respond to the status of
the cell wall during normal growth and in the context of cell-wall
damage (wounding) or infection by pathogens. Thus some WAKS in
plant cells appear to be analogous to adhesion receptors in animal
cells, binding and sensing the ECM and mediating outside-in signaling.
Mechanosensation in Plants
book in 1875 in which he called the plant “one of the most wonderful in
the world” and once noting that he cared more for one of them “than
the origin of all the species in the world.” The Venus flytrap detects the
presence of an insect on its leaf when an insect hits one of its sensory
hairs, which are around 200 μm in diameter at the base and 2 mm long.
The sensory hair is a multicellular organ that, when bent by prey, puts
shear stress on a cluster of cells at its base, which opens
mechanosensitive calcium channels. Calcium entry triggers an action
potential in the leaf (see Chapter 23) that leads to closing the trap and
hormonally induced secretion of digestive enzymes that disassemble
the prey into basic nutrients over a one-to-two-week period.

As with animal cells, plant cells are subject to a variety of mechanical

forces (stresses and strains), both internal (e.g., turgor pressure,
We are all familiar with one aspect of cell-wall–mediated
cytoskeletal forces) and external (e.g., wind), which they sense
adhesion in plants: the softening of fruits, such as tomatoes and
(mechanosensing) and to which they respond (e.g., alterations in
strawberries, during ripening. The ripening of fruits is a complex
cytoskeletal organization, calcium flux, gene expression, wood
process that influences color, aroma, flavor, nutrient content, and
formation, cell wall stiffness, and extent and direction of growth).
texture (including softening and water content). Ripening of seed-
Possible mechanosensors in the cell wall include components whose
bearing fruit is advantageous because it encourages fruit-eating
structures respond to changes in mechanical forces and thus change
animals to eat and disperse the seeds or breaks down the uneaten
the properties of the wall and its interactions with the plasma
fruit’s structure (softening and decay) to release the seeds in situ.
membrane. Potential mechanosensors in the cell’s plasma membrane
However, excessive softening can interfere with successful storage and
are those components that bind to the cell wall (e.g., receptor-like
transport and can reduce shelf life. Expression of a group of cell-wall–
kinases such as pectin-binding WAKs) and mechanosensitive ion
degrading enzymes, including pectate lyase (PL), and their remodeling
channels. There is evidence that suggests inside-out and outside-in
of cell walls by degrading pectin plays a key role in determining the
communication via adhesive molecules contribute to plant responses
rate and extent of softening. Experiments using both strawberries and
to mechanical forces.
tomatoes have suggested that inhibiting the expression of pectate lyase
using either CRISPR gene editing or RNA interference (see Chapter 6)
can substantially delay strawberry and tomato softening and may not
One well-known example of macroscopic plant block other desirable consequences of ripening (changes in color,
mechanosensation is the Venus flytrap (Dionaea muscipula), first flavor, etc.). In the future, targeted genetic modifications of cell-wall
described by the colonial governor of North Carolina, Arthur Dobbs, in metabolism may improve the quality of available fruits while reducing
1759. Charles Darwin studied the Venus flytrap extensively, writing a the costs of production and distribution.

KEY CONCEPTS OF SECTION 20.6

Plant Tissues

 The organization of cells into tissues in plants is fundamentally different from the assembly of animal tissues, primarily because each plant
cell is surrounded by a relatively rigid cell wall.
 The plant cell wall comprises layers of cellulose microfibrils embedded within a matrix of hemicellulose, pectin, extensin, and other less
abundant molecules.
 Cellulose, a large, linear glucose polymer, assembles spontaneously into microfibrils stabilized by hydrogen bonding.
 The cell wall defines the shapes of plant cells and restricts their elongation. Auxin-induced loosening of the cell wall permits cell elongation.
 Adjacent plant cells can communicate through plasmodesmata, junctions formed by tubes of specialized plasma membrane and ER that
connect the cytosols of adjacent cells, and by allowing molecules to pass between cells (see Figure 20-44).
 Plants do not produce homologs of the common adhesion molecules found in animals.
 The molecules mediating cell-matrix adhesion in plants are not as well-defined as those in animals, although both cell wall and plasma
membrane molecules implicated in plant cell adhesion have been identified and are under investigation.
 As in the case of animal cells, mechanotransduction in plants plays an important role in maintaining tissue integrity and directing
differentiation and development.
End of Chapter

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Key Terms

adapter proteins
adherens junction
adhesion receptor
anchoring junction
apical
basal
basal lamina
basolateral
cadherin
cell-adhesion molecule (CAM) or cell-cell adhesion receptor
cell junction
cell-matrix adhesion receptor
cell wall
cellulose
cis (lateral) interactions
classical cadherin
clustered protocadherin
collagen
connective tissue
connexin
desmosomal cadherin
desmosome
elastin
epithelium
epithelial-to-mesenchymal transition
extracellular matrix (ECM)
fibrillar collagen
fibroblast
fibronectin
focal adhesion/focal contact
gap junction
glycosaminoglycan (GAG)
heterophilic binding
heterotypic adhesion
homophilic binding
homotypic adhesion
hyaluronan
inside-out signaling
integrin
isoform
laminin
lateral
matriglycan
matrix metalloproteases (MMPs)
mechanosensor
mechanotransduction
multi-adhesive matrix protein
N-linked oligosaccharide
O-linked oligosaccharide
outside-in signaling
paracellular pathway
PDZ domain
perlecan
plasmodesmata
proteoglycan
RGD motif
selectin
tight junction
talin
trans (intercellular) interactions
transcellular pathway

Review the Concepts

1. Describe the two phenomena that give rise to the diversity of adhesion molecules, such as cadherins. What additional
phenomenon gives rise to the diversity of integrins?
2. Cadherins are known to mediate homophilic interactions between cells. What is a homophilic interaction, and how can it be
demonstrated experimentally for E-cadherins? What component of the extracellular environment is required for the homophilic
interactions mediated by cadherins, and how can this requirement be demonstrated?
3. Together with their role in connecting the lateral membranes of adjacent epithelial cells, adherens junctions play a role in
controlling cell shape. What associated intracellular structure and proteins are involved in this role?
4. What is the normal function of tight junctions? What can happen to tissues when tight junctions do not function properly?
5. Gap junctions between cardiac muscle cells and gap junctions between uterine smooth-muscle cells form connections that
provide for rapid communication. What is this phenomenon called? How is communication among uterine smooth-muscle cells
up-regulated for parturition (childbirth)?
6. What is collagen, and how is it synthesized? How do we know that collagen is required for tissue integrity?
7. Explain how changes in integrin structure mediate outside-in and inside-out signaling.
8. Compare the functions and properties of each of three types of macromolecules that are abundant in the ECM of all tissues.
9. Many proteoglycans have signaling roles. Regulation of feeding behavior by syndecans in the hypothalamic region of the brain is
one example. How is this regulation accomplished?
10. You have synthesized an oligopeptide containing an RGD motif surrounded by other amino acids. What is the effect of this peptide
when added to a fibroblast cell culture grown on a layer of fibronectin adsorbed to the tissue culture dish? Why does this happen?
11. Describe the major activity and possible localization of the three major subgroups of proteins that remodel or degrade the ECM in
physiological or pathological tissue remodeling. Identify a pathological condition in which these proteins play a key role.
12. Blood clotting is a crucial function for mammalian survival. How do the multi-adhesive properties of fibronectin lead to the
recruitment of platelets to blood clots?
13. How do changes in molecular connections between the ECM and the cytoskeleton give rise to Duchenne muscular dystrophy?
14. To fight infection, leukocytes move rapidly from the blood into sites of infection in the tissues. What is this process called? How
are adhesion molecules involved in this process?
15. The structure of a plant cell wall needs to loosen to accommodate cell growth. What signaling molecule controls this process?
16. Compare plasmodesmata in plant cells with gap junctions and tunneling nanotubes in animal cells.