L25 Cells to tissues cell adhesion to extracellular matrix (ECM)

一、Extracellular matrix

Overview

THE EXTRACELLULAR MATRIX OF ANIMALS

Tissues are not made up solely of cells. They also contain a remarkably complex and intricate network of macromolecules constituting the extracellular matrix.

• This matrix is composed of many different proteins and polysaccharides that are secreted locally and assembled into an organized meshwork in close association with the surfaces of the cells that produce them.

The classes of macromolecules constituting the extracellular matrix in different animal tissues are broadly similar, but variations in the relative amounts of these different classes of molecules and in the ways in which they are organized give rise to an amazing diversity of materials

It has an active and complex role in regulating the behavior of the cells that touch it, inhabit it, or crawl through its meshes, influencing their survival, development, migration, proliferation, shape, and function.

The ECM is most diverse: in structure and in function

1. The ECM regulates and influences development & behavior

Integrin signaling controls cell proliferation and survival

• Cells without attachment will die by apoptosis.
• Cells with attachment and activated integrin signaling survive and proliferate → anchorage-dependent growth

2. Different cells produce different ECMs

• The ECM is material that is secreted by cells
• Most of the ECM is connective tissue, produced by fibroblasts

3. ECM contains protein fibers, proteoglycans and hyaluronon

• Proteoglycan（蛋白聚糖,蛋白多糖）: proteins with polysaccharides (glycosaminoglycans)
• Fibrous proteins（纤维蛋白）: proteins with short oligosaccharide side chains
  • Proteoglycan forms hydrated gel-like matrix, in which fibrous proteins are embedded.

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

In most connective tissues, the matrix macromolecules are secreted by cells called fibroblasts（成纤维细胞）

• In certain specialized types of connective tissues, such as cartilage and bone, however, they are secreted by cells of the fibroblast family that have more specific names: chondroblasts（软骨母细胞）, for example, form cartilage, and osteoblasts（格根包尔氏细胞） form bone.

The extracellular matrix is constructed from three major classes of macromolecules:

1. glycosaminoglycans (GAGs，糖胺聚糖), which are large and highly charged polysaccharides that are usually covalently linked to protein in the form of proteoglycans;
2. Fibrous proteins, which are primarily members of the collagen family（胶原蛋白家族）;
3. A large class of noncollagen glycoproteins, which carry conventional asparagine-linked oligosaccharides

Add to this the large number of matrix-associated proteins and enzymes that can modify matrix behavior by cross-linking, degradation, or other mechanisms, and one begins to see that the matrix is an almost infinitely variable material

The proteoglycan molecules in connective tissue typically form a highly hydrated, gel-like “ground substance” in which collagens and glycoproteins are embedded

• The polysaccharide gel resists compressive forces on the matrix while permitting the rapid diffusion of nutrients, metabolites, and hormones between the blood and the tissue cells.

The collagen fibers strengthen and help organize the matrix, while other fibrous proteins, such as the rubberlike elastin（弹性蛋白）, give it resilience.

Glycosaminoglycans (GAGs)

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

• Glycosaminoglycans (GAGs) are unbranched polysaccharide chains composed of repeating disaccharide units
• One of the two sugars in the repeating disaccharide is always an amino sugar (N-acetylglucosamine（N-乙酰葡糖胺） or N-acetylgalactosamine（N-乙酰半乳糖胺）), which in most cases is sulfated.
• The second sugar is usually a uronic acid（糖醛酸） (glucuronic acid（葡萄糖醛酸） or iduronic acid（艾杜糖醛酸）).

Because there are sulfate or carboxyl（羧基） groups on most of their sugars, GAGs are highly negatively charged

Polysaccharide: glycosaminoglycans (GAGs) (黏多糖)

• GAGs are unbranched chains of repeating disaccharides (up to 200)
• Hyaluronan（透明质酸） as special case contains up to 25,000 disaccharide repeats

• High density of negative charges along the chain due to carboxyl (COO-) and sulfate groups (SO3-)
• One of the two sugars is always anamino sugar（氨基糖） frequently sulfated:
  • N-acetylglucosamine (GlcNAc)(based on glucose)
  • or N-acetylgalactosamine(GalNAc) (based on galactose)
• The second sugar is a uronic acid (oxidiation → carboxyl group, COO-).
• Here, glucuronic acid (based on glucose), also iduronic acid (based on idose)

1. Types of glycosaminoglycans (GAGs)

Four main groups of GAGs are distinguished by their sugars, the type of linkage between the sugars, and the number and location of sulfate groups:

1. hyaluronan（透明质酸）,
2. chondroitin sulfate（硫酸软骨素） and dermatan sulfate（硫酸皮肤素）,
3. heparan sulfate（硫酸乙酰肝素）,
4. keratan sulfate（硫酸角质素）.

Polysaccharide chains are too stiff to fold into compact globular structures, and they are strongly hydrophilic

GAGs tend to adopt highly extended conformations that occupy a huge volume relative to their mass (Figure 19–33), and they form hydrated gels（水合凝胶） even at very low concentrations.

Four different groups of GAGs are classified according to

• the type of sugar
• type of linkage between the sugars
• number and location of sulfate groups

1. hyaluronan（透明质酸）,
2. chondroitin sulfate（硫酸软骨素） and dermatan sulfate（硫酸皮肤素）,
3. heparan sulfate（硫酸乙酰肝素）,
4. keratan sulfate（硫酸角质素）.

The weight of GAGs in connective tissue is usually less than 10% of the weight of proteins, but GAG chains fill most of the extracellular space.

Their high density of negative charges attracts a cloud of cations, especially Na+, that are osmotically active, causing large amounts of water to be sucked into the matrix.

This creates a swelling pressure（溶胀压力）, or turgor（肿胀，膨胀）, that enables the matrix to withstand compressive forces (in contrast to collagen fibrils, which resist stretching forces)

Glycosaminoglycans (GAGs): Hyaluronan

Hyaluronan Acts as a Space Filler During Tissue Morphogenesis and Repair

Hyaluronan (also called hyaluronic acid or hyaluronate) is the simplest of the GAGs

Hyaluronan is not a typical GAG because it contains no sulfated sugars, all its disaccharide units are identical, its chain length is enormous, and it is not generally linked covalently to any core protein

Moreover, whereas other GAGs are synthesized inside the cell and released by exocytosis, hyaluronan is spun out directly from the cell surface by an enzyme complex embedded in the plasma membrane.

Hyaluronans:

• it is not a typical GAG:
• Does not contain sulfated sugars.
• only GlcNAc & glucuronic acid dimers
• not linked to proteins at all
• not secreted by cells, it is synthesized by enzymes on the cell surface, instead

Major functions:

• guides cell migration during tissue morphogenesis and repair
• it occupies a large volume compared to it’s mass.
• it provides the space and it fills the space up.
• it is degraded by hyaluronidase

Hyaluronan is thought to have a role in resisting compressive forces（压力） in tissues and joints. It is also important as a space filler during embryonic development, where it can be used to force a change in the shape of a structure, as a small quantity expands with water to occupy a large volume

In the developing heart, for example, hyaluronan synthesis helps in this way to drive formation of the valves and septa that separate the heart’s chambers

When cell migration ends, the excess hyaluronan is generally degraded by the enzyme hyaluronidase

Hyaluronan is also produced in large quantities during wound healing, and it is an important constituent of joint fluid, in which it serves as a lubricant.

(1) Hyaluronic Acid

2. GAGs occupy the space of ECM and resists compression and sequesters water

GAGs adopt highly extended structure, allowing occupation of a large volume relative to their mass

• Due to strong negative charge:
  • GAGs attract Na+ ions on their surface, water is absorbed due to osmosis, causing swelling & turgor
  • GAGs swelling allows therefore withstanding of compressive forces（承受压力）
  • That’s the reason why they are so interesting for the cosmetic industry

Proteoglycans

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

Except for hyaluronan, all GAGs are covalently attached to protein as proteoglycans, which are produced by most animal cells

• Membrane-bound ribosomes make the polypeptide chain, or core protein, of a proteoglycan, which is then threaded into the lumen of the endoplasmic reticulum
• The polysaccharide chains are mainly assembled on this core protein in the Golgi apparatus before delivery to the exterior of the cell by exocytosis
  1. First, a special linkage tetrasaccharide is attached to a serine side chain on the core protein to serve as a primer for polysaccharide growth; then, one sugar at a time is added by specific glycosyl transferases（糖基转移酶）

2. While still in the Golgi apparatus, many of the polymerized sugars are covalently modified by a sequential and coordinated series of reactions
3. Epimerizations（差向异构） alter the configuration of the substituents around individual carbon atoms in the sugar molecule; sulfations（硫酸化） increase the negative charge.

By definition, at least one of the sugar side chains of a proteoglycan must be a GAG.

• Whereas glycoproteins generally contain relatively short, branched oligosaccharide chains that contribute only a small fraction of their weight

1. Structure

Proteoglycans are proteins with long side sugar side chains

• The core protein is synthesized and folded in the ER, linkage and modification occurs later in the Golgi apparatus:
  • Attachment of the “link tetrasaccharide” to a serine side chain (O-linked glycosylation)
  • One by one sugar group addition by glycosyltransferases (modifications occur later)

Proteoglycans: almost limitless heterogeneity

In principle, proteoglycans have the potential for almost limitless heterogeneity.

The core proteins, too, are diverse, though many of them share some characteristic domains such as the LINK domain, involved in binding to GAGs.

Core proteins:

• diverse group of core proteins
• shared feature: the “link” domain

Proteoglycans can be huge. The proteoglycan aggrecan, for example, which is a major component of cartilage, has a mass of about 3 × 10^6^ daltons with over 100 GAG chains

Other proteoglycans are much smaller and have only 1–10 GAG chains; an example is decorin, which is secreted by fibroblasts and has a single GAG chain

• Decorin binds to collagen fibrils and regulates fibril assembly and fibril diameter

The GAGs and proteoglycans of these various types can associate to form even larger polymeric complexes in the extracellular matrix.

GAG side chains:

• diverse in composition and combination of sugars
• variable in modifications (sulfatation)
• a single core protein can carry a highly variable number of different types of GAG side chains
  • sometimes more than 100 GAGs on a core protein (e.g. aggrecan)
  • sometimes only 1-10 (e.g. decorin)

Assembly of polymeric complexes:

• GAGs & proteoglycans can associate to form polymeric complexes:
  • aggrecan and hyaluronan from aggregates in cartilage matrix the size of a bacterium!

2. Common proteoglycans

3. Proteoglycans are NOT simple glycoproteins

Comparison between proteoglycan and glycoproteins

Glycoproteins:

• sugar content 1-60% but usually only few percent
• many short & branched sugar chains
• usually low molecular weight proteins (only few hundred kDa)

Proteoglycans:

• sugar content up to 95%
• contain at least one GAG - a long (up to 25,000), unbranched sugar chains
• usually very high in molecular weight, up to 3000 kDa

4. The function of proteoglycans

Besides associating with one another, GAGs and proteoglycans associate with fibrous matrix proteins such as collagen and with protein meshworks such as the basal lamina

Not all proteoglycans are secreted components of the extracellular matrix.

Some are integral components of plasma membranes and have their core protein either inserted across the lipid bilayer or attached to the lipid bilayer by a glycosylphosphatidylinositol (GPI) anchor.

Among the best-characterized plasma membrane proteoglycans are the syndecans, which have a membrane-spanning core protein whose intracellular domain is thought to interact with the actin cytoskeleton and with signaling molecules in the cell cortex

• Syndecans are located on the surface of many types of cells, including fibroblasts and epithelial cells.
• syndecans can be found in cell–matrix adhesions, where they modulate integrin function by interacting with fibronectin on the cell surface and with cytoskeletal and signaling proteins inside the cell

Form gels (matrices) of varying pore size and charge density

Serve as a selective sieves to regulate traffic of molecules and cells

Matrix Proteoglycans and Glycoproteins Regulate the Activities of Secreted Proteins

The physical properties of extracellular matrix are important for its fundamental roles as a scaffold for tissue structure and as a substrate for cell anchorage and migration.

• En route (在途中) to their targets, the signal molecules encounter the tightly woven meshwork of the extracellular matrix, which contains a high density of negative charges and protein-interaction domains that can interact with the signal molecules, thereby altering their function in a variety of ways

The highly charged heparan sulfate chains of proteoglycans, for example, interact with numerous secreted signal molecules, including fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF), which (among other effects) stimulate a variety of cell types to proliferates

• By providing a dense array of growth factor binding sites, proteoglycans are thought to generate large local reservoirs of these factors, limiting their diffusion and focusing their actions on nearby cells.
• Similarly, proteoglycans might help generate steep growth factor gradients in an embryo, which can be important in the patterning of tissues during development.

FGF activity can also be enhanced by proteoglycans, which oligomerize the FGF molecules, enabling them to cross-link and activate their cell-surface receptors more effectively

The importance of proteoglycans as regulators of the distribution and activity of signal molecules

Regulate signalling through binding e.g. fibroblast growth factor (FGF), Transforming growth factor β (TGF β) and chemical attractants (chemokines（趋化因子）) to allow control of growth and movement of cells

Regulate activities of other proteins in the matrix e.g. proteolytic enzymes

Some cell surface bound proteoglycan such as syndecans acts as coreceptors for growth factors to participate in cell signalling

Binding of chemokines by proteoglycans: immobilization of these substances on the surface of blood vessels during inflammation attracts leucocytes（白细胞） to leave the blood stream and to migrate into the in flamed tissue.

GAGs & proteoglycans can associate with fibrous proteins like collagen and with protein meshworks (basal lamina) to extremely complex structures

Proteoglycans associates with fibrous proteins in the ECM

• GAGs & proteoglycans can associate with fibrous proteins like collagen and with protein meshwork (basal lamina) to extremely complex structures

Collagen

Collagens Are the Major Proteins of the Extracellular Matrix

• The collagens are a family of fibrous proteins found in all multicellular animals.

They are secreted in large quantities by connective-tissue cells, and in smaller quantities by many other cell types.

The primary feature of a typical collagen molecule is its long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains, called α chains, are wound around one another in a ropelike superhelix

• Collagens are extremely rich in proline and glycine, both of which are important in the formation of the triple-stranded helix.

• Collagen: Fibrous proteins

1. Structure: Fibrous proteins

Like a steel cable, collagen fibers bear tremendous strength.

• Major fibrous proteins of ECM in skin and bone
• The most abundant protein in mammals ~25% of total protein mass.
  • 42 genes for distinct collagen a chains
• Multiple types of collagen and they have different properties
  • Two main collagen classes:
    • Fibrillar collagens (form fibrils)
    • Fibril-associated collagens (associate with fibrils)

• Typical collagen molecule is long (~1,000 aa), stiff and triple stranded helical structure
  • 3 amino acid per turn: Gly-X-Y
  • Primary amino acid sequence is rich in proline and glycine.
• Large molecules: the entire molecule is up to 300 nm long

Hydroxylation allows formation of interchain hydrogen bonds

Hydroxyl groups help forming hydrogen bonds that stabilize the triple-stranded helices

• Deficiency in Vitamin C (ascorbic acid（抗坏血酸）) causes defects in proline hydroxylation.
  • This results in failure to assemble stable triple helices
  • This causes blood vessels to be fragile, teeth to be loosen & wounds cease to heal (→ scurvy（坏血病）)

2. Some types of collagen and their properties

Type I is by far the most common, being the principal collagen of skin and bone

It belongs to the class of fibrillar collagens（纤维状胶原）, or fibril-forming collagens（原纤维形成胶原）

• after being secreted into the extracellular space, they assemble into higher-order polymers called collagen fibrils, which are thin structures (10–300 nm in diameter) many hundreds of micrometers long in mature tissues, where they are clearly visible in electron micrographs
• Collagen fibrils often aggregate into larger, cablelike bundles, several micrometers in diameter, that are visible in the light microscope as collagen fibers.

Collagen types IX and XII are called fibril-associated collagens because they decorate the surface of collagen fibrils.

• They are thought to link these fibrils to one another and to other components in the extracellular matrix

Type IV is a network-forming collagen, forming a major part of basal laminae

Type VII molecules form dimers that assemble into specialized structures called anchoring fibrils（锚定原纤维）.

There are also a number of “collagen-like” proteins containing short collagen-like segments.

• These include collagen type XVII, which has a transmembrane domain and is found in hemidesmosomes, and type XVIII, the core protein of a proteoglycan in basal laminae.

3. Biogenesis of collagen fibers

1. Synthesis of precursor pro-α collagen chains in the ER
2. Hydroxylation of some prolines & lysines and glycosylation of some of the hydroxyprolines/hydroxlysines
3. Assembly into a triple helix, the procollagen, in the ER
4. Secretion into the matrix
5. Cleavage by extracellular proteases and assembly into collagen fibrils
6. And cross-linking by lysine

4. Fibril-associated collagens help to organize the fibrils

Secreted Fibril-Associated Collagens Help Organize the Fibrils

In contrast to GAGs, which resist compressive forces, collagen fibrils form structures that resist tensile forces（抵抗拉力）

• The fibrils have various diameters and are organized in different ways in different tissues. In mammalian skin, for example, they are woven in a wickerwork pattern（编织图案） so that they resist tensile stress in multiple directions
• leather consists of this material, suitably preserved.

In tendons, collagen fibrils are organized in parallel bundles aligned along the major axis of tension.

In mature bone and in the cornea（角膜）, they are arranged in orderly plywoodlike（胶合板状） layers, with the fibrils in each layer lying parallel to one another but nearly at right angles to the fibrils in the layers on either side.

• The same arrangement occurs in tadpole skin

Collagen fibrils are arranged in orderly layers

• Fibril-associated type IX and XII collagens differ from fibrillar collagens:
  • Their triple stranded helix is interrupted by short non-helical domains, this results in a more flexible structure.
  • No proteolytic cleavage/processing after secretion
  • No aggregation & formation of fibrils, but they bind to fibrillar collagens:
    • Type IX: binds to type II collagen-containing fibrils in cartilage, cornea & vitreous of the eye
    • Type XII: binds to type I collagen-containing fibrils in tendon and other tissues
  • They mediate the interaction between the fibrils of fibrillar collagens.
  • They help to determine the organization of the fibrils in the matrix!

The connective-tissue cells themselves determine the size and arrangement of the collagen fibrils

cells can regulate the disposition of the collagen molecules after secretion by guiding collagen fibril formation near the plasma membranes

In addition, cells can influence this organization by secreting, along with their fibrillar collagens, different kinds and amounts of other matrix macromolecules.

• In particular, they secrete the fibrous protein fibronectin（纤维连接蛋白）

Type IX and Type XII collagen

Fibril-associated collagens, such as types IX and XII collagens, are thought to be especially important in organizing collagen fibrils

They differ from fibrillar collagens in the following ways.

1. First, their triple-stranded helical structure is interrupted by one or two short nonhelical domains, which makes the molecules more flexible than fibrillar collagen molecules.
2. Second, they do not aggregate with one another to form fibrils in the extracellular space. Instead, they bind in a periodic manner to the surface of fibrils formed by the fibrillar collagens

Type IX molecules bind to type-II-collagen-containing fibrils in cartilage, the cornea, and the vitreous（玻璃状的） of the eye (Figure 19–42), whereas type XII molecules bind to type-I-collagen-containing fibrils in tendons and various other tissues

The fibroblasts influence the alignment of the collagen fibers, and the collagen fibers in turn affect the distribution of the fibroblasts.

• Type IX collagen binds to the surface of fibrillar collagen fibers in a periodic pattern
• Type XII collagen binds to type I-collagen-containing fibers e.g. in tendon

Elastin

Elastin Gives Tissues Their Elasticity

• A network of elastic fibers in the extracellular matrix of these tissues gives them the resilience to recoil after transient stretch

The main component of elastic fibers is elastin, a highly hydrophobic protein (about 750 amino acids long), which, like collagen, is unusually rich in proline and glycine but, unlike collagen, is not glycosylated.

• Soluble tropoelastin（弹性蛋白原） (the biosynthetic precursor of elastin) is secreted into the extracellular space and assembled into elastic fibers close to the plasma membrane, generally in cell-surface infoldings.
• After secretion, the tropoelastin molecules become highly cross-linked to one another, generating an extensive network of elastin fibers and sheets

The elastin protein is composed largely of two types of short segments that alternate along the polypeptide chain

• hydrophobic segments, which are responsible for the elastic properties of the molecule
• alanineand lysine-rich α-helical segments, which are cross-linked to adjacent molecules by covalent attachment of lysine residues

1. Functions

Elastin gives elasticity to blood vessels and lungs

Elastin is the dominant extracellular matrix protein in arteries

Elastic fibers do not consist solely of elastin. The elastin core is covered with a sheath of microfibrils

• The microfibrils appear before elastin in developing tissues and seem to provide scaffolding to guide elastin deposition

Microfibrils are composed of a number of distinct glycoproteins, including the large glycoprotein fibrillin, which binds to elastin and is essential for the integrity of elastic fibers

Elastins are the primary constituent of the ECM in arteries

• Elastic fibers are more than 5 times more elastic than rubber bands with the same cross-section area
• Elastic fibers mainly consist of elastin, but also contain some microfibrils which are composed of glycoproteins, including fibrillin.

Elastin allows for stretching and recoil of ECM

• The molecules are joined together by covalent bonds (red) to generate a cross-linked network.
• In this model, each elastin molecule can extend and contract in a manner resembling a random coil, so that the entire assembly can stretch and recoil like a rubber band

2. Genetic diseases from defects in elastin fiber

Mutation in elastin:

• thinning of arteries and excessive proliferation of smooth muscle cells lining the arteries.

Marfan’s syndrome:

• mutation from fibrillin, easy rupturing aortas,
• displacement of the lens and abnormalities of skeleton and joints

Glycoproteins

Fibronectin and Other Multidomain Glycoproteins Help Organize the Matrix

• 纤连蛋白（fibronecctin）是高分子量（~440kDa）的糖蛋白细胞外基质，和细胞膜内称为集成素的受体蛋白结合。

In addition to proteoglycans, collagens, and elastic fibers, the extracellular matrix contains a large and varied assortment of glycoproteins that typically have multiple domains, each with specific binding sites for other matrix macromolecules and for receptors on the surface of cells

These proteins therefore contribute to both organizing the matrix and helping cells attach to it. Like the proteoglycans, they also guide cell movements in developing tissues, by servings

They can also bind and thereby influence the function of peptide growth factors and other small molecules produced by nearby cells.

The best-understood member of this class of matrix proteins is fibronectin, a large glycoprotein found in all vertebrates and important for many cell–matrix interactions.

Fibronectin is a dimer composed of two very large subunits joined by disulfide bonds at their C-terminal ends

1. Glycoproteins in the ECM

Organization of fibronectin into fibrils on cell surface

Fibronectin Binds to Integrins

Synthetic peptides corresponding to different segments of the integrin-binding domain were then used to show that binding depends on a specific tripeptide sequence (Arg-Gly-Asp, or RGD) that is found in one of the type III repeats

• Even very short peptides containing this RGD sequence can compete with fibronectin for the binding site on cells, thereby inhibiting the attachment of the cells to a fibronectin matrix.

Several extracellular proteins besides fibronectin also have an RGD sequence that mediates cell-surface binding.

Peptides containing the RGD sequence have been useful in the development of anti-clotting drugs

• Some snakes use a similar strategy to cause their victims to bleed: they secrete RGD-containing anti-clotting proteins called disintegrins into their venom.

Each integrin specifically recognizes its own small set of matrix molecules, indicating that tight binding requires more than just the RGD sequence

• In sensing tensions During migration, fibronectin assembles into fibers, in parallel with actin fibers.

Cell help organize collagen fibrils they secreted by exerting tension on the ECM

2. Functions

Fibronectin links cells to extracellular matrix

• RGD: integrin binding
• A large glycoprotein dimer joined by disulfide bond;
• Exists both in soluble or insoluble fibers;
• They don’t self-assemble until sensing tension and cell surface receptors.

Tension Exerted by Cells Regulates the Assembly of Fibronectin Fibrils

Fibronectin can exist both in a soluble form, circulating in the blood and other body fluids, and as insoluble fibronectin fibrils, in which fibronectin dimers are cross-linked to one another by additional disulfide bonds and form part of the extracellular matrix

• fibronectin molecules assemble into fibrils only on the surface of cells, and only where those cells possess appropriate fibronectin-binding proteins

The integrins provide a linkage from the fibronectin outside the cell to the actin cytoskeleton inside it. The linkage transmits tension to the fibronectin molecules

This dependence on tension and interaction with cell surfaces ensures that fibronectin fibrils assemble where there is a mechanical need for them and not in inappropriate locations such as the bloodstream.

Degradation

Cells Have to Be Able to Degrade Matrix, as Well as Make It

Rapid matrix degradation is required in processes such as tissue repair, and even in the seemingly static extracellular matrix of adult animals there is a slow, continuous turnover, with matrix macromolecules being degraded and resynthesized

Matrix degradation is important both for the spread of cancer cells through the body and for their ability to proliferate in the tissues that they invade

In general, matrix components are degraded by extracellular proteolytic enzymes (proteases) that act close to the cells that produce them.

• Many of these proteases belong to one of two general classes. The largest group, with about 50 members in vertebrates, is the matrix metalloproteases（金属蛋白酶）, which depend on bound Ca^2+^ or Zn^2+^ for activity
• The second group is the serine proteases, which have a highly reactive serine in their active site

Some metalloproteases, such as the collagenases, are highly specific, cleaving particular proteins at a small number of sites.

Protease activity is generally confined to the cell surface by specific anchoring proteins, by membrane-associated activators, and by the production of specific protease inhibitors in regions where protease activity is not needed.

Two different classes of proteases:

1. Matrix metalloprotease (MMP) (Ca2+ or Zn2+ dependent)
2. Serine protease

Three ways to activate & regulate these proteases

1. Local activation:
   • The protease is transported as an inactive precursor.
   • It’s activation occurs upon transport at it’s destination
   • Example:
     • The protease plasmin, which helps to break down blood clots.
     • In the capillaries, it is secreted as an inactive precursor, called plasminogen.
     • activation of plasmin occurs by the plasmin-activating proteases in the blood vessel.

二、Basal lamina

The Basal Lamina Is a Specialized Form of Extracellular Matrix

We now describe how some of these components are assembled into a specialized type of extracellular matrix called the basal lamina (also known as the basement membrane)

• This exceedingly thin, tough, flexible sheet of matrix molecules is an essential underpinning of all epithelia.

A sheet of basal lamina not only lies beneath epithelial cells but also surrounds individual muscle cells, fat cells, and Schwann cells (which wrap around peripheral nerve cell axons to form myelin).

• In other locations, such as the kidney glomerulus（肾小球）, a basal lamina lies between two cell sheets and functions as a selective filter

Basal laminae have more than simple structural and filtering roles, however. They are able to determine cell polarity; influence cell metabolism; organize the proteins in adjacent plasma membranes; promote cell survival, proliferation, or differentiation; and serve as highways for cell migration.

This causes a blistering disease called junctional epidermolysis bullosa（大疱性表皮松解症）, a severe and sometimes lethal condition.

All epithelia rest on a basement membrane

• Basal lamina is a structure of the ECM (extracellular matrix)
• Produced by both: epithelia and the stroma
• 40-120 nm thick
• Plays important mechanical role (strength of the epidermis)

Structure of Basal Lamina

1. Basement membrane separates epithelial cells from surrounding tissue

2. Composition of basal lamina

Laminin（层粘连蛋白，基板糖蛋白） and Type IV Collagen Are Major Components of the Basal Lamina

Although the precise composition of the mature basal lamina varies from tissue to tissue and even from region to region in the same lamina, it typically contains the glycoproteins laminin, type IV collagen, and nidogen（巢蛋白）, along with the proteoglycan perlecan

• Other common basal lamina components are fibronectin and type XVIII collagen (an atypical member of the collagen family, forming the core protein of a proteoglycan)

Laminin is the primary organizer of the sheet structure, and, early in development, basal laminae consist mainly of laminin molecules.

• Laminins comprise a large family of proteins, each composed of three long polypeptide chains (α, β, and γ) held together by disulfide bonds and arranged in the shape of an asymmetric bouquet,
• These heterotrimers can self-assemble in vitro into a network, largely through interactions between their heads

Type IV collagen is a second essential component of mature basal laminae, and it, too, exists in several isoforms.

• type IV collagen molecules consist of three separately synthesized long protein chains that twist together to form a ropelike superhelix
• however, they differ from the fibrillar collagens in that the triple-stranded helical structure is interrupted in more than 20 regions, allowing multiple bends

Laminin and type IV collagen interact with other basal lamina components, such as the glycoprotein nidogen and the proteoglycan perlecan

• resulting in a highly cross-linked network of proteins and proteoglycans

The cell-surface receptors are primarily members of the integrin family, but another important type of laminin receptor is dystroglycan, a proteoglycan with a core protein that spans the cell membrane, dangling its GAG chains in the extracellular space.

• The basal lamina consists of 2 main classes of secreted macromolecules:
  • Fibrous proteins (with short oligosaccharide side chains)
  • Proteoglycans (proteins with polysaccharides, glycosaminoglycans, GAGs)

Laminin organizes components of the basement membrane

• Laminin is the primary organizer for the basal lamina structure
• Large heterotrimeric complex, consisting of α-, β-, γ-chains (about 3000 amino acids each subunit), which are hold together by disulfide bonds.
• Can self-assemble via their head domains into a network in vitro

Type IV collagen is the main structural component of the basement membrane

• Second essential component in the basal lamina
• Three separate chains twist together to form rope-like superhelix, with multiple bends.
• Interact with other basal lamina proteins via their terminal domains

3. A model for the formation of the basal lamina

• Laminin and type IV collagen form a meshwork
• Nidogen and perlecan act as linkers:
• they have binding sites for both, laminin and collagen.
• Laminin and type IV collagen have binding sites for cell surface receptors such as Integrin.

The function of basal lamina

Basal Laminae Have Diverse Functions

1. In the kidney glomerulus, an unusually thick basal lamina acts as one of the layers of a molecular filter, helping to prevent the passage of macromolecules from the blood into the urine as urine is formed

2. The basal lamina can act as a selective barrier to the movement of cells, as well as a filter for molecules

   • It does not, however, stop macrophages, lymphocytes, or nerve processes from passing through it, using specialized protease enzymes to cut a hole for their transit

3. The basal lamina is also important in tissue regeneration after injury.

   • When cells in tissues such as muscles, nerves, and epithelia are damaged or killed, the basal lamina often survives and provides a scaffold along which regenerating cells can migrate
   • A particularly striking example of the role of the basal lamina in regeneration comes from studies of the neuromuscular junction, the site where the nerve terminals of a motor neuron form a chemical synapse with a skeletal muscle cell
   • In vertebrates, the basal lamina that surrounds the muscle cell separates the nerve and muscle cell plasma membranes at the synapse, and the synaptic region of the lamina has a distinctive chemical character with special isoforms of type IV collagen and laminin and a proteoglycan called agrin

1. Basal lamina guides tissue regeneration

   • Upon cell damage, basal lamina often survives and guides tissue regeneration.

2. Provides mechanical support.

3. Acts as barriers to keep cells in place.

4. Serves as filters in kidney.

5. Influence cell polarity, differentiation and migration

6. Serve as “highways” for cell migration

三、Cell-extracellular matrix adhesion

CELL–MATRIX JUNCTIONS

Cells make extracellular matrix, organize it, and degrade it. The matrix in its turn exerts powerful influences on the cells

• The influences are exerted chiefly through transmembrane cell adhesion proteins that act as matrix receptors

Several types of molecules can function as matrix receptors or co-receptors, including the transmembrane proteoglycans

The binding of a matrix component to an integrin can send a message into the interior of the cell, and conditions in the cell interior can send a signal outward to control binding of the integrin to the matrix.

integrins can serve not only to transmit mechanical and molecular signals, but also to convert one type of signal into the other.

Hemidesmosome

• Hemidesmosomes spot-weld epithelial cells to the basal lamina
• Hemidesmosomes links laminin outside the cell to keratin filaments inside of the cell

Integrin-mediated cell-ECM adhesion

Integrins Are Transmembrane Heterodimers That Link the Extracellular Matrix to the Cytoskeleton

An integrin molecule is composed of two noncovalently associated glycoprotein subunits called α and β. Both subunits span the cell membrane, with short intracellular C-terminal tails and large N-terminal extracellular domains

• The extracellular domains bind to specific amino acid sequence motifs in extracellular matrix proteins or, in some cases, in proteins on the surfaces of other cells
• The best-understood binding site for integrins is the RGD sequence mentioned earlier (see Figure 19–47), which is found in fibronectin and other extracellular matrix proteins
• Some integrins bind a Leu-Asp-Val (LDV) sequence in fibronectin and other proteins.

The binding of integrins to their matrix ligands is also affected by the concentration of Ca2+ and Mg2+ in the extracellular medium, reflecting the presence of divalent cation-binding domains (二价阳离子结合域) in the α and β subunits

• The divalent cations can influence both the affinity and the specificity of the binding of an integrin to its extracellular ligands.

A large adaptor protein called talin is a component of the linkage in many cases, but numerous additional proteins are also involved.

Like the actin-linked cell–cell junctions formed by cadherins, the actin-linked cell–matrix junctions formed by integrins may be small, inconspicuous（不明显的,不显著的）, and transient, or large, prominent（显著的; 突出的）, and durable.

• Examples of the latter are the focal adhesions（粘连） that form when fibroblasts have sufficient time to establish strong attachments to the rigid surface of a culture dish, and the myotendinous junctions（肌腱连接） that attach muscle cells to their tendons.

In epithelia, the most prominent cell–matrix attachment sites are the hemidesmosomes, where a specific type of integrin anchors the cells to laminin in the basal lamina

• Here, uniquely, the intracellular attachment is to keratin intermediate filaments, via the intracellular adaptor proteins plectin and BP230

• Integrins link extracellular matrix to the intracellular actin cytoskeleton
• Integrins are transmembrane proteins composed of a- and β-subunits
• Bind to extracellular matrix proteins, they are matrix receptors: the N-terminal heads of the integrin chains attach directly to an extracellular matrix protein such as fibronectin
• C-terminal intracellular tail of the integrin b subunit binds to adaptor proteins that interact with actin.
  • Talin is an adaptor, which contains a string of multiple domains for binding actin and other proteins, such as vinculin (helps reinforcing/and regulating actin linkage.
• Play important role in bidirectional signaling between the cell and the matrix (from the cell to the matrix and from the matrix to the cell)
• Defects in integrins signaling cause many genetic diseases

Integrin superfamily

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Integrin	Ligand *Not all ligands are listed	Distribution	Phenotype when α subunit is mutated	Phenotype when β subunit is mutated
α5β1	Fibronectin	Ubiquitous	Death of embryo; defects in blood vessels, somites, neural crest	Early death of embryo (at implantation)
α6β1	Laminin	Ubiquitous	Severe skin blistering; defects in other epithelia also	Early death of embryo (at implantation)
α7β1	Laminin	Muscle	Muscular dystrophy; defective myotendinous junctions	Early death of embryo (at implantation)
αLβ2 (LFA1)	Ig superfamily counterreceptors (ICAM1)	White blood cells	Impaired recruitment of leucocytes	Leukocyte adhesion deficiency (LAD); impaired inflammatory responses; recurrent life-threatening infections
αIIbβ3	Fibrinogen	Platelets	Bleeding; no platelet aggregation (Glanzmann’s disease)	Bleeding; no platelet aggregation (Glanzmann’s disease); mild osteopetrosis
α6β4	Laminin	Hemidesmosomes in epithelia	Severe skin blistering; defects in other epithelia also	Severe skin blistering; defects in other epithelia also

Integrin Defects Are Responsible for Many Genetic Diseases

The β1 subunit forms dimers with at least 12 distinct α subunits and is found on almost all vertebrate cells: α5β1 is a fibronectin receptor and α6β1 is a laminin receptor on many types of cells

The β2 subunit forms dimers with at least four types of α subunit and is expressed exclusively on the surface of white blood cells

• The β2 integrins mainly mediate cell– cell rather than cell–matrix interactions binding to specific ligands on another cell, such as an endothelial cell.
• The ligands are members of the Ig superfamily of cell–cell adhesion molecules.
• an integrin of this class (αLβ2, also known as LFA1) on white blood cells enables them to attach firmly to the Ig family protein ICAM1 on vascular endothelial cells at sites of infection
• People with the genetic disease called leukocyte adhesion deficiency fail to synthesize functional β2 subunits

The β3 integrins are found on blood platelets (as well as various other cells), and they bind several matrix proteins, including the blood clotting factor fibrinogen

Integrin activation

Integrins Can Switch Between an Active and an Inactive Conformation

integrins exist in multiple structural conformations that reflect different states of activity

• In the inactive state, the external segments of the integrin dimer are folded together into a compact structure that cannot bind matrix proteins hooked together, preventing their interaction with cytoskeletal linker proteins.
• In the active state, the two integrin subunits are unhooked at the membrane to expose the intracellular binding sites for cytoplasmic adaptor proteins, and the external domains unfold and extend, like a pair of legs, to expose a high-affinity matrix-binding site at the tips of the subunits.

Thus, the switch from inactive to active states depends on a major conformational change that simultaneously exposes the external and internal ligand-binding sites at the ends of the integrin molecule

Switching between the inactive and active states is regulated by a variety of mechanisms that vary, depending on the needs of the cell.

1. In some cases, activation occurs by an “outside-in” mechanism（“由内而外”的机制）: the binding of an external matrix protein, such as the RGD sequence of fibronectin, can drive some integrins to switch from the low-affinity inactive state to the high-affinity active state.

   • As a result, binding sites for talin and other cytoplasmic adaptor proteins are exposed on the tail of the β chain
   • The chain of cause and effect can also operate in reverse, from inside to outside.
     • This “inside-out” integrin-activation process generally depends on intracellular regulatory signals that stimulate the ability of talin and other proteins to interact with the β chain of the integrin
     • Talin competes with the integrin α chain for its binding site on the tail of the β chain.

2. The regulation of “inside-out” integrin activation is particularly well understood in platelets, where an extracellular signal protein called thrombin bind to a specific G-protein-coupled receptor (GPCR) on the cell surface and thereby activates an intracellular signaling pathway that leads to integrin activation

• Switching from inactive to active states is a major conformational change that simultaneously exposes the external and internal ligand-binding sites at the ends of the integrin molecule.
• External matrix binding and internal cytoskeleton linkages are thereby coupled.

1. Outside-in activation: extracellular ligand binding
2. Inside-out: strong talin binding in response to intracellular signaling molecules such as PIP2, etc.

1. Strength of interaction between integrins and ECM is regulated

Extracellular Matrix Attachments Act Through Integrins to Control Cell Proliferation and Survival

integrins do more than just create attachments. They also activate intracellular signaling pathways and thereby allow control of almost any aspect of the cell’s behavior according to the nature of the surrounding matrix and the state of the cell’s attachments to it.

Many cells will not grow or proliferate in culture unless they are attached to extracellular matrix; nutrients and soluble growth factors in the culture medium are not enough.

• This dependence of cell growth, proliferation, and survival on attachment to a substratum is known as anchorage dependence, and it is mediated mainly by integrins and the intracellular signals they generate

• Integrin activation by intracellular signaling

2. Focal adhesions: Cell-ECM adhesion sites

Integrins Cluster to Form Strong Adhesions

• they usually bind their ligand with lower affinity and are present at a 10–100-fold higher concentration on the cell surface.

Following their activation, integrins cluster together to create a dense plaque in which many integrin molecules are anchored to cytoskeletal filaments.

The assembly of mature cell–matrix junctional complexes depends on the recruitment of dozens of different scaffolding and signaling proteins

• Talin is a major component of many cell–matrix complexes, but numerous other proteins also make important contributions.
• These include the integrin-linked kinase (ILK) and its binding partners pinch and parvin, which together form a trimeric complex that serves as an organizing hub at many junctions.

Cell–matrix junctions also employ several actin-binding proteins, such as vinculin, zyxin, VASP, and α-actinin, to promote the assembly and organization of actin filaments.

• Another critical component of many cell–matrix junctions is the focal adhesion kinase (FAK), which interacts with multiple components in the junction and serves an important function in signaling

Integrins Recruit Intracellular Signaling Proteins at Sites of Cell–Matrix Adhesion

• Integrins and conventional signaling receptors also cooperate to promote cell survival

One of the best-studied modes of integrin signaling depends on a cytoplasmic protein tyrosine kinase called focal adhesion kinase (FAK)

• In studies of cells cultured on plastic dishes, focal adhesions are often prominent sites of tyrosine phosphorylation (Figure 19–59), and FAK is one of the major tyrosine-phosphorylated proteins found at these sites
• When integrins cluster at cell–matrix contacts, FAK is recruited to the integrin β subunit by intracellular adaptor proteins such as talin or paxillin (which binds to one type of integrin α subunit).

• Integrins cluster into focal adhesions that function as signaling platforms (e.g. focal adhesion kinase (FAK), Src etc.)

Cells are mechanoresponsive

Cell–Matrix Adhesions Respond to Mechanical Forces

Like the cell–cell junctions we described earlier, cell–matrix junctions can sense and respond to the mechanical forces that act on them. Most cell–matrix junctions, for example, are connected to a contractile actin network that tends to pull the junctions inward, away from the matrix

• The long C-terminal tail domain of talin, for example, includes a large number of binding sites for the actin-regulatory protein vinculin

1. Stiffness of ECM regulates cell survival and proliferation