Extracellular matrix

Tissues in animals are not just made of cells: they also contain significant quantities of extracellular space, into which an extensive extracellular matrix may have been secreted. Also, don't forget that plants and fungi (and bacteria for that matter) also have an ECM, only we call them 'walls'. Likewise, don't forget chitin in arthropods: chitin and cellulose are by far the most abundant biopolymers on earth.

Connective tissue is a mixture of ECM and cells.

The ECM is anything but an inert glue or scaffolding: components such as laminin and fibronectin can guide the formation of capillary networks and other ordered features when added to homogenous cultures of cells. The ECM in fact plays at least three important roles:

Mechanical: tensile and compressive strength and elasticity.
Protection: buffering against extracellular change and retention of water.
Organisation: control of cell behaviour by binding of growth factors and interaction will cell-surface receptors.

ECM may also be specialised to play particular roles. The matrix of bone and tooth enamel is highly mineralised to withstand compression, whilst the ECM that forms the cornea of the eye is transparent to light. Highly elastic ECM is found in tendons, and it should not be forgotten that everybody contains several litres of liquid ECM, the blood plasma.

The most typical form of ECM is that found in connective tissue, which is the mixture of cells and ECM found in much of the body. The connective tissue is surrounded by specialised ECM called the basal lamina, which underlies epithelial cells. Immune cells are often present too, but these are for defense, not for generation of ECM: connective tissue is primarily generated by fibroblasts. Specialised fibroblasts include chondroblasts (which secrete cartilage) and osteoblasts (which secrete bone).

The ECM is composed of a mixture or water, proteins and carbohydrates. The main macromolecular components of the ECM are glycosaminoglycans (GAGs, acidic polysaccharide derivatives); proteins such as collagen, elastin, fibronectin and laminin; and proteoglycans, which are proteins with GAGs attached to them covalently.

Glycosaminoglycans

GAGs are unbranched polymers of repeated disaccharide derivatives, including amino sugars, sulfated acetylamino sugars and uronic acids.

Galactose.

Galactosamine.

N-acetyl galactosamine-4-sulfate.

Galacturonic acid.

Galactose, galactosamine, N-acetylgalactosamine-4-sulfate and galacturonic acid. The equivalent glucose derivatives are also common components of GAGs.

There are two main types of GAG: hyaluronan, and 'the rest'. Hyaluronan differs from the other GAGs because it is spun out from the cell membrane (rather than being secreted through the Golgi); it is enormous (10⁷ Da - much larger than other GAGs), and is not sulfated.

Hyaluronan.

It is often added to the ECM to hold open areas that would otherwise fill up with cells; it is then removed by hyaluronidase after appropriate cell migration. It is also a 'stand-alone' molecule i.e. unlike most GAGs, it is not attached covalently to protein to form proteoglycans.

Hyaluronan swells to large volumes.

GAGs are acidic and therefore negatively charged, hence they attract ions, especially Na⁺, which osmotically attracts water and causes the molecule to puff up into a gel. This allows GAGs, which only comprise 10% of the ECM mass, to take up 90% of the ECM volume. GAGs (particularly hyaluronan) provide compressive strength, and are a metabolically cheap bulking agent.

The other sorts of GAG are synthesised intracellularly, sulfated, secreted, and usually covalently bound into proteoglycans. They are much smaller than hyaluronan, usually only 20 to 200 sugar residues long. They include:

Chondroitin (shown below).
Dermatan.
Heparan.
Keratan.

Chondroitin.

It is worth distinguishing between proteoglycans and glycoproteins before we go any further. A glycoprotein is a protein with a few small, branched, mannose-rich oligosaccharides added through N-linked (asparagine) glycosylation in the ER and Golgi. A proteoglycan is a serine-rich protein decorated with hundreds of O-linked (usually via serine), acidic, sulfated GAGs.

Proteoglycans have long, unbranched, acidic, O-linked carbohydrate chains added to them.

Aggrecan is a common proteoglycan in the ECM. Its core protein is decorated with c. 100 chondroitin and 30 keratan chains.

Aggrecan.

Aggrecan then binds to hyaluronan (100:1) via adaptor proteins. Aggrecan-hyaluronan aggregates can be as big as bacteria (5 µm long).

Aggrecan aggregate around a central hyaluronan molecule.

Heparan sulfate is a polymer of trisulfated GlcNAc and iduronic acid.

Heparan.

HSPGs have an important roles to play in cell growth. because heparan sulfate proteoglycans (HSPGs) regulate growth factors. HSPGs help to oligomerises FGF (fibroblast growth factor), allowing easier binding to its tyrosine-kinase receptor; they bind chemokines at inflammatory sites, prolonging white-cell attracting activity; and they bind and block certain proteases.

Dally is an HSPG coreceptor for the Wingless growth factor in Drosophila.

Dally is a well known HSPG coreceptor (a glypican) that binds the Wingless protein in Drosophila. Mutations in Dally cause severe developmental defects, similar to those caused by mutations in Wingless or Frizzled themselves (the growth factor and its receptor respectively). Dally (division abnormally delayed) is a membrane-tethered HSPG (see the lecture on the Golgi body). Its GAG chain binds the Wingless (Wg) signalling molecule and helps it bind to the Frizzled receptor, setting off a signalling pathway that eventually increases the expression of particular genes by the transcription factor c-Myc.

Collagen and other proteins

Collagen is a fibrous protein that consists of three α-chains (which are not α-helices, 'helpfully' enough), which form a rope-like triple helix, providing tensile strength to the ECM.

Collagen.

α chains contain GXY repeats: glycine (G) is small, and is the only amino acid that fits in the crowded interior of the triple helix. X is usually proline, which destabilises the formation of a simple α-helix and Y is usually hydroxyproline; the hydrogen-bonding between the OH groups on this hydroxylated form of proline stabilises the triple helix. Hydroxyproline is formed post-translationally by the action of proline hydroxylase. This enzymes has a vitamin-C cofactor, which explains the symptoms of scurvy: tissues containing collagen (gums, skins, capillaries) are weakened, because the unhydroxylated collagen is destroyed without being secreted.

The synthesis of collagen molecules begins on the RER as with all secreted proteins. The pro-α-chains are made on the RER, and are hydroxylated and glycosylated (on hydroxylysines) in the Golgi. Procollagen forms from three α-chains, and possesses terminal 'propeptides'. This procollagen is then secreted from vesicles, and undergoes proteolysis at its ends in the extracellular space, to form mature 100 nm long collagen molecules.

Alpha chains are synthesised on the RER, processed in the Golgi to form procollagen, and then modified in the ECM to form mature collagen molecules.

Collagen molecules are then crosslinked into fibrils: oxidative deamination of hydroxylysine and lysine forms reactive aldehyde groups, which link molecules together (and also link α-chains together too).

Collagen molecules are crosslinked by oxidation.

Collagen fibrils then self-assemble into fibres, which form characteristically straited 'ropes' under EM. Collagen fibrils have a service life of 10 years or so: most enzymes turn-over in about an hour.

Collagen fibrils and fibres form by self-assembly of collagen molecules.

Collagen comes in many different types. Type I collagen is the most common fibrillar collagen (90%), and is found in skin, bone, tendons, etc. Type II collagen provides similar tensile strength to cartilage.

Other sorts of collagen do not form fibres: type IX collagen (and type XII) are fibril-associated collagens, which link type I (or type II) collagen fibrils together. They are more flexible than fibrillar collagens because the GXY-repeats of their α-chains are interrupted more frequently by other amino acids. Type IV and VII collagens are network-forming collagens; they form a meshwork, particularly in basal lamina.

Fibril-associated collagen molecules coat collagen fibres and link them together.

Collagen provides tensile strength to the ECM, but other proteins provide other properties. Elastin forms elastic fibres, which give the ECM its elasticity (surprise!). Elastin is secreted as the tropoelastin precursor. It is then cross-linked in similar way to collagen to form a stretchy net of elastin. Elastic fibres are coated with fibrillin microfibrils. Defects in the fibrillin-1 gene cause Marfan syndrome, which is characterised by weak elastic tissue, causing long fingers, pigeon chest, and the aorta to be very weak. Abraham Lincoln is thought to have had Marfan syndrome.

Elastin forms stretchy networks in elastic fibres.

Fibronectin

Cells bind to the ECM via a basal lamina or via fibronectin (nectere Lat. to tie). Fibronectin-mutant mice die as early embryos from an inability to make blood vessels. Fibronectin is evolutionarily related to fibrinogen (the protein that cross-links platelets together in blood clotting), and provides a similar 'stickiness' to the ECM that this clotting factor provides to blood.

Fibronectin has variable domains that bind other components of the ECM and cell surface receptors.

Negatively stained fibronectin; fibronectin schematic showing binding sites; fibronectin from x-ray crystallograph, showing four type-III repeats, the upper with an RGD binding site for integrin, the second the synergy site, which also binds cells.

Fibronectin has several binding domains, which can be separated and characterised by proteolysis. These different domains can bind heparin, collagen, cells (via integrins), or even itself. Fibronectin a a V-shaped dimer: the two chains differ from one another due to alternative splicing, and splicing is used to generate up to 20 different fibronectins from a single gene. Fibronectin therefore has a modular domain structure: so-called type-III repeats possess the RGD (arginine-glycine-aspartate) sequence and synergy sequence that bind α₅β₁ integrins, a ubiquitous transmembrane cell receptor.

Fibronectin type III repeats.

Disruption of RGD binding prevents binding of cells to the ECM, because this prevents cells from attaching to fibronectin. Competition for RGD binding from e.g. RGD tripeptides causes cells to detach from ECM. Disintegrin proteins from viper venom inhibit the fibrinogen/platelet interaction for the same reason: RGD is required for binding of fibrinogen to platelets.

The ECM - it's not just snot

The ECM is not just snot.

Basal lamina

The basal lamina (BL) is a specialised ECM found underlying epithelial cells: it coats muscle and fat cells, forms the glomerular filter in the kidney, and wraps the myelin sheath formed from Schwann cells on nerve axons. It is usually c. 80 nm thick.

The basal lamina plays several important roles

Structural.
Determines cell polarity.
Organises and binds cells.
Forms a barrier to certain cells.
Forms highways for cell migration.

A specialised basal lamina forms the glomerular basement membrane, which is the filter of the kidney, and confers most of the filter's specificity: note the gaps between the epithelial and endothelial cells.

The glomerular filter of the kidney is a specialised basal lamina.

Basal laminae also act as scaffolding for the repair of neuromuscular junctions: if a muscle cell and its attached nerve cell are cut, they degenerate. If the cells are allowed to regenerate separately, nerve axons are found to reattach at the same point on muscle. Likewise, the muscle cell expresses acetylcholine receptors at the same point. This is directed by a neuromuscular-junction specific protein called agrin, which is not present in the rest of the lamina.

Repair of the neuromuscular junction is aided by the non-random arrangement of the components of the basal lamina.

The basal lamina of stratified squamous epithelia (like skin) binds to the remaining ECM (connective tissue) via type VII collagen fibrils. Together the BL and ECM are termed a basement membrane. Type VII collagen mutations cause epidermolysis bullosa.

Like the rest of the ECM, the basal lamina is composed mostly of GAGs and proteins:

Laminin.
Type IV collagen.
Nidogen (entactin).
Perlecan.
Heparan sulfate is predominant GAG.

Laminin is the pioneer of the basal lamina. This is a 100 nm long sword-shaped trimer that self-assembles via its β/γ arms into a felt-like sheet.

Laminin is a sword-shaped trimer.

The ends of the 'sword' can bind cell receptors, and the crosspieces allow laminin to bind to other laminin molecules. Other sites for nidogen and perlecan binding are also present.

Type IV collagen is a network-forming collagen. This molecule is much bendier than type I, because the triple helix is interrupted repeatedly. There is also no cleavage of its propeptides after secretion, so its ends bind together to form a meshwork.

Type IV collagen forms networks in the basal lamina.

Type IV collagen forms networks. Each collagen IV molecule is shaped somewhat like a tadpole, with a globular propeptide head, and an interrupted triple helix tail.

Collagen IV is not present in the early development of basal lamina, so mutations are not necessarily lethal. Alport syndrome, which causes gross haematuria (blood in the urine) is caused by mutant type IV collagen. Sufferers often have foetal (underdeveloped) glomeruli.

Nidogen (from the Latin nidus nest and Greek γένεσις to create), also known as entactin, is a dumbbell shaped 150 kDa sulphated glycoprotein with three domains. It is not essential for basal lamina structure (mutant mice have a normal-appearing basal lamina), but it is needed for correct functioning.

Nidogen - laminin binding.

Nidogen - perlecan binding.

Nidogen bridges between the laminin and collagen layers of the basal lamina. Nidogen binding involves complex tertiary structures, as shown by these molecular fragments: nidogen-laminin binding with a 6-bladed β-propeller on the nidogen, and nidogen-perlecan binding with a β-barrel (which is very similar to that in green fluorescent protein).

Nidogen bridges the collagen and laminin layers of the basal lamina.

The predominant proteoglycan in the basal lamina is perlecan, which has a 400 kDa core protein with three heparan sulfate chains attached. It binds collagen, laminin, itself and nidogen, but its exact role in the BL is still hazy.

Perlecan is a proteoglycan with three heparan sulfate chains attached.

Perlecan has four pearl-like globular protein domains, with three heparan sulfate tails.

GAGs like perlecan may promote cell adhesion (perlecan crosslinks the vasal lamina); or they may disrupt it (heparin binds antithrombin and inhibits the conversion of prothrombin, so acts as an effective anticoagulant).

The basal lamina: a 3D meshwork.

The basal lamina is a 3D meshwork.
Basal lamina, showing laminin associating with transmembrane integrin receptors. Nidogen acts as a link between the laminin and the other components of the lamina, notably type IV collagen and perlecan.

Integrins and the regulation of cell behaviour

Integrins are adhesive membrane receptors. All integrins are heterodimeric transmembrane proteins. They differ from hormone receptors in that they have lower affinity for their ligands, and are present in higher numbers. They exhibit a Velcro effect: strength in numbers, but individually easy to disrupt. They require Ca²⁺ or Mg²⁺ to bind, and their job is to link the ECM to the cytoskeleton.

Integrins are transmembrane heterodimers.

Integrins are heterodimers. Most cell-matrix adhesion involves the β₁ subunit, and specificity is achieved by different α subunits.

Different integrins bind different components of the ECM.

There are 24 α types and 9 β types - β₁ binds ECM proteins which combine to form many different integrins with varied roles and cell specificities:

α₅β₁ binds fibronectin.
α₆β₁ binds laminin.
α₇β₁ binds laminin in muscle.

β₁ mutant mice die on implantation because their ECM is unable to form. α₇ mutant mice develop muscular dystrophy, because their ECM forms, but is unable to bind muscle cells effectively.

Cell-cell and cell-matrix adhesion involves a vast array of different systems. Tight junctions (formed by claudins) form impermeable barriers. Adhesion belts (adherens junctions) and desmosomes (formed by cadherin proteins) serve to link the actin and intermediate filament components of the cytoskeleton respectively. Gap junctions formed by connexins allow ion exchange between cells. CAMs (immunoglobulin-like receptors), selectins and integrins form non-communicating junctions. Integrins also link the cell to the basal lamina, either alone, or by interaction with actin (focal adhesions), or the intermediate filaments (hemidesmosomes). Membrane proteoglycans also bind the cell to the ECM.

Summary of cell adhesion molecules.

Integrins are mostly found in focal adhesions and hemidesmosomes. The binding of integrins to the ECM promotes activity of FAK (focal adhesion kinase) and the Src tyrosine kinase. This promotes cell survival by altering gene expression. The BL can self-assemble, but this is faster when it is bound to integrins.

Integrins are found in focal adhesions.
Binding of integrins to the basal lamina leads to the formation of focal adhesions, which recruit FAK. FAK recruits Src, a tyrosine kinase which both phosphorylates FAK (activating it), and phosphorylates other proteins (signalling cascades) in the cell. Focal adhesions therefore act as localised phosphorylation 'factories'.

Integrins also participate in cell-cell adhesion. Immunoglobulin-like counter-receptors on other cells (such as epithelial cells) bind the β₂ type integrins found on white blood cells. This recruits WBC to infection sites.

Integrins bind immunoglobulin-like counter-receptors, which aids the recruitment of leucocytes to infection sites on epithelial tissues.

Integrins on white blood cells bind immunoglobulin-like counter-receptors on epithelial cells during response to infection. This slows the migration of WBCs, and keeps them attached to the infected site.

Integrins can also perform inside out signalling: the β subunit binds to cytoplasmic talin, and thence to actin, which is how integrins link the ECM to the cytoskeleton. Phosphorylation of the integrin cytoplasmic tails prevents binding to talin, and dissociates integrin from both cytoskeleton and ECM.

Integrin bridges between the ECM and the actin cytoskeleton.

Integrins integrate the ECM (here fibronectin) and the cytoskeleton (here an actin microfilament). Regulation is possible from both directions: binding of fibronectin to integrins allows the action of FAK; but phosphorylation (probably - the details are still sketchy) of the integrin from within causes it to dissociate from cytoskeleton-binding proteins such as talin. It seem likely that this also causes conformational changes in the extracellular portion of the integrin, loosening its grip on the ECM.

Anchoring to the ECM or basal lamina is critical for cell survival. Cells that fail to anchor often apoptose. For example, during embryogenesis, ectoderm cells are induced to apoptose by chemical signals from endoderm cells in the formation of a proamniotic cavity. Only those endoderm cells attached to the basal lamina survive.

Only cells that anchor to the basal lamina survive in the formation of the proamniotic cavity.

Fibronectin is important for cell survival: finely distributed fibronectin works better than lumps in promoting cell adhesion and survival in vitro: spread-out cells survive better, and proliferate more readily. Cancer cells are often fibronectin-deficient mutants, which makes them fail to adhere to the ECM and thence to metastasise (migrate to other sites). Other mutations cause the cell to fail to apoptose if they don't bind.

Cell migration through the ECM requires proteases. The ECM is continuously degraded by proteases and resynthesised. Degradation can also be inhibited by the presence of protease inhibitors, which obviously inhibit cell migration. Non-adherent paths are cleared through the ECM for cells to migrate using serine proteases and metalloproteases. Proteases are regulated in several ways: plasminogen is secreted as a zymogen called plasminogen, which is only converted to the protease plasmin (which dissolves blood clots) when activated. Activators can be tethered to receptors (like urokinase plasminogen activator - uPA), which is implicated in the metastasis of prostate cancer cells. Alternatively, the activators may be soluble (tissue type plasminogen activators - tPA), which is used to treat strokes and heart attacks.

uPA is a protease activator that is tethered to receptors on migrating cells.

Prostate cancer cell metastasis relies on the cell binding uPA, which allows it to 'drill' through the basal lamina and escape into the capillary circulation.

The ECM and cytoskeleton orient each other: the deposition of the ECM is oriented by the cytoskeleton; but the orientation of the cytoskeleton is then influenced by its binding to talin and thence via integrins to the ECM. Cytoskeletal orientation and cell polarity can therefore propagate from cell to cell via the ECM.

The ECM around a cell and the cytoskeleton within orient one another.

Cells with oriented (polarised) cytoskeletons knit a similarly oriented ECM. Through interactions of this ECM material with the integrins on un-oriented cells, cytoskeletal orientation is propagated through populations of cells.

Summary

The ECM contains GAGs (such as hyaluronan) and proteoglycans, which provide compressive strength and interact with growth factors.
Tensile strength is provided by collagen and elastic fibres: collagen is secreted as a precursor, and is rich in modified amino acids.
Binding to cells via integrins is effected by fibronectin, which also binds collagen, HSPGs and itself.
The basal lamina underlies epithelial cells, and is composed mostly of a mesh of laminin, collagen IV, nidogen and HSPGs such as perlecan.
Binding of the cytoskeleton to ECM and BL is mainly achieved through integrins, and is critical to cell survival.
Migration through the ECM requires proteases and integrin deactivation.

Test yourself

How does hyaluronan differ from the typical GAGs found in proteoglycans?
Which molecules provide the following properties of the ECM?
- Compressive strength;
- Elasticity;
- Tensile strength;
- Cell adhesion;
- Water retention.
- Control of cell behaviour.

Complete the table to show the main differences between connective tissue and the basal lamina.

	Collagen	Anchor	Cell receptor	Secreted by	Proteoglycans
Connective tissue
Basal lamina

Answers

Bibliography

Alberts, B., et al. (2002). Molecular biology of the cell. 4th edition. Garland Science, New York. 1090-1113. "The extracellular matrix of animals"
Alberts, B., et al. (2002). Molecular biology of the cell. 4th edition. Garland Science, New York. 1113-1118. "Integrins"
Critchley, D. R. (2000). Focal adhesions - the cytoskeletal connection. Current Opinion in Cell Biology 12:133-139. http://dx.doi.org/10.1016/S0955-0674(99)00067-8
Mercurio, A. M. (1995). Laminin receptors: achieving specificity through cooperation. Trends in Cell Biology 5:419-423. http://dx.doi.org/10.1016/S0962-8924(00)89100-X

This page has been peer reviewed by 2 people. Thanks to Craig Clifford for his correction.

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