Image: Bochaton-Piallat, M.L. et al. 2000. Invest Ophthalmol Vis Sci. 41:2336-42.

Extracellular Matrix
Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 1

ECM: Definition and Function

The acellular material around cells is called extracellular matrix (ECM)

Function:
• Mechanical support for cells and
tissues.
• Integrates cells into tissues.
• Influences cell shape and cell
movement.
• Influences cell development and
differentiation.
• Coordinates cellular functions
through signaling with cellular
adhesion receptors.
• Reservoir for extracellular
signaling molecules.

Structure of ECM:
• Fibers: collagen and elastin, which provide strength and flexibility.
• Proteoglycans: protein-saccharide complexes, providing a voluminous matrix.
• Adhesive glycoproteins: ‘glue’ cells and ECM, e.g. fibronectin and laminin.

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 2

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 Intermezzo: Connective Tissues
A histological classification:
• Loose connective tissue: supports epithelia and organs.
• Dense connective tissue:
Irregular: forms protective capsules around organs.
Regular: mechanical resistance, connection of tissues
(ligaments and tendons).
• Adipose tissue: insulates, protects, energy store
(subcutis, renal pelvis, kidneys, mammary glands).
• Reticular tissue: framework of lymphatic tissue
(spleen, lymph nodes, bone marrow, liver).
• Blood: transport, immune defense.
• Cartilage: provides support and flexibility
(rib cage, trachea, bronchi, joints).
• Bone: body support.

Specialized cells reside in the ECM:

–blasts: create the ECM
–cytes: maintain the ECM
–clasts: ECM breakdown and remodeling

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 3

Fibrous ECM Proteins: Collagen

• Collagens make up ~25% of total
protein mass in mammals
• Collagen comprises 25 isoforms.
• Collagen type I, II, and III are
organized in fibers.
• Each fiber consists of fibrils.
• Fibrils contain collagen molecules,
made of three coiled polypeptide
α-chains.
• Collagens are rich in proline
(stabilizes helical structure) and
glycine (allows the dense packing
of three α-chains).

• Exists in large quantity in ECM of skin, bone,

tendon, cartilage and other connective tissues for
support and protection.
• Collagen fibers withstand high pulling forces.
• Elastic modulus is ~1 GPa.
• It takes 10 kg to rupture a fiber of 1 mm.
Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 4

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 Fibrous ECM Proteins: Collagen

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 5

Collagen Synthesis and Assembly

• ER/Golgi: Pro-a-chains are produced,
hydroxylated and glycosylated at
selected Lys and Pro residues.
• The lack of vitamin C prevents
hydroxylation → impaired fibril
formation (scurvy).
• Processed pro-peptides assemble into
triple-helical pro-collagen.
• Golgi: Disulphide bonds form between
the N- and C-termini of procollagen.
• After exocytosis, N- and C-termini are
trimmed, allowing fibril assembly

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 6

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 Fibrous ECM Proteins: Elastin
• Elastic fibers permit long-range deformability and passive recoil.
• Elastic modulus is ~0.1 MPa.
• This function is crucial for arteries, lung, skin and other dynamic connective tissues that
undergo cycles of extension and recoil.
• The major component of elastic fibers is the thread-like protein elastin
• Fibrillins provide an outer structure for amorphous, cross-linked elastin.
• During ageing, elastin is degraded
and becomes inflexible.

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Kielty, C.M. et al. 2002. J Cell Sci. 115:2817-28. Slide 7

Adhesive Glycoproteins: Fibronectin (FN)

• Adhesive glycoproteins like fibronectin and laminin mediate
the connection between the ECM and the cell membrane.
• Adhesive proteins bind to proteoglycans, collagens and to
transmembrane ECM-receptors (integrins).

• Fibronectin is a dimer of two identical 250 kDa subunits.

• Alternative splicing of one gene produces ~20 human FNs.
• FN exists as a soluble form (plasma FN) and cellular FN.
• Plasma FN is predominantly produced in the liver.
• Cellular FN is deposited into the ECM by a cell-mediated
process, called FN fibrillogenesis.
• FN binds a variety of other proteins like integrins, heparin,
Fibrin – blood clotting protein collagen, fibrin. It also self-interacts.
Heparin – anti-clotting protein
RGD - Arg-Gly-Asp residues

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 8

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 Intermezzo: Splicing

Genome Transcriptome Proteome

DNA RNA Protein

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 9

Intermezzo: Splicing

Genome: 5’ Intergenic Gene Intergenic 3’

Transcription of pre-mRNA
5’ Exon Intron Exon Intron Exon 3’
GT AG GT AG
Donor Site Acceptor Site

Splicing to mRNA
Transcriptome: 5’ Exon Exon Exon 3’

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 10

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 Adhesive Glycoproteins: Laminin (LN)

• LNs are cross-shaped proteins.

• All LN isoforms contain α, β1 and β2 chains that
are connected by disulfide bonds.
• LN binds to membrane receptors (integrins) of
the overlying cells.
• LN attaches cells to the basal lamina.
• LN contains binding sites for other components
of the basal lamina: type IV collagen, heparin,
Miner, J.H. 2004. Annu Rev Cell Dev Biol. heparan sulfate and entactin.
Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 11

Intermezzo: The Basement Membrane (BM)

The BM (basal lamina) separates epithelial
cells and underlying connective tissue

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Kalluri, R. 2003. Nat Rev Cancer. 3:422-33. Slide 12

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 BM: Collagen IV
• Collagen IV is the most abundant
protein in the BM
• Collagen IV is usually found in
BM’s but also associates with
fibrotic reactions and occurs in
the stroma surrounding tumors
• Like other collagens it forms
triple-helices, which however
contain a non-collageneous (NC1)
globular domain and a 7S domain
• These domains promote self-
assembly into organized networks
• ‘Network-forming collagen’

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Kalluri, R. 2003. Nat Rev Cancer. 3:422-33. Slide 13

Adhesive ECM Proteins: Collagen

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 14

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 Glycosaminoglycans (GAGs)
GAGs are hydrophilic polymers of disaccharides
that form a gelatinous matrix

• GAGs are highly negatively charged.

• Very rigid due to charge repulsion
-> obtain a large volume.
• Attract osmotically active cations;
thus they bind water and swell.
• This results in turgor pressure and allows
connective tissue to resist compression.
• GAGs differ in the type of sugars in the
disaccharide in their chemical bond.

The most common GAGs are:

Hyaluronan (HA) – glucuronate + N-acetyl-glucosamine
Chondrotin sulfate – glucuronate + N-acetyl-galactosamine
Heparan sulfate – glucuronate + N-sulfo-glucosamine
Keratan sulfate – galactose + N-acetyl-glucosamine

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 15

Hyaluronan (HA)

• HA is a large, unbranched and negatively charged

polymer of repeating (2-25K) disaccharides.
• Like other GAGs it may be part of proteoglycans.
• Unlike other GAGs it is synthesized freely at the
plasma membrane by hyaluronan synthases.
• Free HA is found in the ECM of migrating cells.
• HA binds to cell surface receptors
(CD44, RHAMM).
• HA binds proteoglycans.

fibrosarcoma cells + hyaluronase

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Toole, B.P. 2004. Nat Rev Cancer. 4:528-39. Slide 16

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 Proteoglycans
• Proteoglycans consist of a core protein to which GAGs are covalently coupled
• The protein-GAG linkage is always made between Ser and the 3-sugar ‘linker’
Xyl-Gal-Gal, followed by Glucoronic acid.
• Proteoglycans are found both in ECM and attached to the plasma membrane

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Couchman, J.R. 2003. Nat Rev Mol Cell Biol. 4:926-37. Slide 17

ECM Proteoglycans: Aggrecan

• In cartilage the key proteoglycan is aggrecan (MW: 2 x 108)
• The central component of aggrecan is hyaluronan
• At 40 nm intervals aggrecan core proteins are attached (assisted by a linker
protein) to a decasaccharide sequence in hyaluronan
• Attached to the aggrecan core protein are multiple GAGs
• The major GAGs in aggrecan are
chondroitin sulphate and keratin sulphate

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 18

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 Cell Surface Proteoglycans: Syndecan
• Membrane-associated proteoglycans are mostly heparan-sulfate substituted
and are either transmembrane like syndecan or GPI-membrane-anchored
• The core protein spans the membrane with a short cytosolic domain
• The GAGs (heparan sulfate chains) are attached via the trisaccharide linker to
Ser residues
• Syndecan binds extracellularly to collagens and fibronectin and intracellularly
to the cytoskeleton

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Couchman, J.R. 2003. Nat Rev Mol Cell Biol. 4:926-37. Slide 19

ECM Mechanics Influence Cell Behavior

Polyacrylamide gel substrates are used
as compliant culture substrates:

biological tissues

Bao, G., and S. Suresh. 2003. Nat Mater. 2:715-25

Spreading of endothelial cells increases with stiffness:

epithelial cells

fibroblasts
Yeung, T. 2005. Cell Motil Cytoskeleton. 60:24-34.

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Pelham and Wang. 1997. Proc Natl Acad Sci U S A. 94:13661-5. Slide 20

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 ECM Mechanics Influence Differentiation
Fibroblasts in collagen gels:

2h Myotube differentiation on compliant PAG substrates:

less compliant is not necessarily better!
-> Different cell types have different requests on matrix rigidity.

5h
stiffness

72h

Engler, A.J. et al., 2004. J Cell Biol. 166:877-87.

Hinz, B. 2005. Eur J Cell Biol. (in press)

Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Slide 21

Mechanical ECM Properties: Durotaxis

3T3 fibroblasts migrate toward stiff regions:
stiffness gradient in PAG substrates
visualized with mixed microbeads

binding of antibody-covered
microbeads to collagen on the surface

fibroblast eventually fibroblast eventually

continues toward the turns and continues on
stiff substrate the stiff substrate
Extracellular Matrix, 01/07, Boris Hinz, PhD,EPFL/SB/IPMC/LCB Lo, C.M. et al. 2000. Biophys J. 79:144-52 Slide 22

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 Literature
Images and movies appearing in this course have been assembled from the following
books/manuscripts or the corresponding web-pages; they have been used for teaching
purposes only. I apologize for occasionally presenting figures without proper citing.
• Bachmann, A., T. Kiefhaber, S. Boudko, J. Engel, and H.P. Bachinger. 2005. Collagen triple-helix formation in all-trans chains proceeds by a nucleation/growth mechanism with a
purely entropic barrier. Proc Natl Acad Sci U S A. 102:13897-902.
• Bao, G., and S. Suresh. 2003. Cell and molecular mechanics of biological materials. Nat Mater. 2:715-25.
• Bochaton-Piallat, M.L., A.D. Kapetanios, G. Donati, M. Redard, G. Gabbiani, and C.J. Pournaras. 2000. TGF-beta1, TGF-beta receptor II and ED-A fibronectin expression in
myofibroblast of vitreoretinopathy. Invest Ophthalmol Vis Sci. 41:2336-42.
• Bornstein, P., and E.H. Sage. 2002. Matricellular proteins: extracellular modulators of cell function. Curr Opin Cell Biol. 14:608-16.
• Bosman, F.T., and I. Stamenkovic. 2003. Functional structure and composition of the extracellular matrix. J Pathol. 200:423-8.
• Brinckerhoff, C.E., and L.M. Matrisian. 2002. Matrix metalloproteinases: a tail of a frog that became a prince. Nat Rev Mol Cell Biol. 3:207-14.
• Brown, N.H. 2000. An integrin chicken and egg problem: which comes first, the extracellular matrix or the cytoskeleton? Curr Opin Cell Biol. 12:629-33.
• Couchman, J.R. 2003. Syndecans: proteoglycan regulators of cell-surface microdomains? Nat Rev Mol Cell Biol. 4:926-37.
• Egeblad, M., and Z. Werb. 2002. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer. 2:161-74.
• Engler, A.J., M.A. Griffin, S. Sen, C.G. Bonnemann, H.L. Sweeney, and D.E. Discher. 2004c. Myotubes differentiate optimally on substrates with tissue-like stiffness:
pathological implications for soft or stiff microenvironments. J Cell Biol. 166:877-87.
• Georges, P.C., and P.A. Janmey. 2005. Cell type-specific response to growth on soft materials. J Appl Physiol. 98:1547-53.
• Henry, M.D., and K.P. Campbell. 1999. Dystroglycan inside and out. Curr Opin Cell Biol. 11:602-7.
• Kalluri, R. 2003. Basement membranes: structure, assembly and role in tumour angiogenesis. Nat Rev Cancer. 3:422-33.
• Kielty, C.M., M.J. Sherratt, and C.A. Shuttleworth. 2002. Elastic fibres. J Cell Sci. 115:2817-28.
• Lee, J.Y., and A.P. Spicer. 2000. Hyaluronan: a multifunctional, megaDalton, stealth molecule. Curr Opin Cell Biol. 12:581-6.
• Lo, C.M., H.B. Wang, M. Dembo, and Y.L. Wang. 2000. Cell movement is guided by the rigidity of the substrate. Biophys J. 79:144-52.
• Lorand, L., and R.M. Graham. 2003. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol. 4:140-56.
• Miner, J.H., and P.D. Yurchenco. 2004. Laminin Functions in Tissue Morphogenesis. Annu Rev Cell Dev Biol.
• Mott, J.D., and Z. Werb. 2004. Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol. 16:558-64.
• Pankov, R., and K.M. Yamada. 2002. Fibronectin at a glance. J Cell Sci. 115:3861-3.
• Pelham, R.J., Jr., and Y. Wang. 1997. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci U S A. 94:13661-5.
• Sasisekharan, R., Z. Shriver, G. Venkataraman, and U. Narayanasami. 2002. Roles of heparan-sulphate glycosaminoglycans in cancer. Nat Rev Cancer. 2:521-8.
• Schenk, S., and V. Quaranta. 2003. Tales from the crypt[ic] sites of the extracellular matrix. Trends Cell Biol. 13:366-75.
• Sherratt, M.J., C. Baldock, J.L. Haston, D.F. Holmes, C.J. Jones, C.A. Shuttleworth, T.J. Wess, and C.M. Kielty. 2003. Fibrillin microfibrils are stiff reinforcing fibres in
compliant tissues. J Mol Biol. 332:183-93.
• Thery, M., V. Racine, A. Pepin, M. Piel, Y. Chen, J.B. Sibarita, and Bornens. 2005. The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol. 7:947-
53.
• Timpl, R., T. Sasaki, G. Kostka, and M.L. Chu. 2003. Fibulins: a versatile family of extracellular matrix proteins. Nat Rev Mol Cell Biol. 4:479-89.
• Toole, B.P. 2004. Hyaluronan: from extracellular glue to pericellular cue. Nat Rev Cancer. 4:528-39.
• Walpita, D., and E. Hay. 2002. Studying actin-dependent processes in tissue culture. Nat Rev Mol Cell Biol. 3:137-41.
• Wierzbicka-Patynowski, I., and J.E. Schwarzbauer. 2003. The ins and outs of fibronectin matrix assembly. J Cell Sci. 116:3269-76.
• Wight, T.N. 2002. Versican: a versatile extracellular matrix proteoglycan in cell biology. Curr Opin Cell Biol. 14:617-23.
• Yeung, T., P.C. Georges, L.A. Flanagan, B. Marg, M. Ortiz, M. Funaki, N. Zahir, W. Ming, V. Weaver, and P.A. Janmey. 2005. Effects of substrate stiffness on cell morphology,
cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton. 60:24-34.
• Yurchenco, P.D., and W.G. Wadsworth. 2004. Assembly and tissue functions of early embryonic laminins and netrins. Curr Opin Cell Biol. 16:572-9.

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