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Thus, when fibroblasts aremixed with a meshwork of randomly oriented collagen fibrils that form a gel ina culture dish, the fibroblasts tug on the meshwork, drawing in collagen fromtheir surroundings and thereby causing the gel to contract to a small fraction ofits initial volume. By similar activities, a cluster of fibroblasts surrounds itselfwith a capsule of densely packed and circumferentially oriented collagen fibers.If two small pieces of embryonic tissue containing fibroblasts are placed farapart on a collagen gel, the intervening collagen becomes organized into a compact band of aligned fibers that connect the two explants (Figure l9-69). Thefibroblasts subsequently migrate out from the explants along the aligned collagen fibers.
Thus, the fibroblasts influence the alignment of the collagen fibers,and the collagen fibers in turn affect the distribution of the fibroblasts.Fibroblasts may have a similar role in organizing the extracellular matrixinside the body, first of all synthesizing the collagen fibrils and depositing themin the correct orientation, then working on the matrix they have secreted,crawling over it and tugging on it so as to create tendons and ligaments and the tough,dense layers of connective tissue that ensheatheand bind together most organs.5pmFigure19-67 Collagenfibrilsin thetadpoleskin.Thiselectronmicrographshowsthe plywoodlikearrangementoflayersoffibrilsarethe fibrils:successivelaiddown nearlyat right anglesto eachis alsofound inother.Thisorganizationmatureboneand in the cornea.(Courtesyof JeromeGross.)ElastinGivesTissuesTheirElasticityMany vertebrate tissues,such as skin, blood vessels,and lungs, need to be bothstrong and elastic in order to function.
A network of elastic fibers in the extracellular matrix of these tissues gives them the required resilience so that theyFigure19-68TypelX collagen.(A)TypelX collagenmoleculesbindingin aperiodicpatternto the surfaceof a fibrilcontainingtype ll collagen.type-ll-collagen-containing(B)Electronmicrographof a rotary-shadowed(C)An individualsheathedin type lX collagenmolecules.fibrilin cartilage,type lX collagenmolecule.(Band C,from L.Vaughanet al',J. CellBiol.UniversityPress.)With permissionfrom The Rockefeller106:991-997,1988.I 190Chapter19:CellJunction5 Cell Adhesion,and'the ExtracellularMatrixcan recoil after transient stretch (Figure 19-70).
Elastic fibers are at least fivetimes more extensible than a rubber band of the same cross-sectional area.Long, inelastic collagen fibrils are interwoven with the elastic fibers to limit theextent of stretching and prevent the tissue from tearing.The main component of elastic fibers is elastin, a highly hydrophobic protein (about 750 amino acids long), which, like collagen, is unusually rich in proline and glycine but, unlike collagen, is not glycosylated and contains somehydroxyproline but no hydroxylysine. Soluble tropoelastin (the bioslnthetic precursor of elastin) is secreted into the extracellular space and assembled intoelastic fibers close to the plasma membrane, generally in cell-surfaceinfoldings.After secretion, the tropoelastin molecules become highly cross-linked to oneanother, generating an extensive network of elastin fibers and sheets.A mechanism similar to the one that operates in cross-linking collagen molecules formscross-links between the lysines.The elastin protein is composed largely of two types of short segments thatalternate along the polypeptide chain: hydrophobic segments, which areresponsible for the elastic properties of the molecule; and alanine- and lysinerich a-helical segments, which form cross-links between adjacent molecules.Each segment is encoded by a separateexon.
There is still uncertainty concerning the conformation of elastin molecules in elastic fibers and how the structureof these fibers accounts for their rubberlike properties. However, it seems thatparts of the elastin polypeptide chain, like the polymer chains in ordinary rubber, adopt a loose "random coil" conformation, and it is the random coil natureof the component molecules cross-linked into the elastic fiber network thatallows the network to stretch and recoil like a rubber band (Figure 19-71).Elastin is the dominant extracellular matrix protein in arteries, comprising50% of the dry weight of the largest artery-the aorta.
Mutations in the elastingene causing a deficiency of the protein in mice or humans result in narrowingof the aorta or other arteries and excessiveproliferation of smooth muscle cellsin the arterial wall. Apparently, the normal elasticity of an artery is required torestrain the proliferation ofthese cells.Elastic fibers do not consist solely of elastin.
The elastin core is coveredwitha sheath of microftbrils, each of which has a diameter of about l0 nm. Themicrofibrils appear before elastin in developing tissues and seem to providescaffolding to guide elastin deposition. Arrays of microfibrils are elastic in theirov,n right, and in some places they persist in the absenceof elastin: they help tohold the lens in its place in the eye, for example. Microfibrils are composed of anumber of distinct glycoproteins, including the large glycoprotein fibrillin,which binds to elastin and is essential for the integrity of elastic fibers. Mutations in the fibrillin gene result in Marfan's syndrome, a relatively commonhuman genetic disorder.
In the most severely affected individuals the aorta is?Figure 19-69 The shaping of theextracellularmatrix by cells.Thismicrographshowsa regionbetweentwopiecesof embryonicchickheart(richinfibroblastsaswell as heartmusclecells)that were culturedon a collagengel for4 days.A densetract of alignedcollagenfibershasformed betweenthe explants,presumablyas a resultof the fibroblastsin the explantstuggingon the collagen.(FromD. Stopakand A.K.Harris,Dev.Biol.90:383-398,1982.With permissionfromAcademicPress.)Figure 19-70 Elasticfibers.Thesescanningelectronmicrographsshow(A)a low-powerview of a segmentof a dog'saorta and (B)a high-powerview of thedensenetworkof longitudinallyorientedelasticfibersin the outerlayerof thesamebloodvessel.All the othercomponentshavebeen digestedawaywith enzymesand formic acid.(FromK.S.Haaset al.,AnaL Rec.230:86-96,1991.With permissionfrom Wiley-Liss.)TIsSUESTHEEXTRACELLULARMATRIXOFANIMALCONNECTIVEFigure19-71 Stretchinga networkofareThe moleculeselastinmolecules.joined together by covalentbonds (red)network.Into generatea cross-linkedthis model,eachelastinmoleculein thenetworkcan extendand contractin aa randomcoil,so thatmannerresemblingcan stretchand recoilthe entireassemblylikea rubberband.e l a s t i cf i b e rSTRETCH11 9 1RELAXs i n g l ee l a s t i nm o l e c u l eprone to rupture; other common effects include displacement of the lens andabnormalities of the skeleton and joints.
Affected individuals are often unusually tall and lanky: Abraham Lincoln is suspectedto have had the condition.ls an ExtracellularProteinThatHelpsCellsAttachtoFibronectinthe MatrixThe extracellular matrix contains a number of noncollagen proteins that tlpically have multiple domains, each with specific binding sites for other matrixmacromolecules and for receptors on the surface of cells.These proteins therefore contribute to both organizing the matrix and helping cells attach to it. Likethe proteoglycans, they also guide cell movements in developing tissues, byserving as tracks along which cells can migrate or as repellents that keep cellsout offorbidden areas.The first of this class of matrix proteins to be well characterized wasfibronectin, a large glycoprotein found in all vertebrates and important formany cell-matrix interactions.
Thus, for example, mutant mice that are unableto make fibronectin die early in embryogenesis because their endothelial cellsfail to form proper blood vessels.The defect is thought to result from abnormalities in the interactions of these cells with the surrounding extracellular matrix,which normally contains fibronectin.Fibronectin is a dimer composed of two very large subunits joined by disulfide bonds at one end. Each subunit is folded into a seriesof functionally distinctdomains separated by regions of flexible pollpeptide chain (Figure l9-72). Thedomains in turn consist of smaller modules, each of which is serially repeatedand usually encoded by a separate exon, suggestingthat the fibronectin gene,like the collagen genes, evolved by multiple exon duplications.
In the humangenome, there is only one fibronectin gene, containing about 50 exons of simiIar size,but the transcripts can be spliced in different ways to produce many different fibronectin isoforms. Sitesin the main tlpe of module, called the type IIIfibronectin repeat, bind to integrins and thereby to cell surfaces.
This module isabout 90 amino acids long and occurs at least 15 times in each subunit. The tlpeIII fibronectin repeat is among the most common of all protein domains in vertebrates.of Fibronectinthe AssemblyTensionExertedby CellsRegulatesFibrilsFibronectin can exist both in a soluble form, circulating in the blood and otherbodv fluids, and as insoluble fibronectin fibrils, in which fibronectin dimers are1192(A)Chapter19:CellJunctions,CellAdhesion,and the ExtracellularMatrix(c)Figure19-72Thestructureof a fibronectindimer.(A)Electronmicrographsof individualfibronectindimermoleculesshadowedwith platinum;redarrowsmarkthe C-termini.(B)Thetwo polypeptidechainsaresimilarbut generallynotidentical(beingmadefrom the samegenebut from differentlysplicedmRNAs).Theyarejoined by two disulfidebondsnearthe C-termini.Eachchainis almost2500aminoacidslong and isfoldedinto fiveor sixdomainsconnectedby flexiblepolypeptidesegments.Individualdomainscontainmodulesspecializedfor bindingto a particularmoleculeor to a cell,asindicatedfor five of the domains.Forsimplicity,not all of the knownbindingsitesareshown(thereareothercell-binding(C)Thethree-dimensionalsites,for example).structureof two type lll fibronectinrepeatmodulesasdeterminedby x-raycrystallography.Thetype lll repeatis the mainrepeatingmodulein fibronectin.Boththe Arg-Gly-Asp(RGD)and the"synergy/'sequencesshownin redform part of the majorcell-bindingsite,and areimportantfor binding.(A,from J.
Engelet al.'J. Mol.Biol.150:97-120,1981.With permissionfrom AcademicPress;C,from DanielJ.Leahy,Annu.Rev.CellDev.Biol.13:363-393,1997.With permissionfrom AnnualReviews.)cross-linked to one another by additional disulfide bonds and form part of theextracellular matrix. Unlike fibrillar collagen molecules, however, which canself-assembleinto fibrils in a test tube, fibronectin molecules assembleinto fib-molecules to form a fibril (Figure l9-74).This dependence on tension and interaction with cell surfacesensures that fibronectin fibrils assemblewhere there isa mechanical need for them and not in inappropriate locations such as thebloodstream.Figure19-73 Unfoldingof a type lllfibronectin domain in responsetotension.Thestretchingof fibronectininthis way exposescrypticbindingsitesthat causethe stretchedmoleculestoassembleinto filaments,as showninFigure19-74.(FromV.VogelandM.
Sheetz,Nat. Rev.Mol. CellBiol.7:265-275, 2006.With permissionfromMacmillanPublishersLtd.)TISSUEsTHEEXTRACELLULARMATRIXOFANIMALCONNECTIVEr 193Many otherextracellularmatrixproteinshavemultiple repeatssimilarto thetype III fibronectinrepeat,and it is possiblethat tensionexertedon theseproteins alsouncoverscrlptic binding sitesand therebyinfluencestheir polymerization.Throughan RGDMotifFibronectinBindsto IntegrinsOne way to analyze a complex multifunctional protein molecule such asfibronectin is to chop it into pieces and determine the function of its individualdomains. \A/henfibronectin is treated with a low concentration of a proteolytice\zyme, the pollpeptide chain is cut in the connecting regions between thedomains, leaving the domains themselvesintact. One can then show that one ofits domains binds to collagen, another to heparin, another to specific receptorson the surface ofvarious types ofcells, and so on (seeFigure 19-728).
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