Assembly of Fibronectin ECM.pdf

CB26CH16-Schwarzbauer ARI 25 August 2010 21:21

Assembly of FibronectinExtracellular MatrixPurva Singh, Cara Carraher,and Jean E. SchwarzbauerDepartment of Molecular Biology, Princeton University, Princeton,New Jersey 08544-1014; email: [email protected]

Annu. Rev. Cell Dev. Biol. 2010. 26:397–419

First published online as a Review in Advance onJuly 2, 2010

The Annual Review of Cell and DevelopmentalBiology is online at cellbio.annualreviews.org

This article’s doi:10.1146/annurev-cellbio-100109-104020

Copyright c© 2010 by Annual Reviews.All rights reserved

1081-0706/10/1110-0397$20.00

Key Words

integrins, conformational change, insolubility, fibrillar, type Icollagen, microfibrils

Abstract

In the process of matrix assembly, multivalent extracellular matrix(ECM) proteins are induced to self-associate and to interact with otherECM proteins to form fibrillar networks. Matrix assembly is usuallyinitiated by ECM glycoproteins binding to cell surface receptors, suchas fibronectin (FN) dimers binding to α5β1 integrin. Receptor bindingstimulates FN self-association mediated by the N-terminal assemblydomain and organizes the actin cytoskeleton to promote cell contrac-tility. FN conformational changes expose additional binding sites thatparticipate in fibril formation and in conversion of fibrils into a stabi-lized, insoluble form. Once assembled, the FN matrix impacts tissueorganization by contributing to the assembly of other ECM proteins.Here, we describe the major steps, molecular interactions, and cellu-lar mechanisms involved in assembling FN dimers into fibrillar matrixwhile highlighting important issues and major questions that requirefurther investigation.

397

Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


ECM: extracellularmatrix

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 398FIBRONECTIN DOMAIN

ORGANIZATION . . . . . . . . . . . . . . . . 399Domains and Binding Activities . . . . . 399Domains Required for Fibronectin

Matrix Assembly . . . . . . . . . . . . . . . . 399Other Fibronectin Binding Sites . . . . 401

A FIBRONECTIN MATRIXASSEMBLY MODEL . . . . . . . . . . . . . 402Fibronectin Dimer Secretion and

Integrin Binding . . . . . . . . . . . . . . . . 402Fibronectin-Fibronectin

Interactions. . . . . . . . . . . . . . . . . . . . . 404Tethering of Fibronectin at the

Cell Surface . . . . . . . . . . . . . . . . . . . . 405Does Fibronectin Unfold? . . . . . . . . . . . . . 406Fibronectin-Integrin Bond Strength

and Signaling . . . . . . . . . . . . . . . . . . . . . . 407Fibril Maturation and Conversion

to Insolubility. . . . . . . . . . . . . . . . . . . 408Fibronectin Matrix Turnover . . . . . . . 409

FIBRONECTIN-DEPENDENTASSEMBLY OF OTHEREXTRACELLULAR MATRIXPROTEINS . . . . . . . . . . . . . . . . . . . . . . . 410Fibronectin in Microfibril and

Elastic Fiber Assembly . . . . . . . . . . 410Type I Collagen Assembly. . . . . . . . . . 410Fibronectin Matrix and Disease . . . . . 411

INTRODUCTION

The extracellular matrix (ECM) has been rec-ognized as an essential structural componentin multicellular organisms for millennia (Platotrans. 1965). However, the old view of ECMas an inert scaffold is clearly incorrect. ECMis a dynamic network, a reservoir for growthfactors and fluids, and an essential organizer oftissues, cellular microenvironments, and stemcell niches. It shows exquisite tissue specificityand adapts to changes in age, development, anddisease. Even so, the molecular events that as-semble secreted ECM proteins into complex

networks are still not completely understood.Why do we care about ECM assembly? Lossof assembly stops embryogenesis. Deranged as-sembly promotes scarring, tumorigenesis, andfibrotic disease. Delayed assembly underliesbirth defects, chronic wounds, and skeletalmalformations.

Using scanning electron microscopy (EM),two major structural forms of ECM are vis-ible within tissues (Alberts et al. 2008). Theinterstitial matrix or stroma is composed ofthreadlike fibrils that form a fibrous and porousnetwork surrounding cells, whereas the base-ment membrane (basal lamina) has a sheet-likestructure that serves as a platform for cells anda boundary between tissue compartments. Al-though quite different structurally, interstitialmatrices and basement membranes are assem-bled from similar types of proteins (collagens,proteoglycans, cell adhesive glycoproteins). Inaddition, the initial steps of assembly are quitesimilar for these two types of matrices (Mao &Schwarzbauer 2005a, Wierzbicka-Patynowski& Schwarzbauer 2003, Yurchenco & Patton2009): ECM protein processing and secretion,binding to cell surface receptors, ECM proteinself-association, and fibril growth. Thus, under-standing the mechanisms of matrix formationin the interstitium can shed light on basementmembrane assembly and vice versa.

This review will focus on our current under-standing of fibronectin (FN) matrix assembly.FN is a ubiquitous ECM glycoprotein that isassembled into a fibrillar matrix in all tissuesand throughout all stages of life. Its assemblyis a cell-mediated process (McDonald 1988)and is essential for life (George et al. 1993).FN fibrils form linear and branched meshworksaround cells and connect neighboring cells. EMimages of cultured fibroblasts show long, in-terconnected fibrils ranging from more than25 nm down to approximately 5 nm in diameter(Chen et al. 1978), which is approximately thesize of the FN molecule itself at ∼3 nm wide(Engel et al. 1981, Erickson & Carrell 1983,Leahy et al. 1996). Thin fibrils predominate inearly fibroblast cultures, and as the matrix ma-tures, these fibrils are clustered together into

398 Singh · Carraher · Schwarzbauer

Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


FN: fibronectin

GAG:glycosaminoglycan

thicker fibril bundles (Chen et al. 1978, Singer1979). What are the mechanisms and interac-tions that convert FN molecules into fibrils,networks, and bundles?

It has been more than 20 years since thelast Annual Review article on ECM assembly(McDonald 1988). Since then, the amount ofinformation about ECM organization and cell-ECM interactions has grown significantly withthe identification of ECM protein receptors,new ECM components, signal transductiondownstream of ECM, and mechanical proper-ties of ECM (see, for example, Aszodi et al.2006, Bershadsky et al. 2003, Hynes 1990,Schwartz et al. 1995). This information has ledto new insights into the process of FN matrixassembly and how FN matrix affects assemblyof other ECM proteins.

FIBRONECTIN DOMAINORGANIZATION

Domains and Binding Activities

Many ECM proteins are modular and multido-main. This is well illustrated in the diagramof a FN subunit (Figure 1). FN has domainsfor interacting with other ECM proteins, cellsurface receptors, glycosaminoglycans (GAGs),and other FN molecules (Hynes 1990, Mao &Schwarzbauer 2005a). This combination of do-mains allows FNs to bind simultaneously tocells and to molecules within the surroundingmatrix.

FN is encoded by an ∼8-kb mRNA yield-ing FN subunits that range in size from 230–270 kDa depending on alternative splicing(Hynes 1990). FN is a modular protein com-posed of types I, II, and III repeating units(Figure 1). Two intramolecular disulfide bondsform within each type I and type II mod-ule to stabilize the folded structure. Type IIImodules are seven-stranded β-barrel structuresthat lack disulfides (Leahy et al. 1996, Potts &Campbell 1994). Modules are organized intobinding sites for collagen/gelatin, integrins,heparin, FN, and other extracellular molecules(Figure 1). The ∼500-kDa FN dimer forms

through a pair of antiparallel disulfide bonds atthe C terminus.

FN exists in multiple isoforms generated byalternative splicing. The single FN gene tran-script encodes 12 isoforms in rodents and cowsand 20 isoforms in humans. Alternative splic-ing occurs by exon skipping at EIIIA/EDA andEIIIB/EDB and by exon subdivision at the Vregion/IIICS (Schwarzbauer 1991a; Figure 1).Roles have been identified for the V region inFN secretion (Schwarzbauer et al. 1989) and in-tegrin binding (Guan & Hynes 1990, Wayneret al. 1989). EIIIA and EIIIB functions havebeen difficult to decipher using in vitro sys-tems, and mice expressing EIIIA− or EIIIB−

FN are viable and fertile (Muro et al. 2003, Tanet al. 2004). EIIIA/EIIIB double-null mice ex-hibit a requirement for these domains in vas-cular development during embryogenesis, butmatrix incorporation of mutant FN was not af-fected (Astrof et al. 2007). In cell culture ex-periments, recombinant FN containing eitherEIIIA or EIIIB was somewhat more efficientlyincorporated into an existing matrix (Guan et al.1990), and EIIIB-null mouse embryo fibrob-lasts showed a modest reduction in FN matrixlevels (Fukuda et al. 2002). From these results,it seems that these alternative exons are not re-quired for matrix assembly but may affect matrixlevels.

Activities that play critical roles in assem-bly include dimerization of FN subunits, cell-binding activity that localizes FN to the cellsurface, and FN-binding activity that associatesFN dimers into fibrils. However, each FNdimer has multiple integrin- and FN-bindingsites, which raises questions about which sitesdirectly participate in the assembly process andwhich carry out nonessential activities or func-tion in post-assembly processes. Studies of ma-trix assembly have provided answers to some ofthese questions.

Domains Required forFibronectin Matrix Assembly

The gold standard assay for demonstrat-ing FN matrix assembly is conversion from

www.annualreviews.org • Fibronectin Matrix Assembly 399

Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


Type I

I1–5/assemblydomain

Collagen/gelatin

III1–2

Type II

Type III

III4–5

III12–14 /hepII

V

EIIIA

EIIIB

III9 synergy/III10 RGD

Dimercysteines

1 2 3 4 5 6

27

89

1

2

3

45

67

8

9

1011

1213

14

15 10 11 12

1

Figure 1Diagram of a fibronectin (FN) subunit. Each FN subunit consists of three types of repeats: type I (hexagon), type II (square) and type III(cylinder). Based on rotary shadowing electron microscopy images, the two subunits of FN are curved with a contour similar to theshape in this diagram (Engel et al. 1981). Domains required to initiate assembly (red ) include the cell-binding domain (RGD site inIII10 + synergy site in III9), the N-terminal assembly domain (I1−5), and the intermolecular dimer cysteines at the C terminus. The70-kDa fragment extends from I1 through I9, including the assembly and the collagen/gelatin binding domains. The III1−2 domain (redwith stripes) has two FN-binding sites and participates in conformational changes that promote assembly. Other FN-binding sites arelocated in the III4−5 domain and in the III12−14/hepII domain that also binds to heparin and syndecans. Alternatively spliced extradomains EIIIA, EIIIB, and the variable region (V) are shown in white.

DOC: deoxycholatedetergent

deoxycholate detergent (DOC) solubility toDOC insolubility as originally defined byMcKeown-Longo & Mosher (1983). This con-version is an irreversible process that stabi-lizes FN interactions within matrix fibrils toyield a mature fibrillar network. As visualizedby fluorescence microscopy, FN matrix is a cell-associated fibrillar network extending betweenadjacent cells (Figure 2). Binding sites involvedin assembly have been identified using thesemicroscopic and biochemical assays combinedwith blocking reagents (antibodies or peptides),

mutant FNs lacking specific sites or domains,and protein-binding assays.

FN’s dimer structure is mediated by a pairof disulfide bonds at the C terminus of eachsubunit (Figure 1; Hynes 1990). This covalentlink plays an essential role in multimerization ofdimers into fibrils. Expression of recombinantFN lacking these cysteines ablated dimeriza-tion; the resulting monomeric FN was secretedbut did not form fibrils (Schwarzbauer 1991b).Recombinant dimeric proteins containingan FN-binding site but no cell-binding site


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


RGD: Arg-Gly-Aspcell-binding sequence

were efficiently coassembled with full-lengthFN whereas monomeric versions were not(Ichihara-Tanaka et al. 1992, Sottile & Wiley1994), which indicates that the dimer structureis involved in matrix incorporation even inthe absence of cell binding. Interestingly, FNhas endogenous protein disulfide isomeraseactivity that is located near the C-terminaldisulfide bonds (Langenbach & Sottile 1999),where it may be important in forming theantiparallel dimer structure in the endoplasmicreticulum. This activity is partially cryptic andis enhanced by proteolysis (Langenbach &Sottile 1999), so perhaps it has an extracellularrole in stabilizing FN interactions throughdisulfide exchange during matrix remodeling.

Cells mediate FN matrix assembly throughintegrin binding to the RGD (Arg-Gly-Asp)cell-binding domain. The primary receptor forFN matrix assembly is α5β1 integrin, whichbinds to the RGD sequence in III10 (Ruoslahti& Obrink 1996) and the synergy site in III9

(Aota et al. 1994; Figure 1). Antibody block-ade of integrin-cell binding domain interac-tions using anti-integrin or anti-FN antibodiesprevents fibril formation (Fogerty et al. 1990,McDonald et al. 1987). RGD-dependent in-tegrins including α5β1 can bind to FN thatlacks the synergy site (Danen et al. 1995,Sechler et al. 1996). However, both the RGDand synergy sites are required to initiate fibrilformation (Sechler et al. 1997).

The pioneering work of McKeown-Longo& Mosher (1983, 1985) showed that the N-terminal 70-kDa fragment binds to cells inmonolayer culture and, when added in excess,blocks FN matrix assembly. Antibodies to thisregion also block assembly (McDonald et al.1987), and recombinant FN lacking all or partof the first five type I repeats (I1−5, Figure 1)is unable to form fibrils (Schwarzbauer 1991b).Within the 70-kDa fragment, the 40-kDa col-lagen/gelatin binding portion does not appearto play a direct role in assembly, althoughFN binding activity of the 70-kDa fragmentis enhanced over binding of a fragment con-taining only I1−5 (McKeown-Longo & Mosher1985). Functional analyses using recombinant

50 μm

Figure 2Fibronectin (FN) fibrillar matrix surrounds cells in culture. HT1080 cells weregrown on a glass coverslip for 20 hours in medium supplemented with 0.1 μMdexamethasone and 25 μg ml−1 rat plasma FN as described in Brenner et al.(2000). Cells were fixed and stained with anti-FN monoclonal antibody (IC3)followed by fluorescein-tagged goat antimouse immunoglobulin G. Imageshows FN fibrils ( green) around cells with 4’,6-diamidino-2-phenylindole(DAPI)-stained nuclei (blue).

fragments with various mutations showed thatI1−5 functions as a unit to form the pri-mary FN-binding and matrix assembly domain(Sottile et al. 1991).

Other Fibronectin Binding Sites

Affinity chromatography of soluble FNfragments has been used extensively to mapbinding sites for collagen/gelatin, heparin,fibrinogen, and other molecules (Hynes 1990,Ingham et al. 1997). In contrast, most of thebinding sites involved in FN self-associationhave been detected using solid-phase bindingassays. Surface adsorption of FN inducesconformational changes, as demonstrated byexposure of antibody-binding sites (Ugarovaet al. 1995, 1996) or by EM analyses (Engelet al. 1981, Erickson & Carrell 1983), and mayalso expose sites for FN-FN interactions.

Essentially all reported FN-binding sites in-teract with the N-terminal assembly domain,and most of these have been identified using


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


the 70-kDa fragment in binding studies. Bind-ing sites in III2 (Aguirre et al. 1994), III4−5

(Maqueda et al. 2007), III12−14 (Bultmann et al.1998), and heat-denatured III1 (Hocking et al.1994) have been identified (Figure 1), as hasformation of a ternary complex containing the70-kDa fragment, III1, and heat-denatured III10

(Hocking et al. 1996). In addition to FN, I1−5

also binds to fibrinogen, heparin, bacterial pro-teins, and thrombospondin (Hynes 1990).

Accessibility of FN binding sites in III1, III5,and III10 is dependent on denaturation (Hock-ing et al. 1994, 1996), which suggests that thesesites are cryptic in native FN. The site in III4−5

appears to be cryptic in larger FN fragmentsbecause III4−6 does not bind to FN (Maquedaet al. 2007). The cryptic nature of some ofthese sites has raised the hypothesis that typeIII unfolding exposes sites for FN-FN inter-actions during fibril formation. The β-barrelstructures of type III modules are not stabilizedby intramolecular disulfide bonds, thus givingβ-strands some conformational flexibility. Nodeficiencies in matrices assembled from recom-binant proteins lacking these sites (FN�III1 orFN�III4−5) were detected by microscopy orDOC insolubility (Sechler et al. 2001), whichmakes it unlikely that these cryptic sites areindividually essential for FN fibril formation.

Other cryptic sites in FN include a site inI1−5 for tenascin-C binding (Ingham et al. 2004)and a site in III8 that is made available by inclu-sion of the adjacent alternatively spliced exonEIIIB (Figure 1; Ventura et al. 2010). In ad-dition to exposure by conformational changes,cryptic sites might also be made accessible byproteolysis during matrix remodeling. For ex-ample, proteolysis of FN can promote α4β1integrin interactions with FN matrix (Valenicket al. 2005).

Given the number of FN-binding sites, onewonders if they are equivalent. Does the as-sembly domain use all of its binding sites toform fibrils? Our current understanding of therequirements for all of these sites is limitedbecause differences in experimental approachyield different interpretations of function. Forexample, FN�III1 (recombinant FN lacking

only the III1 module) forms a normal ma-trix, but FN�III1−2 does not (Sechler et al.2001). FN�III4−5 also forms a normal ma-trix (Sechler et al. 2001), but addition of theIII4−5 fragment blocks fibril formation by cells(Maqueda et al. 2007). FN�III1−2 does notform DOC-insoluble matrix, but FN�III1−7

(which lacks a larger FN segment) forms DOC-insoluble fibrils at an enhanced rate (Sechleret al. 1996). Addition of fragments spanningIII1, III2, or III12−14 have limited ability toblock matrix assembly (Bultmann et al. 1998,Chernousov et al. 1991) compared with the70-kDa fragment, which is an extremely ef-fective inhibitor of fibril formation (McDonaldet al. 1987, McKeown-Longo & Mosher 1985,Sechler & Schwarzbauer 1998). One interpre-tation of these differential effects is that tem-poral and spatial distributions of FN-bindingsites within the matrix may determine whichdomains interact. Perhaps there is a hierarchi-cal order to FN interactions, or some of theseinteractions may require formation of ternarycomplexes involving other ECM components.

A FIBRONECTIN MATRIXASSEMBLY MODEL

Using the key assays of DOC insolubility,microscopic visualization, and protein-bindingassays, the basic steps of FN matrix assemblyhave been defined (Figure 3). Integrins tetherFN dimers to initiate FN-FN interactionsmediated by the N-terminal assembly domain.Conformational changes expose additionalFN-binding sites to promote further FNinteractions, associations between fibrils, andmatrix insolubility. Evidence for these steps isdescribed below.

Fibronectin Dimer Secretion andIntegrin Binding

FN in solution has a compact conformation,as detected by sedimentation, EM, and othermeasurements (Hynes 1990). FN in solutiondoes not form fibrils even at extremely highconcentrations, a property that is especially


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


i

ii

a

d

b c

N

N

FN dimer

Integrin

Actin

Figure 3Major steps in fibronectin (FN) matrix assembly. Integrin-induced conversion of compact FN to extended fibrils is shown in four steps.(a) A compact FN dimer binds to integrins ( gray). FN subunits of a single dimer are shown in two shades of orange. (b) Intracellularproteins ( pink, yellow, blue) are recruited to integrin cytoplasmic domains and connected to the actin cytoskeleton ( green). Cytoskeletalconnections increase cell contractility (arrows), which induces conformational changes in FN. (c) Integrin clustering and exposedFN-binding sites promote FN-FN interactions and further changes in FN conformation. (d ) Finally, these events trigger formation ofstable insoluble fibrillar matrix. The inset (red box) shows interactions between single subunits of FN dimers. N indicates the Nterminus of an FN subunit. Fibrils form through (i ) end-to-end association of FN dimers, mediated by the N -terminal assemblydomain, followed by (ii ) lateral associations between fibrils that are likely to involve the other FN-binding sites in III1−2, III4−5, andIII12−14. Gray X’s represent interactions between fibrils.

important in body fluids such as blood, whereFN fibril formation could have life-threateningeffects. Soluble FN exhibits selective bindingto cell surface receptors and interacts withα5β1 but not with other RGD-dependentintegrins (Huveneers et al. 2008). FN bindsto α5β1 via its RGD and synergy sites, bothof which are required for de novo FN fibrilformation (Sechler et al. 1996, Sottile et al.2000). However, full-length recombinant FNlacking only the RGD sequence as well assmall dimeric FNs containing the N-terminaldomain but lacking other parts of FN cancoassemble with full-length FN (Ichihara-

Tanaka et al. 1992, Sechler et al. 1996, Sottile &Wiley 1994). Incorporation of these recombi-nant proteins into fibrils must occur primarilythrough FN-FN interactions. Thus, not allFNs in a matrix must directly interact withintegrins. Mouse embryos expressing FN withan inactive RGE sequence in place of RGDdeveloped several days longer than FN-nullembryos and were phenotypically quite similarto α5-null embryos (Takahashi et al. 2007,Yang & Hynes 1996). FN interactions with αvintegrins have been shown to compensate forthe absence of RGD or α5 integrin (Takahashiet al. 2007, Yang & Hynes 1996). These results


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


indicate that, within tissues, some FN matrixcan be assembled in the absence of RGD-α5β1integrin interactions.

Fibronectin-Fibronectin Interactions

FN binding induces integrin clustering, whichbrings receptors together along with theirbound FNs (Figure 3). These clusters pro-vide locally high concentrations of FN at thecell surface, which is probably important topromote FN-FN interactions. The diameterof thin FN fibrils approximates the dimen-sions of individual type III modules (Dzamba &Peters 1991, Engel et al. 1981, Leahy et al.1996), which indicates that compact FN in so-lution is extended during fibril polymerization.Intramolecular interactions between III2−3 andIII12−14 maintain the compact form of FN( Johnson et al. 1999). These regions overlapwith FN-binding sites, which suggests that dis-rupting these interactions to extend compactFN dimers frees these sites to participate in in-termolecular interactions (Figure 3).

A key insight into FN conformationalchanges came from studies of the stimula-tory effects of Rho GTPase on FN assem-bly. Binding of lysophosphatidic acid (LPA) orsphingosine-1-phosphate (S-1-P) to receptorsactivates Rho (Anliker & Chun 2004). Rho-GTP then stimulates Rho kinases to enhancecell contractility by inducing actin-myosin in-teractions and actin rearrangement into stressfibers (Hall 2005). Rho activation also stim-ulates FN incorporation into matrix (Yonedaet al. 2007; Zhang et al. 1994, 1999), and FNmatrix levels are reduced with blockade of Rhoand loss of contractility (Zhong et al. 1998).Mechanistic insights into the role of contractil-ity came from studies with the monoclonal anti-body (MAb) L8 (Chernousov et al. 1987), whichbinds to the I9-III1 region of FN (Chernousovet al. 1991). Inhibition of Rho in cells with anestablished matrix reduced binding of MAb L8,which indicates that accessibility of this epi-tope depends on the contractile effects of cellson the matrix (Zhong et al. 1998). They alsoshowed that binding of FN, the 70-kDa frag-

ment, or MAb L8 to FN attached to a rubbersurface was enhanced by stretching. Therefore,contractility and stretching affect availability ofa III1 FN binding site through effects on FNconformation.

Another key discovery in FN binding wasidentification of a fragment of the III1 module(III1-C) that could induce FN aggregationinto complexes that resemble collapsed fibrils.This III1-C fragment, also known as anastellin,encompasses the C-terminal two-thirds ofthe III1 module and has FN-binding activity(Morla & Ruoslahti 1992). FN treated withthis fragment forms aggregates that can bestretched into structures that appear fibrillar(Morla et al. 1994). Interestingly, this so-calledsuperfibronectin has enhanced cell adhesionactivity, possibly because the aggregates bringmultiple cell-binding domains into closeproximity. Anastellin reportedly can causeloss of FN matrix leading to changes in cellmorphology, cell signaling, and proliferation(Bourdoulous et al. 1998). However, whetherFN matrix is actually lost with anastellin treat-ment has been brought into question by datashowing loss of epitope accessibility withoutloss of FN fibrils upon addition of anastellinto cells in culture (Klein et al. 2003). Theseobservations indicate that anastellin affects FNconformation in solution and within fibrils.

Only recently has the interaction ofanastellin with FN been thoroughly investi-gated. It binds with a stoichiometry of 4:1; threeanastellins bind within III1−3, and one bindsat III11 (Ohashi & Erickson 2005). Anastellininduces aggregation of FN or FN fragmentsthat contain III1−2 or III1−3 and enhances pro-teolytic sensitivity of FN, thus demonstrat-ing that it affects FN conformation (Ohashi &Erickson 2005, Ohashi et al. 2009). The authorspropose that spontaneous unfolding or “breath-ing” of type III modules allows anastellin bind-ing, which then prevents refolding and allowsexposed hydrophobic regions in the module β-strands to interact to form FN aggregates. β-strand swapping has been demonstrated for anindividual type III module, III9, which formedamyloid-like fibrils upon temperature-induced


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


FAK: focal adhesionkinase

partial unfolding (Litvinovich et al. 1998). Itseems likely that β-strand exchange betweenFN molecules contributes to DOC insolubil-ity, but this remains to be demonstrated.

GAG and proteoglycan interactions withFN have been implicated in matrix assem-bly (Chung & Erickson 1997, Galante &Schwarzbauer 2007, Klass et al. 2000, Morla& Ruoslahti 1992). Both syndecan transmem-brane proteoglycans (Woods 2001) and ECMproteoglycans such as perlecan (Chung &Erickson 1997) can participate in this process.Interestingly, many of the FN-binding sitesalso have heparin-binding activity, whichraises the possibility that proteoglycans maycontribute to ternary complexes and mediateinteractions between FN molecules or betweenFN and other ECM proteins or receptors.

Tethering of Fibronectinat the Cell Surface

An FN dimer bound to two integrins providesa way for cell contractility to apply forces thatcould affect FN conformation. Tethering at thecell-binding domains of an FN dimer wouldlikely impact the C-terminal regions of eachsubunit and might also dissociate the interac-tions between III2−3 and III12−14 in the compactform (Figure 3a,b). But can integrin bindingcause long-range conformational changes thataffect the N-terminal assembly domain and theFN-binding sites in III1−2? The finding that the70-kDa fragment is able to bind to cell layersand to block assembly raised the possibility ofan FN matrix assembly receptor (McKeown-Longo & Mosher 1985). Molecules that havebeen implicated in 70-kDa fragment-cell in-teractions include FN itself, heparan sulfateproteoglycans, collagen (Colombi et al. 2003),molecules with large apparent molecular mass(Zhang & Mosher 1996), molecules associatedwith a laminin receptor (Bae et al. 2004), andintegrins α5β1 (Hocking et al. 1998) and αvβ3(Takahashi et al. 2007). Despite much investi-gation, no distinct molecule has emerged as theFN matrix assembly receptor, which suggeststhat it is not a single entity but is instead an

activity and that many different molecules canserve in this role.

Evidence that multiple molecules affectassembly is supported by observations impli-cating substrate composition in the assemblyprocess. Fibroblasts grown on surfaces coatedwith FN will rearrange the FN into fibrillarstructures at matrix assembly sites that can bedetected by 70-kDa fragment binding (Christo-pher et al. 1997, Wierzbicka-Patynowski &Schwarzbauer 2002). Using FN fragments assubstrates, Mosher and colleagues (Xu et al.2009) showed that assembly was limited whencells were grown on FN’s cell-binding domainalone and that optimal assembly occurred onfragments containing III1 plus C-terminalFN domains. Matrix assembly is enhancedwith cells on a 3D fibrillar FN matrix (Mao& Schwarzbauer 2005b). Matrix assembly bycultured cells is facilitated by a rigid substrateand hampered by growth on soft materials(Halliday & Tomasek 1995; C. Carraher andJ.E. Schwarzbauer, unpublished observations).Other ECM protein substrates negatively im-pact assembly, as observed with cells growingon vitronectin (Bae et al. 2004, Hocking et al.1999). One possible interpretation of the sub-strate effects is that FN-binding activity withinthe protein on the substrate provides a tether-ing site for FN, which can then be pulled on tocause FN extension through conformationalchanges. This would explain why fragmentscontaining the III1−2 domain support assemblywhen attached to a surface (Xu et al. 2009).

Integrin binding to FN induces receptorclustering, which brings together cytoplasmicmolecules such as focal adhesion kinase (FAK),Src kinase, paxillin, and others to form protein-rich focal complexes that activate polymeriza-tion of actin filaments and intracellular signal-ing through kinase cascades (Geiger et al. 2001).Differential effects of substrate on FN matrixassembly may be sensed through formation offocal contacts and fibrillar adhesions. By follow-ing integrin movements over time, Yamada andcolleagues (Pankov et al. 2000) showed that fi-broblasts on an FN substrate maintained αvβ3integrins in focal contacts at the cell periphery


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


FRET: fluorescenceresonance energytransfer

whereas α5β1 integrins moved centripetally inparallel with actin filaments. Focal contacts andfibrillar adhesions differ not only in location andmovement but also in the proteins associatedwith the integrin cytoplasmic domains, i.e., pax-illin at focal contacts and tensin at fibrillar ad-hesions moving with α5β1 (Geiger et al. 2001,Pankov et al. 2000, Zamir et al. 1999). Forma-tion of FN fibrils by the movement of fibril-lar adhesions is directional. Ohashi et al. (2002)observed that the ends of fibrils toward the cellperiphery were stationary while elongation oc-curred at the proximal ends, which suggests thatfibrillar adhesions are pulling against the sub-strate. Thus, cell-substrate attachment can pro-mote separation of adhesion sites from sites ofFN assembly. This provides a mechanism forextending compact FN dimers by applying ten-sion through pulling against substrate-attachedprotein and may explain some of the observedeffects of substrate on FN assembly.

Does Fibronectin Unfold?

The III1−2 domain has been the focus of manystudies into FN conformational changes. Itcontains two FN-binding sites, one of themcryptic, and its activity is affected by stretch(Aguirre et al. 1994, Hocking et al. 1994, Zhonget al. 1998). Anastellin, which induces FN ag-gregation, is derived from this region (Morlaet al. 1994). In addition, the III1−2 domain isunique among type III module pairs in thatthere is a flexible 17-residue linker between themodules that extends into the first β-strandof III2 (Vakonakis et al. 2007). This linkermight increase the potential for domain exten-sion. Fluorescence resonance energy transfer(FRET) has been used to follow changes in con-formation of a FRET sensor of III1−2 taggedat opposite ends with cyan fluorescent protein(CFP) and yellow fluorescent protein (YFP). Asignificant change in FRET signal was detectedwith mutation of interfacial residues predictedto form a salt bridge interaction between III1

and III2 (Karuri et al. 2009). Mutant III1−2 wasfurther affected by 70-kDa fragment binding,

which eliminated FRET (Karuri et al. 2009).These results support the idea that a salt bridgebetween III1 and III2 stabilizes this region andcontrols FN binding. Disruption of the saltbridge would then allow separation of the mod-ules, making FN binding sites more accessi-ble and allowing intermolecular interactions tooccur.

The crystal structures of FN fragments showzigzag orientation of type III repeats with tiltangles of up to 62◦ between adjacent mod-ules (Leahy et al. 1996, Sharma et al. 1999).Flattening of the zigzag into a more lineararrangement is one way to extend FN dur-ing fibril formation. Erickson (1994) proposeda model for more dramatic conformationalchanges of type III modules: Application offorce might unravel a β-barrel into a randomcoil conformation. FN fibrils can stretch tofour times their original length when pulled bycells (Ohashi et al. 1999), and fibril elasticityis easily visualized by video microscopy (Mao& Schwarzbauer 2006) (Figure 4 and Sup-plemental Video 1, follow the SupplementalMaterial link from the Annual Reviews homepage at http://www.annualreviews.org). Bothflattening of tilt angles and unraveling of β-barrels would increase the length of an FNdimer and provide some elasticity to fibrils.Single-molecule force spectroscopy measure-ments show that type III modules can un-fold in vitro (Oberhauser et al. 2002). Fur-thermore, fluorescence microscopy with FRETspectroscopy has provided evidence for confor-mational changes in individual FN moleculesduring fibril formation (Baneyx et al. 2001)that have been interpreted as domain unfold-ing (Smith et al. 2007). Breathing of β-strandslikely can briefly expose sites normally buriedinside a β-barrel. However, complete unfold-ing of modules may be problematic, as the con-formational changes could be irreversible, withthe protein prevented from refolding by inter-molecular interactions within fibrils or by ag-gregation, and such changes may also gener-ate immunogenic sites that are not normallypresent in native structures. There is a debate


Supplemental Material

Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.

http://www.annualreviews.org/doi/suppl/10.1146/annurev-cellbio-100109-104020


Figure 4Migration of HT1080 cells on fibrillar fibronectin (FN) matrix. Extraction of a highly confluent fibroblastculture was used to prepare a cell-free fibrillar matrix in which FN is the major protein component. HT1080human fibrosarcoma cells were allowed to attach to the matrix in serum-free medium for 2 h. Fetal bovineserum was then added to the medium to a final concentration of 10% to initiate migration. To watch themigration in action, please see Supplemental Video 1, which was originally published in Mao &Schwarzbauer (2006).

as to whether FN conformational changes andfibril elasticity result from straightening of thezigzag orientation of type III modules, from un-folding, partial unraveling, or breathing of typeIII β structures, or from some combination ofthese events (Erickson 2002, Ohashi et al. 2009,Smith et al. 2007). These questions are likely togenerate interest for the foreseeable future.

Molecular dynamics simulations and mod-eling have been applied to provide insights intopotential conformational changes at the cell-binding site in FN. Simulated application offorce to the III10 module suggests that stepwiseunfolding of β-strands could regulate availabil-ity of the RGD sequence (Gee et al. 2008,Krammer et al. 1999) and may thus provide anovel mechanism for regulating interactions ofcells with FN fibrils.

Fibronectin-Integrin Bond Strengthand Signaling

Many FN receptors can support cell adhesionand migration on FN substrates but lack suf-ficient activity to assemble FN into fibrils. Incontrast to α5β1, which is the primary integrinfor FN matrix assembly, α4β1 (Sechler et al.2000), αvβ1 (Zhang et al. 1993), αvβ3 (Wuet al. 1996), αIIbβ3 (Olorundare et al. 2001),and perhaps others are unable to assemble fib-rils without treatments that increase their ac-tivities. These receptors also lack the ability tobind to soluble FN (Huveneers et al. 2008),which would likely impact recruitment of se-creted FN to assembly sites on the cell surface.The distinction between integrins probably re-lates to FN-binding strength. α5β1-FN bond


Supplemental Material

Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.

http://www.annualreviews.org/doi/suppl/10.1146/annurev-cellbio-100109-104020


strength is dependent on engagement of FN’ssynergy site, which leads downstream to in-creased FAK activation (Friedland et al. 2009).Both FAK activity (Ilic et al. 2004) and the syn-ergy site (Sechler et al. 1997) are required forα5β1-mediated assembly. Integrins can bind toFN lacking the synergy site (Danen et al. 1995,Mao & Schwarzbauer 2006) but require stimu-lation using either Mn2+ or activating antibod-ies to assemble synergy-minus FN into fibrils.Taken together, we conclude that the strengthof integrin binding to FN is a requisite partof initiating and propagating fibril formationand that the ability to achieve sufficient bondstrength for assembly is a property of α5β1 thatdistinguishes it from other integrins.

Facilitating interactions between integrinsand FN is one role of the actin cytoskeleton.Depolymerization of actin with drugs (Hynes1990), deletion of the β integrin cytoplasmicdomain (Wu et al. 1995), or actin rearrange-ment with inhibition of Rho GTPase (Zhonget al. 1998) causes loss of FN matrix, whichdemonstrates the importance of connectionsbetween ECM polymers outside cells withactin polymers inside. Integrin cytoplasmicdomains also stimulate intracellular signaling,and certain pathways are critical for initiationand maintenance of matrix assembly. FAKplays a central role in integrin signaling and isessential for fibroblast assembly of FN fibrils(Ilic et al. 2004). Similarly, loss of talin bindingto β1 integrin reduces integrin activity andFN assembly (Green et al. 2009). Other focaladhesion proteins participate in the assemblyprocess, but their absence does not eliminateassembly. For example, members of the Srckinase family work with FAK in many cell types(Mitra & Schlaepfer 2006); assembly is delayedbut not ablated in cells lacking these kinases(Wierzbicka-Patynowski & Schwarzbauer2002) and is reduced in cells with enhancedSrc activity (Huveneers & Danen 2009).Paxillin phosphorylation is a consequenceof Src activation (Mitra & Schlaepfer 2006)and also contributes to FN matrix formation(Wierzbicka-Patynowski et al. 2007, Zaidel-Bar et al. 2007). Inhibition of Src kinase activity

in cells after matrix accumulation caused arapid loss of that matrix concomitant with areduction in phospho-paxillin (Wierzbicka-Patynowski et al. 2007). Thus, not onlyare focal adhesion proteins involved in theinitiation phase of assembly, but once matrixhas been assembled, these proteins are neededto maintain matrix association with the cellsurface.

Syndecan transmembrane proteoglycanshave been implicated in many of the intracel-lular events that accompany integrin-FN bind-ing. Syndecan-4 is involved in focal adhesionformation (Woods 2001), FAK and Rho acti-vation (Wilcox-Adelman et al. 2002), and cellcontractility (Yoneda et al. 2007). Althoughsyndecan-4 small interfering RNA (siRNA)knockdown does not have a major effect on FNmatrix, knockdown of syndecan-2 causes a sig-nificant reduction in DOC-insoluble FN fibrils(Galante & Schwarzbauer 2007), and deletionof the syndecan-2 cytoplasmic tail prevents as-sembly of FN and other matrix proteins (Klasset al. 2000). Syndecans bind to the hepII do-main of FN (Woods et al. 2000) adjacent tothe α5β1 integrin-binding domain (Figure 1),which may position them to contribute to cell-FN interactions. They also initiate syndecan-specific intracellular signaling cascades that in-volve protein kinase C (for syndecan-4) (Bass& Humphries 2002) and PDZ domain proteins(for syndecan-2) (Essner et al. 2006). Synde-cans might act as coreceptors for ECM alongwith integrins, but more research is needed todetermine the specific roles of syndecans inassembly.

Fibril Maturation and Conversionto Insolubility

Elegant experiments using an anti-FN MAb forimmuno-EM analysis identified an 84-nm spacebetween epitopes, which predicts a 20-nm over-lap of the N termini and suggests that initiallythe fibrils form through end-to-end associationof dimers (Dzamba & Peters 1991; Figure 3).Initial thin fibrils then grow in length and thick-ness as matrix matures. During fibril growth,


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


FN matrix is converted to a DOC-insolubleform. This conversion follows soon after theinitiation of assembly such that more than halfof a given population of FN is DOC insolu-ble within 6 h (McKeown-Longo & Mosher1983).

Insolubility is a critical property of the ECMand provides tissues with stability, rigidity, andshape. But how do FN fibrils become insol-uble? For many years, disulfide exchange wasassumed to account for the insolubility be-cause multimeric FN is dissociated by reduc-ing agents as detected by polyacrylamide gelelectrophoresis, FN has protein disulfide iso-merase activity, and FN has many intrachaindisulfide bonds. However, a tour de force offragmentation analysis failed to identify anydisulfide-bonded fragments, which led to theconclusion that DOC insolubility arises fromstrong, but noncovalent, protein-protein in-teractions (Chen & Mosher 1996). These in-teractions could be related to the interactionsthat form type III modules into amyloid-likefibrils (Litvinovich et al. 1998). Recently, an-other mechanism was proposed to explain therequirement for reducing agents to dissoci-ate FN matrix. Experiments show that FNcan be trapped at the top of the stacking geland appear multimeric simply by adding largeECM proteins to the gel samples (Ohashi &Erickson 2009). These results suggest that FNwithin fibrils exists as covalent dimers but thatother interactions are noncovalent, again fit-ting with the strong protein-protein interac-tions model.

The combination of multiple FN-bindingsites and noncovalent FN-FN interactions inmultimeric fibrils may facilitate fibril elastic-ity. FN interactions initially appear to dependon N-terminal FN binding sites generatingthin fibrils with end-to-end association of FNdimers (Dzamba & Peters 1991). FNs may bepartially in the compact conformation, or typeIII modules may be in a zigzag arrangementin these fibrils (Erickson 2002). Stretching bycell contractility could then cause progressiveextension by first unfolding the compact formand then straightening the zigzag of the mod-

ules as well as perhaps inducing module breath-ing. Binding sites exposed in extended fibril-lar molecules would mediate lateral interactionsbetween thin fibrils. Multiple FN-binding sitesmay be needed so that simultaneous interac-tions involving several sites along a fibril canstabilize relatively weak binding at individualsites (Figure 3). Thus, fibril alignment may de-pend on the sites that are available for interfibrilinteractions. Multiple binding sites might alsocontribute to fibril elasticity. For example, thelength of a fibril bundle could be extended byslippage of individual fibrils along each other.Then, interactions between the N-terminal as-sembly domain and its different binding sitesalong FN could provide stopping points as fib-rils move past each other.

Fibronectin Matrix Turnover

FN matrix assembly is a dynamic and contin-uous process. This point is well illustrated byexperiments showing that FN matrix is lostwhen cells are deprived of FN (Sottile & Hock-ing 2002). FN fibrils are also dissociated whenshear forces are applied to attached cells (Engleret al. 2009). Continuous FN polymerization isneeded for matrix to be stabilized at the cellsurface (Sottile & Hocking 2002, Wierzbicka-Patynowski et al. 2007). If FN polymerizationis inhibited or FN expression is eliminated, theexisting FN matrix subsides, which shows that asteady state exists between FN polymerizationand turnover.

The α5β1 integrin, which carries out matrixassembly, may also be involved in its turnover.Caveolin-1 regulates α5β1 endocytosis, andβ1 integrin is involved in the endocytosis ofsoluble FN (Shi & Sottile 2008, Sottile &Chandler 2005). Thus, our current understand-ing of turnover suggests that events that slowpolymerization of FN into fibrils (such as re-duced integrins or integrin activity, a reducedsource of FN, or increased proteolysis) will in-crease integrin-FN endocytosis. Further inves-tigation is needed to understand the mecha-nisms and regulation of this process.


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


FIBRONECTIN-DEPENDENTASSEMBLY OF OTHEREXTRACELLULAR MATRIXPROTEINS

The list of ECM proteins that depend on FNfor incorporation into the matrix is growingand includes collagens, fibrillin, fibulin, latentTGF-β binding protein (LTBP), and tenascin-C (Chung & Erickson 1997, Dallas et al. 2005,Kadler et al. 2008, Sabatier et al. 2009, Sottile& Hocking 2002, Twal et al. 2001). Some ofthese proteins associate directly with FN fib-rils, whereas others appear to use FN matrix asa scaffold for deposition of independently struc-tured fibers. Initiation of basement membraneassembly does not involve FN but instead re-lies on laminin-integrin interactions, connec-tions to the actin cytoskeleton, and formationof laminin multimers, all steps that are mecha-nistically similar to the early stages of FN ma-trix assembly. This process has been recentlyreviewed by Yurchenco & Patton (2009).

Fibronectin in Microfibriland Elastic Fiber Assembly

Microfibrils assemble through head-to-tail in-teractions of fibrillin that yield a linear fibrilthat resembles beads on a string (Ramirez &Sakai 2010). Assembly of fibrillin-1 into mi-crofibrils occurs in cell culture but requires anextended time of several days for significantmaterial to accumulate. Using human dermalfibroblasts, two groups have applied differentapproaches to show that FN matrix is requiredfor microfibril assembly. In one study, knock-down of FN expression with siRNAs or block-ade of α5β1 integrin with antibodies delayed ac-cumulation of fibrillin-1 in the matrix (Kinseyet al. 2008). Similar reductions were observedin β1 integrin–null cells or with inhibition ofRho GTPase, which acts on the actin cytoskele-ton downstream of integrins. In the other re-port, fibrillin-1 and FN were shown to colocal-ize by immunofluorescence and, importantly,by immuno-EM (Sabatier et al. 2009). Bindingto FN depended on formation of fibrillin mul-timers, which led to the proposal that fibrillin

multimers act as assembly intermediates that ul-timately depend on FN matrix for organizationinto microfibrils (Sabatier et al. 2009).

Microfibrils have an important role in elas-tic fiber assembly. Tropoelastin is secreted andsubsequently formed into aggregates throughassociation with either microfibrils or cells.These elastin aggregates may nucleate furtherassembly. Live cell imaging studies using taggedtropoelastin showed that elastin aggregates firstappeared on the cell surface and then, throughcell motility, the small aggregates moved toand associated with elastin fibers (Czirok et al.2006, Kozel et al. 2006). The link to cellsmay involve FN. Antibody staining of culturesover time detected FN fibrils first, but at latertimes fibrils positive for fibrillin, microfibril-associated glycoprotein-1 (MAGP-1), fibulin-5, and finally elastin appeared in the samelocation as FN (Wagenseil & Mecham 2007).Interestingly, lysyl oxidase (LOX), the enzymethat forms covalent cross-links in elastin fibers,binds to FN, and this interaction stimulatesLOX activity (Fogelgren et al. 2005). To-gether these observations suggest a model inwhich tropoelastin aggregates are cross-linkedby LOX at the cell surface, perhaps facili-tated by LOX-FN interactions. These aggre-gates are associated with fibulins, which mayfacilitate rearrangements of aggregates intofibers through interactions with microfibrils(Wagenseil & Mecham 2007).

Type I Collagen Assembly

Triple-helical type I collagen molecules areassembled into ∼1.5-nm diameter fibrils thathave a repeating pattern with 67-nm period-icity resulting from the head-to-tail and lateralassociations of individual collagen molecules(Alberts et al. 2008, Kadler et al. 2008).Collagen chains assemble into triple-helicalmolecules in the secretory pathway but are pre-vented from further assembly by the presenceof N- and C-terminal procollagen domains.Activation of collagen for assembly into fibrilsoccurs by proteolytic cleavage of procollagendomains.


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


Cells play an important role in type I colla-gen fiber assembly. As shown elegantly by EMstudies of cornea and tendon, there is close as-sociation of collagen molecules within cellularcompartments that surround the forming fibrils(Birk & Trelstad 1984, 1986). These compart-ments facilitate end-to-end association of colla-gen molecules and lateral interactions betweenfibrils. Narrow channels appear to merge togenerate larger compartments in which fibrilsare assembled into fibers, perhaps through re-traction of membrane protrusions (Banos et al.2008, Birk & Trelstad 1986, Zhang et al. 2005).Application of image reconstruction techniquesfurther defined these compartments and, be-cause of their role in collagen fibril deposition,the name “fibripositor” has been coined (Kadleret al. 2008).

One obvious advantage of such com-partments is that they concentrate collagenmolecules in close proximity to promote in-teractions required for fibrillogenesis. In manycases, these assembly compartments are ori-ented parallel to the long axis of the cell, whichwould serve to orient nascent fibrils and maytherefore play a critical role in directional de-position of collagen fibers. FN and type I col-lagen fibers are frequently found together intissues and have been colocalized in the secre-tory pathway of fibroblasts (Ledger et al. 1980).Many groups have shown that collagen fibrilsdo not accumulate in the absence of FN ma-trix (Dallas et al. 2005, McDonald 1988, Sottile& Hocking 2002). In vivo, FN is found in de-veloping tissues prior to the deposition of col-lagen. Because collagen can bind to FN, thetemporal and spatial connections between FNand collagen suggest that FN acts as a scaffoldthat may aid in alignment of collagen fibrils.Perhaps FN matrix orients cells so that colla-gen fibrils are aligned and thus facilitates forma-tion of parallel collagen fibers. FN matrix mightalso guide the retraction of cell processes sothat collagen fibrils are deposited with parallelalignment.

Several reports have also indicated that col-lagen can enhance FN assembly. For example,FN matrix was significantly increased in cells

from the MOV13 mouse (deficient in type Icollagen) by expression of a collagen transgene(Dzamba et al. 1993). Presence of a 13–aminoacid collagen-binding site was required for as-sembly of recombinant FNs in chick embryofibroblasts (Colombi et al. 2003). One possi-ble role for collagen in FN deposition mightbe to provide a rigid collagen network that in-creases tension in the matrix to facilitate FNassembly. This mechanism would be similar tothe tensional effects of cell-cell interactions onFN assembly in Xenopus embryos (Dzamba et al.2009). Although it is possible that collagen con-tributes to FN assembly, much more evidenceshows that FN matrix is a critical scaffold forcollagen assembly.

Fibronectin Matrix and Disease

Despite FN’s abundance and importance in theECM, surprisingly few diseases are caused bydefects in FN matrix assembly. One recentlydescribed example is glomerulopathy with FNdeposits, a kidney disease characterized by non-fibrillar FN aggregates (Castelletti et al. 2008).Gene mapping and sequencing of samples fromseveral pedigrees of affected patients identi-fied mutations in III13 and III4. Interestingly,these mutations map to FN-binding domains

FN dimerbinding to α5β1

integrin

Modulatory moleculesOther ECM components

Multiple FN binding sitesTernary complexes

Matrix turnoverFibrotic disease

FN conformationTissue tension

FN-FNinteractions

Insolublefibrils

Figure 5Future issues. The basic steps of fibronectin (FN) matrix assembly are boxed ingreen. Other proteins, interactions, processes, and mechanisms (blue) mightimpact various steps in matrix assembly as indicated (dashed blue arrows).Additional investigation is required to understand their effects. ECM,extracellular matrix.


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


(Figure 1), which suggests that the mutationsimpact FN interactions during matrix assem-bly. Another possibility is that III13 mutationsdestabilize the compact conformation of FN,which involves III12−14 (Figure 3), and prema-turely expose other binding sites, which leadsto dysregulation of FN-FN interactions.

The possibility exists that FN matrix con-tributes to other diseases. Defective or exces-sive FN matrix is present in fibrotic diseases,keloids, and hypertrophic scars (see, for exam-ple, Pozzi et al. 2009, Wolfram et al. 2009). De-fects in FN matrix may underlie abnormalitiesattributed to other ECM proteins. For exam-

ple, mutations in the diastrophic dysplasia sul-fate transporter (DTDST) cause gross skeletaldefects with undersulfated cartilage proteogly-cans (Rossi et al. 1998). Loss of DTDST alsocauses a significant reduction in FN matrix as-sembly (Galante & Schwarzbauer 2007), whichraises the possibility that the effects of DTDSTmutations may extend to the organization oramount of FN matrix during chondrogenesisand lead downstream to defects in collagen de-position and cartilage formation. Whether FNmatrix has a causal role or is an important con-tributing factor in these diseases remains to bedetermined.

SUMMARY POINTS

1. Initiation of matrix assembly requires binding of FN dimers to integrin receptors followedby binding to other FN molecules via the N-terminal assembly domain.

2. Clustering of receptors bound to FN dimers generates a locally high FN concentrationthat promotes FN-FN interactions.

3. FN subunits change conformation from compact to extended. This process is mediatedby integrins and depends on cell contractility through actin filaments and Rho GTPaseactivity.

4. Conformational changes increase FN-FN interactions by exposing FN-binding sites,some of which are cryptic in native FN.

5. Intracellular signaling downstream of integrins contributes to FN matrix formation; FAKplays a central role in initiating the signals that promote assembly.

6. FN fibrils are converted to a DOC detergent-insoluble form, which provides the matrixwith stability and rigidity.

7. A steady state exists between FN matrix assembly and matrix turnover such that a decreasein fibril formation destabilizes the matrix and induces turnover.

8. Many questions remain about FN matrix assembly mechanisms and the role of FN matrixin tissue homeostasis and disease (Figure 5).

FUTURE ISSUES

1. There are at least four FN-binding sites in each FN subunit. The N-terminal assemblydomain has an essential role in fibril formation. Do the other sites have defined roles,for example, in staggering dimers within fibrils, in lateral interactions between fibrils,or in branching? Do several sites together form ternary complexes to stabilize FN-FNinteractions?


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


2. What molecular interactions convert fibrils to DOC insolubility? Does β-strand ex-change play a role, and are other ECM molecules involved in the process?

3. How does tissue tension regulate matrix assembly? Is it through integrin activity, cy-toskeletal organization, FN conformation, or other mechanisms? Does deposition ofcollagen or other ECM proteins modulate tissue tension?

4. What events are involved in identifying fibrils for turnover and in converting insolublefibrils into a form that can be endocytosed?

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

The authors wish to thank Dan Slone for help with images, NCI and NIGMS for funding (toJ.E.S.), and NIH T32GM007388 for predoctoral support (to C.C.). We also acknowledge thecontributions of current and former members of the Schwarzbauer lab and of all members of theFN matrix assembly community.

LITERATURE CITED

Aguirre KM, McCormick RJ, Schwarzbauer JE. 1994. Fibronectin self-association is mediated by comple-mentary sites within the amino-terminal one-third of the molecule. J. Biol. Chem. 269:27863–68

Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. 2008. Molecular Biology of the Cell. New York:Garland Science. 1268 pp.

Anliker B, Chun J. 2004. Cell surface receptors in lysophospholipid signaling. Semin. Cell Dev. Biol. 15:457–65Aota S, Nomizu M, Yamada K. 1994. The short amino acid sequence Pro-His-Ser-Arg-Asn in human fi-

bronectin enhances cell adhesive function. J. Biol. Chem. 269:24756–61Astrof S, Crowley D, Hynes RO. 2007. Multiple cardiovascular defects caused by the absence of alternatively

spliced segments of fibronectin. Dev. Biol. 311:11–24Aszodi A, Legate KR, Nakchbandi I, Fassler R. 2006. What mouse mutants teach us about extracellular matrix

function. Annu. Rev. Cell Dev. Biol. 22:591–621Bae E, Sakai T, Mosher DF. 2004. Assembly of exogenous fibronectin by fibronectin-null cells is dependent

on the adhesive substrate. J. Biol. Chem. 279:35749–59Baneyx G, Baugh L, Vogel V. 2001. Coexisting conformations of fibronectin in cell culture imaged using

fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 98:14464–68Banos CC, Thomas AH, Kuo CK. 2008. Collagen fibrillogenesis in tendon development: current models and

regulation of fibril assembly. Birth Def. Res. C 84:228–44Bass MD, Humphries MJ. 2002. Cytoplasmic interactions of syndecan-4 orchestrate adhesion receptor and

growth factor receptor signalling. Biochem. J. 368:1–15Bershadsky AD, Balaban NQ, Geiger B. 2003. Adhesion-dependent cell mechanosensitivity. Annu. Rev. Cell

Dev. Biol. 19:677–95Birk DE, Trelstad RL. 1984. Extracellular compartments in matrix morphogenesis: collagen fibril, bundle,

and lamellar formation by corneal fibroblasts. J. Cell Biol. 99:2024–33Birk DE, Trelstad RL. 1986. Extracellular compartments in tendon morphogenesis: collagen fibril, bundle,

and macroaggregate formation. J. Cell Biol. 103:231–40


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


Bourdoulous S, Orend G, MacKenna DA, Pasqualini R, Ruoslahti E. 1998. Fibronectin matrix regulatesactivation of RHO and CDC42 GTPases and cell cycle progression. J. Cell Biol. 143:267–76

Brenner KA, Corbett SA, Schwarzbauer JE. 2000. Regulation of fibronectin matrix assembly by activated Rasin transformed cells. Oncogene 19:3156–63

Bultmann H, Santas AJ, Pesciotta Peters DM. 1998. Fibronectin fibrillogenesis involves the heparin II bindingdomain of fibronectin. J. Biol. Chem. 273:2601–9

Castelletti F, Donadelli R, Banteria F, Hildebrandt F, Zipfel PF, et al. 2008. Mutations in FN1 cause glomeru-lopathy with fibronectin deposits. Proc. Natl. Acad. Sci. USA 105:2538–43

Chen H, Mosher DF. 1996. Formation of sodium dodecyl sulfate–stable fibronectin multimers. J. Biol. Chem.271:9084–89

Chen LB, Murray A, Segal RA, Bushnell A, Walsh ML. 1978. Studies on intercellular LETS glycoproteinmatrices. Cell 14:377–91

Chernousov MA, Faerman AI, Frid MG, Printseva OY, Koteliansky VE. 1987. Monoclonal antibody tofibronectin which inhibits extracellular matrix assembly. FEBS Lett. 217:124–28

Chernousov MA, Fogerty FJ, Koteliansky VE, Mosher DF. 1991. Role of the I-9 and III-1 modules offibronectin in the formation of an extracellular fibronectin matrix. J. Biol. Chem. 266:10851–58

Christopher RA, Kowalczyk AP, McKeown-Longo PJ. 1997. Localization of fibronectin matrix assembly siteson fibroblasts and endothelial cells. J. Cell Sci. 110:569–81

Chung CY, Erickson HP. 1997. Glycosaminoglycans modulate fibronectin matrix assembly and are essentialfor matrix incorporation of tenascin-C. J. Cell Sci. 110:1413–19

Colombi M, Zoppi N, De Petro G, Marchina E, Gardella R, et al. 2003. Matrix assembly induction and cellmigration and invasion inhibition by a 13-amino acid fibronectin peptide. J. Biol. Chem. 278:14346–55

Czirok A, Zach J, Kozel BA, Mecham RP, Davis EC, Rongish BJ. 2006. Elastic fiber macro-assembly is ahierarchical, cell motion–mediated process. J. Cell Physiol. 207:97–106

Dallas SL, Sivakumar P, Jones CJ, Chen Q, Peters DM, et al. 2005. Fibronectin regulates latent transforminggrowth factor-β (TGFβ) by controlling matrix assembly of latent TGFβ-binding protein-1. J. Biol.Chem. 280:18871–80

Danen EH, Aota S, van Kraats AA, Yamada KM, Ruiter DJ, van Muijen GN. 1995. Requirement for thesynergy site for cell adhesion to fibronectin depends on the activation state of integrin α5β1. J. Biol.Chem. 270:21612–18

Dzamba BJ, Jakab KR, Marsden M, Schwartz MA, DeSimone DW. 2009. Cadherin adhesion, tissue tension,and noncanonical Wnt signaling regulate fibronectin matrix organization. Dev. Cell 16:421–32

Dzamba BJ, Peters DM. 1991. Arrangement of cellular fibronectin in noncollagenous fibrils in human fibro-blast cultures. J. Cell Sci. 100:605–12

Dzamba BJ, Wu H, Jaenisch R, Peters DM. 1993. Fibronectin binding site in type I collagen regulate fi-bronectin fibril formation. J. Cell Biol. 121:1165–72

Engel J, Odermatt E, Engel A, Madri JA, Furthmayr H, et al. 1981. Shapes, domain organizations andflexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix. J. Mol. Biol.150:97–120

Engler AJ, Chan M, Boettiger D, Schwarzbauer JE. 2009. A novel mode of cell detachment from fibrillarfibronectin matrix under shear. J. Cell Sci. 122:1647–53

Erickson HP. 1994. Reversible unfolding of fibronectin type III and immunoglobulin domains provides thestructural basis for stretch and elasticity of titin and fibronectin. Proc. Natl. Acad. Sci. USA 91:10114–18

Erickson HP. 2002. Stretching fibronectin. J. Musc. Res. Cell Motil. 23:575–80Erickson HP, Carrell NA. 1983. Fibronectin in extended and compact conformations. Electron microscopy

and sedimentation analysis. J. Biol. Chem. 258:14539–44Essner JJ, Chen E, Ekker SC. 2006. Syndecan-2. Int. J. Biochem. Cell Biol. 38:152–56Fogelgren B, Polgar N, Szauter KM, Ujfaludi Z, Laczko R, et al. 2005. Cellular fibronectin binds to lysyl

oxidase with high affinity and is critical for its proteolytic activation. J. Biol. Chem. 280:24690–97Fogerty FJ, Akiyama SK, Yamada KM, Mosher DF. 1990. Inhibition of binding of fibronectin to matrix

assembly sites by anti-integrin (α5β1) antibodies. J. Cell Biol. 111:699–708Friedland JC, Lee MH, Boettiger D. 2009. Mechanically activated integrin switch controls α5β1 function.

Science 323:642–44


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


Fukuda T, Yoshida N, Kataoka Y, Manabe R, Mizuno-Horikawa Y, et al. 2002. Mice lacking the EDB segmentof fibronectin develop normally but exhibit reduced cell growth and fibronectin matrix assembly in vitro.Cancer Res. 62:5603–10

Galante LL, Schwarzbauer JE. 2007. Requirements for sulfate transport and the diastrophic dysplasia sulfatetransporter in fibronectin matrix assembly. J. Cell Biol. 179:999–1009

Gee EP, Ingber DE, Stultz CM. 2008. Fibronectin unfolding revisited: modeling cell traction-mediatedunfolding of the tenth type-III repeat. PLoS One 3:e2373

Geiger B, Bershadsky A, Pankov R, Yamada KM. 2001. Transmembrane extracellular matrix–cytoskeletoncrosstalk. Nat. Rev. Mol. Cell Biol. 2:793–805

George EL, Georges-Labouesse EN, Patel-King RS, Rayburn H, Hynes RO. 1993. Defects in mesoderm,neural tube and vascular development in mouse embryos lacking fibronectin. Development 119:1079–91

Green JA, Berrier AL, Pankov R, Yamada KM. 2009. β1 integrin cytoplasmic domain residues selectivelymodulate fibronectin matrix assembly and cell spreading through talin and Akt-1. J. Biol. Chem. 284:8148–59

Guan J-L, Hynes RO. 1990. Lymphoid cells recognize an alternatively spliced segment of fibronectin via theintegrin receptor α4β1. Cell 60:53–61

Guan J-L, Trevithick JE, Hynes RO. 1990. Retroviral expression of alternatively spliced forms of rat fi-bronectin. J. Cell Biol. 110:833–47

Hall A. 2005. Rho GTPases and the control of cell behaviour. Biochem. Soc. Trans. 33:891–95Halliday NL, Tomasek JJ. 1995. Mechanical properties of the extracellular matrix influence fibronectin fibril

assembly in vitro. Exp. Cell Res. 217:109–17Hocking DC, Smith RK, McKeown-Longo PJ. 1996. A novel role for the integrin-binding III-10 module in

fibronectin matrix assembly. J. Cell Biol. 133:431–44Hocking DC, Sottile J, McKeown-Longo PJ. 1994. Fibronectin’s III-1 module contains a conformation-

dependent binding site for the amino-terminal region of fibronectin. J. Biol. Chem. 269:19183–91Hocking DC, Sottile J, McKeown-Longo PJ. 1998. Activation of distinct α5β1-mediated signaling pathways

by fibronectin’s cell adhesion and matrix assembly domains. J. Cell Biol. 141:241–53Hocking DC, Sottile J, Reho T, Fassler R, McKeown-Longo PJ. 1999. Inhibition of fibronectin matrix

assembly by the heparin-binding domain of vitronectin. J. Biol. Chem. 274:27257–64Huveneers S, Danen EH. 2009. Adhesion signaling—crosstalk between integrins, Src and Rho. J. Cell Sci.

122:1059–69Huveneers S, Truong H, Fassler R, Sonnenberg A, Danen EH. 2008. Binding of soluble fibronectin to integrin

α5β1—link to focal adhesion redistribution and contractile shape. J. Cell Sci. 121:2452–62Hynes RO. 1990. Fibronectins. New York: Springer-Verlag. 544 pp.Ichihara-Tanaka K, Maeda T, Titani K, Sekiguchi K. 1992. Matrix assembly of recombinant fibronectin

polypeptide consisting of amino-terminal 70 kDa and carboxyl-terminal 37 kDa regions. FEBS Lett.299:155–58

Ilic D, Kovacic B, Johkura K, Schlaepfer DD, Tomasevic N, et al. 2004. FAK promotes organization offibronectin matrix and fibrillar adhesions. J. Cell Sci. 117:177–87

Ingham KC, Brew SA, Erickson HP. 2004. Localization of a cryptic binding site for tenascin on fibronectin.J. Biol. Chem. 279:28132–35

Ingham KC, Brew SA, Huff S, Litvinovich SV. 1997. Cryptic self-association sites in type III modules offibronectin. J. Biol. Chem. 272:1718–24

Johnson KJ, Sage H, Briscoe G, Erickson HP. 1999. The compact conformation of fibronectin is determinedby intramolecular ionic interactions. J. Biol. Chem. 274:15473–79

Kadler KE, Hill A, Canty-Laird EG. 2008. Collagen fibrillogenesis: fibronectin, integrins, and minor collagensas organizers and nucleators. Curr. Opin. Cell Biol. 20:495–501

Karuri NW, Lin Z, Rye HS, Schwarzbauer JE. 2009. Probing the conformation of the fibronectin III1-2domain by fluorescence resonance energy transfer. J. Biol. Chem. 284:3445–52

Kinsey R, Williamson MR, Chaudhry S, Mellody KT, McGovern A, et al. 2008. Fibrillin-1 microfibril depo-sition is dependent on fibronectin assembly. J. Cell Sci. 121:2696–704

Klass CM, Couchman JR, Woods A. 2000. Control of extracellular matrix assembly by syndecan-2 proteo-glycan. J. Cell Sci. 113:493–506


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


Klein RM, Zheng M, Ambesi A, Van De Water L, McKeown-Longo PJ. 2003. Stimulation of extracellularmatrix remodeling by the first type III repeat in fibronectin. J. Cell Sci. 116:4663–74

Kozel BA, Rongish BJ, Czirok A, Zach J, Little CD, et al. 2006. Elastic fiber formation: a dynamic view ofextracellular matrix assembly using timer reporters. J. Cell Physiol. 207:87–96

Krammer A, Lu H, Isralewitz B, Schulten K, Vogel V. 1999. Forced unfolding of the fibronectin type IIImodule reveals a tensile molecular recognition switch. Proc. Natl. Acad. Sci. USA 96:1351–56

Langenbach KJ, Sottile J. 1999. Identification of protein-disulfide isomerase activity in fibronectin. J. Biol.Chem. 274:7032–38

Leahy DJ, Aukhil I, Erickson HP. 1996. 2.0 A crystal structure of a four-domain segment of human fibronectinencompassing the RGD loop and synergy region. Cell 84:155–64

Ledger PW, Uchida N, Tanzer ML. 1980. Immunocytochemical localization of procollagen and fibronectinin human fibroblasts: effects of the monovalent ionophore, monensin. J. Cell Biol. 87:663–71

Litvinovich SV, Brew SA, Aota S, Akiyama SK, Haudenschild C, Ingham KC. 1998. Formation of amyloid-likefibrils by self-association of a partially unfolded fibronectin type III module. J. Mol. Biol. 280:245–58

Mao Y, Schwarzbauer JE. 2005a. Fibronectin fibrillogenesis, a cell-mediated matrix assembly process. MatrixBiol. 24:389–99

Mao Y, Schwarzbauer JE. 2005b. Stimulatory effects of a three-dimensional microenvironment on cell-mediated fibronectin fibrillogenesis. J. Cell Sci. 118:4427–36

Mao Y, Schwarzbauer JE. 2006. Accessibility to the fibronectin synergy site in a 3D matrix regulates engage-ment of α5β1 versus αvβ3 integrin receptors. Cell Commun. Adhes. 13(5–6):267–77

Maqueda A, Moyano JV, Hernandez Del Cerro M, Peters DM, Garcia-Pardo A. 2007. The heparin III–bindingdomain of fibronectin (III4-5 repeats) binds to fibronectin and inhibits fibronectin matrix assembly. MatrixBiol. 26:642–51

McDonald JA. 1988. Extracellular matrix assembly. Annu. Rev. Cell Biol. 4:183–207McDonald JA, Quade BJ, Broekelman TJ, LaChance R, Forsman K, et al. 1987. Fibronectin’s cell-adhesive

domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast peri-cellular matrix. J. Biol. Chem. 262:2957–67

McKeown-Longo PJ, Mosher DF. 1983. Binding of plasma fibronectin to cell layers of human skin fibroblasts.J. Cell Biol. 97:466–72

McKeown-Longo PJ, Mosher DF. 1985. Interaction of the 70,000-mol. wt. amino terminal fragment offibronectin with matrix-assembly receptor of fibroblasts. J. Cell Biol. 100:364–74

Mitra S, Schlaepfer DD. 2006. Integrin-regulated FAK-Src signaling in normal and cancer cells. Curr. Opin.Cell Biol. 18:516–23

Morla A, Ruoslahti E. 1992. A fibronectin self-assembly site involved in fibronectin matrix assembly: recon-struction in a synthetic peptide. J. Cell Biol. 118:421–29

Morla A, Zhang Z, Ruoslahtl E. 1994. Superfibronectin is a functionally distinct form of fibronectin. Nature367:193–96

Muro AF, Chauhan AK, Gajovic S, Iaconcig A, Porro F, et al. 2003. Regulated splicing of the fibronectinEDA exon is essential for proper skin wound healing and normal lifespan. J. Cell Biol. 162:149–60

Oberhauser AF, Badilla-Fernandez C, Carrion-Vazquez M, Fernandez JM. 2002. The mechanical hierarchiesof fibronectin observed with single-molecule AFM. J. Mol. Biol. 319:433–47

Ohashi T, Augustus AM, Erickson HP. 2009. Transient opening of fibronectin type III (FNIII) domains: theinteraction of the third FNIII domain of FN with anastellin. Biochemistry 48:4189–97

Ohashi T, Erickson HP. 2005. Domain unfolding plays a role in superfibronectin formation. J. Biol. Chem.280:39143–51

Ohashi T, Erickson HP. 2009. Revisiting the mystery of fibronectin multimers: the fibronectin matrix iscomposed of fibronectin dimers cross-linked by non-covalent bonds. Matrix Biol. 28:170–75

Ohashi T, Kiehart DP, Erickson HP. 1999. Dynamics and elasticity of the fibronectin matrix in living cellculture visualized by fibronectin-green fluorescent protein. Proc. Natl. Acad. Sci. USA 96:2153–58

Ohashi T, Kiehart DP, Erickson HP. 2002. Dual labeling of the fibronectin matrix and actin cytoskeletonwith green fluorescent protein variants. J. Cell Sci. 115:1221–29

Olorundare OE, Peyruchaud O, Albrecht RM, Mosher DF. 2001. Assembly of a fibronectin matrix by adherentplatelets stimulated by lysophosphatidic acid and other agonists. Blood 98:117–24


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


Pankov R, Cukierman E, Katz BZ, Matsumoto K, Lin DC, et al. 2000. Integrin dynamics and matrix assembly:tensin-dependent translocation of α5β1 integrins promotes early fibronectin fibrillogenesis. J. Cell Biol.148:1075–90

Plato. 1965. Timaeus (English translation). Baltimore: Penguin Books. 121 pp.Potts JR, Campbell ID. 1994. Fibronectin structure and assembly. Curr. Opin. Cell Biol. 6:648–55Pozzi A, Voziyan PA, Hudson BG, Zent R. 2009. Regulation of matrix synthesis, remodeling and accumulation

in glomerulosclerosis. Curr. Pharm. Des. 15:1318–33Ramirez F, Sakai LY. 2010. Biogenesis and function of fibrillin assemblies. Cell Tissue Res. 339:71–82Rossi A, Kaitila I, Wilcox WR, Rimoin DL, Steinmann B, et al. 1998. Proteoglycan sulfation in cartilage and

cell cultures from patients with sulfate transporter chondrodysplasias: relationship to clinical severity andindications on the role of intracellular sulfate production. Matrix Biol. 17:361–69

Ruoslahti E, Obrink B. 1996. Common principles in cell adhesion. Exp. Cell Res. 227:1–11Sabatier L, Chen D, Fagotto-Kaufmann C, Hubmacher D, McKee MD, et al. 2009. Fibrillin assembly requires

fibronectin. Mol. Biol. Cell 20:846–58Schwartz MA, Schaller MD, Ginsberg MH. 1995. Integrins: emerging paradigms of signal transduction. Annu.

Rev. Cell Dev. Biol. 11:549–99Schwarzbauer JE. 1991a. Fibronectin: from gene to protein. Curr. Opin. Cell Biol. 3:786–91Schwarzbauer JE. 1991b. Identification of the fibronectin sequences required for assembly of a fibrillar matrix.

J. Cell Biol. 113:1463–73Schwarzbauer JE, Spencer CS, Wilson CL. 1989. Selective secretion of alternatively spliced fibronectin vari-

ants. J. Cell Biol. 109:3445–53Sechler JL, Corbett SA, Schwarzbauer JE. 1997. Modulatory roles for integrin activation and the synergy site

of fibronectin during matrix assembly. Mol. Biol. Cell 8:2563–73Sechler JL, Cumiskey AM, Gazzola DM, Schwarzbauer JE. 2000. A novel RGD-independent fibronectin

assembly pathway initiated by α4β1 integrin binding to the alternatively spliced V region. J. Cell Sci.113:1491–98

Sechler JL, Rao H, Cumiskey AM, Vega-Colon I, Smith MS, et al. 2001. A novel fibronectin binding siterequired for fibronectin fibril growth during matrix assembly. J. Cell Biol. 154:1081–88

Sechler JL, Schwarzbauer JE. 1998. Control of cell cycle progression by fibronectin matrix architecture. J.Biol. Chem. 273:25533–36

Sechler JL, Takada Y, Schwarzbauer JE. 1996. Altered rate of fibronectin matrix assembly by deletion of thefirst type III repeats. J. Cell Biol. 134:573–83

Sharma A, Askari JA, Humphries MJ, Jones EY, Stuart DI. 1999. Crystal structure of a heparin- and integrin-binding segment of human fibronectin. EMBO J. 18:1468–79

Shi F, Sottile J. 2008. Caveolin-1-dependent β1 integrin endocytosis is a critical regulator of fibronectinturnover. J. Cell Sci. 121:2360–71

Singer II. 1979. The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of5 nm microfilaments in hamster and human fibroblasts. Cell 16:675–85

Smith ML, Gourdon D, Little WC, Kubow KE, Eguiluz RA, et al. 2007. Force-induced unfolding of fi-bronectin in the extracellular matrix of living cells. PLoS Biol. 5:e268

Sottile J, Chandler J. 2005. Fibronectin matrix turnover occurs through a caveolin-1-dependent process. Mol.Biol. Cell 16:757–68

Sottile J, Hocking DC. 2002. Fibronectin polymerization regulates the composition and stability of extracel-lular matrix fibrils and cell-matrix adhesions. Mol. Biol. Cell 13:3546–59

Sottile J, Hocking DC, Langenbach KJ. 2000. Fibronectin polymerization stimulates cell growth by RGD-dependent and -independent mechanisms. J. Cell Sci. 113:4287–99

Sottile J, Schwarzbauer JE, Selegue J, Mosher DF. 1991. Five type I modules of fibronectin form a functionalunit that binds to fibroblasts and Staphylococcus aureus. J. Biol. Chem. 266:12840–43

Sottile J, Wiley S. 1994. Assembly of amino-terminal fibronectin dimers into the extracellular matrix. J. Biol.Chem. 269:17192–98

Takahashi S, Leiss M, Moser M, Ohashi T, Kitao T, et al. 2007. The RGD motif in fibronectin is essentialfor development but dispensable for fibril assembly. J. Cell Biol. 178:167–78


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


Tan MH, Sun Z, Opitz SL, Schmidt TE, Peters JH, George EL. 2004. Deletion of the alternatively splicedfibronectin EIIIA domain in mice reduces atherosclerosis. Blood 104:11–18

Twal WO, Czirok A, Hegedus B, Knaak C, Chintalapudi MR, et al. 2001. Fibulin-1 suppression of fibronectin-regulated cell adhesion and motility. J. Cell Sci. 114:4587–98

Ugarova TP, Ljubimov AV, Deng L, Plow EF. 1996. Proteolysis regulates exposure of the IIICS-1 adhesivesequence in plasma fibronectin. Biochemistry 35:10913–21

Ugarova TP, Zamaroon C, Veklich Y, Bowditch RD, Ginsberg MH, et al. 1995. Conformational transitionsin the cell binding domain of fibronectin. Biochemistry 34:4457–66

Vakonakis I, Staunton D, Rooney LM, Campbell ID. 2007. Interdomain association in fibronectin: insightinto cryptic sites and fibrillogenesis. EMBO J. 26:2575–83

Valenick LV, Hsia HC, Schwarzbauer JE. 2005. Fibronectin fragmentation promotes α4β1 integrin-mediatedcontraction of a fibrin-fibronectin provisional matrix. Exp. Cell Res. 309:48–55

Ventura E, Sassi F, Parodi A, Balza E, Borsi L, et al. 2010. Alternative splicing of the angiogenesis associatedextra-domain B of fibronectin regulates the accessibility of the B-C loop of the type III repeat 8. PLoSOne 5:e9145

Wagenseil JE, Mecham RP. 2007. New insights into elastic fiber assembly. Birth Def. Res. C 81:229–40Wayner EA, Garcia-Pardo A, Humphries MJ, McDonald JA, Carter WG. 1989. Identification and charac-

terization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) inplasma fibronectin. J. Cell Biol. 109:1321–30

Wierzbicka-Patynowski I, Mao Y, Schwarzbauer JE. 2007. Continuous requirement for pp60-Src andphospho-paxillin during fibronectin matrix assembly by transformed cells. J. Cell. Physiol. 210:750–56

Wierzbicka-Patynowski I, Schwarzbauer JE. 2002. Regulatory role for Src and phosphatidylinositol 3-kinasein initiation of fibronectin matrix assembly. J. Biol. Chem. 277:19703–8

Wierzbicka-Patynowski I, Schwarzbauer JE. 2003. The ins and outs of fibronectin matrix assembly. J. CellSci. 116:3269–76

Wilcox-Adelman SA, Denhez F, Goetinck PF. 2002. Syndecan-4 modulates focal adhesion kinase phospho-rylation. J. Biol. Chem. 277:32970–77

Wolfram D, Tzankov A, Pulzl P, Piza-Katzer H. 2009. Hypertrophic scars and keloids—a review of theirpathophysiology, risk factors, and therapeutic management. Dermatol. Surg. 35:171–81

Woods A. 2001. Syndecans: transmembrane modulators of adhesion and matrix assembly. J. Clin. Invest.107:935–41

Woods A, Longley RL, Tumova S, Couchman JR. 2000. Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts. Arch. Biochem. Biophys.374:66–72

Wu C, Hughes PE, Ginsberg MH, McDonald JA. 1996. Identification of a new biological function for theintegrin αvβ3: initiation of fibronectin matrix assembly. Cell Adhes. Commun. 4:149–58

Wu C, Keivenst VM, V O’Toole TE, McDonald JA, Ginsberg MH. 1995. Integrin activation and cytoskeletalinteraction are critical steps in the assembly of a fibronectin matrix. Cell 83:715–24

Xu J, Bae E, Zhang Q, Annis DS, Erickson HP, Mosher DF. 2009. Display of cell surface sites for fibronectinassembly is modulated by cell adherence to 1F3 and C-terminal modules of fibronectin. PLoS One 4:e4113

Yang JT, Hynes RO. 1996. Fibronectin receptor functions in embryonic cells deficient in α5β1 integrin canbe replaced by αv integrins. Mol. Biol. Cell 7:1737–48

Yoneda A, Ushakov D, Multhaupt HAB, Couchman JR. 2007. Fibronectin matrix assembly requires distinctcontributions from Rho kinases I and -II. Mol. Biol. Cell 18:66–75

Yurchenco PD, Patton BL. 2009. Developmental and pathogenic mechanisms of basement membrane assem-bly. Curr. Pharm. Des. 15:1277–94

Zaidel-Bar R, Milo R, Kam Z, Geiger B. 2007. A paxillin tyrosine phosphorylation switch regulates theassembly and form of cell-matrix adhesions. J. Cell Sci. 120:137–48

Zamir E, Katz BZ, Aota S, Yamada KM, Geiger B, Kam Z. 1999. Molecular diversity of cell-matrix adhesions.J. Cell Sci. 112:1655–69

Zhang G, Young BB, Ezura Y, Favata M, Soslowsky LJ, et al. 2005. Development of tendon structure andfunction: regulation of collagen fibrillogenesis. J. Musculoskelet. Neuronal Interact. 5:5–21


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.


Zhang Q, Checovich WJ, Peters DM, Albrecht RM, Mosher DF. 1994. Modulation of cell surface fibronectinassembly sites by lysophosphatidic acid. J. Cell Biol. 127:1447–59

Zhang Q, Mosher DF. 1996. Cross-linking of the NH2-terminal region of fibronectin to molecules of largeapparent molecular mass. J. Biol. Chem. 271:33284–92

Zhang Q, Peyruchaud O, French KJ, Magnusson MK, Mosher DF. 1999. Sphingosine 1-phosphate stimulatesfibronectin matrix assembly through a Rho-dependent signal pathway. Blood 93:2984–90

Zhang Z, Morla AO, Vuori K, Bauer JS, Juliano RL, Ruoslahti E. 1993. The αvβ1 integrin functions as afibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin.J. Cell Biol. 122:235–42

Zhong C, Chrzanowska-Wodnicka M, Brown J, Shaub A, Belkin AM, Burridge K. 1998. Rho-mediatedcontractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly. J. Cell Biol.141:539–51


Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.

AR425-FM ARI 14 September 2010 18:54

Annual Reviewof Cell andDevelopmentalBiology

Volume 26, 2010

ContentsEnzymes, Embryos, and Ancestors

John Gerhart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Control of Mitotic Spindle LengthGohta Goshima and Jonathan M. Scholey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

Trafficking to the Ciliary Membrane: How to Get Acrossthe Periciliary Diffusion Barrier?Maxence V. Nachury, E. Scott Seeley, and Hua Jin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �59

Transmembrane Signaling ProteoglycansJohn R. Couchman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �89

Membrane Fusion: Five Lipids, Four SNAREs, Three Chaperones,Two Nucleotides, and a Rab, All Dancing in a Ring on Yeast VacuolesWilliam Wickner � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 115

Tethering Factors as Organizers of Intracellular Vesicular TrafficI-Mei Yu and Frederick M. Hughson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 137

The Diverse Functions of Oxysterol-Binding ProteinsSumana Raychaudhuri and William A. Prinz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

Ubiquitination in Postsynaptic Function and PlasticityAngela M. Mabb and Michael D. Ehlers � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 179

α-Synuclein: Membrane Interactions and Toxicityin Parkinson’s DiseasePavan K. Auluck, Gabriela Caraveo, and Susan Lindquist � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 211

Novel Research Horizons for Presenilins and γ-Secretases in CellBiology and DiseaseBart De Strooper and Wim Annaert � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 235

Modulation of Host Cell Function by Legionella pneumophilaType IV EffectorsAndree Hubber and Craig R. Roy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 261

A New Wave of Cellular ImagingDerek Toomre and Joerg Bewersdorf � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 285

Mechanical Integration of Actin and Adhesion Dynamicsin Cell MigrationMargaret L. Gardel, Ian C. Schneider, Yvonne Aratyn-Schaus,

and Clare M. Waterman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 315

vii

Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.

AR425-FM ARI 14 September 2010 18:54

Cell Motility and Mechanics in Three-Dimensional Collagen MatricesFrederick Grinnell and W. Matthew Petroll � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 335

Rolling Cell AdhesionRodger P. McEver and Cheng Zhu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 363

Assembly of Fibronectin Extracellular MatrixPurva Singh, Cara Carraher, and Jean E. Schwarzbauer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 397

Interactions Between Nuclei and the Cytoskeleton Are Mediatedby SUN-KASH Nuclear-Envelope BridgesDaniel A. Starr and Heidi N. Fridolfsson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 421

Plant Nuclear Hormone Receptors: A Role for Small Moleculesin Protein-Protein InteractionsShelley Lumba, Sean R. Cutler, and Peter McCourt � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 445

Mammalian Su(var) Genes in Chromatin ControlBarna D. Fodor, Nicholas Shukeir, Gunter Reuter, and Thomas Jenuwein � � � � � � � � � � � � � � 471

Chromatin Regulatory Mechanisms in PluripotencyJulie A. Lessard and Gerald R. Crabtree � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 503

Presentation Counts: Microenvironmental Regulation of Stem Cells byBiophysical and Material CuesAlbert J. Keung, Sanjay Kumar, and David V. Schaffer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 533

Paramutation and DevelopmentJay B. Hollick � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 557

Assembling Neural Crest Regulatory Circuits intoa Gene Regulatory NetworkPaola Betancur, Marianne Bronner-Fraser, and Tatjana Sauka-Spengler � � � � � � � � � � � � � � � 581

Regulatory Mechanisms for Specification and Patterning of PlantVascular TissuesAna Cano-Delgado, Ji-Young Lee, and Taku Demura � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 605

Common Factors Regulating Patterning of the Nervousand Vascular SystemsMariana Melani and Brant M. Weinstein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 639

Stem Cell Models of Cardiac Development and DiseaseKiran Musunuru, Ibrahim J. Domian, and Kenneth R. Chien � � � � � � � � � � � � � � � � � � � � � � � � � � 667

Stochastic Mechanisms of Cell Fate Specification that Yield Randomor Robust OutcomesRobert J. Johnston, Jr. and Claude Desplan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 689

A Decade of Systems BiologyHan-Yu Chuang, Matan Hofree, and Trey Ideker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 721

viii Contents

Ann

u. R

ev. C

ell D

ev. B

iol.

2010

.26:

397-

419.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

b-o

n: U

nive

rsid

ade

do M

inho

(U

Min

ho)

on 0

6/26

/12.

For

per

sona

l use

onl

y.

Documents

Assembly of Fibronectin ECM.pdf