Genetic Analyses of Integrin Signalingcshperspectives.cshlp.org/content/3/2/a005116.full.pdf · 2011. 2. 2. · Genetic Analyses of Integrin Signaling Sara A. Wickstro¨m1, Korana

Genetic Analyses of Integrin Signaling

Sara A. Wickstrom1, Korana Radovanac2, and Reinhard Fassler2

1Paul Gerson Una Group, Skin Homeostasis and Ageing, Max Planck Institute for Biology of Ageing,50937 Cologne, Germany

2Department of Molecular Medicine, Max Planck Institute for Biochemistry, 82152 Martinsried, Germany

Correspondence: [email protected]; [email protected]

The development of multicellular organisms, as well as maintenance of organ architectureand function, requires robust regulation of cell fates. This is in part achieved by conservedsignaling pathways through which cells process extracellular information and translate thisinformation into changes in proliferation, differentiation, migration, and cell shape. Genedeletion studies in higher eukaryotes have assigned critical roles for components of theextracellular matrix (ECM) and their cellular receptors in a vast number of developmentalprocesses, indicating that a large proportion of this signaling is regulated by cell-ECM inter-actions. In addition, genetic alterations in components of this signaling axis play causativeroles in several human diseases. This review will discuss what genetic analyses in miceand lower organisms have taught us about adhesion signaling in development and disease.

Almost all cells in multicellular organisms aresurrounded by a three-dimensional organ-

ized meshwork of macromolecules that consti-tute the extracellular matrix (ECM). The ECMis a dynamic structure that is generated andconstantly remodeled by cells that secrete andmanipulate its components into a precise con-figuration. It functions as a structural frame-work that provides cells with positional andenvironmental information, but also formsspecialized structures such as cartilage, tendons,basement membranes (BM), bone, and teeth.In addition to its structural properties, theECM acts as a signaling platform that regulatesa large number of cellular functions. It is capa-ble of binding growth factors, chemokines,and cytokines thereby modulating their bio-availability and activity. On the other hand,

the ECM is recognized by multiple cell surfacereceptors that transmit information from theextracellular environment by propagating intra-cellular signals (for a review, see Hynes 2009).

The major cell surface receptors that recog-nize and assemble the ECM are integrins. Integ-rins are heterodimeric transmembrane proteinscomposed of a and b subunits. Eighteen a sub-units and eight b subunits can assemble in 24different combinations with overlapping sub-strate specificity and cell-type-specific expres-sion patterns (Hynes 2002; Humphries et al.2006). This, together with the ability of differentheterodimers to assemble specific intracellularsignaling complexes, provides multiple layersof signaling specificity to these receptors. Con-versely, the integrin expression profile of a givencell type determines which ECM components it

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can bind. Signals arising from integrins regulatevirtually all aspects of cell behavior, includingcell migration, survival, cell cycle progression,and differentiation.

Genetics has proven to be a powerful tool todissect the functions of ECM–cell interactionsin complex organisms. To date, all of the integ-rin subunits and their major ligands have beendeleted in mice. Given the large variety of cellu-lar processes regulated by adhesion signaling,it is not surprising that a significant subset ofthese proteins has proven to be essential forembryonic development and/or tissue mainte-nance. However, in addition to underlining theimportance of cell-ECM interactions in devel-opment, genetic studies also revealed criticalroles for tissue- and cell-type-specific modesof adhesion signaling and provided importantinsights into human disease.

THE INTEGRIN-INTERMEDIATEFILAMENT AXIS

Basement membranes (BMs) are dense sheets ofextracellular matrix that function as structuralbarriers that separate epithelial and endothelialcells as well as peripheral nerve axons, fat cells,and muscle cells from the underlying tissuestroma. BMs provide structural support, separatetissues into compartments, and regulate cellbehavior. All cell types are known to producecomponents of BMs, which include type IV col-lagen, laminin, fibronectin, heparan sulfateproteoglycans, and nidogen/entactin as well asseveral minorcomponents (Erickson andCouch-man 2000; Yurchenco 2010). The molecularcom-position of the BM varies among different tissues,conferring signaling specificity important fordefining the specialized functions of epithelialand endothelial cells in different organs.

All BMs contain laminins, a family of 16heterotrimeric glycoproteins generated by thecombination of 5a, 4b, and 3g chains. Whenpresent in sufficient concentrations, lamininnetworks can self-assemble into polymers thatinteract with other ECM components, as wellas cell surface receptors such as integrins anddystroglycan. The laminin isoforms presentin BMs are regulated in a tissue-specific

manner, as well as temporally during develop-ment (Miner 2008; Yurchenco 2010). The cen-tral role of laminins in BM assembly isillustrated by gene deletion studies in mice.Laminin-111 (containing a1, b1, and g1chains) and laminin-511 (containing a5, b1,and g1 chains) are the central constituents ofperi-implantation stage BMs. Mice deficient ing1 or b1 subunits are unable to generate lami-nin trimers, and therefore lack BMs (Smythet al. 1999; Miner et al. 2004).

Laminin-332 (containing a3, b3, and g2chains; previously termed laminin-5) is a com-ponent of epithelial BMs and thus present inskin, stratified squamous mucosa, amnion, andcornea. Its main task is to maintain epithelialintegrity and epithelial-mesenchymal cohesionin tissues exposed to high mechanical forces.This function is facilitated by the uniqueability of laminin-322 to interact with two dis-tinct integrin heterodimers. Its interaction witha3b1 integrin results in the assembly of canonicalfocal adhesions (FA), whereas the interactionwith a6b4 integrin results in the formation ofspecialized adhesion complexes termed hemides-mosomes (Tsuruta et al. 2008).

Hemidesmosomes—Structure and Assembly

Hemidesmosomes were first characterized at theultrastructural level as electron-dense clusters atthe plasma membrane-ECM interphase (Weissand Ferris 1954). Further studies identifiedthem as multiprotein adhesion complexes pre-sent in stratified and simple epithelia. Two typesof hemidesmosomes can be distinguished basedon their molecular composition. Type I or clas-sical hemidesmosomes are found in stratifiedepithelium such as the skin and contain threetransmembrane proteins: a6b4 integrin, tetra-spanin CD151, and type XVII collagen (alsocalled bullous pemphigoid [BP] antigen 180)(Fig. 1). Type II hemidesmosomes are foundin simple epithelia such as intestine, and containonly integrin a6b4 (Uematsu et al. 1994;reviewed in Litjens et al. 2006).

The unique feature of hemidesmosomesis that they connect the ECM to the intermedi-ate filament (IF) network. This interaction is

S.A. Wickstrom et al.

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established by two plakin proteins, plectins andBP230 (also called BPAG1), of which plectin ispresent both in type I and II hemidesmosomes.Plectins are large cytoplasmic proteins, whichat their carboxyl terminus contain six plakinrepeats. A stretch of basic residues linking thefourth and fifth plakin repeat mediates theinteraction of plectin with IFs. The amino ter-minus contains two calponin-homology (CH)domains that constitute an actin-binding do-main. Through the actin-binding domain,plectins can associate with either the cytoplas-mic domain of the b4 integrin subunit or actinfilaments in a mutually exclusive manner, which

explains the absence of actin in hemides-mosomes (Rezniczek et al. 1998; Geerts et al.1999; Litjens et al. 2003). The interaction ofplectin with the cytoplasmic tail of b4 integrinis considered as the initial step of hemidesmo-some assembly (Geerts et al. 1999; Koster et al.2004). The cytoplasmic tail of b4 integrin isunusually large and shares no homology withother b integrin subunits. Its interaction withplectin has been shown to induce a conforma-tional change in the integrin tail (de Peredaet al. 2009). Because the recruitment of typeXVII collagen and the plakin BP230 to hemides-mosomes requires prior interaction of plectin

Stable adhesion

Cell migrationsignaling

Hemidesmosomedisassembly

Collagen XVII

Plectin

BP230

IF

Basalkeratinocyte

CD151

LM 322

α6 Integrinβ4 Integrin

Lamina lucida

Lamina densa

BM

P - S1364P - S1360

P - S1356

Figure 1. Molecular architecture of type I hemidesmosomes. Schematic depiction of a type I hemidesmosomefound in stratified epithelia such as in basal skin keratinocytes. The core component is a6b4 integrin, whichbinds the basement membrane (BM) component laminin (LM)-322. a6b4 integrin recruits the plakin proteinplectin through multiple interactions with the b4 integrin cytoplasmic tail, which initiates the formation ofhemidesmosomes. This is followed by the recruitment of collagen XVII, which interacts both with b4 integrinand plectin as well as with LM 322. Collagen XVII in turn mediates the recruitment of another plakin, bullouspemphigoid antigen 230 (BP 230), which together with plectin provides the connection to intermediate fila-ments (IF). This linkage is essential to stabilize the hemidesmosome and to provide stable adhesion of the basalkeratinocyte to the BM. Also, the transmembrane protein tetraspanin CD151 that interacts with a6 integrin isfound in type I hemidesmosomes. Phosphorylation of serines (S) 1356, 1360, and 1364 on the cytoplasmic tail ofb4 integrin by growth factors induces disassembly of hemidesmosomes, which promotes cell migration and sig-naling. The molecules are not drawn to scale.


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with b4, this conformational change mightfacilitate the interactions of both proteins withthe integrin (Koster et al. 2004).

Deletion of a6b4 leads to the loss of hemi-desmosomes and epithelial detachment in mice(Dowling et al. 1996; van der Neut et al. 1996),indicating that BP180, despite its ability to bindlaminin-332 and plectin in vitro, is not suffi-cient to maintain adhesion of cells to laminin-332 in the absence of a6b4 (Tasanen et al.2004) and identifying a6b4 as the core compo-nent of hemidesmosomes. Another criticalevent in hemidesmosome assembly is the inter-action between b4 integrin tails and plectin.Blocking this interaction compromises hemi-desmosome assembly in vitro (Geerts et al.1999; Koster et al. 2001). In plectin-deficientmice hemidesmosomes appear ultrastructurallynormal, but their number and mechanical sta-bility are reduced (Andra et al. 1997). The factthat type II hemidesmosomes lack both typeXVII collagen and BP230 further shows thata6b4 and plectin alone are sufficient to initiatethe formation of these structures and to main-tain their integrity.

Although a6b4 is the only integrin foundin hemidesmosomes, other integrins can indi-rectly contribute to their assembly. Integrina3b1 has been shown to cluster in “pre-hemi-desmosomal” structures on the basal cell surfaceof human keratinocytes together with CD151.However, as hemidesmosomes mature, a3b1integrins become recruited to cell-cell or focalcontacts, whereas CD151 remains in the hemi-desmosomes (Sterk et al. 2000). The initiala3b1-containing adhesions have been pro-posed to contribute to the recruitment of a6b4to the plasma membrane, and thereby increasethe efficacy with which hemidesmosomes areformed. This is supported by the observationthat b1-deficient mice have reduced numbersof hemidesmosomes in the skin, although itshould be noted that the expression level ofa6b4 is also reduced in these mice, suggestingthat the mechanism could also be more indirect(Brakebusch et al. 2000). Polarity proteinshave also been shown to play a role in hemides-mosome assembly through the regulation ofpolarized targeting of integrins. In the larval

epidermis of the zebrafish, the basolateralidentity protein Lgl2 is required for hemides-mosome formation through recruitment of b4integrin to the basal plasma membrane (Sona-wane et al. 2005, 2009). Taken together, theformation of hemidesmosomes requires theestablishment of a basolateral membrane iden-tity through polarity proteins and possibly alsoother integrin-based adhesion structures to re-cruit a6b4 integrin to these sites. a6b4 integrinthen interacts with BM laminin and togetherwith plectin nucleates stable, IF-bound hemi-desmosomes. Interestingly, however, keratino-cytes expressing a b4 integrin subunit unableto bind laminin can still form structures thatcontain all components of type I hemides-mosomes, which implies that ligand bindingis not necessary for their formation (Nieverset al. 1998, 2000). These complexes, however, dif-fer in their morphology, density, and dynamicsfrom normal hemidesmosomes, suggesting thatthe integrin-laminin interaction plays a role inthe stability and structural organization of hemi-desmosomes (Geuijen and Sonnenberg 2002).

Hemidesmosomes—Guardians ofEpidermal Integrity

A central function of the skin is to act as a bidi-rectional barrier to prevent dehydration and toprotect against injury and pathogens. In addi-tion, it is exposed to constant mechanical stress.On the other hand, this tissue is continuouslyrenewed through a terminal differentiation pro-gram, in which basal keratinocytes detach fromthe BM and move to the upper layers of the epi-thelium where they slough off. Finally, in caseof injury, keratinocytes need to rapidly acquirea motile phenotype and migrate to close thewound. These functions require mechanicallystable but dynamic modes of cell adhesion.Hemidesmosomes are found in the basal plasmamembrane of basal keratinocytes, where theyfunction to attach these cells firmly to the BMseparating the epidermis from the dermis (Fig.1). Interestingly, a6b4 integrins and thus alsohemidesmosomes are not required for skinmorphogenesis or developmental homeostasis(DiPersio et al. 2000). The importance of





hemidesmosomes in maintaining epidermalintegrity is, however, shown by multiple linesof genetic evidence. Ablation of genes encodinga6 integrin,b4 integrin, or plectin in mice resultsin severe blistering of the skin causing neonataldeath because of a severe epithelial barrier defect(Dowling et al. 1996; Georges-Labouesse et al.1996a; van der Neut et al. 1996; Andra et al.1997). In line with their dispensable role in hemi-desmosome assembly, mice lacking type XVIIcollagen or BP230 display only mild forms ofskin blistering (Guo et al. 1995; Nishie et al.2007), whereas deletion of CD151 is dispensablefor both hemidesmosome stability and the integ-rity of skin (Wright et al. 2004).

No experimental data exist on the dynamicbehavior of hemidesmosomes in vivo, but it hasbeen analyzed in keratinocyte culture, in whichhemidesmosomes have been shown to displaya certain dynamics also in nonmigratory cells(Tsuruta et al. 2003). When cells are stimu-lated to migrate, a6b4 integrin is mobilizedfrom hemidesmosomes to actin-based struc-tures such as lamellipodia and filopodia to pro-mote cell motility, resulting in hemidesmosomedisassembly (Geuijen and Sonnenberg 2002;Tsuruta et al. 2003). This type of relocalizationhas also been visualized in human wound edges,where b4 integrins can be seen in trailing-edgehemidesmosomes as well as in lamellipodia ofthe leading edge (Underwood et al. 2009). Invitro analyses have shown that the mobilizationof a6b4 integrin from hemidesmosomes isfacilitated by growth factor-mediated phos-phorylation of multiple residues in the cyto-plasmic tail of b4. Both serines and tyrosinescan be phosphorylated, but the exact sites andtheir relevance remain controversial. The cur-rent data suggest that serine phosphorylationmight be more relevant under physiologicalconditions, leading to hemidesmosome disas-sembly through the release of plectin from theintegrin (Margadant et al. 2008) (Fig. 1)

a6b4 Integrin and Cancer

b4 integrin was originally identified as a “tu-mor-specific” protein (Tumor Surface Protein180) up-regulated in metastatic variants of

mouse lung carcinoma and melanoma cell lines(Kennel et al. 1981), and has later been shown tobe up-regulated in several human cancers, sug-gesting that the expression ofb4 integrin is ben-eficial for tumor cells (Giancotti 2007). The roleof a6b4 integrin signaling in tumorigenesishas been studied in mice carrying a truncatedcytoplasmic tail of b4 lacking the tyrosinephosphorylation sites as well as other potentialinteraction motifs. In contrast to mice lackingthe entire b4 cytoplasmic domain, whichdisplay extensive skin blistering because of theabsence of hemidesmosomes (Murgia et al.1998), mice with a more restricted truncationdo not show these defects. However, they showdelayed wound healing and defective neoangio-genesis in tumor xenografts (Nikolopoulos et al.2004, 2005). In addition, when crossed to theMMTV-Neu mice that carry an activated formof epidermal growth factor receptor-2 (ErbB2)driven by a mouse mammary tumor virus(MMTV) promoter leading to mammary tu-mors, the b4 mutant mice display delayedtumor onset, impaired tumor growth and de-creased metastatic potential (Guo et al. 2006).Whether this applies to other tumor types aswell remains to be shown.

The tumor-promoting properties of a6b4integrin seem to result from both promigratoryand signaling functions. b4 integrin has beenshown to cross-talk with several receptor tyro-sine kinases (RTK), including ErbB2, epidermalgrowth factor receptor (EGF-R), Met and Ron(Mariotti et al. 2001; Trusolino et al. 2001;Santoro et al. 2003; Guo et al. 2006). Followingstimulation, these RTKs induce activation ofphosphatidylinositol 3-kinase (PI3-K) or Srcfamily kinases (SFK), leading to phosphoryla-tion of theb4 integrin tail, disassembly of hemi-desmosomes, and induction of cell motility(Mariotti et al. 2001; Santoro et al. 2003). Onthe other hand, b4 integrin has been shown topromote SFK-dependent phosphorylation ofthe catalytic sites of RTKs and their substrates(Bertotti et al. 2006; Guo et al. 2006), therebyamplifying their signaling ability. Takentogether, a6b4 integrin seems to have a proin-vasive and proangiogenic activity in tumors,but additional genetic studies are needed to





determine whether this role is limited to malig-nancies involving hyperactivation of specificRTKs. In addition, several groups have failedto detect significant tyrosine phosphorylationof b4 integrin in keratinocytes or transformedcell lines in response to stimulation with EGFand hemidesmosome disassembly, making therelevance of tyrosine phosphorylation eventscontroversial (Rabinovitz et al. 1999, 2004; Altet al. 2001; Wilhelmsen et al. 2007). Knockinmice carrying mutations in specific phosphory-lation sites of the b4 integrin subunit wouldprovide more conclusive information on thein vivo significance of the various phosphoryla-tion events in a6b4 integrin signaling, hemi-desmosome turnover, and invasion.

THE INTEGRIN-ACTIN AXIS

In contrast to a6b4, most integrins engage theactin cytoskeleton following ligand binding. Acentral ligand of actin-associated integrins isFN, which is recognized by 11 integrin hetero-dimers in mice and humans, and it will be usedin this section as an example to discuss the prin-ciples of ECM-integrin interactions leading toengagement and remodeling of the actin cytoske-leton. FN is a large, modular glycoprotein thatexists in two forms; cellular FN which is presentin tissues where it is assembled into a fibrillarmatrix, and plasma FN, which is produced byhepatocytes and is secreted into the blood whereit remains in a nonfibrillar, soluble form, or fol-lowing entry into tissues becomes incorporatedinto the ECM (Leiss et al. 2008). FN is foundonly invertebrates, and it has co-evolved togetherwith the cardiovascular system. Consistently, FNis critical for the development of the vasculature,where it localizes between the endothelium andperivascular cells. Deletion of FN in mice resultsin embryonic lethal cardiovascular defects, whichvary depending on the genetic background.FN-null embryos from 129S4 mice are unableto form the dorsal aorta, indicative of an earlydefect already in vasculogenesis, whereas thisstructure is present in C57BL/6 derived embryosthat display defects in vascular lumen formation(George et al. 1993, 1997; Georges-Labouesseet al. 1996b).

FN is secreted as a disulfide-bonded dimer,and its deposition into a fibrillar matrix is acell-driven process that critically depends onintegrins. So fara5b1, avb3, a4b1, and aIIbb3integrins have been shown to induce fibri-llogenesis in vitro. These integrins bind FNthat is secreted as a compact globular structurewhere binding sites for other FN molecules areburied within the protein (see Schwarzbauerand DeSimone 2011). Binding is followed bythe activation of integrin signaling, leading tothe recruitment of cytoplasmic proteins toform focal complexes (FCs). Several FC com-ponents are actin-binding and modulatoryproteins, allowing the recruitment and reorgan-ization of the actin cytoskeleton at these sitesand their maturation into FAs (Geiger andYamada 2011). The major FN-fibril-formingintegrin a5b1 leaves FAs and moves along F-actin to the cell center to form fibrillar adhe-sions and to facilitate the generation of mechan-ical tension via the actin cytoskeleton, leading tostretching of the bound FN molecule, unravel-ing of the cryptic self-association sites, andfinally the binding to other FN moleculesresulting in fibril formation (Mao and Schwarz-bauer 2005; Leiss et al. 2008; Schwarzbauer andDeSimone 2011).

Interactions via the RGD Motif

Cell adhesion to FN critically depends on theArg-Gly-Asp (RGD) motif of FN, which is rec-ognized by a5b1, the av family, a8b1, a9b1,and the platelet-specific aIIbb3 integrins(Fig. 2). a5b1 is considered the major integrinresponsible for FN assembly, and its interactionwith the RGD motif is required for this func-tion. However, although deletion of a5 integrinin mice also leads to embryonic lethality andvascular defects, these mice develop signifi-cantly further than the FN-null mice (Yanget al. 1993). This is attributable to the abilityof cells to assemble FN using other integrins,mainly of the av subfamily. The deletion of allfive av heterodimers also leads to late embry-onic defects in vascular development mainlyin the placenta. A subset of animals can evenproceed through embryonic development and





die after birth because of hemorrhages, indicat-ing that, unlike a5, av integrins are dispensablefor vasculogenesis and partly also for angiogen-esis (Bader et al. 1998). Only a double knockoutof the av and a5 integrin genes results in a lossof FN fibrillogenesis (Yang et al. 1999). Interest-ingly, although the RGD motif is central for theinteraction of FN with a5b1 and avb3, theinactivation of this motif by a RGD to RGEpoint mutation also allows FN fibrillogenesisin vivo. The knockin mice carrying this muta-tion display a phenotype closely resemblingthat of the a5-null mice (Takahashi et al.2007). This highlights the importance of thismotif in FN signaling through a5b1, but alsoshows the ability of other integrin interactionsites on FN to take over the role of RGD in FNfibrillogenesis. Whether these sites play a rolein FN assembly when the RGD sequence isintact, remains open. Together, these resultsindicate that FN fibrillogenesis is not sufficientfor it to carry out its functions during develop-ment. They further illustrate that the majority of

FN signaling in embryogenesis occurs througha5b1 and that av and a5b1 integrins relaydistinct signals on FN binding. The severity ofthe FN-null phenotype in contrast to the integ-rin deletions also suggests that the functionsof FN during development extend beyondthe FN-integrin signaling axis. These functionscould be related to its mechanical role in theECM, or its ability to induce integrin-indepen-dent signaling, for example, through sequester-ing growth factors such as vascular endothelialgrowth factor (VEGF) or transforming growthfactor-b (TGF-b).

Non-RGD Binding Integrins

The FN gene can be alternatively spliced allow-ing the expression of up to 20 possible mo-nomeric isoforms in humans and up to 12 inmouse, potentially giving rise to a larger varietyof FN dimers (Pankov and Yamada 2002). Someof these splice variants can generate additionalintegrin interaction sites on FN. One of them

NGRMotif

Domain

Integrin

Fn1

Fn2

Fn3

Extra domain

Variable region

Reporteddevelopmentalfunction

? Lungmorphogenesis

Lymphangio-genesis

Placentalangiogenesis

SomitogenesisVasculogenesisAngiogenesis

Plateletaggregation

Lymphaticvalvemorphogenesis(α9β1)

Vascular remodellingPericyte recruitment

α4β1α4β7

α4β1

NH2

RGD EDGIHELLDVREDV

S S

COOH

α9β1αIIβ3α9β1α8β1 α5β1

αvβ5αvβ6

α5β1αvβ3

αvβ1αvβ3

Figure 2. Developmental functions of fibronectin-integrin interactions. Fibronectin (FN) is a dimeric glycyo-protein consisting of type I, type II, and type III modular repeats. Dimerization is achieved by disulfide bondsmediated by the two cysteines (S) in the COOH-terminus of the protein. The binding domains and motifs forintegrins, as well as mouse developmental functions reported to depend on the particular interaction are indi-cated. Integrins that have been shown to mediate FN fibrillogenesis in vitro are marked by gray boxes.





is the variable region (v-region) that can inter-act with two non-RGD-binding integrins, a4b1and a4b7 (Wayner et al. 1989; Guan and Hynes1990; Fig. 2). These integrins are mainly ex-pressed by hematopoietic cells, although a4b1is also found in several other cell types, suchas neural crest cells and cellular componentsof the cardiovascular and peripheral nervoussystems. The two a4 integrins have many im-portant roles during development. Deletion ofthe a4 integrin gene results in embryonic le-thality because of early placental and cardiovas-cular defects (Yang et al. 1995). However, thesedefects are probably not attributed to the FN-a4b1/a4b7 integrin interaction, as these twointegrins also bind cell counter receptors suchas vascular-cell adhesion molecule-1 (VCAM-1)and MadCAM, respectively. The developmen-tal defects in the a4 integrin-null mice arevery similar to those found in mice lackingVCAM-1, indicating that the observed defectsare caused by an abrogated interaction betweena4b1 integrin and VCAM-1 (Kwee et al. 1995;Yang et al. 1995). From a clinical point of view,it is particularly interesting that the interactionbetween a4b1 and VCAM-1 is important forthe homing of autoreactive T cells to the centralnervous system during the pathogenesis of anautoimmune disease called multiple sclerosis(Yednock et al. 1992; Vajkoczy et al. 2001; Baueret al. 2009). This disease is characterized byaxon demyelination and damage, leading tonumbness, weakness, paresis as well as cogni-tive problems. Blocking antibodies against a4integrin have proven to be an effective treatmentof multiple sclerosis (Steinman 2009), andgenetic analysis in mice showed that a4b1 isrequired for the arrest of T cells on the brainendothelial surface (Bauer et al. 2009).

a4b1 integrins can also interact with thealternative spliced extra domain A (EDA) of FN(Fig. 2), which is highly expressed during de-velopment and during pathological conditionssuch as tumorigenesis. This “reactivation” ofthe embryonic splice pattern is interesting, asa4b1 has recently been shown to play a criti-cal role in tumor lymphangiogenesis in a FN-dependent manner. Both a4b1 and FN arehighly expressed in lymphatic endothelial cells

of tumors, whereas other integrin subunitsseem not to be up-regulated (Garmy-Susiniet al. 2010). Whether this function reallydepends on the EDA domain was, however,not analyzed. In addition, it is not clear whethera4b1 integrin is also regulating developmentallymphangiogenesis, as the constitutive knock-out mice die before the initiation of this process.The EDA domain itself, however, plays a rolein developmental lymphangiogenesis throughintegrin a9b1. Interestingly, this integrin ishighly expressed in lymphatic but not bloodendothelial cells (Huang et al. 2000). Deletionof a9 integrin gene leads to a severe chylothoraxcaused by defects in lymphatic valve morpho-genesis and subsequent respiratory failure(Huang et al. 2000; Bazigou et al. 2009). Thisphenotype was partially recapitulated by dele-tion of EDA. Furthermore, both a9b1 integrinas well as the EDA domain were shown to becritical for FN assembly in lymphatic endothe-lial cells in vitro (Bazigou et al. 2009), in con-trast with previous studies showing that EDAis not required for FN assembly or mouse devel-opment because of compensation by EDB (Tanet al. 2004; Astrof et al. 2007). This study sug-gests that matrix assembly might be regulatedby integrins that are expressed in a tissue-spe-cific manner to allow more stringent controlof distinct anatomical structures.

Linkage to the Actin Cytoskeleton

In addition to catalyzing matrix assembly, thebinding of integrins to FN or other ligands leadsto the induction of multiple modes of intracel-lular signaling. These include activation of clas-sical signaling pathways through tyrosine andserine phosphorylation of specific substrates,resulting ultimately in the regulation of cell sur-vival, growth and differentiation. This signal-ing operates in close collaboration with growthfactor signaling, making it difficult to dissectwhich signals specifically emanate from ligatedintegrins (Legate et al. 2009; Ivaska and Heino2010). The other central signaling mode is therecruitment of filamentous (F-) actin to integ-rin adhesion sites, a process tightly coupled tothe active remodeling of the actin cytoskeleton.





The integrin-actin linkage is central to integrinfunction in several respects. First, active actinpolymerization, crosslinking, and subsequentforce generation are critical for the maturationof nascent and short-lived integrin adhesionsinto signaling-competent and stable FAs. Sec-ond, it is required for the precise spatiotemporalcontrol of cell protrusion and retraction duringcell migration. Third, it allows cells to adopt orchange their characteristic shape, for exampleto polarize. Finally, it is critical for FN matrixassembly. Integrins do not bind actin directly,but regulate this linkage by recruiting a largenumber of actin-binding and regulatory pro-teins. A recent study based on database miningidentified 156 signaling, structural and adaptormolecules that can be found in integrin adhe-sions (Zaidel-Bar et al. 2007). Only a small sub-set of these proteins binds directly to integrins,and the large majority is recruited throughprotein-protein interactions between the vari-ous scaffold proteins (for reviews, see Legateand Fassler 2009; Geiger and Yamada 2011). Alarge body of structural, biochemical, andgenetic evidence points to talin, kindlin, andthe integrin-linked kinase (ILK)-pinch-parvin(IPP) complex as some of the central FA com-ponents regulating the integrin-actin linkage.All of these proteins are ubiquitously expressedand regulate a wide range of integrin hetero-dimers. Deletion of any of these proteins leadsto early embryonic lethality, which resultsfrom compromised integrin function anddefects in their connection to the cytoskeleton.On the cell-biological level these defects spanthe entire range of functions attributed to theintegrin-actin linkage. Hence, they can beviewed as global regulators of integrin function.The precise functions of these proteins will bediscussed in the next section.

IN VIVO MECHANISMS OF INTEGRINSIGNALING THROUGH INTRACELLULAREFFECTORS AND SCAFFOLDS

Talin and kindlin are two FA proteins thatdirectly bind integrins and thereby are involvedin the very early events of integrin signaling.They are distinguished from the large group

of FA proteins by their ability to regulate bothintegrin activation (inside-out signaling) andintracellular signaling downstream of ligandbinding (outside-in signaling). Talin is presentin all multicellular eukaryotes; lower eukary-otes encode only a single talin isoform, whereasvertebrates have two talin isoforms, talin-1and talin-2. Talin consists of a large carboxy-terminal rod and an amino-terminal headdomain composed of a FERM domain withfour subdomains: F0, F1, F2, and F3. The F3subdomain contains a phosphotyrosine-bind-ing motif, which harbors a high affinity bindingsite for b-integrin tails. The rod domain con-tains multiple binding sites for the actin–bind-ing protein vinculin as well as for actin itself. Inaddition it contains a second integrin-bindingsite (Critchley and Gingras 2008). The talinhead has been shown to be sufficient for integ-rin binding and activation, whereas the rod isrequired for the scaffolding function of talinin outside-in signaling (Garcia-Alvarez et al.2003; Tanentzapf and Brown 2006; Tanentzapfet al. 2006).

Kindlins are an evolutionarily conservedfamily of multidomain proteins, which in mam-mals consists of three members: kindlin-1 (alsoknown as FERMT1), kindlin-2 (also knownas FERMT2 or MIG-2), and kindlin-3 (alsoknown as FERMT3 or URP2). Although en-coded by separate genes, they show identicaldomain architecture and high sequence simi-larity (Ussar et al. 2006). Like talin, kindlinscontain a FERM domain through which theyinteract with b1, b2, and b3 integrins, butthis FERM domain is unique in that it is inter-rupted by a pleckstrin homology (PH) domain,which provides a putative binding motif formembrane lipids.

Consequences of Loss of IntegrinInside-Out Signaling

Integrins are present on the plasma membranein low-, intermediate-, and high-affinity states.Structural studies suggest that a low-affinity stateis characterized by a bent, “closed” conformationof the extracellular domains and a high-affinitystate by an extended, “open” conformation





(Campbell and Humphries 2011). The extendedconformation is thought to be achieved byseparation of the a and b cytoplasmic tailsthrough binding of talin to the b subunit(Moser et al. 2009b; Shattil et al. 2010). Studiesin cultured cells have shown that the bindingof talin-1 to the cytoplasmic domain of theb-integrin subunit is a common step in b1and b3 integrin activation in vitro (Tadokoroet al. 2003). In contrast, kindlin alone is notsufficient to activate these integrins, but itsco-operation with talin is required to generatefully active integrins (Montanez et al. 2008;

Moser et al. 2008, 2009a). The molecular detailsof this cooperation are not clear.

Regulation of the ligand binding affinity isfundamental for various cellular functions.For example, the platelet integrin aIIbb3 needsto be maintained inactive to prevent it frombinding fibrinogen present in the blood streamthus prohibiting platelet aggregation andthrombus formation. On the other hand, thisintegrin needs to be rapidly activated to mediateplatelet adhesion on vascular injury to preventbleeding (Fig. 3). Similarly, leukocytes requireintegrin activation to adhere to and migrate

GPI

1

2

3 4

5

54321

Talin

Kindlin

Adaptor proteins

F-Actin

Resting platelet Inside-out signaling Outside-in signaling Platelet spreading Platelet aggregation

Inactive integrins Talin and kindlinrecruitment

Activation of integrin

Ligation of active integrinsRecruitment of adaptors

Cytoskeletal rearrangementAdhesion strengthening

Binding to soluble fibrinogenThrombus formation

αβ Integrin

Figure 3. Talin and kindlin regulate bidirectional integrin signaling. Bidirectional integrin signaling is essentialfor platelets (tan) to seal the injured blood vessel endothelium (red) and stop bleeding. Integrins in circulating,resting platelets exist in a low affinity state indicated by a bent confirmation (1). Following vessel injury, von Wil-lebrand factor (vWF) and collagen are exposed to bind their receptors GPIb and GPVI that are expressed on thesurface of platelets. Together with locally produced thrombin these receptors trigger the activation of aIIbb3integrin. This is achieved by promoting the association of talin-1 and kindlin-3 with the cytoplasmic tail ofb3 integrin, facilitating a conformational change in the integrin (inside-out signaling) (2). The conformationalchange allows integrins to bind fibrinogen, vWF, and fibronectin with high affinity. As a result, platelets adhere tothe vessel wall. Integrin ligation subsequently initiates signaling through kindlin and talin (outside-in signaling)(3) resulting in the recruitment of adaptor proteins and rearrangement of the cytoskeleton to promote cellspreading (4). This, together with integrin-mediated binding of soluble fibrinogen, results in platelet aggrega-tion and formation of a stable clot (5). The molecules are not drawn to scale.





across the endothelium to combat pathogens.The importance of this process is illustrated bythe phenotypes of talin- and kindlin-deficientmice. Deletion of talin-1 or kindlin-3 in plate-lets blocks activation of b1 and b3 integrins,leading to the inability of platelets to bind fibri-nogen and to form clots. As a result, these micesuffer from severe bleeding (Nieswandt et al.2007; Petrich et al. 2007; Moser et al. 2008).Similarly, neutrophils lacking kindlin-3 areunable to activate b2 integrins, resulting inloss of neutrophil adhesion to activated endo-thelial cells (Moser et al. 2009a). Talin has alsobeen shown to bind and activate neutrophilintegrin b2 in vitro (Simonson et al. 2006).The importance of integrin affinity regulationin adherent cell types is not as clear. Althoughdeletion of talin and kindlin in mesenchymalor epithelial cells leads to defects in integrinactivation, these cells are still capable of adher-ing, albeit to a reduced extent (Ussar et al.2008; Zhang et al. 2008), suggesting that underconditions of less stringent requirements forrapid induction of adhesion, high concentra-tions of ligands, and the absence of high shearflow, avidity regulation might be the predomi-nant mechanism of integrin regulation. Thefact that both talin and kindlin are also impor-tant in regulation of outside-in signaling makesthe in vivo dissection of these two processesdifficult.

Consequences of Loss of Outside-InSignaling

A central piece of evidence that talin has impor-tant functions besides integrin activation comesfrom studies performed with talin-null flies,which show that in the absence of talin, integ-rins are able to associate with the ECM, butare unable to connect to the cytoskeleton, lead-ing to muscle detachment at the integrin-actininterphase (Brown et al. 2002). Consistent withthis finding, mouse fibroblasts lacking bothtalin-1 and talin-2 are able to adhere, but notto link integrins to the cytoskeleton (Priddleet al. 1998; Zhang et al. 2008). This functionrequires the talin rod domain, which containsbinding sites for F-actin and vinculin

indicating that talin provides an essential linkageto the cytoskeleton independent of its ability toactivate integrins. Kindlins do not bind actindirectly, but connect to the actin cytoskele-ton via a migfilin-filamin interaction as wellas through the IPP complex (Montanez et al.2008). Platelets lacking kindlin-3 expressionare unable to organize their cytoskeleton orto establish stable lamellipodia (Moser et al.2008), which probably contributes to theirinability to spread and form a crosslinked clot(see Fig. 3). Loss of kindlin-2 impairs actinorganization and FA formation in endodermcells, which together with defective integrinactivation leads to early embryonic lethality.Interestingly, the rudimentary FAs in kindlin-2-deficient cells do not contain ILK, suggestingthat kindlin acts as a nucleator of FA formation(Montanez et al. 2008).

Despite the central role of talin and kindlinin outside-in signaling, they are not sufficient toestablish a stable integrin-actin connection. Animportant scaffold also required for this func-tion is the IPP complex, which is a constituentof at least b1 and b3 integrin-containing adhe-sion sites. The core component of this complexis ILK, a pseudokinase that is also capable ofbinding these integrins directly, at least in vitro(Wickstrom et al. 2010). Whether the bindingis actually relevant for the function of this com-plex, or whether it occurs in vivo, has not beenconclusively shown. The other two membersof this complex are pinch and parvin. Pinch,which interacts with the amino-terminalankyrin repeats of ILK, is a family of proteinscontaining only LIM domains and consistingof two members, pinch-1 and pinch-2 (Tuet al. 1999, 2001; Chiswell et al. 2008). The par-vins, which interact with the carboxy-terminalkinase-homology domain of ILK, are a familyof CH-domain-containing proteins with threemembers; the ubiquitously expressed a-parvin(also known as actopaxin or CH-ILKBP), b-parvin (also known as affixin), which is highlyexpressed in heart and skeletal muscle, and g-parvin, which is restricted to the hematopoieticsystem (Nikolopoulos and Turner 2000; Olskiet al. 2001; Tu et al. 2001; Yamaji et al. 2001;Chu et al. 2006). The IPP complex is thought





to be preassembled in the cytoplasm and is sub-sequently recruited to integrin adhesions in anILK-dependent manner (Zhang et al. 2002).The stability of each individual component de-pends on the assembly of the complex, as deple-tion of ILK or pinch leads to a decrease in theprotein levels of the other two complex mem-bers (Fukuda et al. 2003; Li et al. 2005).

The central biological function of the IPPcomplex is the regulation of the cytoskeletondownstream of integrins, although a numberof additional functions for this complex aswell as its individual components have beenassigned by in vitro studies. The importanceof these proteins in the integrin-actin connec-tion is highlighted by deletion studies in mice,where deletion of ILK or pinch-1 results in peri-implantation lethality caused by severe defectsin F-actin organization at adhesion sites, lead-ing to a failure in epiblast polarization (Sakaiet al. 2003; Li et al. 2005). The role of parvinsis more complex because of the presence ofthree structurally very similar isoforms. Micelacking b- or g-parvin show no obvious pheno-types, whereas a-parvin-null mice survive upto embryonic day 14.5 and die because ofdefects in cardiovascular development (Chuet al. 2006; Montanez et al. 2009; I Thievessenand R Fassler, unpubl.). Thea/b-parvin doubleknock-out results in early embryonic lethality,suggesting that parvins can compensate foreach other during early development (E. Mon-tanez and R. Fassler, unpubl.). Caenorhabditiselegans expresses only single orthologs for pinchand parvin, and is thus a suitable model organ-ism for a more straightforward analysis of theIPP complex. Studies in C. elegans have shownthat ILK (PAT-4), pinch (UNC-97), and parvin(PAT-6) colocalize with b integrin (PAT-3) atmuscle attachment sites, where a robust con-nection of cells to the cytoskeleton is requiredduring muscle contraction. Deletion of b in-tegrin or any member of the IPP complex leadsto detachment of muscles from the body walland embryonic lethality (Mackinnon et al.2002; Lin et al. 2003; Norman et al. 2007).

On the cellular level, ILK-deficiency leadsto compromised cell adhesion, spreadingand migration. This is because of defective

recruitment of the cytoskeleton to adhesionsites, resulting in defects in FA maturation andcytoskeletal remodeling. The defective mat-uration of ILK-deficient FAs into fibrillaradhesions subsequently leads to impaireddeposition of the FN matrix (Sakai et al. 2003;Stanchi et al. 2009). The precise molecularmechanism by which ILK regulates the cyto-skeleton is not clear. Parvins are capable ofinteracting with F-actin through their two CHdomains, but as these domains also regulatethe interaction of parvins with ILK and paxillin,they might not be available for actin binding.Therefore, it is likely that the IPP complexrequires additional downstream partners forthe regulation of the actin cytoskeleton, andthe identification of these proteins is a centralfocus of future research. It is, however, clearthat the presence of parvin in the IPP complexis critical for the regulation of the actin cyto-skeleton. The absence of a-parvin in micecauses impaired migration of vascular smoothmuscle cells (vSMC) toward developing vesselsresulting in defective stabilization of the vascu-lature and subsequent dilation of vessels, forma-tion of microaneurysms and vessel rupture. Themigration defect is caused by aberrant actomyo-sin contractility (Montanez et al. 2009), and canbe phenocopied by vSMC-specific ablation ofILK (Kogata et al. 2009). However, this functionseems to be restricted to certain cell types, sug-gesting that although the central role of theIPP complex in integrin outside-in signaling isubiquitous, the precise molecular mechanismsmight be cell type-specific to accommodatethe specialized needs of various cell and tissuetypes.

GENETIC DISEASES OF CELL–MATRIXINTERACTIONS

Aberrant cell–matrix interactions are involvedin a large number of pathological conditionssuch as cancer and various inflammatory dis-eases. Altered function of this signaling axis ishowever rarely the actual cause of these disor-ders. In contrast, recent studies have identifieda panel of genetic diseases whose etiology canbe pinpointed to a mutation in a component





of the adhesive machinery. The high degree ofconservation in the components of this pathwayhas allowed the generation of mouse models forthese diseases that can be analyzed to generatemechanistic insights and therapeutic modalitiesfor clinical applications.

Integrin Activation Diseases

As discussed earlier, leukocyte and plateletadhesion are cellular events in which integrinaffinity regulation is of key importance. Inher-ited human diseases with defects in these proc-esses have been described more than 25 yearsago, but only recently, after the discovery ofthe central role of talin and kindlin in integrinactivation, the causes for these diseases havebeen identified.

There are currently three distinct syndromesthat together constitute the leukocyte adhesiondeficiency (LAD) family of diseases. They affectdistinct phases of the leukocyte adhesion cas-cade and therefore cause symptoms of variableseverity. LAD I, a disease that affects severalhundreds of patients worldwide, results fromimpaired firm adhesion of leukocytes, leadingto recurrent severe infections and impairedwound healing. This disease is caused by a rangeof mutations in the b2 integrin (ITGB2) gene,including deletions, truncations, substitutions,frame shifts, and intronic mutations, resultingin loss of protein expression or expression ofa truncated protein. LAD III (also known asLAD I/variant), which has been more recentlydescribed, is characterized by similar symptomsas LAD I. Interestingly, these patients also sufferfrom a bleeding tendency, indicating that addi-tional b subunits are involved. Indeed, thesepatients show defects in b1, b2, and b3 integrinactivation (Kuijpers et al. 1997; Alon andEtzioni 2003). In contrast to LAD I, no muta-tions in integrin genes have been identified,and the cause of this disease remained unknownuntil the role of kindlin in integrin activationwas discovered. Subsequently, genetic sequenc-ing of LAD III patients has revealed mutationsin the kindlin 3 (FRMT3) gene in all patientstested so far, leading to expression of a trun-cated protein, or reduction or loss of protein

expression (Mory et al. 2008; Kuijpers et al.2009; Malinin et al. 2009; Svensson et al.2009). Transfection of patient’s lymphocyteswith wild-type kindlin-3 was shown to restoreintegrin activation (Malinin et al. 2009; Svens-son et al. 2009). In addition, kindlin-3-defi-cient mice recapitulate all symptoms of LADIII, firmly identifying defective kindlin-3 asthe cause of LAD III (Moser et al. 2008; Moseret al. 2009a). In this respect, reconstitution ofkindlin-3 expression provides a possible thera-peutic modality for this disease. Interestingly,the naturally occurring mutations have alsoprovided information on the structure-func-tion aspects of kindlin-3. A homozygous stopcodon identified in three patients occurs distalto the PTB-containing integrin-binding subdo-main of kindlin-3, suggesting that the carboxylterminus of kindlin is required for its functionin integrin activation (Mory et al. 2008). A pointmutation leading to a truncation in the middleof the PH-domain was further shown to impairmembrane localization of kindlin-3 and therebyblock both lymphocyte adhesion and migra-tion, whereas a point mutation in the F2 sub-domain inhibited only the migration of thesecells, demonstrating that the functions of kind-lin in inside-out and outside-in signaling can beuncoupled (McDowall et al. 2010).

The bleeding tendency of LAD III patientsclosely resembles that of Glanzmann’s throm-basthenia (GT), a rare, autosomal recessivebleeding disorder caused by mutations leadingto quantitative or qualitative defects in plateletaIIbb3 integrin. The genetic background isvery heterogeneous: more than 100 differentmutations in either the aIIb (ITGA2B) or b3integrin (ITGB3) genes have been reported sofar (Kannan and Saxena 2009). Genetic ablationof the b3 integrin in mice almost fully reca-pitulates the human disease, including gastroin-testinal and cutaneous hemorrhage, increasedbleeding time, reduced platelet aggregation,and clot retraction (Hodivala-Dilke et al.1999). However, the heterogeneity in themutation spectrum of the integrins found inGT patients also applies to the symptoms; sib-lings sharing the same mutation can displaysymptoms of varying severity, suggesting that





polymorphisms in other genes involved inhemostasis could influence the phenotype. Inaddition, several GT patients lacking a mutationin the aIIbb3 integrin have been identified(Kannan and Saxena 2009). As the bleedingphenotype of the kindlin-3-deficient mice andthe LADIII patients closely resemble GT, it ispossible that altered function of this proteinmight play a role in this disease as well. How-ever, no polymorphisms in the kindlin-3 genein GT patients have been reported so far. Takentogether, integrin activation plays a central rolein immune function and hemostasis. Theprominent role of kindlin-3 in these processestogether with its restricted expression patternmakes it a promising candidate for anti-inflam-matory and antithrombotic therapies.

Adhesion Strengthening Diseases

Another “class” of diseases involving impairedcell–matrix interactions can be found affectingtissues that are subjected to high levels ofmechanical stress such as the skin and skeletalmuscle. The common denominator of thesedisorders, namely muscular dystrophies andskin-blistering diseases, is that the symptomsare caused by weakened interactions of cellswith their extracellular environment, leadingto tissue dysfunction and disruption. Anothercommon feature is that causative mutations fora single disorder can be found in either compo-nents of the ECM, their integrin receptors, oradhesion-strengthening scaffold proteins. Inthe case of congenital muscular dystrophies,although the dystrophin-dystroglycan-laminin/agrin axis is a common target (Yurchenco2010), other mutations affect the laminin-211/a7b1 integrin/plectin axis, leading to defectsin organization and migration of myoblasts,subsequent myofiber degeneration and progres-sive muscle weakness (for more detailed reviewsee Kanagawa and Toda 2006; Mendell et al.2006). Clinical features, cellular defects and theircorresponding causative mutations of musculardystrophies resulting from defective cell-ECMinteraction are summarized in Table 1.

The same principles are true for epidermol-ysis bullosa (EB), the prototype of human

skin-blistering diseases (Uitto 2009). EB is agroup of highly variable disorders characterizedby fragility of skin leading to blistering and ero-sions on mechanical trauma. Genetic studies ondifferent variants of EB have so far shown muta-tions in 12 distinct genes encoding structuralcomponents of the BM and adhesion proteins(Table 2). Interestingly, some of the EB patientsalso develop muscular dystrophy, underliningthe pathophysiological link between these twodiseases. As the histopathology of these diseasesis very complex, the identification of the causa-tive mutation has become a central diagnostictool of EB (Nagy and McGrath 2010). Geneticmouse models for the different EB variantsare available, and they have been used to workout therapeutic strategies for these diseases(Natsuga et al. 2010). As a result, gene delivery,ectopic protein replacement as well as cellulartherapies have already been tested on EBpatients (Uitto 2009).

CONCLUDING REMARKS

Two decades of genetic studies on cell-ECM in-teractions have illustrated the importance ofadhesion signaling in the development of mul-ticellular organisms as well as in disease. It isnow clear that these interactions not only pro-vide structural support and positional cues toguide morphogenesis, but also act as signalingplatforms to regulate cell fate in a highly tis-sue-specific manner. It is also evident that therelationship between ECM proteins and theirintegrin receptors is complex: Integrins trans-mit information from the ECM to the cell, butalso regulate the deposition and remodeling ofthe matrix itself. In addition, the ECM func-tions as a reservoir for growth factors, whereasintegrins cross talk with growth factor signal-ing on various levels. Because of this complex-ity, mechanistic interpretations of the variousphenotypes caused by genetic ablation of indi-vidual components of the adhesion signalingmachinery have proven to be difficult. Thus, amajor challenge for the future is to understandwhich types of intracellular signals directly ema-nate from integrin adhesions, and how thesesignals are propagated. This requires generation





Table 1. Selected mutations causing muscular dystrophy and their corresponding mouse models

Human disease Central clinical features Affected gene (protein) Cellular defect Mouse model Reference

Limb-Girdlemusculardystrophy

Early adulthood onsetproximal muscle weakness,nasal pattern of speech

MYOT (myotilin) Sarcomeric organization N/A –

Severe, early onset muscleweakness

SGCA (a sarcoglycan) Muscle membraneintegrity

KO (Duclos et al. 1998)SGCB (b sarcoglycan) KO (Durbeej et al. 2000)SGCG (g sarcoglycan) KO (Sasaoka et al. 2003)SGCD (d sarcoglycan) KO (Coral-Vazquez et al. 1999)

Late onset muscle weaknessand atrophy,cardiomyopathy

TTN (titin) Sarcomere contraction/relaxation

N/A –

Relatively mild muscleweakness, variable clinicalfeatures

TCAP (telethonin) Sarcomere stability N/A –

Congenitalmusculardystrophy

Early onset with variablesymptoms, hypotonia,torticollis

ITGA7 (integrin a7) Impaired musclecell–matrix interactions

KO (Mayer et al. 1997)

Early onset hypotonia andmuscle weakness

COL6A1COL6A2COL6A3 (type VI

collagen)

Stability of extracellularmatrix

KOa (Bonaldo et al. 1998)

Severe, early onset muscleweakness, white matterhypodensity, mentalretardation

LAMA2 (laminin 211) Attachment, migrationand organization ofmyoblasts

Dy/Dy (naturally occurringmutant)

(Sunada et al. 1994)

Dy2j/Dy2j (naturallyoccurring mutant)

(Xu et al. 1994)

DyPas/DyPas (naturallyoccurring null allele)

(Besse et al. 2003)

Dy3K/Dy3K (KO) (Miyagoe et al. 1997)DyW/DyW (hypomorph) (Kuang et al. 1998)

Duchennemusculardystrophy

Severe, early onset muscleweakness with respiratory,orthopedic and cardiaccomplications

DMD (dystrophin) Stabilization of membraneduring contraction

Mdx (naturally occurring nullallele)

(Bulfield et al. 1984)

Mdx 2Cv, Mdx 3CvMdx 4Cv, Mdx 5Cvb

(Chapman et al. 1989)

Mdx 52 (deletion of exon 52) (Araki et al. 1997)aAblation of the col6a1 gene.bMutants recovered from ENU chemical mutagenesis screen.

Abbreviations: N/A, not analyzed; KO, knockout mouse; Dy, Dystrophin; mdx, X chromosome-linked muscular dystrophy.

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Table 2. Mutations causing epidermolysis bullosa (EB) and their corresponding mouse models

Human diseaseSite of tissueseparation Affected gene (protein) Mouse model Mouse skin phenotype Reference

Epidermolysis

bullosa simplex(EBS)

Intraepidermal PKP1 (plakophilin-1) N/A – –

DSP (desmoplakin) N/A – –KRT5 (keratin-5) KOa Severe blistering, loss of keratin

filaments(Peters et al. 2001)

KRT14 (keratin-14) KO Blistering, decreased keratinfilaments

(Lloyd et al. 1995)

K14-R131C KIa Severe blistering (Cao et al. 2001)K14-DCT TG Blistering (Vassar et al. 1991)

PLEC1 (plectin) KOa Blistering, reduced stability andnumber of hemidesmosomes

(Andra et al. 1997)

Conditional KO Blistering, skin fragility (Ackerl et al. 2007)Junctional

epidermolysisbullosa (JBS)

Lamina lucida ITGA6 (integrin a6) KOa Severe blistering, loss ofhemidesmosomes

(Georges-Labouesse et al. 1996a)

ITGB4 (integrin b4) KOa Severe blistering, loss ofhemidesmosomes

(Dowling et al. 1996; van der Neutet al. 1996)

ITGB4-DCT KIa Severe blistering, loss ofhemidesmosomes,hypoproliferation

(Murgia et al. 1998)

Conditional KO Blistering, loss of hemidesmosomes (Raymond et al. 2005)LAMA3 (laminin-332) KOa Severe blistering, abnormal

hemidesmosomes, reduced cellsurvival

(Ryan et al. 1999)

LAMB3 (laminin-332) Spontaneous null allele Blistering, abnormalhemidesmosomes

(Kuster et al. 1997)

LAMC2 (laminin-332) KOa Blistering, rudimentary

hemidesmosomes, apoptosis

(Meng et al. 2003)

Spontaneoushypomorphic allele

Blistering, ulcerations,hyperkeratosis, rudimentaryhemidesmosomes

(Bubier et al. 2010)

Dystrophicepidermolysisbullosa (DBS)

Sub-lamina densa COL7A1 (type VII collagen) KOa Severe blistering, absence ofanchoring fibrils

(Heinonen et al. 1999)

COL7 hypomorph Severe blistering, decreasedanchoring fibrils

(Fritsch et al. 2008)

Kindler syndrome Primarily lamina

lucida

FERMT1 (kindlin-1) KOa Skin atrophy (Ussar et al. 2008)

aLeads to early postnatal lethality.

Abbreviations used: N/A, not analyzed; KO, knock-out mouse; K14-R131C, Arginine-131 to Cysteine substitution in the Keratin-14 gene; KI, knock in mouse; K14-DCT, carboxy-

terminal truncation of Keratin-14; ITGB4-DCT, deletion of the entire cytoplamsic domain of integrin b4; TG, transgenic mouse.

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of more sophisticated mouse models, such asknock-in mice carrying point mutations thatonly partially disrupt protein function togetherwith reporter mice for specific signaling events,transcriptional changes, or changes in protein-protein interactions or conformations. In addi-tion, a large majority of the cell-biological anal-yses on integrin signaling are still performedon rigid 2D-surfaces, which very poorly reca-pitulate the more compliant and complex 3Denvironment of tissues. As our understandingof the biophysical properties of the ECM in tis-sues steadily increases, a major goal of futureresearch is to translate this knowledge into gen-eration of more relevant in vitro models for cellbiological studies of integrin signaling.

ACKNOWLEDGMENTS

The authors apologize to all those whose workcould not be cited because of space restrictionsand for not always citing all the primary litera-ture. The authors would like to thank MaxIglesias for artwork. The work of the Wickstromand Fassler laboratories are funded by the MaxPlanck Society.

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December 30, 20102011; doi: 10.1101/cshperspect.a005116 originally published onlineCold Spring Harb Perspect Biol

Sara A. Wickström, Korana Radovanac and Reinhard Fässler Genetic Analyses of Integrin Signaling

Subject Collection Extracellular Matrix Biology

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Pengfei Lu, Ken Takai, Valerie M. Weaver, et al.

ThrombosisExtracellular Matrix Proteins in Hemostasis and

Wolfgang Bergmeier and Richard O. HynesExtracellular Matrix Constituents and Functions

An Inventory of−−Overview of the Matrisome

Richard O. Hynes and Alexandra NabaThe Thrombospondins

Josephine C. Adams and Jack LawlerIntegrins in Cell Migration

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John S. Munger and Dean SheppardFunctionsFibronectins, Their Fibrillogenesis, and In Vivo

Jean E. Schwarzbauer and Douglas W. DeSimoneHeparan Sulfate Proteoglycans

Jeffrey D. EskoStephane Sarrazin, William C. Lamanna and System

Extracellular Matrix: Functions in the Nervous

MüllerClaudia S. Barros, Santos J. Franco and Ulrich

The Collagen FamilySylvie Ricard-Blum Adhesions

Molecular Architecture and Function of Matrix

Benjamin Geiger and Kenneth M. Yamada

ModulationTenascins and the Importance of Adhesion

Ruth Chiquet-Ehrismann and Richard P. Tuckerand Diseased SkinCell-Extracellular Matrix Interactions in Normal

Fiona M. Watt and Hironobu FujiwaraIntegrin Structure, Activation, and Interactions

Iain D. Campbell and Martin J. HumphriesGenetic Analyses of Integrin Signaling

Reinhard FässlerSara A. Wickström, Korana Radovanac and

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