Proc. Natl. Acad. Sci. USAVol. 92, pp. 9161-9165, September 1995Cell Biology

Targeted disruption of vinculin genes in F9 and embryonic stemcells changes cell morphology, adhesion, and locomotion

(gene targeting/cytoskeleton)

J.-L. COLL*t, A. BEN-ZE'EVt§, R. M. EZZELLII, J. L. RODRfGUEZ FERNANDEZt, H. BARIBAULT*, R. G. OSHIMA*,AND E. D. ADAMSON**La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, CA 92037; tDepartment of Molecular Genetics and Virology, WeizmannInstitute of Science, 76100, Rehovot, Israel; and I1Surgery Research Laboratory, Massachusetts General Hospital, Department of Surgery, Harvard MedicalSchool, Charlestown, MA 02129

Communicated by Elizabeth D. Hay, Harvard Medical School, Boston, MA, June 26, 1995 (received for review March 10, 1995)

ABSTRACT Vinculin, a major constituent of focal adhe-sions and zonula adherens junctions, is thought to be involvedin linking the microfilaments to areas of cell-substrate andcell-cell contacts. To test the role of vinculin in cell adhesionand motility, we used homologous recombination to generateF9 embryonal carcinoma and embryonic stem cell cloneshomozygous for a disrupted vinculin gene. When compared towild-type cells, vinculin-mutant cells displayed a roundermorphology and a reduced ability to adhere and spread onplastic or fibronectin. Decreased adhesion of the mutant cellswas associated with a reduction in lamellipodial extensions, asobserved by time-lapse video microscopy. The locomotiveactivities of control F9 and the vinculin-null cells were com-pared in two assays. Loss of vinculin resulted in a 2.4-foldincrease in cell motility. These results demonstrate an impor-tant role for vinculin in determining cell shape, adhesion,surface protrusive activity, and cell locomotion.

Vinculin, a 117-kDa protein that localizes to focal contacts (1)and intercellular adherens junctions (2), is part of a complexof proteins that form a submembranal plaque connecting theactin cytoskeleton to the integrin or cadherin transmembraneadhesion receptors. Vinculin was shown to bind in vitro to talinthrough its N-terminal domain (3) and to paxillin via theC-terminal tail (4). The usual representation of the integrin-based cell-matrix junctions is that the actin filaments binda-actinin, which in turn can bind to 13i integrin either directly(5) or via vinculin and talin (1, 2, 6).The sequence of vinculin is highly conserved among nem-

atodes (7), chicken (8), and humans (9). Mutations in thevinculin of Caenorhabditis elegans result in muscle paralysisand arrest of development at the larval stage (10). Whilevinculin is found in all cells, its expression is regulated afterchanges in cell shape and cell culture density (11, 12), duringcell migration in vivo (13), after growth stimulation by serumfactors (14, 15), and in regenerating liver (16). Vinculinorganization and expression are also regulated during differ-entiation of a variety of cell types (17-19) and in embryonicmorphogenesis (20). Moreover, vinculin is absent, or dramat-ically reduced, in highly malignant and metastatic tumor cells(21).To determine whether vinculin plays a role in these cellular

processes, we used homologous recombination to inactivatethe vinculin gene** in F9 and embryonic stem (ES) cells. TheF9 mouse embryonal carcinoma cell line is a useful model tostudy differentiation, as it can differentiate into parietalendoderm (PE) or into more complex structures called em-bryoid bodies (22). In addition, F9 cells have been used totarget the j31 integrin genes (23). On the other hand, gene

targeting in ES cells is the first step toward investigating therole(s) of a gene in vivo. In this study, we describe the isolationof F9 and ES clones in which both alleles of the vinculin genewere disrupted. The loss of vinculin resulted in effects on cellshape, adhesion, and locomotion but not on the expression ofdifferentiation markers.

MATERIALS AND METHODSCell Culture and Differentiation. F9 cells were cultured on

gelatin-coated or Primaria (Becton Dickinson Labware) tissueculture dishes. Differentiation into PE or embryoid bodieswith an outer layer of visceral endoderm was as described byGrover and Adamson (24). The culture of ES cells (clone E14)was as described (25).Gene Targeting Vector. The vinculin gene fragment used to

construct the targeting vector was isolated from a 129SvJmouse genomic library in A DASHII, provided by T.Doetschman (26). The library was screened with a mousevinculin cDNA (14), and an 8-kb EcoRI fragment of a ADASHII clone was selected (Fig. 1, WT). The DNA sequencesoverlapping exons 3 and 4 were determined (GenBank acces-sion nos. L13299 and L13300). The targeting vector containeda 2.3-kb Bgl II-HindIII fragment ending at the beginning of thethird exon of vinculin, followed by 1.2 kb of the neor gene(pMClneo polA Stratagene) inserted in the HindIlI site ofexon 3. The diphtheria toxin-A gene was separated from theneor gene by 950 bp of vinculin DNA. The insertion of the neorgene removed a 0.8-kb HindIII fragment containing the 3'region of exon 3 and the beginning of the following intron.

Transfection. Cells grown as a monolayer were harvested at-60% confluence. Approximately 3 x 106 cells in 1 ml ofmedium were electroporated at 350 mV for 5 msec in thepresence of 40 ,ug of linear vector by using a BTX (San Diego)transfector 100 and plated in five dishes (100 mm). After 24 h,G418 (0.3 mg/ml) was added, and selection was maintainedduring the cloning procedure. Two double knock-out clonesfrom F9 and one from ES cells were obtained by growing 5 x105 single knock-out cells in medium containing G418 (2mg/ml) for 2 weeks (27).

Southern Blot Hybridization. Genomic DNA (4 ,tg) was cutwith EcoRI, electrophoresed on 0.7% agarose gel, and trans-ferred to a Zeta-Probe membrane (Bio-Rad). Hybridizationsto a 32P-labeled vinculin or neor probe (Fig. 1) were performedas described by the manufacturer.

Abbreviations: ES, embryonic stem; PE, parietal endoderm; neor,neomycin resistance; wt, wild type.tPresent address: Centre Regional Leon Berard, Laboratoire deBiologie Cellulaire, 28 rue Laennec, 69373 Lyon Cedex 08, France.§To whom reprint requests should be addressed.**The sequences reported in this paper have been deposited in theGenBank data base (accession nos. L18880, L13299, and L13300).

9161

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 92 (1995)

BamHI Hincli Hindill Hindi EcoRI

exon 3 exon 4 exon 5

_~-2.1 kb _EcoRI Xhol EcoRI

BamH| HincoRl Hindill BamHI KpnlI k PIpnI.n

exon A3 neo exon 4 DTA Bluescript

F.coRI

Mutant

EcoRIBamHI HHndil X I EcoRi

HincjE coRI Hincil HincIl~

exon A3 neo exon 4

neo probe

exon 5

Vin probe

HI

500 bp

FIG. 1. Structures of the 8-kb EcoRI fragment of the wild-type (wt) nmouse vinculin gene region overlapping exons 3, 4, and 5 (WT); the NotI-linearized targeting vector (Vector) derived from this portion of the gene; and the mutant fragment (Mutant). The mutant structure is a targetedallele corresponding to the wt gene in which a 0.8-kb HindIII-HindIII fragment containing part of exon 3 has been replaced by theneomycin-resistance (neor) gene. Negative selection of targeted clones was provided by DNA that encodes the A subunit of the diphtheria toxin(DTA) gene.

Immunoblot Analysis. Cells were lysed in Laemmli's samplebuffer. Protein concentration was determined by using the DCprotein assay (Bio-Rad), and 200 ,ug of protein (or the amountsindicated) was analyzed by SDS/PAGE on 7% polyacrylamidegels and electrotransferred onto poly(vinylidene difluoride)membranes. Vinculin was detected by using anti-human vin-culin monoclonal antibodies from Chemicon or Sigma, fol-lowed by anti-mouse IgG coupled to an enhanced chemilumi-nescent probe (ECL, Amersham). Vinculin levels on immu-noblots were determined from samples containing serialdilutions of protein lysates (equal loading) from wt and mutantcells by scanning the x-ray films with an LKB ultroscan XLlaser densitometer.

Immunofluorescence. Cells plated 24 h before use on gel-atin/fibronectin-coated glass coverslips (5 ,g/cm2) werewashed with PBS, fixed for 8 min in 4% (wt/vol) paraform-aldehyde in PBS, and permeabilized for 2 min with 0.1%Triton X-100 in PBS. Double fluorescence staining of vinculinand filamentous actin was as described (28). The cells wereviewed in a Zeiss LSM 410 laser scanning confocal microscope.Images were printed with a Sony UP/D7Q00 digital printer.Time-Lapse Video Microscopy, Sparse F9 cell cultures on

25-mm-diameter glass coverslips coated with human fibronec-tin (Collaborative Research) at 5 gg/cm2 were followed bytime-lapse video microscopy using Nomarski differential con-trast optics, starting 30 min after plating and continuing for 2h, and pictures were taken every 15 sec (28).Adhesion Assays. Aliquots (100 ,ul) of cell suspensions (5 x

104 cells) in a-modified minimum essential medium and 1%ITS premix (Collaborative Research) were dispensed into96-well (0.6 mm diameter) culture dishes coated with bovineserum albumin (0.6 mg/cm2), gelatin (0.3 mg/cm2), poly(D-lysine) (5 gg/cm2), fibronectin (3 /tg/cm2), or vitronectin (3gg/cm2). The adhesion protocol was established by TeliosPharmaceuticals (San Diego). After 1 h at 37°C, the mediumand unattached cells were removed by aspiration, and the

cultures were washed twice with 200 ,ul of PBS, fixed withparaformaldehyde, stained with toluidine blue overnight,washed extensively with water, and air-dried. The cells weredissolved in 100 ,ul of 2% (wt/vol) SDS and the OD600 wasmeasured. Differences between means were tested for signif-icance by using the t test.

Cell Migration and Phagokinetic Tracks. The lengths ofphagokinetic tracks produced by individual cells on colloidalgold-coated coverslips were determined 24 h after seeding thecells, after viewing the tracks by dark,field microscopy (29).Artificial wound healing, by introduping a "wound" in aconfluent monolayer, was measured by counting the numberof cells migrating into a 1-mm2 area of the wound after 24 h(29). Differences between means were tested for significanceby using the Dunnet test.

RESULTS

Targeted Replacement of the Vinculin Gene By neor. Thetargeting vector DNA (Fig. 1) was introduced by electropo-ration into 3 x 106 F9 and ES cells. After 2 weeks, 25 F9 neorclones were obtained. One F9 clone (-y2) had incorporated thevector by homologous recombination and presented a vin(+ -) genotype, demonstrated by Southern blot hybridization(Fig. 2A) with a vinculin or a neor probe (for location of theprobes, see Fig. 1). After digestion of the DNAwithEcoRI andhybridization with the vinculin probe, an 8-kb fragment was

obtained for the wt gene, whereas the targeted allele appearedas a 4-kb fragment. Hybridization with the neor probe (Fig. 2A)confirmed the previous result and also indicated that only onecopy of the neor was integrated.The (+/-) clones were further selected with a higher

concentration of G418 (2 mg/ml) (27). Fifty thousand cellswere plated, and after 2 weeks, 16 G418-resistant colonieswere isolated. Southern blot hybridization (Fig. 2A) revealedthat two of these 16 clones had no wt copy of the 8-kb EcoRI

EcoRi

WT

Vector

i a . E2==:]. m m

,,S-i n ... AIM

n mmp Mmum -II

9162 Cell Biology: Coll et al.

Proc. Natl. Acad. Sci. USA 92 (1995) 9163

A F9 ES+/+ +/- -1- -/- +1+ +1- -/- +1- +1- +/+

t-)a LL e c0) ~~0) CI3(OU)U)n

8kb -

4kb - vinculin

1 2 3 4 5 6 7 8 9 10,-- / / I \ I I I I I

4kb- neo

B F9 i - -/-I ES +/+ ES-/-.2 .2 .2 .2 .2 .1 .05 .02j .6 .4 .2

125- - _94 -

72- ilL

51 -

CellProt.(mg)

-vin

uw-___ a-act

1 2 3 4 5 6 7 8 9 10 11

FIG. 2. (A) Southern blot analysis of genomic DNA after digestionwithEcoRI. The 8-kb fragment represents the wt vinculin gene and the4-kb fragment is the targeted allele. The blots were hybridized to a32P-labeled vinculin probe (Fig. 1, Vin probe) or a neo probe (Fig. 1,neo probe). (B) Immunoblot with anti-vinculin antibody showing lossof vinculin protein in homozygous (-/-) F9 (lane 3, y227; lane 4,y229) and ES (lanes 9-11) vinculin-targeted clones. Lanes: 1, wt F9;2, y2 heterozygote. F9 vin (+/-) cells expressed 52 ± 8% (n = 7) ofthe vinculin expressed by wt F9. Lanes 5-11 show immunoblotdetection of vinculin (vin) and a-actinin (a-act) in wt ES (ES +/+)and mutant (ES -/-) cells, demonstrating absence of vinculin in(-/-) lines, even at high protein (Prot.) concentrations. ES vin (+/-)cells expressed 59 ± 4% (n = 4) of the vinculin expressed by wt EScells. The molecular mass markers in kDa are indicated on the left.a-Actinin was detected on the same blot to compare the amounts ofprotein loaded.

vinculin fragment (clones y227 and -y229). These two cloneswere negative for vinculin in Western blots (Fig. 2B, lanes 3and 4) by using either of two monoclonal antibodies againsthuman vinculin. Heterozygote and homozygpte vinculin-nullclones were similarly obtained with ES cells (Fig. 2 A, lanes7-10, and B, lanes 5-11).

Localization of Vinculin and Filamentous Actin. Immuno-fluorescence staining with anti-vinculin antibody showed nospecific signal for vincplin, above background, in the mutantcells (Fig. 3B), whereas strong vinculin staining in adhesionplaques was seen in wt F9 cells (Fig. 34). Double staining forfilamentous actin with phalloidin revealed that the mutant cells(Fig. 3D) assembled an actin cytoskeleton that was similar tothat of wt F9 cells (Fig. 3C). Similar results were obtained bycomparing wt and vinculin-null ES cells (data not shown).

Morphological Changes in Vinculin-Null Mutant Cells. F9cells formed flat colonies with cells that spread and extendedlong projections at the periphery of the colony on noncoated"Primaria" dishes (Fig. 4A and C). On the same substrate, themutant F9 cells were more rounded (Fig. 4B) and formedcompact colonies without cell spreading at the colony periph-ery (Fig. 4D). Cell diameter was also reduced significantly inmutant ES cells (wt ES, 16 ± 3 ,um, n = 20; ES vinc(-/-),11.68 ± 2.4 ,um, n = 20; P < 0.0002). The heterozygous (+/-)F9 and ES cells, expressing =50% the normal vinculin level(Fig. 2B), were indistinguishable from the wt cells in theirmorphology and adhesion (data not shown).

FIG. 3. Vinculin and actin staining in double knock-out F9 cells. (Aand B) Immunofluorescence confocal microscopy of F9 cells immu-nostained with a monoclonal anti-human vinculin antibody followedby 5-[(4,6-dichlorotriazin-2-yl)amino]fluorescein (DTAF)-labeled an-ti-mouse IgG. (C and D) F-actin was visualized in the same cells withrhodamine-labeled phalloidin. (A and C) wt F9 cells. (B and D)Vinculin-null cells. (Bar = 25 ,um.)

Cell morphology and the time course of cell spreading onfibronectin were followed by using differential contrast (No-marski) optics and time-lapse video microscopy. Video imagestaken 2 h after plating showed that the mutant cells have analtered cell spreading. The F9 cell shown in Fig. SA had largelamellipodia that even increased in size after 2 min (Fig. SB),while the mutant cell (Fig. SC) was not spread well and hadsmall lamellipodia, some of which retracted during this time(Fig. SD). The filopodia of the mutant cells also appearedunstable when compared to those of wt cells.Changes in Adhesion of Vinculin-Mutant Cells. The adhe-

sion of cells, 1 h after seeding, on tissue culture multiwell dishesuncoated or coated with fibronectin, vitronectin, polylysine,gelatin, or bovine serum albumin was determined (Fig. 6).Adhesion of the vinculin-mutant cells to plastic and fibronectin

F9 wt F9 mut

FIG. 4. Morphological changes in F9 vinculin-null cells. Phase-contrast micrographs of F9 (A and C) and vinculin-null F9 (B and D)cells cultured at low (A and B) and high (C andD) density on positivelycharged plastic tissue culture dishes. (Bar in A = 50 pLm.)

Cell Biology: Coll et al.

Proc. Natl. Acad. Sci. USA 92 (1995)

FIG. 5. Surface protrusive activity of wt F9 (A and B) and F9 vin(-/-) (C and D) live cells. Cells cultured on fibronectin-coatedcoverslips were examined by differential contrast (Nomarski) opticsand video-enhanced images, photographed at 2-min intervals, areshown 2 h after plating the cells. The wt F9 cell had filopodia andextending lamellipodia (arrows in A and B). The mutant cell was lessspread and had irregular lamellipodia (arrows in C and D) that do notextend as far as those in the wt cell. About 50 cells of each type wereanalyzed and gave differences similar to those shown in the figure. (Barin D = 5 ,um.)

was significantly decreased compared to that ofwt F9 cells. Nosignificant differences were observed between the adhesion ofwt and mutant cells to polylysine (Fig. 6) or vitronectin orgelatin (data not shown).

Cell Locomotion. The effect of vinculin loss on locomotionof F9 cells was examined by determining the length of phago-kinetic tracks produced by individual cells plated on colloidalgold-coated coverslips. In addition, artificial wound healingwas measured as the rate at which cells migrated into a

"wound" introduced in a confluent monolayer (Table 1). Theanalysis of phagokinetic tracks and wound healing indicatedthat the mutant F9 cells migrated -2.4 times faster than wt.

Un

C-

c

:0e

c.i

co'a

c.ci)

a:

600

500

400

300

200

100

0

CD 0 0)

cN CM CMCM 0) CM 0) C )

Plastic Fibronectin Polylysine

FIG. 6. Decreased adhesiveness of vinculin-null F9 cells. Cells wereincubated for 1 h in serum-free medium in 96-well tissue culture platesuncoated or coated with fibronectin or polylysine. Bars representstandard deviations from 6 or 12 wells from five experiments. Thedifferences between wt and mutant cells were significant on plastic (wt,149 + 25; mutant, 34 + 44; n = 12; P = 0.0002) and on fibronectin (wt,351 + 74; mutant, 212 + 50; n = 12; P < 0.0001) but not on polylysine(wt, 545 ± 77; mutant, 463 + 94; n = 12; P = 0.0289).

Table 1. Increased motility in vinculin-null F9 cells

Phagokinetic tracks, Wound healing, no.Cells j,m/24 h cells per mm2

F9 48 ± 33 (60) 7 ± 2 (12)y/229 115 ± 46 (70) 17 ± 4 (10)F9 (PE) 74 ± 31(34) NDy227 (PE) 190 ± 85 (27) NDy229 (PE) 170 ± 80 (28) ND

Numbers in parentheses indicate the number of tracks or woundareas analyzed. PE indicates cells differentiated to PE. y227 and 'y229are vinculin-null clones. ND, not determined. The difference betweenwt and mutant cells was significant (P < 0.0001).

When F9 cells were induced to differentiate into PE, a similarincrease in the locomotion of PE cells derived from mutant F9cells was also observed compared to wt cells. As expected, PEcells moved 1.5 times faster than undifferentiated cells. Thus,the loss of vinculin in both F9 and differentiated (PE) cellsresulted in increased cell motility.

Differentiation of Vinculin-Null Clones. The induction ofdifferentiation of mutant F9 cells into PE and visceralendoderm cells, by retinoic acid (24), was followed by deter-mining the expression of the marker genes laminin, type IVcollagen, a-fetoprotein, and cytokeratins K8 and K18 (30).These markers were induced with similar kinetics in mutantand wt cells (data not shown). In addition, when seeded onnonadhesive bacteriological dishes, the mutant cells formedcystic embryoid bodies with an outer epithelial layer that weresimilar in size to those from wt cells (data not shown).

DISCUSSIONThis study describes the isolation of F9 and ES vinculin-nullcells by targeting both alleles of the vinculin gene by homol-ogous recombination. The targeting vector was designed toreplace the 3' end of exon 3 and part of the following intronby the neor gene. By using this vector, several homozygousmutant clones carrying the expected homologous recombina-tion were isolated. These clones were identified as vinculin-nullclones based on Southern blot hybridization with vinculin andneor probes and by immunoblot and immunofluorescenceanalyses.The effects of vinculin loss on cell morphology (Fig. 4) and

adhesion (Fig. 6) were striking in cells seeded on substrateswith intermediate adhesiveness for F9 cells, such as plastic orfibronectin. The decrease in adhesion and spreading of thevinculin-deficient F9 cells on these substrates suggest that theloss of vinculin resulted in destabilization of the actin cytoskel-eton-membrane interactions at cell substrate contact sites.Since cell adhesion to fibronectin is mediated by integrinreceptors, the results of this study imply that adhesion invinculin-null cells is established by alternative mechanism(s)involving a-actinin or talin, both of which can bind to actin andthe cytoplasmic tail of 31 integrin (5, 6, 31). This is supportedby the finding that the vinculin-null F9 cells can form focaladhesions containing a-actinin, paxillin, talin, and phospho-tyrosinated components (32). However, more completespreading apparently requires that actin filaments are linked tointegrin via a-actinin, vinculin, and talin. Previous studies arein agreement with this hypothesis, because cells with nodetectable vinculin (21) or in which vinculin levels werereduced by antisense transfection (33) are still adherent butspread very poorly. Moreover, 3T3 cells reattaching in theabsence of serum are able to spread without mobilizingvinculin into adhesion plaques (14), and quiescent 3T3 cellsstimulated with growth factors release vinculin from focaladhesions while talin localization remains unchanged (34).Thus, cells can attach and form focal adhesions in the absenceof vinculin.

9164 Cell Biology: Coll et al.

Proc. Natl. Acad. Sci. USA 92 (1995) 9165

Decreased adhesion and spreading of vinculin-null cellswere associated with increased cell motility (Table 1). Theseresults are in agreement with recent studies showing maximallocomotion of smooth muscle cells at intermediate attachmentstrength to the substrate (35). Changes in the level of vinculinexpression were also correlated with cell locomotion in 3T3cells in which vinculin overexpression results in decreasedmotility (29), while the suppression of vinculin levels leads toincreased cell movement (33).The vinculin-null F9 cells also displayed a decreased ability

to extend and stabilize lamellipodia and filopodia (Fig. 5). Therapidly forming and retracting filopodia may have enabledgreater locomotion, but further studies will be needed to testthis hypothesis. Unstable filopodia and lamellipodia and re-duced cell attachment and neurite outgrowth were also de-scribed for PC12 cells in which vinculin expression was reducedby using antisense vinculin transfection (36). These findingsfurther support a strong interrelationship between vinculinand the stability of cell surface protrusions, adhesion, and cellshape.The loss of vinculin in mutant F9 and ES cells did not affect

the ability of the cells to express differentiation markers forparietal or visceral endoderm. Both the time course and thelevel of expression of the marker genes were the same invinculin-null and wt cells. Similar results were reported for /31integrin-null F9 cells (23). Interestingly, the elimination ofeither vinculin or (31 integrin (23) in F9 cells resulted indecreased adhesion and spreading on the substrate, thuspointing to the importance of individual structural compo-nents that link the actin cytoskeleton to the extracellular matrixin defining the strength of adhesion and spreading on thesubstrate.The availability of the heterozygous mutant vinculin ES

cells, described herein, should enable in vivo studies on the roleof vinculin in cell adhesion, motility, signaling, and embryonicmorphogenesis.

We are grateful to Dr. M. Schibler and Mr. M. Hasham forinvaluable assistance with confocal microscopy and photography. Thiswork was supported by grants from the Public Health Service (CA54233 and P30 CA 30199 to E.D.A., CA 42302 to R.G.O.), MuscularDystrophy Association, Massachusetts General Hospital Center forthe Study of Inflammatory Bowel Disease (DK43351), and a CareerDevelopment Award from the Crohn's and Colitis Foundation ofAmerica to R.M.E. J.-L.C. was supported in part by a fellowship fromAssociation pour la Recherche sur le Cancer (ARC, France). A.B.-Z.was supported by grants from The Council For Tobacco Research,from the United States-Israel Binational Foundation (BSF), from theIsrael Cancer Research Fund (ICRF), and from the Leo and JuliaForchheimer Center for Molecular Genetics.

1. Burridge, K., Fath, K., Kelly, T., Nuckolls, G. & Turner, C. (1988)Annu. Rev. Cell Bio. 4, 487-525.

2. Geiger, B. & Ginsberg, D. (1991) Cell Motil. Cytoskel. 20, 1-6.3. Jones, P., Jackson, P., Price, G. J., Patel, B., Ohanion, V., Lear,

A. L. & Critchley, D. R. (1989) J. Cell Bio. 109, 2917-2927.4. Turner, C. E., Glenney, J. R., Jr., & Burridge, K. (1990) J. Cell

Biol. 111, 1059-1068.5. Otey, C. A., Pavalko, F. M. & Burridge, K. (1990)J. CellBiol. 111,

721-729.

6. Horwitz, A., Duggan, E., Buck, C., Beckerle, M. C. & Burridge,K. (1986) Nature (London) 320, 531-533.

7. Barstead, R. J. & Waterson, R. H. (1989) J. Biol. Chem. 264,10177-10185.

8. Coutu, M. D. & Craig, S. W. (1988) Proc. Natl. Acad. Sci. USA 85,8535-8539.

9. Weller, P. A., Ogryzko, E. P., Corben, E. B., Zhidkova, N. I.,Patel, B., Price, G. J., Spurr, N. K., Koteliansky, V. E. & Critch-ley, D. R. (1990) Proc. Natl. Acad. Sci. USA 87, 5667-5671.

10. Barstead, R. J. & Waterston, R. H. (1991) J. Cell Biol. 114,715-724.

11. Ungar, F., Geiger, B. & Ben-Ze'ev, A. (1986) Nature (London)319, 787-791.

12. Bendori, R., Salomon, D. & Geiger, B. (1987) EMBO J. 6,2897-2905.

13. Zieske, J. D., Bukusoglu, G. & Gipson, I. K. (1989) J. Cell Biol.109, 571-576.

14. Ben-Ze'ev, A., Reiss, R., Bendori, R. & Gorodecki, B. (1990) CellRegul. 1, 621-636.

15. Bellas, R. E., Bendori, R. & Farmer, S. R. (1991) J. Biol. Chem.266, 12008-12014.

16. Gliick, U., Rodriguez Fernandez, J. L., Pankov, R. & Ben-Ze'ev,A. (1992) Exp. Cell Res. 202, 477-486.

17. Lehtonen, E., Lehto, V.-P., Badley, R. A. & Virtanen, I. (1983)Exp. Cell Res. 144, 191-197.

18. Ben-Ze'ev, A. & Amsterdam, A. (1987) J. Biol. Chem. 262,5366-5376.

19. Rodriguez Fernandez, J. L. & Ben-Ze'ev, A. (1989) Differentia-tion 42, 65-74.

20. Lehtonen, E. & Reima, I. (1986) Differentiation 32, 125-134.21. Rodriguez Fernandez, J. L., Geiger, B., Salomon, D., Sabanay, I.,

Zoller, M. & Ben-Ze'ev, A. (1992) J. Cell Biol. 119, 427-438.22. Strickland, S. & Mahdavi, V. (1978) Cell 15, 393-403.23. Stephens, L. E., Sonne, J. E., Fitzgerald, M. L. & Damsky, C. H.

(1993) J. Cell Biol. 123, 1607-1620.24. Grover, A. & Adamson, E. D. (1986) Dev. Biol. 114, 492-503.25. Robertson, E. J. (1987) Teratocarcinomas and Embryonic Stem

Cells: A Practical Approach (IRL, Oxford).26. Shull, M. M., Ormsby, I., Kier, A. B., Pawlowski, S., Diebold,

R. J., Yin, M., Allen, R., Sidman, C., Proetzel, G., Calvin, D.,Annunziata, N. & Doetschman, T. (1992) Nature (London) 359,693-699.

27. Mortensen, R. M., Conner, D. A., Chao, S., Geisterfer-Low-rance, A. A. & Seidman, J. G. (1992) Mol. Cell. Biol. 12, 2391-2395.

28. Samuels, M., Ezzell, R. M., Cardozo, T. J., Critchley, D. R.. Coll,J.-L. & Adamson, E. D. (1993) J. Cell Biol. 121, 909-921.

29. Rodriguez Fema'ndez, J. L., Geiger, B., Salomon, D. & Ben-Ze'ev, A. (1992) Cell Motil. Cytoskel. 22, 127-134.

30. Oshima, R. G. (1982) J. Biol. Chem. 257, 3414-3421.31. Goldmann, W. H., Bremer, A., Haner, M., Aebi, U. & Isenberg,

G. (1994) J. Struct. Biol. 112, 3-10.32. Volberg, T., Geiger, B., Kam, Z., Pankov, R., Simcha, I., Sabanay,

H., Coll, J.-L., Adamson, E. & Ben-Ze'ev, A. (1995) J. Cell Sci.108, 2253-2260.

33. Rodriguez Fernandez, J. L., Geiger, G., Salomon, D. & Ben-Ze'ev, A. (1993) J. Cell Biol. 122, 1285-1294.

34. Herman, B. & Pledger, W. J. (1985) J. Cell Biol. 100, 1031-1040.35. DiMilla, P. A., Stone, J. A., Quinn, J. A., Albeda, S. M. &

Lauffenburger, D. A. (1993) J. Cell Biol. 122, 729-737.36. Varnum-Finney, B. & Reichardt, L. F. (1994) J. Cell Biol. 127,

1071-1084.

Cell Biology: Coll et al.