2263Research Article

IntroductionTetraspanins, a family of proteins with 33 members in mammals,play important roles in gamete fusion, brain development andfunction, immunity, photoreceptor morphology, and the maintenanceof epidermal integrity (Hemler, 2005; Levy and Shoham, 2005). Inaddition to these physiological roles, tetraspanins are also implicatedin a variety of pathological settings, notably tumor cell metastasis.For example, whereas tetraspanins CD9 and CD82 are potentialmetastasis suppressors (Boucheix et al., 2001; Lazo, 2007; Liu andZhang, 2006; Tonoli and Barrett, 2005), tetraspanin CD151 has beenlinked to enhanced metastasis of colon, prostate and lung cancer(Ang et al., 2004; Hashida et al., 2003; Tokuhara et al., 2001). Themechanisms whereby tetraspanins might regulate metastasis can begrouped into several classes, including regulation of (i) pericellularmatrix proteolysis (Gesierich et al., 2006; Hasegawa et al., 2007;Hong et al., 2006; Hong et al., 2005; Huang et al., 2007; Saito etal., 2006; Sugiura and Berditchevski, 1999), (ii) integrin cell-surfaceexpression and trafficking (Berditchevski and Odintsova, 2007; Liuet al., 2007; Winterwood et al., 2006), (iii) integrin-dependentmigration, invasion and signaling (Berditchevski, 2001; Hemler,2005), (iv) integrin crosstalk with growth factor receptors (Sridharand Miranti, 2006) and (v) pathological angiogenesis (Gesierich etal., 2006; Takeda et al., 2007). Most of these proposed tetraspaninfunctions center around regulation of cell-extracellular matrixinteractions and downstream signaling events.

In addition to alterations in cell-matrix interactions, the metastasisof carcinomas, which arise in epithelial layers, often entails changescell-cell adhesion. A prevailing view is that carcinoma cellmetastasis might be triggered by aberrant reactivation of a

developmental switch that includes a downregulation of E-cadherin-dependent cell-cell adhesion, similar to the epithelial-mesenchymaltransitions of embryogenesis (Brabletz et al., 2005; Hugo et al.,2007; Thiery and Sleeman, 2006). However, the invasive capacityof certain E-cadherin-positive carcinomas (Cowin et al., 2005;Cowin and Welch, 2007; Jang et al., 2007) indicates that additionalregulatory mechanisms, beyond simply extinguishing E-cadherinexpression, are likely to come into play in some settings.

Several lines of research suggest that regulation of cell-cellinteractions could be an important additional mechanism by whichCD151 might influence metastasis. Humans and mice lackingCD151 show loss of epithelial integrity in the skin and kidney(Karamatic Crew et al., 2004; Sachs et al., 2006), and CD151-nullmice also show defective wound healing (Cowin et al., 2006). Inkidney epithelial cells from mice lacking α3β1 integrin (a majorCD151 partner), E-cadherin localization and function appearedperturbed, and association of α3β1 integrin with CD151 might beimportant for the ability of α3β1 integrin to regulate E-cadherin inthis system (Chattopadhyay et al., 2003). Conversely,overexpression of CD151 enhanced carcinoma cell-cell association(Shigeta et al., 2003), whereas an anti-CD151 antibody interferedwith E-cadherin localization in HaCat cells and promoted theirdispersal (Chometon et al., 2006). Collectively, these data suggestthat CD151 might regulate cell-cell interactions between tumor cells;however, the effect of CD151 loss of function on E-cadherin intransformed cells has not been determined.

We recently reported that near-total, RNAi-mediated silencingof CD151 in epidermal carcinoma cells resulted in impairedadhesion and migration mediated by the CD151-associated integrins,

Tetraspanins regulate integrin-dependent tumor cellinteractions with the extracellular matrix. Here we show thattetraspanin CD151, which plays critical roles in regulating theadhesion and motility of individual tumor cells, is also animportant regulator of collective tumor cell migration. Neartotal silencing of CD151 destabilizes E-cadherin-dependentcarcinoma cell-cell junctions and enhances the collectivemigration of intact tumor cell sheets. This effect does not dependon reduced E-cadherin cell-surface expression or intrinsicadhesivity, or on obvious disruptions in the E-cadherinregulatory complex. Instead, the loss of CD151 causes excessiveRhoA activation, loss of actin organization at cell-cell junctions,and increased actin stress fibers at the basal cell surface. Cell-cell contacts within CD151-silenced monolayers display a nearlythreefold increase in remodeling rate and a significant reductionin lifespan as compared to cell-cell contacts within wild-type

monolayers. CD151 re-expression restores junctional stability,as does acute treatment of CD151-silenced cells with a cell-permeable RhoA inhibitor. However, a CD151 mutant withimpaired association with α3β1 integrin fails to restorejunctional organization. These data reveal that, in addition toits roles in regulating tumor cell-substrate interactions, CD151is also an important regulator of the stability of tumor cell-cellinteractions, potentially through its interaction with α3β1integrin. This could help to explain the phenotypes in humanpatients and mice lacking CD151.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/13/2263/DC1

Key words: Tetraspanin, CD151, E-cadherin, Rho, α3β1 integrin

Summary

Tetraspanin CD151 regulates RhoA activation and thedynamic stability of carcinoma cell-cell contactsJessica L. Johnson1, Nicole Winterwood1, Kris A. DeMali2 and Christopher S. Stipp1,*1Department of Biology and 2Carver College of Medicine, Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA*Author for correspondence (e-mail: christopher-stipp@uiowa.edu)

Accepted 20 March 2009Journal of Cell Science 122, 2263-2273 Published by The Company of Biologists 2009doi:10.1242/jcs.045997

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α3β1 and α6β4 (Winterwood et al., 2006). In this previous study,we examined the behavior of individual, dissociated tumor cells.Here, we extended our analysis to conditions where cell-cellcontacts were maintained. Our data uncover an important role forCD151 in promoting the stability of carcinoma cell-cell junctions,and reveal complexities that would have to be considered in anypotential strategy that targets CD151 to inhibit tumor cell motility.In addition, the data help to illuminate a mechanism whereby theorganization of intercellular junctions can be perturbed even thoughE-cadherin cell surface expression is maintained.

ResultsEnhanced migration of CD151-silenced carcinoma cell sheetsTo determine how CD151 might regulate tumor cell motility whencell-cell contacts are maintained, we compared parental A431epidermal carcinoma cells (WT) to CD151-silenced cells (A431sh3 cells) in gap-filling assays. Freshly confluent monolayers wereinscribed with a gap using a micropipet tip, and gap closure wasmonitored by time-lapse microscopy (Fig. 1A; supplementarymaterial Movie 1). Both cell types closed the gap as continuoussheets. However, in contrast to the reduced single-cell velocity thatwe previously observed for the CD151-silenced sh3 cells(Winterwood et al., 2006), the sh3 cell sheets closed the gapsignificantly faster than their wild-type counterparts. Quantificationindicated that the gap closure rate for sh3 cells was over twice asfast as for wild-type cells (Fig. 1B). The enhanced closure rate ofthe sh3 cell sheets was not due to a difference in proliferation,because frame-by frame analysis of mitoses occurring during assayrevealed no differences (Fig. 1C).

We used a spheroid assay to further investigate how CD151 mightregulate collective tumor cell migration. Spheroids, created byculturing cells overnight in tubes coated with non-adhesive poly(2-hydroxyethyl methacrylate) (poly-HEMA), were plated on laminin-5, an α3β1 integrin ligand. Both wild-type and sh3 cell spheroidsexpanded as intact sheets, and once again the sh3 cell sheetsmigrated significantly farther than wild-type cell sheets (Fig. 1D).Re-expressing CD151 in sh3 cells (creating cells designated A431sh3 Rx) reversed the enhanced migration of the sh3 cell spheroids(Fig. 1D). Quantification showed that sh3 cell spheroids expandedover twice as far as wild-type or Rx cell spheroids (Fig. 1E). Thus,despite the fact that the motility of individual cells is significantlyreduced upon silencing CD151 (Winterwood et al., 2006), thecollective migration of intact, CD151-silenced monolayers issignificantly enhanced. These results are summarized as linearvelocities in Table 1.

The data in Table 1 indicate that all three cell types migratedsignificantly slower as intact monolayers than as single cells.However, whereas wild-type and Rx monolayer velocities were only~6% of their respective single-cell velocities, the sh3 monolayervelocity was nearly 50% of the sh3 single-cell velocity. An analysisof wild-type and sh3 collective migration velocity in the gap-fillingexperiments yielded very similar results (data not shown). Thesedata suggested that: (i) the maintenance of cell-cell contacts during

collective migration imposes a restraint on migration velocity, and(ii) this restraint on velocity is manifested much more in wild-typeand Rx monolayers than in sh3 monolayers.

To compare our results to a scenario where cell-cell contacts areexplicitly disrupted, we treated wild-type spheroids with an anti-E-cadherin function-blocking antibody. In contrast to the intactsheets observed in the previous experiments, treatment of spheroidswith anti-E-cadherin caused cells to emigrate as individuals, creating

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Fig. 1. Enhanced migration of CD151-silenced carcinoma cell sheets.(A) Time-lapse microscopy of gap closure by wild-type (WT) and CD151-silenced (sh3) A431 cells. Scale bar: 125 μm. (B) Average gap closure rate inthree independent trials ± s.e.m. WT closure rate was significantly lower than insh3 cells; *P<0.005, unpaired t-test. (C) The cumulative number of mitoses wasscored for wild-type and sh3 cells during the assay in 175-μm wide strips oneither side of initial gap. (D) Tumor cell spheroids formed from wild-type(WT), CD151-silenced (sh3) or CD151 rescue (Rx) cells were plated in serum-free medium in laminin-5-coated wells. Photographs were taken 1 hour and 18hours after plating; the extent of spheroid dispersal at 18 hours is indicated withblack outlines. (E) Pooled data from eight spheroids per cell type ± s.e.m. sh3spheroids dispersed over twofold more than either wild-type or Rx cellspheroids. *P<0.001, ANOVA with Bonferroni t-test. (F) Wild-type spheroids(as in D) were cultured on laminin-5 in the presence of 10 μg/ml anti-E-cadherin antibody, SHE78-7. (G) Pooled data from wild-type, sh3 or Rxspheroids (at least nine spheroids per cell type, mean ± s.e.m.) plated oncollagen I. *P<0.01, ANOVA with Bonferroni t-test.

Table 1. Single cell vs intact sheet linear velocities

Single cell velocities Intact sheet velocitiesCell type (μm/h) (μm/h)

WT 116.7±5.3 (n=5) 7.0±0.9 (n=8)sh3 52.8±7.5 (n=3) 24.4±2.8 (n=8)Rx 113.0±8.3 (n=3) 7.0±1.7 (n=8)

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a large halo of single cells around the spheroids (Fig. 1F; see alsosupplementary material Fig. S1 for an expanded view). Althoughthese data were consistent with the view that cell-cell contact canrestrain cell motility, they suggested that wholesale disruption ofE-cadherin adhesive function was unlikely to explain the phenotypeof CD151-silenced monolayers. Instead, the enhanced migration ofintact sh3 monolayers suggested that cell-cell contacts weremaintained, but might be perturbed, in sh3 cell monolayers.

Lastly, to test whether regulation of collective migration byCD151 depends on the extracellular substrate, we analyzedspheroids on collagen I, a ligand for α2β1 integrin. The loss ofCD151 enhanced collective migration on collagen I to a similarextent as on laminin-5 (Fig. 1G). These data indicate that the abilityof CD151 to act as a negative regulator of collective migration inthis assay might not be strongly dependent on the exogenousextracellular substrate supplied.

E-cadherin and its partners are mis-localized in CD151-silenced carcinoma cellsWe hypothesized that CD151 might regulate collective migrationby regulating the organization of carcinoma cell-cell junctions.Therefore, we next examined the localization of E-cadherin andassociated proteins in wild-type, sh3 and Rx cells. Whereas E-cadherin strongly localized to cell-cell contact sites in wild-typecells (Fig. 2A), its junctional localization was substantially perturbedin CD151-silenced cells (Fig. 2B). Re-expression of CD151 in A431sh3 Rx cells restored normal E-cadherin localization (Fig. 2C). Thejunctional localization of E-cadherin regulatory proteins β-catenin(Fig. 2D-F), α-catenin (Fig. 2G-I), plakoglobin (Fig. 2J-L) andp120ctn (Fig. 2M-O) were also perturbed in the sh3 cells. CD151itself localized to cell-cell contact sites (Fig. 2P,R), which wasconsistent with a role in regulating cell-cell contacts, and wasspecifically absent in the sh3 cells, as expected (Fig. 2Q). Lastly,the receptor protein tyrosine phosphatase PTPμ, whose expressionmight be regulated by integrin-tetraspanin complexes in some cells(Chattopadhyay et al., 2003), appeared equally expressed in all threecell types (Fig. 2S-U). These data indicated that E-cadherin-basedcell-cell junctions are less well organized in CD151-silenced cells.

The E-cadherin regulatory complex appears intact in CD151-silenced carcinoma cellsTo begin to explore the basis of the altered cell-cell junctions inCD151-silenced cells, we examined E-cadherin association withcomponents of the cadherin regulatory complex. In E-cadherinimmunoprecipitations, the amounts of co-precipitating β-catenin,α-catenin, p120ctn, α-actinin and plakoglobin all appearedunchanged in CD151-silenced cells (Fig. 3A-E). In addition, theamounts of E-cadherin or p120ctn that co-precipitated with β-cateninwere unchanged, as was the amount of E-cadherin that co-precipitated with p120ctn (Fig. 3F,G). Because many E-cadherinpartners are regulated by phosphorylation, we examined the profileof phosphoproteins co-precipitating with E-cadherin, focusing ona size range that encompasses α-, β- and p120 catenin, as well asplakoglobin and α-actinin. No obvious differences were observedin any of the cell types (Fig. 3H, lanes 2-4). However, treatmentof cells with epidermal growth factor (EGF) confirmed thatincreased phosphorylation of protein bands corresponding to β-catenin and plakoglobin (Hoschuetzky et al., 1994) could bedetected by our assay system (Fig. 3H, lane 1). Moreover, repeatedexperiments that directly assessed β-catenin phosphorylation failedto reveal any differences (data not shown).

We also observed that E-cadherin surface expression wasmaintained at similar levels in CD151-silenced cells as in wild-typeor Rx cells (Fig. 3I), and we observed no changes in the detergentextractability of E-cadherin in CD151-silenced cells (Fig. 3J). Next,we tested for a possible association between E-cadherin and CD151or other tetraspanins. As shown in Fig. 3K, although E-cadherinwas readily detected in a β-catenin immunoprecipitate (lane 2), noE-cadherin could be detected co-precipitating with any of severaldifferent tetraspanins (lanes 3-6) or with α2- or α3-integrin subunits(lanes 7 and 8). Conversely, α3 integrin was detected in tetraspaninimmunoprecipitates (Fig. 3L, lanes 1-3), but not in a β-cateninimmunoprecipitate (lane 4). Lastly, we tested for cell-surfaceexpression of PTPμ and observed no differences in wild-type, sh3or Rx cells (Fig. 3M), consistent with the staining data in Fig. 2S-U. Collectively, these data indicate that major biochemicaldisruptions in the E-cadherin regulatory complex or changes in

Fig. 2. E-cadherin and associated proteins are mis-localized in CD151-silencedcells. (A-U) Wild-type, CD151-silenced sh3 cells and CD151-rescued Rx cellswere stained with antibodies specific for the indicated proteins, followed byCy2 goat anti-mouse secondary antibody. Actin staining was with Alexa 594phalloidin. CD151 staining was performed on non-permeabilized cells.

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PTPμ expression are unlikely to explain the disorganized junctionsin our CD151-silenced cells. In addition, E-cadherin itself mightnot directly interact with CD151 or other tetraspanins.

E-cadherin-dependent adherens junctions initially form, but failto remain organized in CD151-silenced cellsTo further define how E-cadherin localization and function mightbe altered in CD151-silenced cells, we tested wild-type and CD151-silenced sh3 cells in a short-term adhesion assay on purified E-cadherin-Fc fusion protein. As shown in Fig. 4, both cell typesadhered equally well to E-cadherin-Fc, and adhesion could beblocked substantially with anti-E-cadherin antibody and completelywith the calcium chelator, EGTA. These data suggested that initialE-cadherin-dependent adhesion events might proceed normally inCD151-silenced cells. To further test this, we examined thelocalization of E-cadherin during a calcium switch assay. Tofacilitate these experiments, we introduced an E-cadherin-GFPfusion protein (EcadGFP) into A431 wild-type and sh3 cells andsorted GFP-positive cells. In cells cultured overnight, EcadGFPstrongly localized to cell-cell contacts in wild-type cells, but muchless so in sh3 cells (Fig. 5A,B). After 15 minutes of exposure tolow calcium, both cell types rounded up and cell-cell junctions werelargely disrupted (Fig. 5C,D). Strikingly, 30 minutes after calciumrestoration, EcadGFP was strongly localized to the re-forming cell-cell junctions in sh3 cells, very similar to the situation in wild-typecells (Fig. 5E,F). However, after 3 hours, EcadGFP localization insh3 cells had begun to deteriorate (Fig. 5G,H). Collectively, thesedata indicated that E-cadherin-based adherens junctions can formin CD151-silenced cells but they appear unstable and fail to remainorganized. The data also suggested that the adhesive function of E-cadherin, per se, might not be impaired in our CD151-silenced cells.

Excessive RhoA activity and formation of stress fibers inCD151-silenced cellsThe formation and maintenance of E-cadherin-based adherensjunctions is known to depend on the activity levels of Rho familysmall GTPases, which regulate the organization of the actincytoskeleton (Arthur et al., 2002; Braga, 2002; Fukata and Kaibuchi,2001; Yap and Kovacs, 2003). Therefore, we examined F-actinlocalization in relation to E-cadherin using confocal microscopy.We observed copious actin stress fibers near the basal surface of

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Fig. 3. The E-cadherin regulatory complex is intact in CD151-silenced cells.(A-G) The indicated proteins were immunoprecipitated from NP-40 extracts ofwild-type (WT), CD151-silenced (sh3) or CD151 rescued (Rx) cells, followedby immunoblotting with a polyclonal anti-E-cadherin antibody and monoclonalantibodies recognizing cadherin-associated proteins. Cadherin and associatedproteins were detected simultaneously with Alexa-680 goat-anti-rabbit andIRdye 800 goat-anti-mouse secondary antibodies using a LiCor near-infraredgel imager. (H) E-cadherin immunoprecipitates were blotted forphosphotyrosine. Wild-type cells in lane 1 were treated with 50 nM EGF for 10minutes prior to lysis. Bands corresponding to β-catenin and plakoglobin areindicated. (I) E-cadherin was immunoprecipitated from extracts of biotinylatedcells and visualized with IRdye-800 streptavidin. (J) Indicated cell types wereeither extracted directly with Laemmli buffer (T, total) or sequentially with0.2% NP-40 (S, soluble) followed by Laemmli buffer (I, insoluble). E-cadherinin each fraction was quantified by immunoblotting. (K) The indicated proteinswere immunoprecipitated from a Brij 96V extract of wild-type cells, and co-precipitating E-cadherin was assayed by immunoblotting. Arrowhead indicatesintact E-cadherin. (L) The indicated proteins were immunoprecipitated fromBrij 96V lysates of wild-type cells, and co-precipitating α3 integrin wasdetected by immunoblotting. (M) PTPμ was immunoprecipitated from extractsof biotinylated cells and visualized with IRdye-800 streptavidin. E-cad, E-cadherin; β-catenin, β-ctn; plakoglobin, pkgb.

Fig. 4. Initial E-cadherin-dependent adhesion is normal in CD151-silencedcells. Wild-type and CD151-silenced cells were plated on E-cadherin-Fc fusionprotein in the presence or absence of anti-E-cadherin function-blockingantibodies (10 μg/ml SHE78-7 and 20 μg/ml DECMA-1 in combination) or 5mM EGTA. Adhesion was measured as described in the Materials andMethods. Non-specific adhesion in BSA-coated control wells is also indicated.Values are mean ± s.e.m. of four wells per condition.

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the sh3 cells, whereas F-actin was more concentrated at the cellperimeter in wild-type cells (Fig. 6A’,E’). In addition, E-cadherinwas fairly evenly distributed over the entire basal cell surface ofsh3 cells, whereas in wild-type cells it was more concentrated at

cell-cell boundaries (Fig. 6A,E, and overlaid with actin in Fig.6A”,E”). At more lateral and apical planes in sh3 cells, some F-actin and E-cadherin was observed at some cell-cell junctions (Fig.6B-D,B’-D’, overlaid in Fig. 6B”-D”), but overall the junctionalstaining of both proteins was less prominent and less well-organizedthan in wild-type cells (Fig. 6F-H,F’-H’, overlaid in Fig. 6F”-H”).Confocal analysis of β-catenin revealed a similar pattern: in wild-type cells, β-catenin was prominently localized at most cell-celljunctions (supplementary material Fig. S2A-C), whereas in sh3 cells,β-catenin localized to some junctions, but in general appeared morediffuse and less well-organized (supplementary material Fig. S2D-F). Re-expression of CD151 in Rx cells restored the junctionallocalization of β-catenin (supplementary material Fig. S2G-I).

Because of the abundant stress fibers in sh3 cells, we nextcompared sh3 and wild-type cells for the levels of active RhoA,which controls formation of stress fibers. As shown in Fig. 7A, inconfluent wild-type cell cultures, the level of active, GTP-loadedRhoA was significantly reduced (lane 3) compared to that in roundedwild-type cells (lane 1), which served as a positive control for RhoAactivation. By contrast, the level of active RhoA in confluent sh3cell cultures (lane 4) was significantly elevated and similar to therounded cell positive control (lane 2). Restoring CD151 expressionin Rx cells reversed the elevation of levels of active RhoA observedin sh3 cells (Fig. 7B). These data suggested that elevated RhoAactivity is probably responsible for the elevated formation of stressfibers in CD151-silenced cells.

Monolayers of CD151-silenced cells are more dynamic thanwild-type monolayersElevated RhoA activity has been associated with a destabilizationof adherens junctions (Jou and Nelson, 1998; Zhong et al., 1997)and cell scattering in response to factors such as hepatocyte growthfactor (Wells et al., 2005). We therefore examined the behavior ofindividual cells within intact wild-type and CD151-silencedmonolayers by time-lapse microscopy. These experiments revealedthat sh3 cell monolayers appeared much more dynamic, withindividual cells moving within the monolayer to a much greater

Fig. 5. Adherens junctions form but fail to remain organized in CD151-silencedcells. Wild-type and CD151-silenced sh3 cells expressing E-cadherin-GFP werecompared in a calcium switch assay. (A,B) Cells cultured overnight in normalcalcium (con, control). (C,D) Cells placed in low Ca2+ (5 μM) for 15 minutes(after overnight culture under normal conditions). (E-H) Cells treated with lowCa2+ and then restored to normal conditions for 30 or 180 minutes.

Fig. 6. Elevated stress fiber formation anddisorganized junctions in CD151-silencedcells. (A-H”) CD151-silenced cells (sh3)and wild-type cells (WT) were double-labeled for F-actin and E-cadherin andexamined by confocal microscopy. Foreach cell type, optical sections spanningfrom basal to apical surfaces are displayedfrom top to bottom.

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extent than in wild-type monolayers (Fig. 8A,B; supplementarymaterial Movie 2). Quantification showed that sh3 cells moved witha threefold higher velocity than wild-type cells (Fig. 8C). Indeed,we sometimes observed cell-cell junctions that were literally pulledapart by the highly dynamic movements within the sh3 cellmonolayers (Fig. 8D; supplementary material Movie 2). To capturethe remodeling of junctions more quantitatively, we measured thesize of individual cell-cell contacts every 5 minutes for 2 hours.Compared to wild-type cell-cell contacts, whose size remainedrelatively constant, the size of sh3 cell-cell contacts oscillateddramatically as cells alternately pushed together and pulled awayfrom one another within the monolayer (Fig. 8E,F). As a result, theaverage rate of change of cell-cell contact size was nearly threefoldhigher in the sh3 cell monolayers (Fig. 8G). The elevated dynamicswithin the sh3 cell monolayer corresponded to a significant decreasein the lifespan of individual sh3 cell-cell contacts (Fig. 8H).Together with the previous experiments, these data suggested thata consequence of elevated RhoA activity in sh3 cells is a moredynamic monolayer in which individual cell-cell contacts undergocontinuous remodeling and display a significantly shortenedlifespan. To begin to test this, we monitored intra-monolayer cellmotility before and after the acute addition of recombinant, cell-permeable C3 transferase, a selective Rho inhibitor. Upon C3treatment, the motility of individual cells within the CD151-silenced monolayer slowed to wild-type levels (Fig. 8I). C3treatment of wild-type cells did not cause any further reduction inintra-monolayer motility (Fig. 8I).

Suppression of RhoA activity in CD151-silenced cells restoresa more normal E-cadherin localizationWe next tested whether the reduced motility within CD151-silencedmonolayers upon C3 treatment corresponded to a restored junctionalorganization. Untreated sh3 cells displayed actin stress fibers and

poorly organized junctions at the basal cell surface (Fig. 9A-C). Bycontrast, C3-treated cells showed fewer stress fibers and a morenormal junctional localization of F-actin and E-cadherin (Fig. 9D-F, compared to untreated wild-type cells in Fig. 9G-I). Wild-typecells treated with C3 retained normal F-actin and E-cadherinlocalization, although F-actin at cell-cell boundaries appearedsomewhat depleted, perhaps because RhoA activity in C3-treatedwild-type cells was suboptimal for maintenance of F-actin at

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Fig. 7. Elevated RhoA activity in CD151-silenced cells. (A) Confluent wild-type or CD151-silenced sh3 cells were detached and held in suspension for 5minutes (susp.) or left attached (attach.) prior to lysis. Active RhoA, recoveredby pull-down with GST-Rhotekin (upper panel), and total RhoA in the lysates(lower panel) were quantified by immunoblotting. (B) The levels of activeRhoA in lysates of confluent A431 wild-type cells, CD151-silenced sh3 cells orCD151-rescued Rx cells were measured as in A. The numbers above each laneindicate the amount of active RhoA corrected for total RhoA in each lysate,with the amount of active RhoA in lane 1 set to 1.0.

Fig. 8. Enhanced dynamics and decreased junctional stability within CD151-silenced monolayers. (A,B) Individual cells within wild-type and sh3monolayers were tracked by time-lapse microscopy and their trajectoriesoverlaid, setting the initial location of each cell at the center of the graph.Fifteen tracks per cell type are shown for an 8.5-hour experiment. Scale bar:100 μm. (C) Mean velocities ± s.e.m. for wild-type and sh3 cells moving withinconfluent monolayers; 35 cells of each type were measured in two separatetrials. The sh3 cells migrated three- to fourfold faster than wild-type cells.*P<0.0001, unpaired t-test. (D) A sequence of frames 30 minutes apartdepicting two cells within an sh3 cell monolayer that initially shared a junction,but which pulled apart during the observation period. Asterisks mark the twocells, and an arrowhead tracks the junction as it is disrupted. (E,F) The lengthsof individual cell-cell contacts within wild-type and sh3 cell monolayers weremeasured every 5 minutes for 2 hours. Fluctuations in relative cell-cell contactsize are plotted, with the original size of each contact set to 1.0. (G) Theaverage rate of change of cell-cell contact length ± s.e.m. The sh3 cell junctionschanged size at nearly three times the rate of wild-type junctions. *P<0.0001,unpaired t-test, n=10 junctions/cell type. (H) The fate of 30 wild-type and sh3cell-cell junctions was followed for 12 hours, and the percentage of the originaljunctions remaining intact at 15 minutes intervals is plotted. The sh3 celljunctions had a significantly reduced lifetime, with a median survival time of525 minutes versus >720 minutes for wild-type cells. P=0.0112, log rank test.(I) The mean velocities of individual cells within wild-type and sh3 cellmonolayers were measured for 3 hours before and 3 hours after the addition of2 μg/ml cell-permeable C3 transferase. *P<0.01, ** P<0.001, ANOVA withBonferroni t-test. Values are mean ± s.e.m. of 17 cells per condition.

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junctions (Fig. 9J-L). At a more lateral plane, some F-actin and E-cadherin did localize to some sh3 cell junctions (Fig. 9A’-C’), butthis was more diffuse and less organized than in C3-treated sh3cells (Fig. 9D’-F’) or untreated wild-type cells (Fig. 9G’-I’).Treatment of wild-type cells with C3 again partially depleted F-actin from the junctions, but did not perturb their organization (Fig.9J’-L’). Collectively, the data in Figs 8 and 9 show that a RhoAinhibitor can suppress the elevated motility within CD151-silencedmonolayers and restore a more normal junctional organization.

A CD151 mutant with impaired association of α3β1 integrinfails to restore junctional organizationGiven that α3β1 integrin is a prominent CD151 partner (Kazarovet al., 2002; Sterk et al., 2002; Yauch et al., 1998) and might regulatejunctional stability in non-transformed cells (Chattopadhyay et al.,2003; Wang et al., 1999), we next began to explore the extent towhich α3β1 integrin might be involved in the ability of CD151 topromote junctional organization in carcinoma cells. Using a FLAG-tagged, wild-type CD151 Rx cDNA as a template, we constructeda FLAG-tagged CD151 mutant, CD151VR, in which the ‘variableregion’ in the CD151 large extracellular loop (EC2 domain) wasreplaced with that of tetraspanin TM4SF2 (see the Materials andMethods). In relatively mild Brij detergent lysates (1:1 ratio of Brij99 to Brij 96), wild-type CD151 readily co-precipitated with α3integrin (Fig. 10A, lane 1). By contrast, virtually no CD151VR wasdetected co-precipitating with α3 integrin (Fig. 10A, lane 3).Conversely, α3 integrin co-precipitated with CD151, but only a traceof α3 integrin co-precipitated with the CD151VR mutant (Fig. 10A,lanes 4 and 6). In CD151-silenced sh3 cell lysates, no specific signalwas detected in FLAG blots, and no α3 integrin was retrieved byFLAG immunoprecipitation (Fig. 10A, lanes 2 and 5). The CD151VR

mutant migrated as a broad band of ~45 kDa versus the ~32 kDaband observed for wild-type FLAG-tagged CD151, probablyreflecting the presence of four glycosylation sites in the TM4SF2variable region, as compared to the single site in the CD151 variableregion.

We next immunoprecipitated FLAG-tagged wild-type CD151 andthe CD151VR mutant from extracts of biotin-labeled cells.Simultaneous detection of the biotin label and the FLAG epitoperevealed that wild-type CD151 and CD151VR could both be cell-surface-labeled (Fig. 10B). In addition, CD151 and CD151VR bothassociated with biotin-labeled species with apparent molecularmasses (~20-22 kDa) that correspond to tetraspanins CD9 andCD81. Thus, the CD151VR mutant is expressed on the cell surfaceat a comparable level to wild-type CD151, and appears to retainassociation with other tetraspanins.

In contrast to wild-type CD151, which was concentrated at cell-cell junctions (Fig. 10C; see also Fig. 2P,R), the CD151VR mutantdisplayed a more dispersed cellular localization (Fig. 10D).Moreover, E-cadherin, which again strongly localized to junctionsin cells rescued with wild-type CD151 (Fig. 10E), was severelyperturbed in cells rescued with CD151VR (Fig. 10F). Collectively,these data suggest that the ability of CD151 to associate with α3β1integrin might be important for its ability to promote junctionalorganization in carcinoma cells.

DiscussionCD151 regulates RhoA activation, E-cadherin junctionalstability and cell monolayer dynamicsWe have shown that CD151-silenced carcinoma cells displayenhanced collective migration, despite our earlier observation thatthe same CD151-silenced cells display reduced single-cell migrationvelocities (Winterwood et al., 2006). This apparent paradox mightbe resolved if the factors that limit the much faster single-cellvelocity [such as efficient detachment at the lateral or trailing edge(Winterwood et al., 2006)] are not the velocity-limiting factors inslower, collective cell migration. The collective migration velocityof wild-type cells is only ~6% of their single-cell velocity, whereasthat of CD151-silenced cells is a full 50% of the single-cell velocity.Thus, for collective cell migration, a major velocity-limiting factormight be the restraint imposed by being part of the collective, arestraint that appears significantly reduced for CD151-silenced cells.

Fig. 9. A RhoA inhibitor restoresjunctional organization in sh3monolayers. (A-L) CD151-silenced sh3cells (A-F) or wild-type cells (G-L) weretreated for 1 hour with 2 μg/ml cell-permeable C3 transferase (D-F, J-L) orleft untreated (A-C, G-I). Cells weredouble-labeled for F-actin and E-cadherin and analyzed by confocalmicroscopy. A-L show staining at thebasal level and A’-L’ show staining of thesame fields at a mid-lateral level.

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We provide evidence that the basis of this effect lies with theexcessive activation of the RhoA small GTPase in CD151-silencedcells, leading to elevated actin stress fiber formation, and with afar more dynamic cell monolayer in which cell-cell contactsundergo constant remodeling and have a significantly shorter meanlifespan.

The role of RhoA in the regulation of epithelial morphology iscomplex. Although Rho activity is required for the formation andstability of E-cadherin-dependent cell-cell junctions (Braga et al.,1997; Takaishi et al., 1997; Yamada and Nelson, 2007), as epithelialcell monolayers reach confluence or re-establish contacts aftercalcium switch, RhoA activity is progressively reduced to a lowbasal level over a time-course of hours (Arthur et al., 2002; Norenet al., 2001). Elevated RhoA activity appears not to interfere withthe early steps of junction formation (Braga et al., 1997), butexcessive Rho activity can be disruptive of steady-state junctionalstability and organization (Jou and Nelson, 1998; Zhong et al.,1997). These studies are consistent with our calcium switch data,in which junctions initially formed in CD151-silenced cells butfailed to remain organized. Cell-cell contact-dependent regulationof RhoA can involve signaling through p190RhoGAP to reduceRhoA activity (Nimnual et al., 2003; Noren et al., 2003; Wildenberg

et al., 2006). Suppression of RhoA activity via p190RhoGAP canalso result from integrin ligand binding (Bradley et al., 2006; DeMaliet al., 2003; Peacock et al., 2007). Thus, convergent signaling tolimit or localize RhoA activity might be an important form ofintegrin-cadherin cross-regulation. Our data are consistent with thisview and suggest that tetraspanin CD151 could play an importantrole in this regulatory mechanism. The inability of the CD151VR

mutant to rescue E-cadherin localization suggests that CD151 mightregulate junctional organization via association with α3β1 integrinand/or other integrin partners.

Kreidberg and colleagues (Chattopadhyay et al., 2003; Wang etal., 1999) observed that E-cadherin localization was perturbed inα3-integrin-null kidney epithelial cells, an obsevation similar to thatdescribed here. Reconstituting the cells with an α3 mutant with areduced ability to associate with CD151 in harsh detergents failedto rescue the E-cadherin phenotype (Chattopadhyay et al., 2003).In addition, CD151-silenced kidney cells showed a loss of corticalactin organization (Chattopadhyay et al., 2003). However, themechanism suggested by these studies entailed a loss of E-cadherin–α-actinin association, elevated β-catenin phosphorylation,a loss of PTPμ expression and reduced E-cadherin-dependentadhesive strength. By contrast, we observed no change in E-cadherin–α-actinin interaction, no increase in β-cateninphosphorylation, no reduction in PTPμ and no obvious reductionin the adhesive function, per se, of E-cadherin. Instead, we foundthat E-cadherin-dependent junctions initially form normally in ourCD151-silenced cells but fail to remain organized, probably due tothe highly dynamic nature of the CD151-silenced monolayer. Thus,there could be different mechanisms operating in our humancarcinoma cells than in murine embryonic kidney cells.

In another study, Shigeta et al. observed that overexpression ofa CD151-GFP fusion protein in A431 cells accelerated the rate atwhich the cells re-aggregated after dissociation, and slowedmigration rate in gap-filling experiments (Shigeta et al., 2003). Inaddition, an anti-CD151 antibody perturbed F-actin and E-cadherinlocalization. Rac and Cdc42 activities were elevated in CD151-overexpressing cells, but no change in Rho activation was observed.However, Shigeta et al. performed Rac, Cdc42 and Rho pulldownassays in subconfluent cultures, in which the basal Rho activationlevel was likely to be high (Noren et al., 2001) and might maskany effect of CD151 overexpression on Rho activation. Inpreliminary experiments, we did not observe any obvious changesin Rac or Cdc42 activation in our CD151-silenced cell cultures (datanot shown). Potentially, Rac and Cdc42 regulation by CD151 aremore easily uncovered by CD151 overexpression in A431 cells.Overall, our data generally agree with those of Shigeta andcolleagues, with the consensus being that overexpression of CD151enhances junctions and loss of CD151 destabilizes them.

Further support for this view comes from another recent studyin which CD151 was silenced in H5SC carcinoma cells, resultingin redistribution of cortical actin and enhanced stress fibers(Hasegawa et al., 2007). Lastly, our results contrast with arecently published study that demonstrated decreased gap fillingin vitro by MCF-10A cells treated with a CD151 siRNA (Yang etal., 2008). Multiple factors could account for this difference,including the use of immortalized instead of tumorigenic cell types,the presence of exogenous EGF in the MCF-10A assay, andpossible differences in the balance between the ability of CD151to enhance cell-substrate adhesion strengthening (Lammerding etal., 2003) and its ability to regulate junctional stability in MCF-10A cells versus A431 cells.

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Fig. 10. A CD151 mutant with impaired α3β1 integrin association fails torescue junctional organization. (A) Brij 99/96 detergent lysates were preparedfrom CD151-silenced cells rescued with FLAG-tagged wild-type CD151 (Rx)cells, un-rescued cells (sh3) or CD151-silenced cells reconstituted with theCD151VR mutant (VR) (1:1 Brij 99:Brij 96, 1% total detergent by volume). Theα3 integrin subunit (lanes 1-3) or FLAG-tagged CD151 constructs (lanes 4-6)were immunoprecipitated, followed by blotting with polyclonal anti-α3antibody (top panels) or polyclonal anti-FLAG antibody (bottom panels).(B) Rx, sh3, or VR cells were biotinylated and lysed in Brij 99/96, as in A.FLAG-tagged CD151 constructs were immunoprecipitated, and blots weredeveloped simultaneously with IRdye 800-streptavidin and polyclonal anti-FLAG antibody followed by Alexa 680-goat anti-rabbit secondary. Top panelshows biotinylated species, middle panel shows the FLAG blot, and the bottompanel shows with overlay with CD151, CD151VR and CD9/CD81 bandsindicated by arrowheads. (C,D) Rx (WT) and VR cells were fixed,permeabilized and stained with anti-FLAG polyclonal antibody followed byAlexa 594 goat anti-rabbit antibody. (E,F) E-cadherin staining of Rx (WT) andVR cells.

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Implications for CD151 function in development and diseaseGenetic evidence from mice and humans has identified importantroles for CD151 in epithelial integrity, wound healing andpathological angiogenesis elicited by tumor formation (Cowin etal., 2006; Karamatic Crew et al., 2004; Sachs et al., 2006; Takedaet al., 2007). How might the Rho-suppressive activity of CD151be involved in these processes? Elevated Rho activity upon loss ofCD151 could diminish the stability of epithelial adherens junctions,contributing to the structural failure in skin and kidney epithelia ofhuman patients and mice lacking CD151 (Karamatic Crew et al.,2004; Sachs et al., 2006). Wound healing could also be impairedby an overly dynamic epithelium in which RhoA was deregulatedand adherens junctions were destabilized. This could help toexplain the seeming paradox that CD151-silenced cells can fill agap in vitro more rapidly than wild-type cells (Fig. 1), whereaswound healing in vivo is impaired in CD151-null mice (Cowin etal., 2006). A less stable, CD151-null epithelium on a provisionalwound matrix might be more easily disrupted by in vivo mechanicalstresses than in the relatively quiescent environment of a tissueculture monolayer. Interestingly, genetic deletion of α3 integrin inkeratinocytes led to an enhanced rate of wound healing in vivo andof gap closure in vitro, and this was ascribed to a loss of anti-migratory α3-integrin-dependent adhesion on laminin-5 (Margadantet al., 2009). However, in contrast to α3-integrin-null keratinocytes,which also migrated more rapidly than wild-type keratinocytes insingle-cell assays (Margadant et al., 2009), single-cell migration ofour CD151-silenced carcinoma cells was slower than that of wild-type cells (Winterwood et al., 2006). Thus, an enhanced intrinsicmigration rate of individual cells due to reduced α3-integrin-dependent adhesion would be unlikely to explain the increased rateof collective migration that we observed for the CD151-silencedmonolayers.

Disruptions in Rho family GTPase signaling could also interferewith pathological angiogenesis. For example, excessive RhoAsignaling, such as might occur upon loss of CD151, has been linkedto loss of bipolarity, detachment, cell death and sprout retractionin an organotypic model of angiogenesis (Mavria et al., 2006). Sucha mechanism could help to explain the reduced pathologicalangiogenesis observed in CD151-null mice (Takeda et al., 2007).

The above considerations indicate that the role of CD151 inmetastasis is probably be complex. Several studies have identifiedCD151 as a potential promoter of metastasis (Ang et al., 2004;Hashida et al., 2003; Kohno et al., 2002; Testa et al., 1999; Tokuharaet al., 2001; Yang et al., 2008). However, if downregulation ofCD151 activates RhoA, destabilizes adherens junctions andenhances collective migration, then the potential exists for CD151to be a suppressor of invasion in some settings. Recently, an anti-metastatic CD151 antibody was shown to prevent tumor-celldetachment from the primary tumor site (Zijlstra et al., 2008).Activation of the ability of CD151 to stabilize cell-cell contactsmight represent another mode of action by this anti-metastaticantibody, in addition to its ability to upregulate integrin-dependentadhesion at the trailing edge (Zijlstra et al., 2008).

The potentially opposing roles for CD151 in metastasis couldhelp to explain why its major partner, α3β1 integrin, might act aseither a promoter or a suppressor of metastasis, depending on thecontext (Giannelli et al., 2002). Thus, future studies in which theroles of α3β1 integrin and CD151 are carefully examined in specificstages and types of malignancy will be important to determine inwhich contexts interfering with either protein might beadvantageous. It will also be important to determine whether the

ability of CD151 to promote integrin-dependent cell motility andits ability to promote stable cell-cell junctions are separablefunctions. If so, factors regulating the balance between theseopposing functions might determine whether CD151-integrincomplexes act as promoters or suppressors of metastasis in specificsettings.

Materials and MethodsAntibodies and laminin-5Monoclonal antibodies used were anti-E-cadherin (BD Biosciences #610182), SHE78-7(Zymed Laboratories, Invitrogen), DECMA-1 (Sigma), anti-β-catenin (BD Biosciences#610154), anti-α-catenin (BD Biosciences #610194), anti-p120 catenin (BDBiosciences #610134), anti-α-actinin (BD Biosciences #612577), anti-plakoglobin(BD Biosciences #610253), anti-phosphotyrosine PY20 (BD Biosciences #610000),anti-CD9, ALB6 (Chemicon International), anti-PTPμ BK2 (Santa Cruz Biotechnology#sc-33651), anti-FLAG epitope tag M2 agarose (Sigma) and anti-RhoA (Cytoskeleton#ARHO1). Monoclonal antibodies specific for CD81 (M38), CD151 (5C11 and11B1.G4), α2 integrin (A2-IIE10) and α3 integrin (A3-X8 and A3-IIF5) werepreviously referenced (Winterwood et al., 2006). Also used were rabbit polyclonalantibodies recognizing E-cadherin (gift of Jack Lilien and Janne Balsamo, Universityof Iowa Department of Biology, Iowa City, IA), α3 integrin (Kazarov et al., 2002) andFLAG epitope tag (Sigma). Secondary reagents were Alexa 680-goat anti-rabbit(Invitrogen), IRdye 800-goat anti-mouse and IRDye 800-streptavidin (Rockland), andCy2-goat anti-mouse and FITC-goat anti-rabbit antibodies (Jackson ImmunoResearch).Human laminin-5 was purified from SCC-25 cell-conditioned medium as described(Winterwood et al., 2006).

RNA interference and rescue with wild-type and mutant CD151The production of fluorescence activated cell sorting (FACS)-sorted A431 epithelialcarcinoma cells, with near-total silencing of CD151 (A431 sh3 cells), and the A431sh3 Rx rescue cells, in which wild-type CD151 is re-expressed, was described previously(Winterwood et al., 2006). The wild-type CD151 Rx cDNA contains two silent mutationsnear the center of the site targeted by the sh3 CD151 shRNA. Using the CD151 RxcDNA as a template, the CD151VR mutant was created with two rounds of recombinantPCR to (i) add a FLAG tag at the amino terminus, (ii) convert the endogenous initiatormethionine to leucine, (iii) retain the silent Rx mutations that allow re-expression inCD151-silenced cells and (iv) swap the region of the CD151 EC2 domain from serine158 to glycine 207 with the corresponding region from human tetraspanin TM4SF2/A15.This region corresponds to the ‘variable region’ of CD151 and contains sequences criticalfor CD151 interaction with α3β1 integrin (Stipp et al., 2003). The CD151VR mutantis not bound by anti-CD151 antibodies 5C11 or 11B1.G4, and was recognized weaklyor not at all by available anti-TM4SF2 antibodies. CD151VR is readily detected by anti-FLAG antibody. We also created a FLAG-tagged version of wild-type CD151 for usein comparison with the CD151VR mutant.

Time-lapse video microscopyA431 cells were cultured in standard medium (DME with 10% FBS, 2 mM L-glutamine,100 U/ml penicillin and 100 μg/ml streptomycin). Gaps were inscribed in freshlyconfluent monolayers using a yellow micropipet tip, and the cultures were refed withserum-free medium [SFM; DME with 5 mg/ml cell culture grade BSA (Sigma-Aldrich)and 25 mM HEPES pH 7.2]. Images were acquired every 5 minutes for 18-24 hours,using a stage incubator and video-microscope system described in our previous study(Winterwood et al., 2006). Gap area at 1-hour intervals was measured using NIH Image1.63 software to calculate the gap closure rate in μm2/h. To measure mitoses duringthe assays, 175-μm wide strips on either side of the initial gap were analyzed frame-by-frame. Mitotic events were clearly visualized as cells rounded up, divided and spreadback into the monolayer. Single-cell velocities on laminin-5 were measured for wild-type, CD151-silenced sh3 cells and Rx rescue cells in 3-hour videos, as previouslydescribed (Winterwood et al., 2006). In another series of experiments, unwoundedmonolayers in standard growth medium were imaged as described above, and NIHImageJ 1.63 software was used to record the XY position of cell centroids in frames30 minutes apart, for 35 cells per experiment. Custom Java software was then used tocalculate the mean velocity of cell migration within the monolayers. The change in thesize of individual cell-cell contact was followed every 5 minutes for 2 hours in oneexperiment. The lifespan of individual cell-cell contacts was assessed by examiningframes 15 minutes apart for a total of 12 hours in another experiment.

Spheroid dispersal assaysEppendorf tubes (0.5 ml) were prepared by adding 100 μl of 10 mg/ml cell culturegrade poly-HEMA [poly(2-hydroxyethyl methacrylate), Sigma] in 95% ethanol. Asmall hole was made in the lid of each tube with a sterile 26G needle, and the poly-HEMA solution was dried down in a speedvac. Tubes were rinsed with SFM and250 μl of a suspension containing 1.0�105 cells/ml was placed in each tube andincubated overnight in standard growth medium. Spheroids were rinsed into SFMand placed into wells of a 24-well plate that had been coated with 2 μg/ml humanlaminin-5 and blocked with SFM. Spheroids were photographed at 1 hour and 18 hours

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after plating, and their areas were measured using NIH ImageJ 1.63. The followingequation was used to estimate linear velocity (in μm/h) of cell sheet migration inspheroid dispersal assays:

where Area18h and Area1h are the areas (in μm2) of the spheroids measured at 18hours and 1 hour, respectively.

ImmunostainingCells cultured on coverslips were fixed in 10% formalin in HEPES-buffered saline(HBS) with 4% sucrose and 1 mM CaCl2, rinsed twice with TBS, and blocked with10% goat serum in PBS. For intracellular epitopes, cells were permeabilized witheither 0.2% NP-40 or 0.1% saponin during the blocking step. Cells were stained for1 hour with primary antibodies in blocking buffer, and then for 45 minutes with Cy2-goat anti-mouse antibody. For F-actin staining, Alexa 594-phalloidin (Invitrogen)diluted 1:40 in PBS was added to cells after the secondary antibody step. Cells weremounted in FluorSave (Calbiochem) or Prolong Gold (Invitrogen) and analyzed bystandard fluorescence microscopy as previously described (Winterwood et al., 2006),or by laser scanning confocal microscopy on a Leica SP2 AOBS inverted microscope.

Immunoprecipitation and immunoblottingCells were lysed in PBS with 1% detergent, protease inhibitors (2 mM PMSF, 10μg/ml aprotinin, 5 μg/ml leupeptin and 5 μg/ml E-64) (Roche Diagnostics), and HALTphosphatase inhibitors (Pierce Biotechnology). Detergents were NP-40, Brij 96V ora 1:1 mixture of Brij 96V and Brij 99 (all from Sigma-Aldrich). In some experiments,cells were biotinylated with 0.1 mg/ml Sulfo-NHS-LC biotin (Pierce Biotechnology)in HBS (20 mM HEPES pH 7.2, 150 mM NaCl) for 1 hour at room temperature andthen rinsed three times with HBS prior to lysis. Between 2�106 and 6�106 cellswere used for each experiment. Lysates were clarified and normalized for proteinconcentration. Immune complexes were collected, separated by SDS-PAGE, andblotted to nitrocellulose as previously described (Winterwood et al., 2006). Blotswere blocked with AquaBlock (East Coast Bio, North Berwick, ME) and probedwith primary antibodies diluted in 10% AquaBlock in TBST (20 mM Tris pH 7.5,150 mM NaCl, 0.1% Tween-20). Blots were developed with Alexa 680-goat anti-rabbit and/or IRDye 800-goat anti-mouse secondary antibodies and analyzed with aLiCor infrared fluorescence blot imager (LI-COR, Lincoln, NE). Biotinylated proteinswere detected with IRDye 800-streptavidin.

E-cadherin-Fc adhesion assaysE-cadherin-Fc protein (gift of W. James Nelson, Stanford University, Stanford, CA)was purified from transfected CHO cell-conditioned medium on a protein G-Sepharosecolumn; size, purity and dimeric structure were confirmed by SDS-PAGE andCoomassie blue staining. For adhesion assays, wells of a 96-well plate were coatedovernight at 4°C with 40 μg/ml of E-cadherin-Fc fusion protein diluted in HBS with2 mM CaCl2 (HBSC), and then blocked with 10% heat-inactivated BSA in HBSC.To prepare cells for the assay, 7.5�105 of each cell type were plated at low densityin a T75 flask the day before the assay. On the day of the assay, the cells were harvestedwith 0.25% trypsin with 0.38 g/l EDTA•4Na (Invitrogen) and then replated at 7.5�105

cells per T75 flask for 4 hours to allow for recovery. Subsequently, the cells wereharvested again, this time with 0.1% trypsin without EDTA in HBSC. Harvested cellswere rinsed with, and resuspended in, DME with 2% FBS. This protocol was designedto yield a single-cell suspension of contact-naïve cells with E-cadherin cell-surfaceexpression preserved; 1.8�104 cells/ml were plated per well. Some samples weretreated with 5 mM EGTA or with anti-E-cadherin function-blocking antibodies for10 minutes prior to plating. After 30 minutes, non-adherent cells were removed byrinsing; adherent cells were quantified by crystal violet staining and compared tototal cells plated (as measured in poly-L-lysine-coated wells) and non-specificadhesion in BSA-coated wells, as previously described (Winterwood et al., 2006).

Expression and analysis of E-cadherin-GFP fusion proteinAn E-cadherin-GFP (EcadGFP) fusion construct (gift of W. James Nelson) wastransfected into A431 wild-type and CD151-silenced sh3 cells. Stable transfectantswere selected with G418, and EcadGFP-expressing cells were sorted by FACS. Forcalcium switch experiments, cells growing on coverslips were placed in low-calciummedium (CaCl2-free DME with 2% extensively dialyzed FBS and 5 μm CaCl2) for15 minutes. Then, calcium was restored to 1.8 mM for 30 minutes, 1 hour or 3hours,and cells fixed and examined by immunofluorescence microscopy.

Analysis of the role of RhoA activationFor RhoA activity assays, 5�106 cells were plated in 100 mm dishes and culturedfor 3 days to achieve complete confluence. Cells were then lysed with 600 μl of 25mM HEPES pH 7.2, 150 mM NaCl, 1% NP-40, 10 mM MgCl2, 10% glycerol andprotease inhibitors, followed by centrifugation at 16,000 � g for 4 minutes at 4°C.Active RhoA was recovered for 30 minutes at 4°C with 30 μg of GST-Rhotekinbound to glutathione Sepharose and analyzed by SDS-PAGE followed by blotting

Linear velocity =Area18h / π − Area1h / π( )

17 , (1)

for RhoA. Blots were quantified using a LiCor infrared-fluorescence blot imager. Asa positive control for RhoA activation, some dishes were harvested with trypsin plusEDTA, and the cells were held in suspension for 5 minutes prior to lysis and RhoApulldown.

To assess the role of RhoA activity on junctional stability in CD151-silenced sh3cells, cells plated on coverslips were treated for 1 hour with 2 μg/ml cell-permeableC3 transferase (Cytoskeleton, Denver, CO). Treated and untreated cells were thenfixed and stained for E-cadherin and F-actin, as above. In parallel experiments, freshlyconfluent monolayers were analyzed by time-lapse microscopy, as described above,for 3 hours before and 3 hours after the addition of 2 μg/ml cell-permeable C3transferase.

We thank Janne Balsamo and Jack Lilien for advice, suggestions andantibody reagents, and Mary Herndon and members of the Universityof Iowa Tumor Biology and Genetics Group for comments and critique.This study was supported by a grant from the Roy J. Carver CharitableTrust (07-2869) and an American Cancer Society Research ScholarAward (#RSG-07-043-01-CSM).

ReferencesAng, J., Lijovic, M., Ashman, L. K., Kan, K. and Frauman, A. G. (2004). CD151 protein

expression predicts the clinical outcome of low-grade primary prostate cancer betterthan histologic grading: a new prognostic indicator? Cancer Epidemiol. Biomarkers Prev.13, 1717-1721.

Arthur, W. T., Noren, N. K. and Burridge, K. (2002). Regulation of Rho family GTPasesby cell-cell and cell-matrix adhesion. Biol. Res. 35, 239-246.

Berditchevski, F. (2001). Complexes of tetraspanins with integrins: more than meets theeye. J. Cell Sci. 114, 4143-4151.

Berditchevski, F. and Odintsova, E. (2007). Tetraspanins as regulators of proteintrafficking. Traffic 8, 89-96.

Boucheix, C., Thien Duc, G. H., Jasmin, C. and Rubinstein, E. (2001). Tetraspaninsand malignancy. Exp. Rev. Mol. Med. 2001, 1-17.

Brabletz, T., Jung, A., Spaderna, S., Hlubek, F. and Kirchner, T. (2005). Opinion:migrating cancer stem cells-an integrated concept of malignant tumour progression. Nat.Rev. Cancer 5, 744-749.

Bradley, W. D., Hernandez, S. E., Settleman, J. and Koleske, A. J. (2006). Integrinsignaling through Arg activates p190RhoGAP by promoting its binding to p120RasGAPand recruitment to the membrane. Mol. Biol. Cell 17, 4827-4836.

Braga, V. M. (2002). Cell-cell adhesion and signalling. Curr. Opin. Cell Biol. 14, 546-556.

Braga, V. M., Machesky, L. M., Hall, A. and Hotchin, N. A. (1997). The small GTPasesRho and Rac are required for the establishment of cadherin-dependent cell-cell contacts.J. Cell Biol. 137, 1421-1431.

Chattopadhyay, N., Wang, Z., Ashman, L. K., Brady-Kalnay, S. M. and Kreidberg,J. A. (2003). alpha3beta1 integrin-CD151, a component of the cadherin-catenin complex,regulates PTPmu expression and cell-cell adhesion. J. Cell Biol. 163, 1351-1362.

Chometon, G., Zhang, Z. G., Rubinstein, E., Boucheix, C., Mauch, C. and Aumailley,M. (2006). Dissociation of the complex between CD151 and laminin-binding integrinspermits migration of epithelial cells. Exp. Cell Res. 312, 983-995.

Cowin, A. J., Adams, D., Geary, S. M., Wright, M. D., Jones, J. C. and Ashman, L.K. (2006). Wound healing is defective in mice lacking tetraspanin CD151. J. Invest.Dermatol. 126, 680-689.

Cowin, P. and Welch, D. R. (2007). Breast cancer progression: controversies andconsensus in the molecular mechanisms of metastasis and EMT. J. Mammary GlandBiol. Neoplasia 12, 99-102.

Cowin, P., Rowlands, T. M. and Hatsell, S. J. (2005). Cadherins and catenins in breastcancer. Curr. Opin. Cell Biol. 17, 499-508.

DeMali, K. A., Wennerberg, K. and Burridge, K. (2003). Integrin signaling to the actincytoskeleton. Curr. Opin. Cell Biol. 15, 572-582.

Fukata, M. and Kaibuchi, K. (2001). Rho-family GTPases in cadherin-mediated cell-celladhesion. Nat. Rev. Mol. Cell. Biol. 2, 887-897.

Gesierich, S., Berezovskiy, I., Ryschich, E. and Zoller, M. (2006). Systemic inductionof the angiogenesis switch by the tetraspanin D6.1A/CO-029. Cancer Res. 66, 7083-7094.

Giannelli, G., Astigiano, S., Antonaci, S., Morini, M., Barbieri, O., Noonan, D. M. andAlbini, A. (2002). Role of the alpha3beta1 and alpha6beta4 integrins in tumor invasion.Clin. Exp. Metastasis 19, 217-223.

Hasegawa, M., Furuya, M., Kasuya, Y., Nishiyama, M., Sugiura, T., Nikaido, T.,Momota, Y., Ichinose, M. and Kimura, S. (2007). CD151 dynamics in carcinoma-stroma interaction: integrin expression, adhesion strength and proteolytic activity. Lab.Invest. 87, 882-892.

Hashida, H., Takabayashi, A., Tokuhara, T., Hattori, N., Taki, T., Hasegawa, H., Satoh,S., Kobayashi, N., Yamaoka, Y. and Miyake, M. (2003). Clinical significance oftransmembrane 4 superfamily in colon cancer. Br. J. Cancer 89, 158-167.

Hemler, M. E. (2005). Tetraspanin functions and associated microdomains. Nat. Rev. Mol.Cell. Biol. 6, 801-811.

Hong, I. K., Kim, Y. M., Jeoung, D. I., Kim, K. C. and Lee, H. (2005). Tetraspanin CD9induces MMP-2 expression by activating p38 MAPK, JNK and c-Jun pathways in humanmelanoma cells. Exp. Mol. Med. 37, 230-239.

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Hong, I. K., Jin, Y. J., Byun, H. J., Jeoung, D. I., Kim, Y. M. and Lee, H. (2006).Homophilic interactions of Tetraspanin CD151 up-regulate motility and matrixmetalloproteinase-9 expression of human melanoma cells through adhesion-dependentc-Jun activation signaling pathways. J. Biol. Chem. 281, 24279-24292.

Hoschuetzky, H., Aberle, H. and Kemler, R. (1994). Beta-catenin mediates the interactionof the cadherin-catenin complex with epidermal growth factor receptor. J. Cell Biol.127, 1375-1380.

Huang, H., Sossey-Alaoui, K., Beachy, S. H. and Geradts, J. (2007). The tetraspaninsuperfamily member NET-6 is a new tumor suppressor gene. J. Cancer Res. Clin. Oncol.133, 761-769.

Hugo, H., Ackland, M. L., Blick, T., Lawrence, M. G., Clements, J. A., Williams, E.D. and Thompson, E. W. (2007). Epithelial-mesenchymal and mesenchymal-epithelialtransitions in carcinoma progression. J. Cell Physiol. 213, 374-383.

Jang, A. C., Starz-Gaiano, M. and Montell, D. J. (2007). Modeling migration andmetastasis in Drosophila. J. Mammary Gland Biol. Neoplasia 12, 103-114.

Jou, T. S. and Nelson, W. J. (1998). Effects of regulated expression of mutant RhoA andRac1 small GTPases on the development of epithelial (MDCK) cell polarity. J. CellBiol. 142, 85-100.

Karamatic Crew, V., Burton, N., Kagan, A., Green, C. A., Levene, C., Flinter, F., Brady,R. L., Daniels, G. and Anstee, D. J. (2004). CD151, the first member of the tetraspanin(TM4) superfamily detected on erythrocytes, is essential for the correct assembly ofhuman basement membranes in kidney and skin. Blood 104, 2217-2223.

Kazarov, A. R., Yang, X., Stipp, C. S., Sehgal, B. and Hemler, M. E. (2002). Anextracellular site on tetraspanin CD151 determines alpha 3 and alpha 6 integrin-dependentcellular morphology. J. Cell Biol. 158, 1299-1309.

Kohno, M., Hasegawa, H., Miyake, M., Yamamoto, T. and Fujita, S. (2002). CD151enhances cell motility and metastasis of cancer cells in the presence of focal adhesionkinase. Int. J. Cancer 97, 336-343.

Lammerding, J., Kazarov, A. R., Huang, H., Lee, R. T. and Hemler, M. E. (2003).Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening. Proc. Natl.Acad. Sci. USA 100, 7616-7621.

Lazo, P. A. (2007). Functional implications of tetraspanin proteins in cancer biology. CancerSci. 98, 1666-1677.

Levy, S. and Shoham, T. (2005). The tetraspanin web modulates immune-signallingcomplexes. Nat. Rev. Immunol. 5, 136-148.

Liu, L., He, B., Liu, W. M., Zhou, D., Cox, J. V. and Zhang, X. A. (2007). TetraspaninCD151 promotes cell migration by regulating integrin trafficking. J. Biol. Chem. 282,31631-31642.

Liu, W. M. and Zhang, X. A. (2006). KAI1/CD82, a tumor metastasis suppressor. CancerLett. 240, 183-194.

Margadant, C., Raymond, K., Kreft, M., Sachs, N., Janssen, H. and Sonnenberg, A.(2009). Integrin {alpha}3{beta}1 inhibits directional migration and wound re-epithelialization in the skin. J. Cell Sci. 122, 278-288.

Mavria, G., Vercoulen, Y., Yeo, M., Paterson, H., Karasarides, M., Marais, R., Bird,D. and Marshall, C. J. (2006). ERK-MAPK signaling opposes Rho-kinase to promoteendothelial cell survival and sprouting during angiogenesis. Cancer Cell 9, 33-44.

Nimnual, A. S., Taylor, L. J. and Bar-Sagi, D. (2003). Redox-dependent downregulationof Rho by Rac. Nat. Cell Biol. 5, 236-241.

Noren, N. K., Niessen, C. M., Gumbiner, B. M. and Burridge, K. (2001). Cadherinengagement regulates Rho family GTPases. J. Biol. Chem. 276, 33305-33308.

Noren, N. K., Arthur, W. T. and Burridge, K. (2003). Cadherin engagement inhibitsRhoA via p190RhoGAP. J. Biol. Chem. 278, 13615-13618.

Peacock, J. G., Miller, A. L., Bradley, W. D., Rodriguez, O. C., Webb, D. J. and Koleske,A. J. (2007). The Abl-related gene tyrosine kinase acts through p190RhoGAP to inhibitactomyosin contractility and regulate focal adhesion dynamics upon adhesion tofibronectin. Mol. Biol. Cell 18, 3860-3872.

Sachs, N., Kreft, M., van den Bergh Weerman, M. A., Beynon, A. J., Peters, T. A.,Weening, J. J. and Sonnenberg, A. (2006). Kidney failure in mice lacking the tetraspaninCD151. J. Cell Biol. 175, 33-39.

Saito, Y., Tachibana, I., Takeda, Y., Yamane, H., He, P., Suzuki, M., Minami, S., Kijima,T., Yoshida, M., Kumagai, T. et al. (2006). Absence of CD9 enhances adhesion-dependent morphologic differentiation, survival, and matrix metalloproteinase-2production in small cell lung cancer cells. Cancer Res. 66, 9557-9565.

Shigeta, M., Sanzen, N., Ozawa, M., Gu, J., Hasegawa, H. and Sekiguchi, K. (2003).CD151 regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actincytoskeletal reorganization. J. Cell Biol. 163, 165-176.

Sridhar, S. C. and Miranti, C. K. (2006). Tetraspanin KAI1/CD82 suppresses invasionby inhibiting integrin-dependent crosstalk with c-Met receptor and Src kinases. Oncogene25, 2367-2378.

Sterk, L. M., Geuijen, C. A., van den Berg, J. G., Claessen, N., Weening, J. J. andSonnenberg, A. (2002). Association of the tetraspanin CD151 with the laminin-bindingintegrins alpha3beta1, alpha6beta1, alpha6beta4 and alpha7beta1 in cells in culture andin vivo. J. Cell Sci. 115, 1161-1173.

Stipp, C. S., Kolesnikova, T. V. and Hemler, M. E. (2003). Functional domains intetraspanin proteins. Trends Biochem. Sci. 28, 106-112.

Sugiura, T. and Berditchevski, F. (1999). Function of alpha3beta1-tetraspanin proteincomplexes in tumor cell invasion: evidence for the role of the complexes in productionof matrix metalloproteinase 2 (MMP-2). J. Cell Biol. 146, 1375-1389.

Takaishi, K., Sasaki, T., Kotani, H., Nishioka, H. and Takai, Y. (1997). Regulation ofcell-cell adhesion by rac and rho small G proteins in MDCK cells. J. Cell Biol. 139,1047-1059.

Takeda, Y., Kazarov, A. R., Butterfield, C. E., Hopkins, B. D., Benjamin, L. E.,Kaipainen, A. and Hemler, M. E. (2007). Deletion of tetraspanin Cd151 results indecreased pathologic angiogenesis in vivo and in vitro. Blood 109, 1524-1532.

Testa, J. E., Brooks, P. C., Lin, J. M. and Quigley, J. P. (1999). Eukaryotic expressioncloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis. Cancer Res. 59,3812-3820.

Thiery, J. P. and Sleeman, J. P. (2006). Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev. Mol. Cell. Biol. 7, 131-142.

Tokuhara, T., Hasegawa, H., Hattori, N., Ishida, H., Taki, T., Tachibana, S., Sasaki,S. and Miyake, M. (2001). Clinical significance of CD151 gene expression in non-small cell lung cancer. Clin. Cancer Res. 7, 4109-4114.

Tonoli, H. and Barrett, J. C. (2005). CD82 metastasis suppressor gene: a potential targetfor new therapeutics? Trends Mol. Med. 11, 563-570.

Wang, Z., Symons, J. M., Goldstein, S. L., McDonald, A., Miner, J. H. and Kreidberg,J. A. (1999). (Alpha)3(beta)1 integrin regulates epithelial cytoskeletal organization. J.Cell Sci. 112, 2925-2935.

Wells, C. M., Ahmed, T., Masters, J. R. and Jones, G. E. (2005). Rho family GTPasesare activated during HGF-stimulated prostate cancer-cell scattering. Cell Motil.Cytoskeleton 62, 180-194.

Wildenberg, G. A., Dohn, M. R., Carnahan, R. H., Davis, M. A., Lobdell, N. A.,Settleman, J. and Reynolds, A. B. (2006). p120-catenin and p190RhoGAP regulatecell-cell adhesion by coordinating antagonism between Rac and Rho. Cell 127, 1027-1039.

Winterwood, N. E., Varzavand, A., Meland, M. N., Ashman, L. K. and Stipp, C. S.(2006). A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5. Mol. Biol. Cell 17, 2707-2721.

Yamada, S. and Nelson, W. J. (2007). Localized zones of Rho and Rac activities driveinitiation and expansion of epithelial cell-cell adhesion. J. Cell Biol. 178, 517-527.

Yang, X. H., Richardson, A. L., Torres-Arzayus, M. I., Zhou, P., Sharma, C., Kazarov,A. R., Andzelm, M. M., Strominger, J. L., Brown, M. and Hemler, M. E. (2008).CD151 accelerates breast cancer by regulating alpha 6 integrin function, signaling, andmolecular organization. Cancer Res. 68, 3204-3213.

Yap, A. S. and Kovacs, E. M. (2003). Direct cadherin-activated cell signaling: a viewfrom the plasma membrane. J. Cell Biol. 160, 11-16.

Yauch, R. L., Berditchevski, F., Harler, M. B., Reichner, J. and Hemler, M. E. (1998).Highly stoichiometric, stable, and specific association of integrin alpha3beta1 with CD151provides a major link to phosphatidylinositol 4-kinase, and may regulate cell migration.Mol. Biol. Cell 9, 2751-2765.

Zhong, C., Kinch, M. S. and Burridge, K. (1997). Rho-stimulated contractility contributesto the fibroblastic phenotype of Ras-transformed epithelial cells. Mol. Biol. Cell 8, 2329-2344.

Zijlstra, A., Lewis, J., Degryse, B., Stuhlmann, H. and Quigley, J. P. (2008). Theinhibition of tumor cell intravasation and subsequent metastasis via regulation of in vivotumor cell motility by the tetraspanin CD151. Cancer Cell 13, 221-234.

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