1478 Research Article CD151 restricts the a6 integrin diffusion mode

Xiuwei H. Yang1,*,`,§, Rossen Mirchev2,§, Xinyu Deng3, Patrick Yacono2, Helen L. Yang3, David E. Golan2,4 and Martin E. Hemler1,` 1Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA 3Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, KY 40536-0298, USA 4Hematology Division, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA *Present Address: Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, KY 40536-0298, USA `Authors for correspondence ([email protected]; [email protected]) §These authors contributed equally to this work

Accepted 11 November 2011 Journal of Cell Science 125, 1478–1487 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.093963

Summary Laminin-binding integrins (a3b1, a6b1, a6b4, a7b1) are almost always expressed together with tetraspanin CD151. In every coexpressing cell analyzed to date, CD151 makes a fundamental contribution to integrin-dependent motility, invasion, morphology, adhesion and/or signaling. However, there has been minimal mechanistic insight into how CD151 affects integrin functions. In MDA- MB-231 mammary cells, tetraspanin CD151 knockdown impairs a6 integrin clustering and functions without decreasing a6 integrin expression or activation. Furthermore, CD151 knockdown minimally affects the magnitude of a6 integrin diffusion, as measured using single particle tracking. Instead, CD151 knockdown has a novel and unexpected dysregulating effect on the mode of a6 integrin diffusion. In control cells a6 integrin shows mostly random-confined diffusion (RCD) and some directed motion (DMO). In sharp contrast, in CD151-knockdown cells a6 integrin shows mostly DMO. In control cells a6 diffusion mode is sensitive to actin disruption, talin knockdown and phorbol ester stimulation. By contrast, CD151 knockdown cell a6 integrin is sensitive to actin disruption but desensitized to talin knockdown or phorbol ester stimulation, indicating dysregulation. Both phorbol ester and EGF stimulate cell spreading and promote a6 RCD in control cells. By contrast, CD151-ablated cells retain EGF effects but lose phorbol-ester-stimulated spreading and a6 RCD. For a6 integrins, physical association with CD151 promotes a6 RCD, in support of a6-mediated cable formation and adhesion. By comparison, for integrins not associated with CD151 (e.g. av integrins), CD151 affects neither diffusion mode nor av function. Hence, CD151 support of a6 RCD is specific and functionally relevant, and probably underlies diverse CD151 functions in skin, kidney and cancer cells.

Key words: Integrin, Tetraspanin, CD151, Single particle tracking, Laminin Journal of Cell Science

Introduction xenograft model (Sadej et al., 2009; Yang et al., 2008). Among the 24 different ab heterodimers in the integrin family, Consistent with the multiple roles for CD151 in cancer, anti- the laminin-binding integrins (a3b1, a6b1, a6b4, a7b1) are a CD151 antibodies that can inhibit tumor growth and metastasis are distinct subgroup, based on functional and structural similarities being evaluated for potential clinical application (Haeuw et al., (Belkin and Stepp, 2000) and their close association with cell 2011). surface proteins in the tetraspanin family (Sterk et al., 2002; Stipp CD151 appears to function largely through its effects on et al., 2003b). Among the tetraspanin proteins, CD151 shows the laminin-binding integrins. A human CD151 mutation has been most robust association with laminin-binding integrins, in terms found to be associated with end-stage kidney failure, regional skin of stability and stoichiometry. CD151 association with a3 and a6 blistering and other defects in two individuals (Kagan et al., 1988; integrins occurs through direct protein–protein interaction, occurs Karamatic Crew et al., 2004). Consistent with this, laminin- early in biosynthesis, and can affect integrin glycosylation binding integrin a3, a6, and b4 subunits also contribute to skin and (Baldwin et al., 2008; Berditchevski et al., 2001; Kazarov et al., kidney development (Belkin and Stepp, 2000). As further evidence 2002; Yauch et al., 1998; Yauch et al., 2000). for CD151 working through laminin-binding integrins, ablation or CD151 expression on cancer cells correlates with poor clinical mutation of CD151 markedly disrupts integrin-dependent effects outcome and/or high grade in non-small cell lung (Tokuhara et al., on cell migration, proliferation, cable formation, morphology and 2001), prostate (Ang et al., 2004), hepatocellular (Liu et al., 2007), signaling (Berditchevski et al., 2002; Johnson et al., 2009; Kazarov breast (Novitskaya et al., 2010; Sadej et al., 2009; Yang et al., 2008) et al., 2002; Lammerding et al., 2003; Novitskaya et al., 2010; and other cancers (Romanska and Berditchevski, 2011). Sadej et al., 2009; Stipp et al., 2003a; Winterwood et al., 2006; Furthermore, CD151 is enriched on prostate-tumor-initiating cells Yang et al., 2002; Zevian et al., 2011; Zhang et al., 2002; Zuo et al., (Rajasekhar et al., 2011), contributes functionally to tumor cell 2010). metastasis (Kohno et al., 2002; Zijlstra et al., 2008), supports breast Although CD151 is known to modulate a6b1-, a6b4- and cancer cell resistance to ErbB2 antagonists (Yang et al., 2010) and a3b1-integrin-dependent cell morphology, motility and neurite accelerates primary breast cancer growth in a human-mouse outgrowth (Ashman, 2002; Hemler, 2005), mechanistic details CD151 influence on a6 integrins 1479

are lacking. Despite its close association, CD151 is not needed only minimally affected a6 integrin diffusion magnitude. Instead, for a3ora6 integrin expression (Sachs et al., 2006; Takeda et al., there was a substantial and unexpected effect on diffusion mode, 2007; Wright et al., 2004). A suggested CD151 support of a3b1 as a6 shifted away from random-confined diffusion (RCD) and integrin ‘activation’ neoepitopes (Nishiuchi et al., 2005) is towards directed motion (DMO). The shift towards DMO was counterbalanced by a report that a3b1 integrin is remarkably accompanied by loss of diffusion sensitivity to talin knockdown resistant to changes in neoepitopes, even when saturated with and phorbol ester stimulation, and by diminished phorbol-ester- manganese (Bazzoni et al., 1998). CD151 also might affect stimulated cell spreading function. These results suggest that integrin trafficking (Liu et al., 2007; Winterwood et al., 2006), CD151 supports a6 integrin functions by preventing unregulated and supports (Nishiuchi et al., 2005; Winterwood et al., 2006) or DMO while favoring RCD. Hence, a6 integrins can properly does not support (Berditchevski, 2001; Testa et al., 1999) translate outside-in signals and extracellular laminin cues into integrin-mediated adhesion. In a case where CD151 did not appropriate cell adhesion, migration, morphology, signaling and influence initial cell binding to laminin-coated beads, it did affect other events. subsequent adhesion strengthening (Lammerding et al., 2003). Laminin-binding integrins are linked, through CD151, to Results tetraspanin-enriched microdomains (Nydegger et al., 2006; CD151 affects cell cable formation and adhesion Takeda et al., 2007; Yanez-Mo et al., 2009; Yang et al., 2008), To gain new insights into functions of CD151 in MDA-MB-231 which probably participate in the regulation of integrin functions. mammary cells, we first used a three-dimensional (3D) Matrigel Studies of CD151 in breast cancer (Sadej et al., 2009; Yang ‘cable formation’ assay, which involves laminin adhesion and et al., 2008) have relied on mammary cell lines (MDA-MB-231 adhesion strengthening (Zhang et al., 2002) while modeling and/or MCF-10A) with basal-like properties (Neve et al., 2006). branching morphogenesis (Michaelson et al., 2005; Stahl In these cells, CD151 affects a6-integrin-dependent migration, et al., 1997). Alignment of MDA-MB-231 cells into a invasion, spreading and/or signaling in vitro, and both ectopic branching network of cellular cables was markedly diminished and orthotopic tumor growth in vivo. However, insights are when CD151 was knocked down (Fig. 1A). CD151 surface needed into how CD151 fundamentally and specifically affects expression was decreased by .80–90% upon siRNA treatment a6 integrin functions in basal-like mammary cell lines. Here, we (Fig. 1B). Next we examined mechanisms by which CD151 show that CD151 ablation does not affect a6 integrin expression might affect MDA-MB-231 cell invasion (Yang et al., 2008) and or activation. Instead, CD151 supports a6-integrin-dependent morphology (Fig. 1A) in Matrigel, which is largely composed of morphology, adhesion (especially at low laminin or integrin laminin-1. MDA-MB-231 cells treated with control or CD151- density) and antibody-induced clustering – events all suggestive specific siRNAs showed little adhesion to a surface coated with of possible effects on integrin diffusion. To characterize a6 BSA, and similar adhesion to laminin-1 (higher dose). However, integrin diffusion, we utilized single particle tracking (SPT), a cells lacking CD151 showed significantly less static adhesion to technique with suitable sensitivity for studying slowly diffusing surfaces coated with a lower dose of laminin-1 (mediated by receptors such as integrins (Cairo et al., 2006). Several studies a6b1 integrin) or coated with two different doses of laminin-5 have focused on the regulation of integrin diffusion magnitude (Fig. 1C). Two different CD151 siRNAs yielded similar

Journal of Cell Science (see Chen et al., 2007; Gaborski et al., 2008; Hirata et al., 2005; decreases in MDA-MB-231 adhesion (not shown). By contrast, Kucik et al., 1996; Yauch et al., 1997). However, CD151 ablation knockdown of CD151 (by .90%) did not affect adhesion of

Fig. 1. CD151 effects on mammary cell morphology and adhesion. (A) MDA-MB-231 cells treated with siRNAs were seeded at 56104 cells/well on Matrigel-coated 48-well plates and photographed after 18 hours. Scale bar: 5 mm. Results are representative of many experiments. Numbers indicate nodes with three or more branches. (B) MDA-MB-231 cells were treated with siRNAs for 5 days, and then the surface expression of a6 integrin and CD151 was determined by flow cytometry, counting at least 5000 cells/experiment. MFI, mean fluorescence intensity. (C) After treatment with siRNAs for 5 days, MDA-MB-231 cells were seeded in triplicate into 96-well plates precoated with ECM proteins. After 45 minutes, static cell adhesion was assessed. Values are means 6 s.e.m.; n53; *P,0.02. (D) After siRNA treatment the MCF-10A cells were seeded in triplicate on the indicated ECM proteins for 30 minutes, and then static cell adhesion was assessed. 1480 Journal of Cell Science 125 (6)

MCF-10A cells to laminin-5 (mediated by a3b1 and a6b4 integrins) or fibronectin (Fig. 1D). MCF-10A adhesion to laminin-1 was insufficiently elevated above background to allow for evaluation of CD151-knockdown effects (not shown). To gain insight into the mechanism of the different CD151 effects on MDA-MB-231 and MCF-10A cell adhesion, we analyzed integrin–CD151 complexes in those cells, using metabolic labeling with [3H]palmitate. Immunoprecipitation of a3 and a6 (but not a2) integrins yielded [3H]palmitate-labeled CD151 (supplementary material Fig. S1, lanes 1–4), and immunoprecipitation of CD151 yielded a mixture of a3 and a6 integrins (supplementary material Fig. S1, lanes 5, 6*). Hence, CD151 is associated with a3 and a6 integrins, as expected. Relevant to the cell adhesion results (Fig. 1C,D), fewer a6 integrins were present in MDA-MB-231 (supplementary material Fig. S1, lane 4) than in MCF-10A cells (lane 3). Cells containing fewer a6 integrins may be more dependent on CD151 for optimizing adhesion avidity on laminin-1 and laminin-5, especially at lower coating doses (Fig. 1C). By contrast, high levels of a6b4 integrin, together with a3b1 integrin, appear to support adhesion of MCF-10A cells to laminin-5, even when CD151 was ablated (Fig. 1D). The abundance of a3b1 integrin in MDA-MB-231 cells (supplementary material Fig. S1, lane 2) should not affect adhesion to laminin-1, because a3b1 integrin interacts minimally with laminin-1 (Delwel et al., 1994). The relative abundance of a3 and a6 subunits was confirmed by immunoblotting (supplementary material Fig. S1, lower panels). In a control experiment, no [3H]palmitate-labeled proteins associated with a2 integrin (supplementary material Fig. S1, lane 7), even though a2 is as abundant as a3 and a6 integrins in MCF-10A cells (not shown).

CD151 does not affect a6 integrin expression or activation, but may affect diffusion

Journal of Cell Science Despite nearly complete removal of CD151 from MDA-MB-231 Fig. 2. CD151 affects integrin clustering. (A-H) To induce a6b4 integrin cells, surface levels of integrin a6, a3 and b1 subunits and clustering, siRNA-treated MCF-10A cells were detached and incubated with tetraspanin CD82 did not decrease (supplementary material Fig. anti-b4 antibody (ASC-8) for 45 minutes (at 4˚C), followed by staining with S2). Depletion of tetraspanin CD82 also did not diminish surface Alexa-Fluor-594-conjugated red secondary antibody at 37˚C for 10 minutes (A,B). After washing, these cells were further stained with FITC-conjugated expression of CD151 or integrin subunits. Furthermore, the anti-CD151 (11G5A; C,D) or CD9 (MM2-57; G,H), allowed to adhere to a percentage of b1 integrin expressing the 9EG7 neoepitope poly-lysine-coated coverslip, and then mounted with Prolong anti-fade [sometimes associated with integrin activation (Nilsson et al., reagent for examination by confocal microscopy. (E) Merged image of A and 2006)] was also not decreased (9.9% increased to 11.5%; C; (F) merged image of B and D. Scale bar: 20 mm. supplementary material Fig. S2). Because CD151 can affect the molecular organization of integrin complexes (Takeda et al., 2007; Yang et al., 2008) and arrowheads, Fig. 2B). In a control experiment, CD151 absence affect laminin adhesion selectively in a cell expressing low levels did not affect CD9 staining (Fig. 2G,H). of a6 integrin (Fig. 1C,D), and we found that it especially affected adhesion to lower doses of laminin (Fig. 1C) and did not CD151 minimally affects diffusion rates affect integrin expression or activation, we considered that Next, we used SPT (Cairo et al., 2006) to assess CD151 effects CD151 might affect integrin lateral diffusion. To test this on a6 integrin diffusion in the plasma membrane. We used hypothesis, we first analyzed antibody-induced capping, which MDA-MB-231 cells because the lower density of a6 integrin on depends on integrin diffusion (Yauch et al., 1997). For capping MDA-MB-231, compared with MCF-10A cells (supplementary experiments, we used MCF-10A cells, which express a6b4 material Fig. S1), facilitates bead tracking. Both short-range (Dm) integrin at sufficiently high levels (supplementary material Fig. and long-range (DM) diffusion coefficients showed minimal S1) to be readily detected upon antibody-induced capping. When change when CD151 was ablated stably (Fig. 3, groups 1, 2) or CD151 (green) was present in MCF-10A cells (Fig. 2A,C,E), transiently (group 7). Dm is determined over a short time interval .40% of cells showed large clusters of a6b4 integrin (red), and DM over a longer time interval, and therefore measure the colocalized with CD151 (yellow). However, upon CD151 local and long-range mobility of a6 integrin, respectively silencing, ,10% of cells showed integrin clusters (Mirchev and Golan, 2001). Diffusion coefficients measured at –12 2 –12 (Fig. 2B,D,F). Cells with little remaining CD151 showed the 37˚C(Dm53.360.48610 cm /second, DM54.061.2610 most diffuse (and therefore less obvious) a6b4 staining (white cm2/second; n596) were similar to those measured at room CD151 influence on a6 integrins 1481

Fig. 3. CD151 has little effect on diffusion coefficients. Diffusion

coefficients (Dm and DM), determined as described in the Materials and Methods, are indicated for each condition. Data supporting these bar graphs are shown in supplementary material Table S1, Table S2 and Fig. S3. Left panel: effect of CD151 shRNA; right panel: effect of CD151 siRNA (plus talin siRNA). Prior to adding beads, cells were either unstimulated, or treated with cytochalasin D (cytoD, 10 mM, 1 hour), phorbol 12-myristate 13-acetate (PMA, 0.2 mg/ml, 30 minutes), epidermal growth factor (EGF, 0.02 mg/ml, 1 hour) or methyl-b- cyclodextrin (MbCD, 2 mM, 1 hour). Values are means 6 s.e.m. The numbers 1–8 indicate the sample groups.

temperature (Fig. 3, groups 1, 2, 7). Treatment of cells with Control cells showed 21% confined diffusion, 33% random cytoD increased diffusion rates (Fig. 3, group 3; supplementary diffusion and 46% directed motion of a6 integrins on the cell material Fig. S3), consistent with integrin uncoupling from the surface (Fig. 4A; Table 1). In MDA-MD-231 cells lacking actin cytoskeleton (Kucik et al., 1996; Yauch et al., 1997). CD151, the confined population dropped to 5% and the Diffusion rates for a6 integrin were less affected by other agents directed-motion population rose to 71% (Fig. 4B; Table 1), [phorbol 12-myristate 13-acetate (PMA), epidermal growth factor indicating that CD151 enhances confined diffusion while (EGF), methyl-b-cyclodextrin (MbCD)]. Diffusion of a6 integrin diminishing DMO. Upon siRNA knockdown (targeting a in CD151-knockdown cells was either slightly lower (groups 3, different CD151 RNA sequence), confined diffusion of a6 6), not different (group 4), or elevated (group 5), compared with integrin again decreased (from 38 to 9%), whereas random (from controls. Mean Dm values from knockdown cells (Fig. 3, first 22 to 36%) and directed (from 40 to 55%) motion increased white bar, groups 1–7) were ,20% lower than in control cells (Fig. 5A; Table 2). CD151 did not affect av integrin diffusion in (first black bar, groups 1–7). Similarly, the mean DM in MDA-MB-231 cells. Both with and without CD151 ablation, av knockdown cells (second white bar, groups 1–7) was only diffusion was mostly confined (84–88%), with a small random ,7% lower than in controls (second black bar, groups 1–7). fraction (12–16%) and no directed motion (supplementary Hence, CD151 is not a major regulator of the a6 integrin material Fig. S5A,B). CD151 ablation also did not change av diffusion rate. diffusion coefficients (Dm and DM; supplementary material Fig. S5C–F).

Journal of Cell Science CD151 affects diffusion mode The effect of the cytoskeleton on diffusion mode CD151-knockdown effects were much more pronounced when the Directed motion of a6 integrin in the cell membrane implies a mode of integrin diffusion was analyzed. SPT analysis discerned role for the cytoskeleton in regulating integrin diffusion. three distinct modes of a6 integrin diffusion (representative tracks Disruption of the actin cytoskeleton organization by shown in supplementary material Fig. S4). Correspondingly, we cytochalasin D (cytoD) removed almost completely the define three populations (I, II, III) with mean a-values of 0.4–0.6, directed-motion population (leaving 1–2%), while increasing 0.9–1.1 and 1.3–1.5. Population I trajectories represent weakly confined (50–55%) and random (43–49%) populations confined diffusion; population II includes random (Brownian) (Fig. 4C,D; Table 1). Hence, directed motion may involve a6 diffusion trajectories; and population III contains trajectories that integrin binding to the cytoskeleton and translocation along actin show diffusion combined with directional (assisted) motion. fibers. Removal of the cytoskeletal protein talin promoted DMO Fractional populations from one experiment (Table 1) were and diminished confined diffusion, thus mimicking removal of statistically identical to mean fractional populations from three CD151 (Fig. 5A,C; Table 2). However, cells lacking CD151 experiments (supplementary material Table S3), indicating were altered such that talin knockdown showed no additional reproducibility of results. DMO-inducing effects (Table 2; compare Fig. 5B and 5D). Thus,

Table 1. Mean values of a and fractional populations for confined-diffusion (aI), random-diffusion (aII), and directed-motion (aIII) trajectories Control CD151 knockdown Mean a (percentage) Mean a (percentage)

aI aII aIII n aI aII aIII n No treatment 0.55 (21) 1.04 (33) 1.48 (46) 181 0.60 (5) 0.92 (24) 1.43 (71) 149 cytoD 0.56 (50) 1.03 (49) 1.40 (1) 100 0.60 (55) 1.04 (43) 1.40 (2) 113 MbCD 0.56 (45) 1.10 (23) 1.47 (32) 53 0.63 (36) 1.10 (16) 1.51 (48) 53 PMA 0.56 (54) 0.96 (25) 1.40 (21) 50 0.40 (3) 1.03 (19) 1.31 (78) 50 EGF 0.59 (32) 0.99 (21) 1.45 (47) 59 0.50 (45) 0.93 (25) 1.54 (30) 43 1482 Journal of Cell Science 125 (6)

Fig. 4. Effects of stable CD151 knockdown on a6 integrin diffusion mode. Distribution of the diffusion-mode- parameter a in control MDA-MB-231 cells (left panels) and in CD151-knockdown cells (right panels) that were untreated (A,B) or treated with cytoD (C,D), PMA (E,F), EGF (G,H) or MbCD (I,J). The thick line in each panel represents the envelope (smoothing) curve of each distribution, and was calculated using all measured a-values as described in the text. The thick curve was fit with a sum of three Gaussian curves (thin lines), which produced the dotted line. Visibility of dotted lines is partially limited because of the excellent

Journal of Cell Science agreement between experimental data and curve-fit data. For comparison, a histogram of the experimental data is plotted in the background. The numbers of a values included in each analysis were: A, n5181; B, n5149; C, n5100; D, n5113; E, n550; F, n550; G, n559; H, n543; I, n553; J, n553.

DMO in CD151-ablated cells has the unusual property of being Integrin engagement with extracellular matrix ligands leads to actin-dependent but having no apparent contribution from talin. PKC-dependent cell spreading, which exerts major control over cell proliferation and signaling (Assoian and Klein, 2008; Loss of response to phorbol ester stimulation Defilippi et al., 1999). Integrin PKC-dependent signaling can Although CD151 knockdown and control cells stimulated with also overlap with EGF–EGFR signaling events to affect cell PMA did not differ much in the rate of integrin diffusion (Fig. 3), morphology (Cabodi et al., 2004; Rabinovitz et al., 2004; they differed markedly in diffusion mode. In control MDA-MB- Wilhelmsen et al., 2007). We found that differential CD151 231 cells, PMA decreased a6 integrin DMO (from 46 to 21%) and effects on PMA- and EGF-induced DMO translate to differential increased confined diffusion (from 21 to 54%; Fig. 4A,E; Table 1). effects on cell spreading. Ablation of CD151 caused a However, CD151-knockdown cells became unresponsive to pronounced decrease in PMA-induced [i.e. cAMP-dependent PMA. As indicated, DMO was essentially unchanged (from 71 protein kinase (PKC)-dependent] MDA-MB-231 cell spreading to 78%) and confined diffusion did not increase (from 5 to 3%; on laminin-1, but minimally affected EGF-induced cell spreading Fig. 4B,F; Table 1). In sharp contrast to PMA, other agents (i.e. (Fig. 6A,B). Thus, the inability of PMA (but not EGF) to reverse EGF, MbCD) reversed DMO in CD151-knockdown cells. EGF a6 integrin DMO may provide a mechanism for the loss of cell decreased DMO (from 71 to 30%), with a corresponding major spreading function in PMA-treated (but not EGF-treated) CD151- increase in confined diffusion (from 5 to 45%; Fig. 4B,H; knockdown cells. Table 1). Treatment with MbCD (a cholesterol-depleting agent) decreased DMO for both control cells (from 46 to 32%) and Discussion CD151-ablated cells (from 71 to 48%), decreased random It is well established that CD151 associates closely with laminin- diffusion, and caused corresponding increases in confined binding integrins to fundamentally affect their functions (see populations (Fig. 4I,J; Table 1). Introduction). However, at a basic molecular level it has been CD151 influence on a6 integrins 1483

Fig. 5. Effects of CD151 and talin siRNAs on a6 integrin diffusion mode. (A–D) Distribution of the diffusion-mode-parameter a in control-siRNA-treated MDA-MB-231 cells (left panels) and in CD151-siRNA-treated cells (right panels), which were further untreated (A,B) or treated with talin knockdown siRNA (C,D). The curve designation is as in Fig. 4. The numbers of a-values included in each analysis were: A, n587; B, n557; C, n5148; D, n559. (E) Cells were treated with siRNAs to knockdown talin 1, CD151, talin 2 and talin 1+CD151. Talin 1, CD151 and actin were then western blotted from whole cell lysates.

unclear how this occurs. Because CD151 does not affect integrin Yang et al., 2008). Five observations led to the investigation of expression or activation, we considered that it might affect CD151 effects on integrin diffusion. First, we observed decreased integrin diffusion, which is known to affect integrin functions cable formation by MDA-MB-231 cells, which is caused by (Kucik et al., 1996; Yauch et al., 1997). Unexpectedly, CD151 decreased adhesion and/or adhesion strengthening on the had minimal effect on a6 integrin diffusion magnitude, but laminin-1 Matrigel matrix. Consistent with this, CD151 instead markedly affected diffusion mode. Our results suggest knockdown diminished MDA-MB-231 cell adhesion to that CD151 restricts a6 integrin to random and confined (RCD) laminin-1. modes of diffusion, thereby making the integrin more available to Second, the contribution of CD151 to cell adhesion was more participate in adhesion, spreading and other activities. That is, evident upon adhesion to lower doses of laminin-1 and laminin-5, Journal of Cell Science RCD confers on a6 integrin more freedom to reach newly formed in agreement with laminin adhesion results seen elsewhere adhesion sites and enforce and/or enlarge them, as it is released (Winterwood et al., 2006). Under suboptimal integrin and/or from other sites of disrupted adhesion; in contrast, DMO ligand density conditions, integrin diffusion would become more indicates inability of a6 integrin to be ‘recycled’ through crucial. Conversely, CD151 knockdown did not affect MCF-10A diffusion in the membrane because it can serve in only a single cell adhesion to laminin-5, probably because of the abundance of or limited number of binding sites. In CD151-knockdown cells, a6 integrins on those cells. This would elevate adhesion to a level a6 integrin showed dysregulated actin-dependent ‘directed’ above the threshold at which CD151 effects on diffusion may be motion, which could not be reversed by phorbol ester detected in this type of assay. treatment. Hence, the absence of CD151 not only causes Third, CD151 removal did not affect a6 integrin expression or dysregulated a6-directed motion, but also appears to activation. As seen here and elsewhere (Baleato et al., 2008; dysregulate conventional PKC isoforms proximal to a6 integrins. Sachs et al., 2006; Takeda et al., 2007; Wright et al., 2004), a6 expression remained unchanged. Furthermore, in contrast to Initial clues pointing to altered diffusion another report (Iwase et al., 2003), the presence of CD151 did not We focused on MDA-MB-231 and MCF-10A basal-type promote the appearance of integrin ‘activation’ neoepitopes, mammary epithelial cells because CD151 markedly affects suggesting little effect on the conformation and activation several a6 integrin functions in those cells (Sadej et al., 2009; of laminin-binding integrins. In this regard, laminin-binding

Table 2. Mean values of a and fractional populations in siRNA-treated cells Mean a (percentage)

aI aII aIII n Control siRNA 0.64 (38) 1.04 (22) 1.47 (40) 87 CD151 siRNA 0.60 (9) 0.90 (36) 1.41 (55) 57 Control siRNA 0.60 (44) 1.01 (18) 1.43 (38) 119 Talin siRNA 0.54 (16) 0.96 (28) 1.41 (56) 148 Talin+CD151 siRNA 0.60 (11) 0.86 (34) 1.40 (55) 59 1484 Journal of Cell Science 125 (6)

Diffusion measured for a6 integrin in MDA-MB-231 cells (2–10610–12 cm2/second) is relatively slow compared with diffusion rates (50–1000610–12 cm2/second) for other integrins in various cells (Barreiro et al., 2008; Grabham et al., 2000; Kucik et al., 1996; Yauch et al., 1997). However, in at least one other situation (Chen et al., 2007), rates of integrin diffusion approach the low levels seen here. Similar diffusion coefficients were measured at room temperature and 37˚C, indicating that protein–lipid interactions are unlikely to be responsible for the low magnitude of a6 diffusion. More likely, a6 diffusion is constrained by the actin-based cytoskeleton (as mentioned above) and/or by b4 interactions with the intermediate filament cytoskeleton (Rabinovitz et al., 2004; Wilhelmsen et al., 2007).

CD151 ablation results in dysregulated DMO Removal of CD151, using both shRNA and siRNA, consistently shifted a6 integrin from RCD to DMO. Conversely, disruption of the cytoskeleton with cytoD almost completely removed moderate DMO (from control cells) and excess DMO (from Fig. 6. CD151-knockdown cells show an aberrant spreading response to CD151-ablated cells), while enhancing RCD in both cases. PMA. (A) MDA-MB-231 cells, stably expressing control vector or CD151 Hence, a6 integrin DMO occurs along actin fibers, as typically shRNA, were allowed to spread on plastic surfaces coated with laminin-1, for seen for DMO of other integrins (Grabham et al., 2000; Bauer 1 hour, and then for 1 additional hour in the presence of 20 ng/ml PMA or et al., 1993). 10 ng/ml EGF. Cells were then photographed. Results from two independent However, control and CD151-ablated cells showed notable experiments are shown for the PMA treatment. Scale bar: 20 mm. (B) The differences in the regulation of a6 integrin DMO. Talin appears percentage of cell spreading for each condition was determined. Values are to play an active role in maintaining a6 in RCD mode (when means 6 s.d. n53; *P,0.002, t-test. The difference between the EGF-treated CD151 is present) because talin ablation caused a6 integrin to samples is not significant. shift away from RCD mode. Because talin links integrins with the actin cytoskeleton (Critchley, 2009), we expected that talin integrins are more stable than other integrins, and therefore less ablation would especially affect a6 integrin DMO in both control likely to undergo conformational changes during activation and CD151-ablated cells, because DMO is so dependent on the (Bazzoni et al., 1998). It was also previously noted that CD151 actin cytoskeleton. However, only control cells were sensitive to can affect a6 integrin adhesion strengthening without affecting talin ablation, as seen by an increase in DMO trajectories. When ligand binding (Lammerding et al., 2003), again pointing to a CD151 is absent, talin no longer appears to play a role, because mode of action independent of the effects on integrin ligand Journal of Cell Science knockdown of talin had no effect on the percentage of trajectories binding affinity and/or activation. in DMO mode. The insensitivity of DMO trajectories in CD151- Fourth, ablation of CD151 caused a reduction in antibody- ablated cells to talin ablation identifies an atypical case of an induced a6b4 clustering in MCF-10A cells. As shown previously, integrin–actin connection independent of talin, and supports the diminished clustering may arise as a result of altered integrin idea that a6 is undergoing dysregulated DMO in CD151-ablated lateral diffusion (Yauch et al., 1997). cells. Loss of talin sensitivity and dysregulated DMO are both Fifth, removal of CD151 altered the spectrum of cell-surface consistent with a6 being functionally less available to contribute partner proteins for a6 integrins, indicative of disconnection from to cell adhesion and spreading (see next section). tetraspanin-enriched microdomains (TEMs). As seen elsewhere, CD151-knockdown cells were also impervious to PMA, residence within TEMs can affect cell surface diffusion (Espenel showing essentially no shift of DMO towards RCD. By et al., 2008). contrast, a6 integrin DMO in control cells was shifted mostly to RCD upon PMA treatment. PMA activates and translocates Diffusion rate is minimally affected novel and conventional PKC isozymes (e.g. PKCa and b). Most integrin diffusion studies have focused on factors affecting CD151 can recruit conventional PKCs to proximity with a6 diffusion magnitude (i.e. diffusion coefficients). However, integrins (Zhang et al., 2001). Consistent with this, CD151 knockdown of CD151 only marginally affected a6integrin removal decreases the association of PKCa with a6 integrins diffusion coefficients (Dm and DM), as seen from results compiled (Q. Li, X. H. Yang, F. Xu, C. Sharma, H.-X. Wang, K. Knoblich, using seven different experimental conditions. Stimulation with I. Rabinovitz, S. R. Granter and M. E. Hemler, unpublished). This cytoD and PMA increased diffusion rates for both control and helps to explain why a6 integrin DMO becomes insensitive to CD151-knockdown cells. Stimulation of a6 integrin diffusion rate PMA in CD151-ablated cells. By contrast, control cell CD151 by cytoD and PMA is consistent with results seen for other integrins can recruit PKC, which may lead to localized disruption of the (Kucik et al., 1996; Yauch et al., 1997; Zhou and Li, 2000). EGF actin cytoskeleton (Cuchillo-Ibanez et al., 2004), thus resulting in stimulation differentially affected diffusion rates for control and diminished a6 integrin DMO. CD151-knockdown cells, which may help to explain altered Integrin a6 DMO in CD151-knockdown cells remained responses to EGF by CD151-knockdown cells (Yang et al., somewhat sensitive to cholesterol depletion, perhaps because 2008). Nonetheless, despite a few small differences, the overall cholesterol depletion can disrupt cellular actin (Kwik et al., 2003) effect of CD151 knockdown on diffusion rates was minimal. in a PKC-independent manner. Control cells, with constitutively CD151 influence on a6 integrins 1485

more RCD and less DMO, were less affected by cholesterol association with the actin cytoskeleton, as evidenced by loss of depletion. In this regard, RCD of tetraspanin CD9 was also sensitivity to both talin ablation and PMA stimulation. Normal mostly unaffected by cholesterol depletion (Espenel et al., 2008). integrin adhesion and spreading functions are typically dependent CD151-knockdown cells also retained sensitivity to EGF, with on an integrin–talin–actin connection, and stimulated by PMA. In respect to shifting DMO towards RCD. This makes sense because the absence of CD151, a6 integrin appears to have lost both talin EGF stimulates cell migration partly by signaling the transient and PMA sensitivity, as well as the ability to ‘explore’ the cell disassembly of the actin cytoskeleton (Chang et al., 1995), in a membrane, thus explaining diminished adhesion and spreading PKC-independent manner. functions. We predict that results seen here with MDA-MD-231 cells will extrapolate to most, if not all, of the many other cell Functional consequences of dysregulated a6 DMO types where a6 integrin is associated with CD151. Control of a6 Three sets of results support the functional relevance of integrin diffusion mode is probably also a principal factor in the dysregulated a6 integrin DMO. First, the increased appearance subsequent effects of CD151 in kidney and skin development, of a6 integrin DMO in CD151-ablated MDA-MB-231 cells skin wound healing, tumor growth, metastasis, angiogenesis and correlates with diminished adhesion to laminin-1 and diminished other functions. Our new mechanistic understanding also should cable formation. Second, PMA stimulated CD151-knockdown help to explain why cells and mice lacking CD151 show cells failed to shift a6 away from DMO and failed to spread on substantial alterations in phorbol-ester-stimulated cell signaling laminin-1. By contrast, EGF stimulation of CD151-ablated cells and oncogenesis (Q. Li, X. H. Yang, F. Xu, C. Sharma, H.-X. triggered a substantial DMO to RCD shift, in parallel with Wang, K. Knoblich, I. Rabinovitz, S. R. Granter and M. E. abundant spreading on laminin-1. Third, ablation of CD151, Hemler, unpublished). which is not a partner for integrin av and does not affect av function (Winterwood et al., 2006), did not change either the Materials and Methods diffusion mode or diffusion magnitude of av integrin. These Cells and reagents Immortalized MCF-10A and malignant MDA-MB-231 cells were cultured in results help to rule out non-specific membrane perturbation Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Park Memorial effects, and reinforce the functional connection between CD151 Institute (RPMI) 1640 with 10% FCS (Life Sciences Technologies, Inc.), and a6 integrin diffusion mode. 10 mmol/l HEPES and antibiotics (penicillin and streptomycin). Anti-CD151 antibodies included 5C11 (Yauch et al., 1998), 1A5 (Testa et al., 1999) and FITC- For technical reasons, a6 integrin diffusion was measured on conjugated 11G5A (GeneTex, Inc.). Other tetraspanin antibodies recognized CD9 the cell apical surface. Results obtained are indicative of global (MM2/57 unconjugated and FITC-conjugated; from Biosource) and CD82 (M104, cellular responses to ‘outside-in’ signaling (e.g. as a result of from Osamu Yoshi, Shionogi Institute, Osaka, Japan). Anti-integrin antibodies treatment with EGF or MCD) or ‘inside-out’ signaling (e.g. as a (Bazzoni et al., 1995; Bergelson et al., 1994; Lee et al., 1995; Weitzman et al., 1993) recognized a3 (A3-X8), a6 (GoH3, A6-ELE), a2 (IIE10), b1 (TS2/16), result of perturbation by cytoD or talin siRNA). Furthermore, in activated b1 (9EG7), and b4 (ASC-8) subunits. The anti-av antibody was RMV-7 dynamic situations, such as cell spreading and migration, the (eBiosciences). Targeting of CD151 (using siRNA#4 and siRNA#2) and CD82 mode of apical a6 integrin diffusion should markedly affect (Yang et al., 2008) was achieved using oligonucleotides purchased from Dharmacon, and shRNA knockdown of CD151 was described previously (Yang function. In particular, RCD should confer more freedom for a6 et al., 2008). integrin to reach newly formed adhesion sites and reinforce and/

Journal of Cell Science or enlarge them; by contrast, DMO could indicate the inability of Cell cable formation and adhesion assays a6 integrin to diffuse to new adhesion sites because the For cell cable formation, cells were plated on the surface of a layer of 3D Matrigel as described previously (Zhang et al., 2002). Cell images were acquired with a directionality of the diffusion could indicate strong integrin monochrome CCD camera (RT SPOT, Diagnostic Instruments, Sterling Heights, attachment to cytoskeletal binding sites. Thus, both RCD and MI) on an Axiovert 135 inverted microscope (Zeiss). To measure static cell DMO are likely to be necessary for normal cell function, and adhesion, 96-well plates were coated (at 5–20 mg/ml, for 12 hours) with laminin-1, CD151 appears to have a crucial role in this regulation. laminin-5, BSA or fibronectin; and then cells were added, and attached cells were quantified using a Cytofluor 2300 measurement system (Millipore) as described previously (Bazzoni et al., 1995). Summary and further functional implications CD151 promotion of a6 integrin diffusion in RCD modes may be Immunoprecipitation and [3H]palmitate labeling at least partly due to recruitment of a6 integrins into tetraspanin- Because tetraspanins (e.g. CD151) and many of their partner proteins (e.g. a3, a6 and b4 integrin subunits) undergo post-translational palmitoylation, labeling with enriched microdomains (TEMs) (Hemler, 2005; Nydegger et al., [3H]palmitate has been useful for evaluation of tetraspanin complexes (Takeda 2006; Yanez-Mo et al., 2009). Within TEMs, CD151 can et al., 2007; Yang et al., 2004). Cells (siRNA-treated, 80–90% confluent) were associate with many other tetraspanins, including CD9 and washed in PBS, serum starved for 3–4 hours, pulsed for 1–2 hours in medium 3 CD82, which themselves display RCD, but not DMO (Danglot containing 0.2–0.3 mCi/ml [ H]palmitate plus 5% dialyzed fetal bovine serum, and then lysed in 1% Brij-96 for 5 hours at 4˚C. Immunoprecipitation and protein et al., 2010; Espenel et al., 2008). Localization of CD9 within detection were then carried out as described previously (Takeda et al., 2007; Yang TEMs correlates with confined diffusion (Espenel et al., 2008), et al., 2002; Yang et al., 2004). whereas tetraspanin CD82 appears to help activated EGFR switch from confined to random diffusion (Danglot et al., 2010). These Single particle tracking (SPT) Beads (1 mm carboxylated polystyrene microspheres) were coated with anti-a6 results reinforce the idea that TEMs may promote RCD while antibody (A6-ELE) as described previously (Karnchanaphanurach et al., 2009). preventing DMO. Briefly, beads in MES buffer, pH 6.1, were sonicated for 5 minutes and the Molecules with type I (confined) and II (random) trajectories carboxyl groups were sensitized by incubation in the presence of 100 mM N- hydroxysuccinimide and 0.4% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide should be capable of ‘exploring’ larger areas in the cell hydrochloride for 20 minutes at room temperature. After washing in MES, a membrane. Hence, by supporting RCD, CD151 may make a6 mixture of anti-a6 antibody (A6-ELE) and isotype control IgG was incubated with integrins more available to participate in cell adhesion, spreading the beads under constant shaking for 2 hours at room temperature. We used the and other functions. By contrast, a6 integrin that is diverted to minimum amount of specific antibody required to achieve selective binding of beads to cells, mixed with a sufficient amount of isotype IgG to ensure complete type III (directed) trajectories, because of CD151 ablation, is in coverage of the bead surface. The conjugation reaction was terminated by adding a dysregulated state. This state is characterized by aberrant ethanolamine to 100 mM final concentration for 15 minutes, followed by addition 1486 Journal of Cell Science 125 (6)

of BSA to 1% for 15 minutes. The beads were washed twice in PBS with 0.1% Baleato, R. M., Guthrie, P. L., Gubler, M. C., Ashman, L. K. and Roselli, S. (2008). BSA and resuspended at 0.05% (m/v). Then, the beads were incubated for 1 hour Deletion of CD151 results in a strain-dependent glomerular disease due to severe with MDA-MB-231 cells, yielding 15–20% of cells with one or two attached alterations of the glomerular basement membrane. Am. J. Pathol. 173, 927-937. beads. Under identical conditions, control beads coated with isotype IgG bound to Barreiro, O., Zamai, M., Yanez-Mo, M., Tejera, E., Lopez-Romero, P., Monk, P. N., less than 2% of cells. In some experiments, cells were also treated with Gratton, E., Caiolfa, V. R. and Sanchez-Madrid, F. (2008). Endothelial adhesion cytochalasin D (cytoD; 10 mM, 1 hour), methyl-b-cyclodextrin (MbCD; 2 mM, receptors are recruited to adherent leukocytes by inclusion in preformed tetraspanin 1 hour), phorbol 12-myristate 13-acetate (PMA; 0.2 mg/ml, 30 minutes) or nanoplatforms. J. Cell Biol. 183, 527-542. epidermal growth factor (EGF; 0.02 mg/ml, 1 hour) prior to adding the beads. Bauer, J. S., Varner, J., Schreiner, C., Kornberg, L., Nicholas, R. and Juliano, R. L. (1993). Functional role of the cytoplasmic domain of the integrin a5 subunit. J. Cell For single particle tracking (Cairo et al., 2006), video recordings at 30 frames/ Biol. 122, 209-221. , second were obtained for 70 seconds, and 30 beads were tracked in each Bazzoni, G., Shih, D.-T., Buck, C. A. and Hemler, M. E. (1995). MAb 9EG7 defines a experiment. Video data were processed with MetaMorph (Molecular Devices) and novel b1 integrin epitope induced by soluble ligand and manganese, but inhibited by converted to trajectories, then analyzed with mean square displacement (MSD) calcium. J. Biol. Chem. 270, 25570-25577. analysis implemented in a custom MATLAB (MathWorks) program. The Bazzoni, G., Ma, L., Blue, M.-L. and Hemler, M. E. (1998). Divalent cations and microdiffusion coefficient (Dm) was calculated by performing a linear fit to ligands induce conformational changes that are highly divergent among b1 integrins. MSD54Dmt, using the first four increments of the MSD versus time interval curve. J. Biol. Chem. 273, 6670-6678. The macrodiffusion coefficient (DM) was calculated by fitting the initial third of Belkin, A. M. and Stepp, M. A. (2000). Integrins as receptors for laminins. Microsc. a the MSD versus time interval curve to the equation MSD54DMt . The parameter a Res. Tech. 51, 280-301. classifies the mode of diffusion (Mirchev and Golan, 2001). Diffusion trajectories Berditchevski, F. (2001). Complexes of tetraspanins with integrins: more than meets the were grouped based on population analysis as described previously (Cairo et al., eye. J. Cell Sci. 114, 4143-4151. 2006). Briefly, a kernel-smoothing probability density calculation was used to Berditchevski, F., Gilbert, E., Griffiths, M. R., Fitter, S., Ashman, L. and Jenner, smooth the normalized distribution of a-values for each experimental condition. S. J. (2001). Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin This envelope (smoothing) curve was then fitted to the sum of three Gaussian interactions by mutagenesis. J. Biol. Chem. 276, 41165-41174. distributions, which represented three populations of diffusion trajectories. For all Berditchevski, F., Odintsova, E., Sawada, S. and Gilbert, E. (2002). Expression of the of the a-value distributions obtained in control cells under the various palmitoylation-deficient CD151 weakens the association of alpha 3beta 1 integrin experimental conditions, the 3-Gaussian fit gave a better fit than a 2-Gaussian with the tetraspanin-enriched microdomains and affects integrin-dependent signalling. fit, as determined by applying the F-statistic at 95% to test the significance of the J. Biol. Chem. 277, 36991-37000. goodness of fit. For consistency, we applied a 3-Gaussian fit to all experimental Bergelson, J. M., St. John, N. F., Kawaguchi, S., Pasqualini, R., Berdichevsky, F., Hemler, M. E. and Finberg, R. W. (1994). The I domain is essential for echovirus 1 conditions (Tables 1,2; Figs 4,5; supplementary material Tables S1–S3, Figs. interaction with VLA-2. Cell Adhes. Commun. 2, 455-464. S3,S5). The three Gaussians had intersection points in the ranges of 0.7–0.9 Cabodi, S., Moro, L., Bergatto, E., Boeri, E. E., Di Stefano, P., Turco, E., Tarone, G. (leftmost and middle Gaussian curves) and 1.1–1.2 (middle and rightmost and Defilippi, P. (2004). Integrin regulation of epidermal growth factor (EGF) Gaussian curves), giving experimental thresholds to classify trajectories based on receptor and of EGF-dependent responses. Biochem. Soc. Trans. 32, 438-442. their a-values. Thus, a,0.8 (leftmost Gaussian curve) represented confined or Cairo, C. W., Mirchev, R. and Golan, D. E. (2006). Cytoskeletal regulation couples corralled motion, 0.8,a,1.2 (middle Gaussian curve) was consistent with LFA-1 conformational changes to receptor lateral mobility and clustering. Immunity Brownian diffusion, and a.1.2 (rightmost Gaussian curve) represented directed 25, 297-308. 2 diffusion. Two normal distributions were considered statistically different if: [(s1 Chang, J. H., Gill, S., Settleman, J. and Parsons, S. J. (1995). c-Src regulates the 2 2 2 2 + s2 )/(s1 )(s2 )](m1–m2) .8, where m and s denote the mean and standard simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP deviation of each distribution (Johnson et al., 2004). The fractional percentage of following epidermal growth factor stimulation. J. Cell Biol. 130, 355-368. each population was calculated from the normalized weighting factor by which Chen, H., Titushkin, I., Stroscio, M. and Cho, M. (2007). Altered membrane dynamics each Gaussian is multiplied in the best-fitted sum. If, instead of using empirically of quantum dot-conjugated integrins during osteogenic differentiation of human bone determined thresholds derived directly from experimental data, we used fixed marrow derived progenitor cells. Biophys. J. 92, 1399-1408. thresholds (a,0.8; 0.8,a,1.2; a.1.2) throughout, the fractional percentages Critchley, D. R. (2009). Biochemical and structural properties of the integrin-associated assigned to each population (confined, Brownian, directed; Table 1, Table 2; cytoskeletal protein talin. Annu. Rev. Biophys. 38, 235-254. supplementary material Fig. S5) would change only slightly (typically 3–4%), and Cuchillo-Ibanez, I., Lejen, T., Albillos, A., Rose, S. D., Olivares, R., Villarroya, M., none of the conclusions of the study would be affected. Garcia, A. G. and Trifaro, J. M. (2004). Mitochondrial calcium sequestration and protein kinase C cooperate in the regulation of cortical F-actin disassembly and Journal of Cell Science secretion in bovine chromaffin cells. J. Physiol. 560, 63-76. Cell spreading Danglot, L., Chaineau, M., Dahan, M., Gendron, M. C., Boggetto, N., Perez, F. and Cell spreading was recorded using a Nikon Eclipse Ti Series inverted microscope, Galli, T. (2010). Role of TI-VAMP and CD82 in EGFR cell-surface dynamics and equipped with a humidified 37˚CCO2 chamber, automated mobile stage and signaling. J. Cell Sci. 123, 723-735. focusing system, and capability for simultaneously capturing cell movements in Defilippi, P., Olivo, C., Venturino, M., Dolce, L., Silengo, L. and Tarone, G. (1999). real-time in 24 bright fields (in a 24-well plate) under a 206 objective. Cells, in Actin cytoskeleton organization in response to integrin-mediated adhesion. Microsc. the presence of 0.1% fetal bovine serum, were plated for 1 hour on laminin-1- Res. Tech. 47, 67-78. coated surfaces, in the presence of either 10 ng/ml EGF or 20 ng/ml PMA, as Delwel, G. O., de Melker, A. A., Hogervorst, F., Jaspars, L. H., Fles, D. L., indicated. Kuikman, I., Lindblom, A., Paulsson, M., Timpl, R. and Sonnenberg, A. (1994). Distinct and overlapping ligand specificities of the alpha 3A beta 1 and alpha 6A beta 1 integrins: recognition of laminin isoforms. Mol. Biol. Cell 5, 203-215. Funding Espenel, C., Margeat, E., Dosset, P., Arduise, C., Le Grimellec, C., Royer, C. A., This work was supported by National Institutes of Health [grant Boucheix, C., Rubinstein, E. and Milhiet, P. E. (2008). Single-molecule analysis of numbers CA42368 to M.E.H. and HL32854 to D.E.G.]; and by a CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web. J. Cell Biol. 182, 765-776. S.G. Komen Career Catalyst Award [grant number KG081481 to Gaborski, T. R., Clark, A., Jr, Waugh, R. E. and McGrath, J. L. (2008). Membrane X.H.Y.] and a DOD Concept Award [grant number W81XWH-07-1- mobility of beta2 integrins and rolling associated adhesion molecules in resting 0568 to X.H.Y.]. Deposited in PMC for release after 12 months. neutrophils. Biophys. J. 95, 4934-4947. Grabham, P. W., Foley, M., Umeojiako, A. and Goldberg, D. J. (2000). Nerve growth factor stimulates coupling of beta1 integrin to distinct transport mechanisms in the Supplementary material available online at filopodia of growth cones. J. Cell Sci. 113, 3003-3012. http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.093963/-/DC1 Haeuw, J. F., Goetsch, L., Bailly, C. and Corvaia, N. (2011). Tetraspanin CD151 as a target for antibody-based cancer immunotherapy. Biochem. Soc. Trans. 39, 553-558. Hemler, M. E. (2005). Tetraspanin functions and associated microdomains. Nat. Rev. References Mol. Cell Biol. 6, 801-811. Ang, J., Lijovic, M., Ashman, L. K., Kan, K. and Frauman, A. G. (2004). CD151 Hirata, H., Ohki, K. and Miyata, H. (2005). Mobility of integrin alpha5beta1 measured protein expression predicts the clinical outcome of low-grade primary prostate cancer on the isolated ventral membranes of human skin fibroblasts. Biochim. Biophys. Acta better than histologic grading: a new prognostic indicator? Cancer Epidemiol. 1723, 100-105. 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