CD151 Restricts the A6 Integrin Diffusion Mode

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 ß 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

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