|CD81 regulates cell migration through its association with Rac GTPase.|
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|PMID: 23264468 Owner: NLM Status: Publisher|
|CD81 is a member of the tetraspanin family that has been described to have a key role in cell migration of tumor and immune cells. To unravel the mechanisms of CD81-regulated cell migration, we performed proteomic analyses that revealed an interaction of the tetraspanin C-terminal domain with the small GTPase Rac. Direct interaction was confirmed biochemically. Moreover, microscopy cross-correlation analysis demonstrated the in situ integration of both molecules into the same molecular complex. Pull-down experiments revealed that CD81-Rac interaction was direct and independent of Rac activation status. Knockdown of CD81 resulted in enhanced protrusion rate, altered focal adhesion formation and decreased cell migration, correlating with increased active Rac. Re-expression of wild type CD81, but not its truncated form lacking the C-terminal cytoplasmic domain, rescued these effects. The phenotype of CD81 knockdown cells was mimicked by treatment with a soluble peptide with the C-terminal sequence of the tetraspanin. Our data show that the interaction of Rac with the C-terminal cytoplasmic domain of CD81 is a novel regulatory mechanism of the GTPase activity turnover. Furthermore, they provide a novel mechanism for tetraspanin-dependent regulation of cell motility and open new avenues for tetraspanin-targeted reagents by the use of cell permeable peptides.|
|Emilio Tejera; Vera Rocha-Perugini; Soraya López-Martín; Daniel Pérez-Hernández; Alexia I Bachir; Alan Rick Horwitz; Jesús Vázquez; Francisco Sánchez-Madrid; María Yáñez-Mo|
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|Type: JOURNAL ARTICLE Date: 2012-12-21|
|Title: Molecular biology of the cell Volume: - ISSN: 1939-4586 ISO Abbreviation: Mol. Biol. Cell Publication Date: 2012 Dec|
|Created Date: 2012-12-24 Completed Date: - Revised Date: -|
Medline Journal Info:
|Nlm Unique ID: 9201390 Medline TA: Mol Biol Cell Country: -|
|Languages: ENG Pagination: - Citation Subset: -|
|†Unidad de Investigación, Hospital Santa Cristina, Instituto de Investigación Sanitaria La Princesa, Madrid, Spain £Servicio de Inmunología, Hospital de la Princesa, Instituto de Investigación Sanitaria La Princesa, Madrid, Spain $Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain. *Cardiovascular Proteomics Laboratory, Centro de Biología Molecular "Severo Ochoa"-Consejo Superior de Investigaciones Científicas, 28049 Cantoblanco, Madrid, Spain. #Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22908.|
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Journal ID (nlm-ta): Mol Biol Cell
Journal ID (iso-abbrev): Mol. Biol. Cell
Journal ID (hwp): molbiolcell
Journal ID (pmc): mbc
Journal ID (publisher-id): Mol. Bio. Cell
Publisher: The American Society for Cell Biology
© 2013 Tejera et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Received Day: 04 Month: 9 Year: 2012
Revision Received Day: 16 Month: 11 Year: 2012
Accepted Day: 05 Month: 12 Year: 2012
Print publication date: Day: 01 Month: 2 Year: 2013
Volume: 24 Issue: 3
First Page: 261 Last Page: 273
PubMed Id: 23264468
Publisher Id: E12-09-0642
|CD81 regulates cell migration through its association with Rac GTPase|
|Alexia I. Bachird|
|Alan Rick Horwitzd|
Role: Monitoring Editor
aUnidad de Investigación, Hospital Santa Cristina, Instituto de Investigación Sanitaria La Princesa, 2006 Madrid, Spain
eServicio de Inmunología, Hospital de la Princesa, Instituto de Investigación Sanitaria La Princesa, 2006 Madrid, Spain
bDepartamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, 28029 Madrid, Spain
cCardiovascular Proteomics Laboratory, Centro de Biología Molecular “Severo Ochoa”–Consejo Superior de Investigaciones Científicas, 28049 Cantoblanco, Madrid, Spain
dDepartment of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22908
|Johns Hopkins University
|Correspondence: 1Address correspondence to: María Yáñez-Mó (firstname.lastname@example.org).
Tetraspanins are involved in adhesion and migration processes, such as leukocyte extravasation and cancer invasion (Yáñez-Mó et al., 2001a, 2001b, 2009; Hemler, 2003; Tarrant et al., 2003; Barreiro et al., 2005; Charrin et al., 2009), and are promising targets for cancer therapeutics (Sala-Valdes et al., 2012).
Multiple studies have provided evidence that tetraspanins regulate cell migration. Initial reports used antitetraspanin antibodies (Lagaudriere-Gesbert et al., 1997; Yáñez-Mó et al., 1998): anti-CD81 antibodies inhibited α6β1-induced migration (Domanico et al., 1997), and anti-CD151 and anti-CD81 antibodies reduced migration of endothelial or epithelial cells (Yáñez-Mó et al., 1998; Penas et al., 2000). More recently, anti-CD81 antibodies were found to ameliorate autoimmune encephalomyelitis by blocking monocyte transmigration (Dijkstra et al., 2008).
Genetic studies also support this role for tetraspanins. Small interfering RNA (siRNA)-mediated deletion of CD9 and CD151 enhanced primary melanocyte motility (Garcia-Lopez et al., 2005), whereas targeting of CD151 enhanced melanoma cell migration (Hong et al., 2006). CD82 overexpression suppressed the migration of oligodendrocyte precursors (Mela and Goldman, 2009). In the immune system, CD81 regulates the migration and trafficking of natural killer (Kramer et al., 2009) and dendritic (Nattermann et al., 2006) cells, while CD9/CD81 double-knockout mice show defects in macrophage cell motility (Takeda et al., 2008).
Tetraspanins are highly hydrophobic proteins that cross the cellular membrane four times, displaying N-terminal and C-terminal cytoplasmic domains. Tetraspanins structure tetraspanin-enriched microdomains (TEMs) at the plasma membrane that organize transmembrane proteins, including integrins, immunoglobulin-like adhesion molecules and metalloproteinases, and signaling effectors (Yáñez-Mó et al., 2009, 2011). Integrin insertion in TEMs enhances integrin’s avidity for ligands and regulates outside-in signaling (Berditchevski and Odintsova, 1999), including the activation of FAK, Src, p130cas and paxillin (Yamada et al., 2008), protein kinase B, endothelial nitric-oxide synthase, and the small GTPases Rac1 and Cdc42 (Takeda et al., 2007).
Emerging evidence suggests that tetraspanins also interact with different cytoplasmic molecules, facilitating the activation of signaling cascades. At least five tetraspanins—CD9, CD63, CD81, CD151, and A15/TALLA/Tspan7—associate with type II phosphatidylinositol 4-kinase (Berditchevski et al., 1997; Yauch and Hemler, 2000). Tetraspanin association to PI4K may play a role in cell migration (Mazzocca et al., 2008), bacterial infection (Tham et al., 2010), and tumor cell proliferation (Carloni et al., 2004). When activated, protein kinase C (PKC) associates with CD9, CD53, CD81, CD82, and CD151 (Zhang et al., 2001a), and PKC-mediated phosphorylation was shown to be necessary for α3-integrin−dependent signaling to FAK, p130cas, and paxillin during spreading and migration (Zhang et al., 2001b). The C-terminal cytoplasmic domain of CD63 binds to the adaptor protein syntenin-1 (Latysheva et al., 2006) and that of CD81 to 14-3-3 proteins (Clark et al., 2004). Tetraspanins connect to the actin cytoskeleton through ezrin, radixin, and moesin (ERM) proteins (Sala-Valdes et al., 2006), and tetraspanin-mediated signaling may control actin polymerization and remodeling. However, the precise mechanisms through which tetraspanins control cell migration remain largely unexplored.
In this study, we performed pull-down assays using the cytoplasmic C-terminus tail of CD81 as bait, followed by high-throughput protein identification by mass spectrometry, to identify new intracellular interacting partners of tetraspanins implicated in the regulation of actin dynamics and cell migration. We found that tetraspanin CD81 associates with the small GTPase Rac. We also demonstrate that CD81 interaction with Rac limits the GTPase activation within the plasma membrane, providing an unexpected novel regulatory mechanism of Rac activity turnover.
Searching for novel interactions of CD81 implicated in cell migration, we performed mass spectrometry using pull downs of T-lymphoblast cell lysates with biotinylated peptides corresponding to the C-terminal cytoplasmic region of CD81 as bait. The analysis identified two peptides belonging to the GTPase Rac1 and five corresponding to Rac2 (Figure 1A) in the bound fraction. The association of CD81 with Rac was also detected by pull-down assays with adherent cells (Figure 1B). Interestingly, the C-terminal cytoplasmic regions of the tetraspanins CD9, CD151, and CD82 did not associate with Rac (Figure 1B; unpublished data). Similarly, biotinylated peptides corresponding to the cytoplasmic sequence of other tetraspanin-associated membrane molecules (EWI-2, ICAM-1, VCAM-1) and other membrane receptors (CD147, CD69, CXCR4, CCR7) were unable to pull down Rac (Figure 1B; unpublished data). Direct interaction with the C-terminal sequence of CD81 was confirmed by direct binding of Rac1–glutathione S-transferase (GST) protein obtained in Escherichia coli cultures (Figure 1C). Interaction between the endogenous molecules was confirmed by coimmunoprecipitation in serum-starved, serum-induced, or epidermal growth factor (EGF)-stimulated primary human umbilical vein endothelial cells (HUVEC) or SUM159 breast carcinoma cells (Figure 1D).
CD81-Rac molecular complexes were detected in situ by total internal reflection microscopy (TIRFM)-based fluorescence image cross-correlation analysis of mCherry-CD81 and green fluorescent protein (GFP)-tagged wild-type Rac (WT-Rac1; Figure 2A). Correlation studies rely on the analysis of fluorescence intensity fluctuations from fluorescently tagged molecules in an image time series. The fluctuations, in this case, likely arise from diffusion and/or membrane binding–unbinding kinetics. The decay of the autocorrelation function for CD81 and wild type Rac (WT-Rac1) indicates that both molecules are producing fluorescence fluctuations over the timescale of the measurement (Figure 2B, top panels). This observation is typical for transmembrane receptors such as CD81, which diffuse in the cell membrane on the seconds timescale (faster than cytosolic proteins), or proteins with slow exchange with the membrane, and suggests that we are measuring a Rac population that is either diffusing in the membrane or exchanging with a membrane-bound complex (Moissoglu et al., 2006).
As we have previously demonstrated, correlation analysis can also be applied to detect molecular complexes (Choi et al., 2011). If two molecules tagged with different fluorophores (e.g., GFP and mCherry) reside within the same molecular complex, they will produce similar temporal fluorescence intensity fluctuation patterns, which will be revealed by calculating the cross-correlation function (see Materials and Methods; Wiseman et al., 2004; Brown et al., 2006; Digman et al., 2009). In the absence of a complex, the intensity fluctuations are independent, and the cross-correlation function will be featureless and indistinguishable from the noise level. The cross-correlation function of CD81 and WT-Rac-1 therefore confirms that the membrane-targeted Rac1 resides with CD81 in the same complex. CD81 also showed high cross-correlation with V12-Rac, a constitutively active mutant of Rac (Figure 2B, middle panels). Coexpression of WT-Rac with a CD81 mutant lacking the cytoplasmic region (CD81-ΔCyt) resulted in reduced Rac autocorrelation, suggesting a faster exchange of the GTPase with the membrane. Moreover, both molecules showed no detectable cross-correlation, further indicating that Rac specifically binds to the CD81 C-terminal region (Figure 2B, bottom panels).
We next studied the subcellular localization of CD81/Rac complexes in live cells. Stimulation of SUM159 cells or HUVEC with EGF induced partial colocalization of endogenous Rac and CD81 at the cell edge (Figure 3, A and B), and similar results were observed during cell spreading (unpublished data). Colocalization at the leading lamella was not observed for tetraspanin CD151, which concentrated at cell–cell contacts, or for the glycophosphatidylinositol (GPI)-linked receptor CD59 (Figure 3A). Analysis of similar samples using TIRFM, illuminating only ∼100 nm of the ventral surface of the cell, revealed the colocalization of CD81 and Rac1 at the edges of HUVEC cells (Figure 3C).
Loading of Rac with GTP is normally concomitant with its translocation to the plasma membrane. To determine whether association of CD81 with Rac depends on the activation state of the GTPase, we loaded SUM159 cell lysates with an excess of GDP or the nonhydrolyzable GTP analogue GTP-γS. CD81-biotinylated peptides pulled down similar amounts of Rac regardless of the GTP or GDP load, suggesting that CD81 association with Rac is independent of the Rac activation state (Figure 4A).
The dynamic behavior of the CD81/Rac complexes in migrating cells was analyzed with a Förster resonance energy transfer (FRET)-based Raichu-Rac plasmid that allows the dynamic visualization of Rac activity (Ouyang et al., 2008). In human microvascular endothelial cells cotransfected with DsRed-CD81 and Raichu-Rac, EGF stimulation induced a clear colocalization of Rac activity with CD81 at the leading edges of the cells (Figure 4B and Supplemental Video S1). In resting cells, FRET signal was almost undetectable. To define more precisely the location of CD81-Rac complexes at the leading lamella, we cotransfected cells with AqGFP-CD81 and orange-paxillin and monitored them using time-lapse TIRFM. CD81 concentrated at the edges of the cells (Figure 4C, arrowhead, and Video S2), ahead of the nascent adhesions marked by paxillin (Figure 4C, arrow) suggesting that CD81 may prime these foremost areas for Rac activation.
The functional significance of the CD81/Rac interaction was explored in cells with reduced endogenous CD81 expression by transfection of two different siRNA sequences: one against the coding sequence and one against the 3′ untranslated region (3′UTR; see Material and Methods). After 48 h of siRNA transfection, down-regulation was assessed by flow cytometry and Western blotting (Figure 5, A and B); experiments were performed only when down-regulation was higher than 50%. We monitored the subcellular location of GFP-coupled Rac1 during migration on fibronectin in control cells and CD81-depleted cells (Figure 5C). Rac concentrated at the leading edge in both conditions, suggesting that CD81 is not required for Rac translocation to the plasma membrane (Figure 5C and Video S3).
Interestingly, active (GTP-bound) Rac levels, as detected by pull down with GST-PAK-CRIB, were significantly higher in unstimulated CD81-silenced cells (p < 0.05 in Student’s t test). Moreover, in CD81-silenced cells, Rac-GTP levels remained largely unaffected by EGF stimulation. Indeed, Rac activity remained high and almost constant, being also significantly higher at 30 min of EGF stimulation compared with control cells (p < 0.05 in Student’s t test). In contrast, no significant differences were observed in RhoA activity (detected with GST-C21), which was only slightly reduced in CD81-silenced cells (Figure 5D).
Cell protrusion during spreading depends mainly on Rac-induced actin polymerization (Choi et al., 2008). Consistent with the higher levels of activated Rac, CD81-deficient cells displayed a faster rate of protrusion and spreading than control cells (Figure 6, A and B). Importantly, this effect was rescued using full-length CD81 but not with the C-terminus truncated form (CD81-ΔCyt) fused to GFP. Moreover, overexpression of WT-CD81 had the opposite effect, slowing cell protrusion (Figure 6A).
The effects of CD81 depletion in actin polymerization and adhesion formation were visualized by staining F-actin and the adhesion protein paxillin in control and CD81-deficient cells. To normalize cell morphology, we used micropatterned substrates (Thery et al., 2006) that permit averaging the staining of multiple cells with the same morphology, as well as analyzing the response to geometric cues (Tan et al., 2004; Figure 6C). CD81-depleted cells showed increased paxillin staining of focal adhesions (Figure 6C). Interestingly, adhesions in control cells were restricted to the rim of the lamellar edge, but formed in a broader region in CD81-silenced cells. CD81 depletion also induced a defective cell polarization in both F-actin and focal adhesions in polarizing micropatterns. CD81 silencing in primary HUVECs plated onto fibronectin also increased the size of focal adhesions (Figure 6D), an effect reverted by cotransfection with full-length CD81, but not with CD81-ΔCyt-GFP. It has been reported that protrusion rate presents a direct correlation with adhesion assembly rate (Choi et al., 2008). We analyzed adhesion assembly and disassembly rates in cells interfered for CD81, with or without reconstitution of the expression with a complete or truncated CD81 form. For adhesion assembly rate, results were consistent with protrusion rates: silencing of CD81 accelerated adhesion assembly compared with control cells, and this phenotype was rescued by complete CD81 but not by the truncated form (Figure 6E). In contrast, no statistically significant differences were found between any of the groups for the rate of adhesion disassembly, indicating CD81 is able to modify the turnover of focal adhesions, exerting an effect on the assembly of the adhesions, but not on the disassembly.
To directly corroborate that CD81 regulated Rac activation by their interaction through the C-terminal cytosolic domain of the tetraspanin, we incubated cells with a fluorescently labeled, cell-permeable peptide corresponding to the C-terminal sequence of CD81 or with a scrambled combination of the same eight amino acids as a control. These peptides were readily internalized; at 1 h after incubation, 100% of the cells were fluorescently labeled. The mean fluorescence intensity of the cells increased up to 3 h and declined thereafter, although 100% of the cells retained detectable fluorescence after 24 h (Figure 7A; unpublished data). Both peptides faintly labeled the plasma membrane and strongly accumulated at intracellular vesicles (Figure 7B). Incubation with the CD81 C-terminal peptide, but not with the control, increased the basal levels of Rac activity, but prevented its activation in response to EGF (Figure 7C), similar to what was observed when silencing CD81 in these cells. In the same manner, CD81 C-terminal peptides induced a higher protrusion rate (Figure 7D) and increased the cell area after spreading (Figure 7E).
Therefore our data suggest that perturbation of the Rac-CD81 interaction alters the normal Rac activation/inactivation cycle, resulting in increased but delocalized protrusion and increased adhesion formation. Time-lapse video microscopy experiments revealed that these alterations in Rac activity by CD81 depletion decreased the rate of cell migration, an effect avoided by cotransfection with full-length CD81 but not with the CD81ΔCyt mutant (Figure 8, A and B). Again, overexpression of WT-CD81 had the opposite effect, facilitating cell migration (Figure 8C), while treatment of cells with the permeable peptides recapitulated the phenotype of CD81 siRNA depletion, slowing cell motility (Figure 8D).
In sum, the binding of Rac to the C-terminal cytoplasmic domain of CD81 is an important mechanism responsible for tetraspanin regulation of cell migration. Furthermore, insertion into TEM emerges as a novel regulatory mechanism for Rac activity turnover.
Tetraspanins have been implicated in the regulation of cell migration in different biological scenarios. The current view favors an interpretation of these results based on the interaction of tetraspanins with other membrane molecules in tetraspanin-enriched microdomains (Berditchevski, 2001; Yáñez-Mó et al., 2009). Our study provides the first evidence that CD81 and Rac associate in the same multimolecular complex and emphasizes the role of tetraspanins as direct regulators of signals that control cell migration.
Our results show that CD81 directly binds to Rac proteins. Mass spectrometry analyses of CD81 pull downs show a preference for Rac2, which is enriched in T-lymphoblasts, over Rac1 (Guo et al., 2008). These results were corroborated in adherent cells with Rac-1 and by in vitro direct binding of Rac-1 protein produced in bacterial lysates to the C-terminal sequence of CD81. Previous data showed that interaction of tetraspanin CD9 with α2β1-integrin interferes with membrane anchorage of Rac (Cailleteau et al., 2010), while deletion of CD151 increases RhoA activation (Johnson et al., 2009). CD151 overexpression induces PKC-dependent activation of Rac and Cdc42, but not Rho, promoting cell adhesion (Shigeta et al., 2003). Also, resting levels of Rac activity are lowered in CD81-deficient dendritic cells (Quast et al., 2011). Our current findings thus provide a molecular explanation for the regulation of Rac by TEMs, indicating that CD81 regulates Rac1 dynamics and localization at the cell membrane during membrane protrusion and adhesion formation and establishing a mechanism through which this tetraspanin regulates cell migration.
CD81-Rac complexes were most prominent at the cell leading edge. In this region, Rac promotes actin polymerization and the formation of dendritic actin branches through the activation of the actin-related proteins (Arp2/3) complex via Wiskott–Aldrich syndrome protein (WASP) family Verprolin-homologous protein (WAVE) proteins (Burridge and Wennerberg, 2004). CD81 and other tetraspanins are also commonly used as protein markers of endosomes, lysosomes and exosomes (Simons and Raposo, 2009; Ostrowski et al., 2010). Therefore it is plausible that this interaction also occurs along the export/recycling route.
Most importantly, our data indicate that rather than being necessary for Rac activation or translocation to the membrane, CD81 absence delays its inactivation. Compartmentalization of Rac at the plasma membrane by insertion into TEM might facilitate its binding to a Rho-GDP-dissociation inhibitor (Rho-GDI) protein that targets it for recycling or to a specific Rac-GTPase activating protein (Rac-GAP) or Rac effector. Compartmentalization of Rac at caveolin-rich membrane domains is implicated in its internalization (del Pozo et al., 2005) and degradation (Nethe et al., 2010). Rac interacts directly with caveolin, and depletion of caveolin results in Rac overexpression and hyperactivation (Nethe et al., 2010). Association of Rac with CD81 does not appear to require caveolin, since caveolin was not detected among the proteins pulled down by CD81 C-terminal domain bait peptides. CD81 may thus provide an alternative mode of Rac compartmentalization at the membrane in cells with low or negligible caveolin expression. Importantly, the reduction in CD81 expression may slow Rac deactivation but does not lead to a constitutive activation of the GTPase, since a clear decay is observed at late time points of EGF stimulation. Therefore compartmentalization into TEM emerges as a novel regulatory mechanism of Rac activity turnover.
The increase in basal Rac activity in CD81-silenced cells or in cells pretreated with the permeable peptide correlates with higher protrusion rates and altered adhesion dynamics. Tetraspanins associate with integrins and partially colocalize with small focal adhesions in the cellular periphery (Berditchevski and Odintsova, 1999). Our results suggest that CD81 may localize Rac to the leading edge anterior to nascent adhesions, thereby controlling Rac availability and favoring the initial steps in adhesion formation. CD81-deficient cells show a facilitated formation of adhesions that is similar to that observed in constitutively active Rac-expressing cells (del Pozo et al., 1999; Rottner et al., 1999; Webb et al., 2004; Choi et al., 2008). However, in contrast with cells expressing a constitutive active form of Rac, the subsequent events of adhesion maturation are not grossly altered in CD81-silenced cells. Thus the combination of faster protrusion and spreading and the facilitation of the initial steps of adhesion formation leads, at later time points, to an increased focal adhesion formation that slows cell motility. Moreover, adhesions in CD81 knocked-down cells are formed beyond the outer rim of the cell, and cytoskeletal polarity is lost, suggesting that CD81-dependent compartmentalization may also be crucial for the spatial regulation of Rac activity and cell polarity. Nevertheless, although cell polarity is perturbed in CD81-silenced cells, the persistence of migration in the absence of any directional cue was very low and no significant differences were observed between the experimental conditions (unpublished data).
Our results map the regulatory effect of CD81 to its C-terminal cytoplasmic region, which strongly suggests that the role of CD81 in controlling cell migration is due to its interaction with Rac rather than its lateral association with other membrane receptors in the context of tetraspanin-enriched microdomains. Moreover, our data with cell-permeable peptides with the sequence of CD81 C-terminal domain suggest that these peptides may be an exciting tool to affect tetraspanin-dependent biological processes in vivo, offering a new, therapeutically valuable application of tetraspanin-targeted reagents for the treatment of malignancies or immune-related diseases.
HUVEC were obtained and cultured as described elsewhere (Barreiro et al., 2008). SUM159 breast carcinoma cell line was cultured in DMEM/F-12 (Life Technologies, Invitrogen, Carlsbad, CA), supplemented with 5% fetal bovine serum (FBS), 1% penicillin/streptomycin, nonessential amino acids, 5 μg/ml insulin, and 1 μg/ml hydrocortisone. HEK and U2OS cells were cultured in DMEM (Lonza, Basel, Switzerland) or McCoy’s 5A medium (Life Technologies), respectively, both supplemented with 10% FBS and 1% penicillin/streptomycin. HMEC-1 microvascular endothelial cell line was grown in MCDB131 medium (Life Technologies) supplemented with 20% FBS, 1% penicillin/streptomycin, 20 mM HEPES, 2.5 μg/ml fungizone, 10 ng/ml EGF, and 1 μg/ml hydrocortisone.
Control siRNA was purchased from Dharmacon (Thermo Scientific, Lafayette, CO). Two different siRNAs for CD81 were purchased from Eurogentec (Seraign, Belgium): CD81b (CACCTTCTATGTAGGCATC) and CD81c (CACGTCGCCTTCAACTGTA). CD81c sequence corresponds to a nontranslated (3′UTR) region of CD81 mRNA, which does not interfere with the expression of exogenous CD81. This sequence was used in rescue experiments.
VJ1/20 (anti-CD9), LIA1/1 (anti-CD151), VJ1/12 (anti-CD59), and HP2/9 (anti-CD44) monoclonal antibodies have been previously described (Yáñez-Mó et al., 1998; Barreiro et al., 2005). Anti-CD81 5A6 was kindly provided by S. Levy (Stanford University, CA), and I.33.22 by R.Vilella (Hospital Clinic, Barcelona). Monoclonal anti-Rac1 (clone 23A8) was purchased from Upstate (now Millipore, Billerica, MA); rabbit policlonal anti-Rac antibody was from Santa Cruz Biotechonology (Santa Cruz, CA), anti-vimentin clone VIM 3B4 from Millipore (Billerica, MA); monoclonal antibody anti-paxillin, reference 03-6100, was from Zymed Laboratories (now Invitrogen, Carlsbad, CA), and clone 177 anti-paxillin was from BD Biosciences (Franklin Lanes, NJ).
The truncated CD81-ΔCyt-GFP was obtained using the specific primers: CTCGAGATGGGAGTGGAGGGCTGC (5′) and AAGCTTACAGCACAGCACCATGCT (3′) by PCR onto a CD81 cDNA plasmid. The PCR product was first introduced into the TopoTA vector (Invitrogen) and then subcloned into pAcGFP-N1 or mRFP-N1 (Invitrogen). GFP-wild-type-Rac1, GFP-V12-Rac1 and Orange-paxillin have been previously described (del Pozo et al., 1999).
GST-Rac was provided by X.R. Bustelo (Centro de Investigación del Cancer, Salamanca, Spain); GST-PAK-Crib and C21-GST by J.G. Collard (The Netherlands Cancer Institute, Amsterdam, The Netherlands); and Enhanced Cyan Fluorescent Protein (ECFP)/Ypet-based Raichu-Rac by Y. Wang (University of Illinois, Urbana–Champaign, IL; Ouyang et al., 2008). pTRIP-CD81 (DsRed-CD81) and AqGFP-CD81 (Harris et al., 2008) were provided by J. McKeating (University of Birmingham, UK) and mEmerald-GFP-CD81 (GFP-CD81) and mCherry-CD81 by M.W. Davidson (Florida State University, Tallahassee, FL). All CD81 plasmids had the fluorescent tag in the N-terminal region of the tetraspanin to avoid interference with C-terminal cytoplasmic region conformation or putative function. pEGFP-N1 was from Clontech (Mountain View, CA). EGF was purchased from R&D (Minneapolis, MN) and used at 100 ng/ml at the indicated times. Fibronectin, GTP-γS and GDP were from Sigma-Aldrich (St. Louis, MO).
Tetramethylrhodamine (TAMRA) N-terminal–labeled peptides with the sequences RRRRRRRCCGIRNSSVY (CD81) or RRRRRRRYSVNICRGCSS (Scrambled) were purchased from LifeTein (South Plainfield, NJ).
N-terminally biotinylated peptides containing a SGSG linker sequence connected to the cytoplasmic C-terminal domains of the proteins of interest were purchased from Ray Biotech, (Norcross, GA): CD81, biotin-SGSG-CCGIRNSSVY; CD9, biotin-SGSG-CCAIRRNREMV; CD151, biotin-SGSG-YRSLKLEHY; CD82, biotin-SGSG-CRHVHSEDYSKVRKY; EWI-2, biotin-SGSG-CCFMKRLRKR; ICAM-1, biotin-SGSG-RQRKIKKYRLQQAQKGTPMKPTQATPP. Each peptide (30 nmol) was conjugated to 40 μl streptavidin-Sepharose (GE Healthcare, Uppsala, Sweden). Pull-down assays with T-lymphoblasts or HEK extracts were carried out as previously described (Sala-Valdes et al., 2006) and analyzed by Western blotting or mass spectrometry. Briefly, cells were washed once with ice-cold phosphate-buffered saline (PBS) and then lysed in 1% NP-40 in PBS containing protease and phosphatase inhibitors (Complete, PhosSTOP; Roche, Basel, Switzerland). Lysates were precleared for 2 h at 4ºC with streptavidin-Sepharose (GE Healthcare) and then incubated for 2 h at 4ºC with biotinylated peptides immobilized on streptavidin-Sepharose beads.
For preferential loading of Rac with GDP or GTP, cells were lysed in 5 mM EDTA-containing lysis buffer, and the lysates were incubated with 50 nM of GTP-γS or GDP at 30ºC for 5 min before addition of 6 mM MgCl. Alternatively, cells treated, or not, with EGF were lysed in the presence of 2 mM MgCl before incubation with the Sepharose beads coated with the biotinylated peptides, PAK-GST or C21-GST.
Sepharose beads from the pull-down assays were directly resuspended in Laemmli buffer, applied onto a SDS–PAGE gel, and subjected to the one-step in-gel trypsin digestion method (Bonzon-Kulichenko et al., 2011). The resulting peptides were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using a Surveyor LC system coupled to an LTQ linear ion-trap mass spectrometer (Thermo Fisher, San Jose, CA), as previously described (Lopez-Ferrer et al., 2004; Jorge et al., 2009). The LTQ was operated in a data-dependent MS/MS mode using the 15 most intense precursors detected in a survey scan from 400 to 1600 m/z, as previously described (Lopez-Ferrer et al., 2004). Proteins were identified using the SEQUEST algorithm (Bioworks 3.2 package; Thermo Finnigan, Cambridge, MA); the raw MS/MS files were searched against the Human Swissprot database (Uniprot release 14.0, 19929 sequence entries for human) supplemented with the sequence of porcine trypsin. SEQUEST results were validated using the probability ratio method (Martinez-Bartolome et al., 2008), and false discovery rates (FDR) were calculated by the refined method (Navarro and Vázquez, 2009). Peptide and scan counting was performed, assuming as positive events those with a FDR equal to or lower than 5%.
Cells were plated onto 2 μg/ml of fibronectin and serum-starved for at least 6 h when indicated. EGF (100 ng/ml) was applied at the indicated times before lysis in 0.5% Triton-X-100, 60 mM octylglucoside (Sigma-Aldrich) in Tris 10 mM (pH 8), 150 mM NaCl supplemented with protease and phosphatase inhibitors. Antibodies were precoupled to protein G–Sepharose (GE Healthcare), incubated with cell lysates, and washed several times with lysis buffer before being loaded onto an SDS–PAGE gel.
For immunofluorescence, cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), permeabilized with 0.5% Triton X-100 (5 min), and stained with corresponding primary antibodies followed by species-matching secondary antibodies (Invitrogen). Samples were analyzed in a Leica TCS-SP5 confocal laser-scanning unit equipped with Ar and He/Ne laser beams and attached to a Leica DMIRBE inverted epifluorescence microscope (Leica Microsystems, Heidelberg, Germany); in a TIRF microscope (Leica Microsystems, on a Leica DMI 6000B) using ∼100-nm depth penetration; or a wide-field fluorescence Leica DMIRE2 microscope coupled to a monochromator (Polychrome IV; Till Photonics, Munich, Germany) and a CCD camera (CoolSNAP HQ; Photometrics, Tucson, AZ). For time-lapse fluorescence microscopy, cells were maintained at 37ºC in 5% CO2.
For quantification of focal adhesions, subconfluent HUVEC cultures were plated onto 2 μg/ml fibronectin 72 h after transfection, fixed, permeabilized, and stained for paxillin. The area of focal adhesions was selected and measured by Image J using a thresholding method.
For DNA or siRNA transfection, cells were electroporated with 2 μM of either negative siRNA or CD81 siRNA duplexes or 20 μg of plasmid DNA, in a Gene Pulser II device (Bio-Rad, Hercules, CA). Conditions used were 200 V, 0.975 × 10−9 Faradays, in 200 μl of standard medium (5 μl of 1.5 M NaCl had been previously supplemented). For rescue experiments, the siRNA duplexes were electroporated together with 20 μg of GFP, GFP-CD81, or CD81-ΔCyt-GFP.
CD81 membrane expression was routinely analyzed 48 or 72 h after transfection with siRNA duplexes by immunostaining with anti-CD81 mAb in a FACScan Canto II cytofluorometer (BD Biosciences, Franklin Lanes, NJ), as previously described (Yáñez-Mó et al., 2008). Silencing of CD81 resulted in a reduction of mean fluorescence intensity consistently greater than 50%. In rescue experiments, membrane expression of CD81, stained with an APC-labeled goat anti-mouse, was analyzed after gating GFP-positive cells.
Incorporation of cell-permeable, fluorescently TAMRA-labeled peptides was analyzed in trypsinized unstained cells by flow cytometry.
U2OS cells were cotransfected with mCherry-CD81 (wild-type or cytoplasmic deletion mutant) and GFP-Rac1 (wild-type or V12 active mutant). TIRF images were acquired on an Olympus IX71 microscope using a 60×/1.45 numerical aperture (NA) Plan-Apo oil objective and a Ludl controller (Ludl Electronic Products, Hawthorne, NY), and controlled by MetaMorph software (Molecular Devices, Downingtown, PA). GFP and mCherry were excited using a 488-nm line of an argon ion laser (Melles Griot, Albuquerque, NM) and a 561-nm Cobolt Jive ion laser (Market Tech, Scotts Valley, CA), respectively. For simultaneous dual-color imaging, a polychroic mirror (Z488/568 rpc) and dual-emission filter (Z488/568 nm; Chroma Technology, Bellows Falls, VT) were used. Image time series were acquired with a charge-coupled device camera (Retiga Exi; QImaging, Surrey, Canada). Image processing and correlation analysis was performed in MATLAB 7.7.0 (MathWorks, Natick, MA). Images were corrected for background and photobleaching intensity effects. Cell regions (25 × 25 – 64 × 64 pixels) from both channels were selected for analysis. Single-channel auto- (a = b = 1 or 2) and cross-correlation (a = 1; b = 2) functions were calculated following Wiseman et al. (2000):
GTP-Rac loading was measured by pulling down GTP-loaded Rac using a GST-coupled PAK-CRIB construct immobilized on Sepharose beads, as described elsewhere (Sander et al., 1999).
Alternatively, endothelial cells were cotransfected with ECFP/Ypet Raichu-Rac and DsRed-CD81 and plated onto 2 μg/ml fibronectin. Cells either untreated or stimulated with 100 ng/ml EGF were imaged using time-lapse confocal microscopy. FRET efficiency is presented as the yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP) ratio after CFP stimulation, as previously described (Ouyang et al., 2008).
SUM159 cells were either transfected with GFP or CD81-GFP, cotransfected with control or CD81 siRNA and GFP, or transfected with GFP and incubated 2 h with 1 μM permeable CD81 or scrambled peptides. Cells were then trypsinized, washed, and plated onto 35-mm dishes (MatTek) coated with 10 μg/ml of fibronectin. Images were acquired for 10 min with 1 frame every 5 s using a Leica AM TIRF MC mounted on a Leica DMI6000B microscope fitted with a 100×/1.46 NA oil-immersion objective with 1× magnification and ∼90-nm penetration depth. The protrusion rate was measured with Image J’s Multiple Kymograph plug-in, as described elsewhere (Choi et al., 2008).
For the study of focal adhesion turnover, cells cotransfected with Orange-paxillin, together with control or CD81siRNA and GFP, GFP-CD81, or CD81-ΔCyt-GFP, were allowed to spread, and adhesion assembly and disassembly rates were analyzed with Image J, as previously described (Webb et al., 2004; Choi et al., 2008).
Micropatterned slides were purchased from CYTOO (Grenoble, France). Cells were trypsinized and replated onto micropatterned slides (CYTOO Starter), according to the manufacturer’s instructions. Cells were allowed to attach and spread for 3 h and were then fixed with paraformaldehyde, permeabilized, and stained for paxillin or F-actin. Images were acquired using wide-field fluorescence microscopy and analyzed with the Image J Reference-cell macro. Briefly, images were cropped following the micropatterned image and micropatterns containing multiple cells were discarded by nuclei counting. Average projections of the label image of several individual cells (paxillin or F-actin) are presented in a pseudocolor intensity-scale image.
SUM159 cells were plated on fibronectin (2 μg/ml) 2 d after transfection and serum-starved for 12 h. Alternatively, SUM159 were plated on fibronectin (2 μg/ml) and incubated for 1 h with 1 μM permeable CD81 or scrambled peptides. Images were acquired at 10-min intervals for 24 h in a Nikon Eclipse Ti-E video microscope (Tokyo, Japan). Tracking of transfected cells was performed with Image J and MetaMorph software.
Statistical significance was calculated using Student’s t test or one-way analysis of variance (ANOVA), and significant differences were labeled as: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.
This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E12-09-0642) on December 21, 2012.
We thank M. Vicente-Manzanares and M. Gómez for critical reading of the manuscript. Microscopy was partially conducted at the CNIC-Microscopy & Dynamic Imaging Unit. This work was supported by grants PI080794 and PI11/01645 from the Instituto de Salud Carlos III to M.Y.-M.; SAF2011-25834 and ERC AdG-2011 to F.S.M; BIO2009-07990 from the Ministerio de Educación y Ciencia, CAM BIO/0194/2006 from Comunidad de Madrid, and RECAVA RD06/0014 from the Fondo de Investigaciones Sanitarias (Ministerio de Sanidad y Consumo, Instituto Salud Carlos III) to J.V. and F.S.-M. A.R.H. was supported by National Institutes of Health grant GM23244 and the Cell Migration Consortium (U54 GM064346).
|ANOVA||analysis of variance|
|EGF||epidermal growth factor|
|FBS||fetal bovine serum|
|FDR||false discovery rate|
|FRET||fluorescence resonance energy transfer|
|GFP||green fluorescent protein|
|HUVEC||human umbilical vein endothelial cells|
|LC-MS/MS||liquid chromatography–tandem mass spectrometry|
|PKC||protein kinase C|
|siRNA||small interfering RNA|
|TIRFM||total internal reflection microscopy|
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