A crucial role of beta 1 integrins for keratinocyte migration in vitro and during cutaneous wound repair

Integrins are ubiquitous transmembrane receptors that play crucial roles in cell-cell and cell-matrix interactions. In this study, we have determined the effects of the loss of beta 1 integrins in keratinocytes in vitro and during cutaneous wound repair. Flow cytometry of cultured beta 1-deficient keratinocytes confirmed the absence of beta 1 integrins and showed downregulation of alpha 6 beta 4 but not of alpha v integrins. beta 1-null keratinocytes were characterised by poor adhesion to various substrates, by a reduced proliferation rate and by a strongly impaired migratory capacity. In vivo, the loss of beta 1 integrins in keratinocytes caused a severe defect in wound healing. beta 1-null keratinocytes showed impaired migration and were more densely packed in the hyperproliferative epithelium. Surprisingly, their proliferation rate was not reduced in early wounds and even increased in late wounds. The failure in re-epithelialisation resulted in a prolonged inflammatory response, leading to dramatic alterations in the expression of important wound-regulated genes. Ultimately, beta 1-deficient epidermis did cover the wound bed, but the epithelial architecture was abnormal. These findings demonstrate a crucial role of beta 1 integrins in keratinocyte migration and wound re-epithelialisation. Movies available on-line     

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.     

Biological function of laminin-5 and pathogenic impact of its deficiency

The basement membrane glycoprotein laminin-5 is a key component of the anchoring complex connecting keratinocytes to the underlying dermis. It is secreted by keratinocytes as a cross-shaped heterotrimer of alpha3, beta3 and gamma2 chains and serves as a ligand of various transmembrane receptors, thereby regulating keratinocyte adhesion, motility and proliferation. In intact skin, laminin-5 provides essential links to both the hemidesmosomal alpha6beta4 integrin and the collagen type VII molecules which form the anchoring fibrils inserting into the dermis. If the basement membrane is injured, laminin-5 production increases rapidly. It then serves as a scaffold for cell migration, initiates the formation of hemidesmosomes and accelerates basement membrane restoration at the dermal-epidermal junction. Mutations of the laminin-5 genes or auto-antibodies against one of the subunits of laminin-5 may lead to a significant lack of this molecule in the epidermal basement membrane zone. The major contributions of laminin-5 to the resistance of the epidermis against frictional stress but also for basement membrane regeneration and repair of damaged skin are reflected by the phenotype of Herlitz junctional epidermolysis bullosa, which is caused by an inherited absence of functional laminin-5. This lethal disease becomes manifest in widespread blistering of skin and mucous membranes, impaired wound healing and chronic erosions containing exuberant granulation tissue. Here, we discuss current understanding of the biological functions of laminin-5, the pathogenic impact of its deficiency and implications on molecular approaches towards a therapy of junctional epidermolysis bullosa.     

Integrin beta4 regulates migratory behavior of keratinocytes by determining laminin-332 organization

Whether alpha6beta4 integrin regulates migration remains controversial. beta4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express beta4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type beta4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with alpha6beta4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the alpha6beta4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.     

Keratinocyte growth factor (KGF) promotes keratinocyte cell attachment and migration on collagen and fibronectin

Keratinocyte growth factor (KGF) induction of keratinocyte attachment and migration on provisional and basement membrane proteins was examined. KGF-treated keratinocytes showed increased attachment to collagen types I and IV and fibronectin, but, not to laminin-1, vitronectin, or tenascin. This increase was time- and dose-dependent. Increase in attachment occurred with 2 10 microg/ml of ECM proteins. This KGF-stimulated cell attachment was beta1 integrin-dependent but was not associated with stimulation of the cell surface expression nor affinity (activity) of the collagen integrin receptor (alpha2beta1) nor the fibronectin integrin receptors (alpha5beta1 or alphav). At the basal layer of KGF-treated cells significant accumulation of beta1 integrins was found at the leading edges, and actin stress fibers colocalized with beta1. KGF also induced migratory phenotype and stimulated keratinocyte migration on both fibronectin and collagen types I and IV but not on laminin-1, vitronectin nor tenascin. The results suggest that in addition to its proliferation promoting activity. KGF is able to modulate keratinocyte adhesion and migration on collagen and fibronectin. Our data suggest that KGF induced integrin avidity (clustering), a signaling event, which is not dependent on the alteration of cell surface integrin numbers.     

Structure-function analysis of tetraspanin CD151 reveals distinct requirements for tumor cell behaviors mediated by α3β1 versus α6β4 integrin

The basement membrane protein laminin-332 (laminin-5) mediates both stable cell adhesion and rapid cell migration and thus has the potential to either restrain or promote tumor cell metastasis. The major cellular receptors for laminin-332 are integrin α3β1, which mediates rapid tumor cell migration, and integrin α6β4, which often mediates stable cell attachment. Tetraspanin protein CD151 interacts directly with both α3β1 and α6β4 integrins and with other tetraspanins, thereby promoting α3β1 and α6β4 association with tetraspanin-enriched microdomains on the cell surface. To explore the possibility of selectively modulating tumor cell responses to laminin-332, we re-expressed a series of CD151 mutants in epidermoid carcinoma cells with near total, RNAi-mediated silencing of endogenous CD151. The interactions of CD151 with its integrin partners or its interactions with other tetraspanins were selectively disrupted by specific mutations in the CD151 large extracellular loop (EC2 domain) or in intracellular CD151 palmitoylation sites, respectively. CD151-integrin association and CD151-tetraspanin association were both important for α3β1 integrin-dependent initial adhesion and rapid migration on laminin-332. Remarkably, however, only CD151-integrin association was required for stable, α6β4 integrin-dependent cell attachment on laminin-332. In addition, we found that a QRD amino acid motif in the CD151 EC2 domain, which had been thought to be crucial for CD151-integrin interaction, is not essential for CD151-integrin association or for the ability of CD151 to promote several different integrin functions. These new data suggest potential strategies for selectively modulating migratory cell responses to laminin-332, while leaving stable cell attachment on laminin-332 intact.     

Targeted deletion of the integrin beta4 signaling domain suppresses laminin-5-dependent nuclear entry of mitogen-activated protein kinases and NF-kappaB, causing defects in epidermal growth and migration

The alpha6beta4 integrin-a laminin-5 receptor-mediates assembly of hemidesmosomes and recruitment of Shc and phosphoinositide 3-kinase through the unique cytoplasmic extension of beta4. Mice carrying a targeted deletion of the signaling domain of beta4 develop normally and do not display signs of skin fragility. The epidermis of these mice contains well-structured hemidesmosomes and adheres stably to the basement membrane. However, it is hypoplastic due to reduced proliferation of basal keratinocytes and undergoes wound repair at a reduced rate. Keratinocytes from beta4 mutant mice undergo extensive spreading but fail to proliferate and migrate in response to epidermal growth factor (EGF) on laminin-5. EGF causes significant phosphorylation of extracellular signal-regulated kinase (ERK) and Jun N-terminal protein kinase (JNK) and phosphorylation and degradation of IkappaB in beta4 mutant cells adhering to laminin-5. Unexpectedly, however, ERK, JNK, and NF-kappaB remain in the cytoplasm in beta4 mutant cells on laminin-5, whereas they enter effectively into the nucleus in the same cells on fibronectin or in wild-type cells on both matrix proteins. Inhibitor studies indicate that alpha6beta4 promotes keratinocyte proliferation and migration through its effect on NF-kappaB and P-JNK. These findings provide evidence that beta4 signaling promotes epidermal growth and wound healing through a previously unrecognized effect on nuclear translocation of NF-kappaB and mitogen-activated protein kinases.     

Role of plasma-derived fibrin on keratinocyte and fibroblast wound healing

Fibrin has excellent biocompatibility and biological properties to support tissue regeneration and promote wound healing. However, the role of diluted fibrin in wound healing has yet to be elucidated as it is commonly used in high concentration. This study was aimed to examine the effects of diluted plasma-derived fibrin (PDF) on keratinocyte and fibroblast wound healing in term of cell proliferation, migration, extracellular matrix (ECM) production and soluble factor secretion. Two PDF concentrations, 10 and 20% (v/v) were tested on keratinocytes and fibroblasts indirectly co-cultured in the transwell system. The control group was cultured with 5% FBS. Results showed that PDF reduced the keratinocyte growth rate and fibroblast migration, and increased the fibroblast ECM gene expression whereby significant differences were found between the 20% PDF group and the 5% FBS group. Similar trend was seen for the 10% PDF group but the differences were not significant. Comparison of the soluble factors between the PDF groups demonstrated that the level of growth-related oncogene alpha, interleukin-8 and epithelial neutrophil-activating peptide-78 were significantly higher in the 10% PDF group, whilst interleukin-1 alpha and granulocyte–macrophage colony stimulating factor were significantly more concentrated in the 20% PDF group. Our results suggested that PDF selectively elevated the expression of collagen type 1 and collagen type 3 in fibroblasts but slowed down the migration in concentration-dependent manner. These novel findings provide new insight into the role of PDF in wound healing and may have important implications for the use of fibrin in skin tissue engineering.     

The tetraspanin CD151 regulates cell morphology and intracellular signaling on laminin-511

The tetraspanin CD151 forms a stable complex with integrin alpha3beta1, a widely expressed laminin receptor, and is implicated in the regulation of integrin alpha3beta1-mediated cellular responses, including cell attachment, spreading and migration. However, the molecular mechanism by which CD151 regulates integrin alpha3beta1 functions remains unclear. To address this issue, we knocked down CD151 expression in A549 human lung adenocarcinoma cells by RNA interference. When plated on laminin-511 (laminin-10), the CD151-knocked-down cells showed aberrant membrane protrusions and exhibited reductions in the tyrosine phosphorylation of focal adhesion kinase, Src, p130Cas and paxillin. The formation of membrane protrusions was attenuated when the cells were either plated on surfaces coated with higher concentrations of laminin-511 or treated with the integrin beta1-activating mAb TS2/16; however, neither treatment could rescue the reduced tyrosine phosphorylation. These results indicate that CD151 knockdown weakens the integrin alpha3beta1-mediated adhesion to laminin-511 and thereby provokes an aberrant morphology, but this reduced adhesive activity is not involved in the decline of signaling events in CD151-knocked-down cells. Thus, our results suggest that CD151 regulates integrin alpha3beta1 functions in two independent aspects: potentiation of integrin alpha3beta1-mediated cell adhesion and promotion of integrin alpha3beta1-stimulated signaling events involving tyrosine phosphorylation.     

Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro

We investigated the effect of synthetic antimicrobial decapeptide KSL-W (KKVVFWVKFK) on normal human gingival fibroblast growth, migration, collagen gel contraction, and α-smooth muscle actin protein expression. Results show that in addition to promoting fibroblast adhesion by increasing F-actin production, peptide KSL-W promoted cell growth by increasing the S and G2/M cell cycle phases, and enhanced the secretion of metalloproteinase (MMP)-1 and MMP-2 by upregulating MMP inhibitors, such as tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in fibroblasts. An in vitro wound healing assay confirmed that peptide KSL-W promoted fibroblast migration and contraction of a collagen gel matrix. We also demonstrated a high expression of α-smooth muscle actin by gingival fibroblasts being exposed to KSL-W. This work shows that peptide KSL-W enhances gingival fibroblast growth, migration, and metalloproteinase secretion, and the expression of α-smooth muscle actin, thus promoting wound healing.     

The MSP receptor regulates alpha6beta4 and alpha3beta1 integrins via 14-3-3 proteins in keratinocyte migration

Growth factors, integrins, and the extracellular matrix (ECM) are known to play key roles in epidermal wound healing, although the interplay between these proteins is not fully understood. We show that growth factor macrophage stimulating protein (MSP)- and its receptor Ron-mediated PI3K activation in keratinocytes induces phosphorylation of both Ron and alpha6beta4 integrin at specific 14-3-3 binding sites. Consequently, a Ron/alpha6beta4 complex formed via 14-3-3 binding displaces alpha6beta4 from its location at hemidesmosomes (structures supporting cell adhesion) and relocalizes it to lamellipodia. Concomitant activation of alpha3beta1 and keratinocyte spreading/migration on laminin-5 occurs. Further, MSP-dependent beta4 tyrosine phosphorylation evokes p38 and NF-kappaB signaling required for keratinocyte wound closure. Based on these results, we propose a mechanism based on MSP-Ron-dependent phosphorylation and 14-3-3 association, whereby the function of alpha6beta4 switches from a mechanical adhesive device into a signaling component, and might be critically involved in human epidermal wound healing.     

Angiogenic laminin-derived peptides stimulate wound healing

Acceleration of the wound healing process by using angiogenic peptides has been demonstrated previously. Here we used select laminin-111 peptides, A13 and C16, from the laminin alpha1 and gamma1 chain, respectively, to test whether they are able to stimulate wound healing in a rat full thickness wound model. The 12-mer peptides C16 and A13 are highly angiogenic and bind to integrins alphavbeta3 and alpha5beta1. We show that A13 increases wound re-epithelialization as much as 17% over controls by day 4 and C16 increases coverage by 11%. Contraction of the treated wounds was increased as much as 11% for A13 and 8% for C16 at day 4. No differences were observed at day 7 with either peptide. The peptides also stimulated fibroblast migration in Boyden chamber assays. A13 increased cell migration as much as 2.4-fold on uncoated filters and as much as 16-fold on collagen type IV-coated filters over negative controls. Similarly, C16 also stimulated migration 1.8-fold on uncoated filters and as much as 12-fold on collagen-coated filters. A13 and C16 significantly decreased expression of the pro and active forms of matrix metalloproteinase 2 in foreskin fibroblasts indicating their role in collagen accumulation. We conclude that small bioactive angiogenic peptides can promote dermal wound healing and may offer a new class of stable and chemically manipulable therapeutics for wound healing.     

Characterization of mice lacking the tetraspanin superfamily member CD151

The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially alpha3beta1, alpha6beta1, alpha7beta1, and alpha6beta4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of alpha3 and alpha6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.     

Transforming growth factor-beta1 stimulates collagen matrix remodeling through increased adhesive and contractive potential by human renal fibroblasts

Renal tubulointerstitial fibrosis is the common final pathway leading to end-stage renal failure. Tubulointerstitial fibrosis is characterized by fibroblast proliferation and excessive matrix accumulation. Transforming growth factor-beta1 (TGF-beta1) has been implicated in the development of renal fibrosis accompanied by alpha-smooth muscle actin (alpha-SMA) expression in renal fibroblasts. To investigate the molecular and cellular mechanisms involved in tubulointerstitial fibrosis, we examined the effect of TGF-beta1 on collagen type I (collagen) gel contraction, an in vitro model of scar collagen remodeling. TGF-beta1 enhanced collagen gel contraction by human renal fibroblasts in a dose- and time-dependent manner. Function-blocking anti-alpha1 or anti-alpha2 integrin subunit antibodies significantly suppressed TGF-beta1-stimulated collagen gel contraction. Scanning electron microscopy showed that TGF-beta1 enhanced the formation of the collagen fibrils by cell attachment to collagen via alpha1beta1 and alpha2beta1 integrins. Flow cytometry and cell adhesion analyses revealed that the stimulation of renal fibroblasts with TGF-beta1 enhanced cell adhesion to collagen via the increased expression of alpha1 and alpha2 integrin subunits within collagen gels. Fibroblast migration to collagen was not up-regulated by TGF-beta1. Furthermore, TGF-beta1 increased the expression of a putative contractile protein, alpha-SMA, by human renal fibroblasts in collagen gels. These results suggest that TGF-beta1 stimulates fibroblast-collagen matrix remodeling by increasing both integrin-mediated cell attachment to collagen and alpha-SMA expression, thereby contributing to pathological tubulointerstitial collagen matrix reorganization in renal fibrosis.     

Keratinocyte-derived laminin-332 promotes adhesion and migration in melanocytes and melanoma

Melanocytes are highly motile cells that play an integral role in basic skin physiological processes such as wound healing and proper skin pigmentation. It has been postulated that surrounding keratinocytes contribute to melanocyte migration, but underlying mechanisms remain rather vague so far. In this study, we set out to analyze the specific potential contribution of keratinocyte components to melanocytes and melanoma cell migration-related processes. Our studies revealed that A375 human melanoma cell attachment, spreading, and migration are interestingly better supported by HaCaT keratinocyte extracellular matrix (ECM) than by self-derived A375 ECM. Moreover, HaCaT ECM caused increased integrin α6 expression, adhesion-mediated focal adhesion kinase phosphorylation, and focal adhesion formations. Similar effects were confirmed in human melanocytes. Furthermore, we found that keratinocyte-derived soluble factors did not appear to significantly contribute to these processes. Specific extrinsic factors that promoted melanoma migration were attributed to keratinocyte-derived laminin-332, whereas alternative ECM component such as laminin-111 and fibronectin functions appeared to have insignificant contributions. Taken together, these studies implicate extrinsic laminin-332 in promoting the high mobility property and perhaps invasiveness inherently characteristic of, and that are the menace of, melanocytes and melanomas, respectively.     

Lumican Accelerates Wound Healing by Enhancing α2β1 Integrin-Mediated Fibroblast Contractility

Lumican is a dermatan sulfate proteoglycan highly expressed in connective tissue and has the ability to regulate collagen fibril assembly. Previous studies have shown that lumican is involved in wound healing, but the precise effects of lumican on reepithelialization and wound contraction, the two pivotal aspects of skin wound healing, have not been investigated. Here we explored the roles of lumican in fibroblast contractility, a main aspect of skin wound healing, by adopting mice skin wound healing model and the corresponding in vitro cellular experiments. Our results showed that lumican can promote skin wound healing by facilitating wound fibroblast activation and contraction but not by promoting keratinocyte proliferation and migration. Silencing of integrin α2 completely abolished the pro-contractility of lumican, indicating lumican enhances fibroblast contractility via integrin α2. Our study for the first time demonstrated that lumican can affect fibroblast’s mechanical property, which is pivotal for many important pathological processes, such as wound healing, fibrosis, and tumor development, suggesting that lumican might have a potential to be used to modulate these processes.     

E2F-1 is essential for normal epidermal wound repair.

E2F factors are involved in proliferation and apoptosis. To understand the role of E2F-1 in the epidermis, we screened wild type and E2F-1(-/-) keratinocyte mRNA for genes differentially expressed in the two cell populations. We demonstrate the reduced expression of integrins alpha(5), alpha(6), beta(1), and beta(4) in E2F-1(-/-) keratinocytes associated with reduced activation of Jun terminal kinase and Erk upon integrin stimulation. As a consequence of altered integrin expression and function, E2F-1(-/-) keratinocytes also show impaired migration, adhesion to extracellular matrix proteins, and a blunted chemotactic response to transforming growth factor-gamma1. E2F-1(-/-) keratinocytes, but not dermal fibroblasts, exhibit altered patterns of proliferation, including significant delays in transit through both G(1) and S phases of the cell cycle. Recognizing that proliferation and migration are key for proper wound healing in vivo, we postulated that E2F-1(-/-) mice may exhibit abnormal epidermal repair upon injury. Consistent with our hypothesis, E2F-1(-/-) mice exhibited impaired cutaneous wound healing. This defect is associated with substantially reduced local inflammatory responses and rates of re-epithelialization. Thus, we demonstrate that E2F-1 is indispensable for a hitherto unidentified cell type-specific and unique role in keratinocyte proliferation, adhesion, and migration as well as in proper wound repair and epidermal regeneration in vivo.     

Tetraspanin CD151 regulates glycosylation of (alpha)3(beta)1 integrin

The tetraspanin CD151 forms a stoichiometric complex with integrin alpha3beta1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of alpha3beta1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. alpha6beta1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucalpha1-2Gal and bisecting GlcNAc-beta(1-->4) linkage on N-glycans of the alpha3 integrin subunit. The modulatory activity of CD151 toward alpha3beta1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of alpha3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward alpha3beta1. We also found that glycosylation of CD151 is also critical; Asn(159) --> Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or alpha3beta1 but negated its modulatory function. Changes in the glycosylation pattern of alpha3beta1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of alpha3beta1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin.