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• W1973012238 abstract "Tetraspanins CD9 and CD151 have been implicated in cellular motility and intercellular adhesion in several cellular types. Here, we have studied the subcellular localization and the functional role of these molecules in primary melanocytes. We found that endogenous tetraspanins preferentially clustered in areas of melanocyte homotypic intercellular contacts and at the tips of dendrites. These observations were further confirmed using time-lapse fluorescence confocal microscopy of melanocytes transfected with CD9– and CD151–GFP (green fluorescent protein) constructs, suggesting an involvement of these proteins in cellular contacts and migration. Cell adhesion and migration assays performed using blocking monoclonal antibodies against CD9 and CD151 showed no significant effect on cell–extracellular matrix adhesion, whereas the migration of melanocytes was significantly enhanced. The regulation of the migratory capacity of melanocytes by CD9 and CD151 was further confirmed knocking down the endogenous expression of these tetraspanins with small interference RNA oligonucleotides. Therefore, tetraspanin molecules are localized at motile structures in primary human melanocytes regulating the migratory capacity of these cells. Tetraspanins CD9 and CD151 have been implicated in cellular motility and intercellular adhesion in several cellular types. Here, we have studied the subcellular localization and the functional role of these molecules in primary melanocytes. We found that endogenous tetraspanins preferentially clustered in areas of melanocyte homotypic intercellular contacts and at the tips of dendrites. These observations were further confirmed using time-lapse fluorescence confocal microscopy of melanocytes transfected with CD9– and CD151–GFP (green fluorescent protein) constructs, suggesting an involvement of these proteins in cellular contacts and migration. Cell adhesion and migration assays performed using blocking monoclonal antibodies against CD9 and CD151 showed no significant effect on cell–extracellular matrix adhesion, whereas the migration of melanocytes was significantly enhanced. The regulation of the migratory capacity of melanocytes by CD9 and CD151 was further confirmed knocking down the endogenous expression of these tetraspanins with small interference RNA oligonucleotides. Therefore, tetraspanin molecules are localized at motile structures in primary human melanocytes regulating the migratory capacity of these cells. extracellular matrix green fluorescent protein monoclonal antibody small interference RNA Tetraspanins comprise a numerous group of proteins that contain four putative membrane-spanning domains and, characteristically, the presence of a large divergent extracellular loop between the third and fourth membrane-spanning domains (Wright and Tomlinson, 1994Wright M.D. Tomlinson M.G. The ins and outs of the transmembrane 4 superfamily.Immunol Today. 1994; 15: 588-594Abstract Full Text PDF PubMed Scopus (329) Google Scholar). They have been implicated in the regulation of cell development, proliferation, activation, and motility and have been shown to couple to signal transduction pathways (Berditchevski, 2001Berditchevski F. Complexes of tetraspanins with integrins: More than meets the eye.J Cell Sci. 2001; 114: 4143-4151Crossref PubMed Google Scholar; Boucheix and Rubinstein, 2001Boucheix C. Rubinstein E. Tetraspanins.Cell Mol Life Sci. 2001; 58: 1189-1205Crossref PubMed Scopus (514) Google Scholar; Hemler, 2001Hemler M.E. Specific tetraspanin functions.J Cell Biol. 2001; 155: 1103-1107Crossref PubMed Scopus (313) Google Scholar; Stipp et al., 2003Stipp C.S. Kolesnikova T.V. Hemler M.E. Functional domains in tetraspanin proteins.Trends Biochem Sci. 2003; 28: 106-112Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). In this regard, it has been suggested that the main role of tetraspanins is to organize other proteins into signal-transducing complexes at the cell surface (Berditchevski, 2001Berditchevski F. Complexes of tetraspanins with integrins: More than meets the eye.J Cell Sci. 2001; 114: 4143-4151Crossref PubMed Google Scholar; Yáñez-Mó et al., 2001Yáñez-Mó M. Mittelbrunn M. Sanchez-Madrid F. Tetraspanins and intercellular interactions.Microcirculation. 2001; 8: 153-168Crossref PubMed Scopus (47) Google Scholar; Tarrant et al., 2003Tarrant J.M. Robb L. van Spriel A.B. Wright M.D. Tetraspanins: Molecular organisers of the leukocyte surface.Trends Immunol. 2003; 24: 610-617Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Tetraspanins have been shown to coprecipitate with several transmembrane proteins, and with β1 integrins in particular (Berditchevski et al., 1996Berditchevski F. Zutter M.M. Hemler M.E. Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins).Mol Biol Cell. 1996; 7: 193-207Crossref PubMed Scopus (250) Google Scholar; Hemler et al., 1996Hemler M.E. Mannion B.A. Berditchevski F. Association of TM4SF proteins with integrins: Relevance to cancer.Biochim Biophys Acta. 1996; 1287: 67-71PubMed Google Scholar; Rubinstein et al., 1996Rubinstein E. Le Naour F. Lagaudriere-Gesbert C. Billard M. Conjeaud H. Boucheix C. CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins.Eur J Immunol. 1996; 26: 2657-2665Crossref PubMed Scopus (322) Google Scholar; Maecker et al., 1997Maecker H.T. Todd S.C. Levy S. The tetraspanin superfamily: Molecular facilitators.FASEB J. 1997; 11: 428-442Crossref PubMed Scopus (788) Google Scholar). In epidermal cell adhesion and migration, β1 integrins play a critical role (Watt and Hertle, 1994Watt F.M. Hertle M.D. Keratinocyte integrins.in: Leight I.M. Lane E.B. Watt F.M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 153-164Google Scholar). To participate in those functions, integrins may not only bind to intracellular proteins and extracellular ligands (Clark and Brugge, 1995Clark E.A. Brugge J.S. Integrins and signal transduction pathways: The road taken.Science. 1995; 268: 233-239Crossref PubMed Scopus (2758) Google Scholar) but may also laterally associate with other transmembrane proteins such as those from the tetraspanin superfamily (Berditchevski, 2001Berditchevski F. Complexes of tetraspanins with integrins: More than meets the eye.J Cell Sci. 2001; 114: 4143-4151Crossref PubMed Google Scholar; Boucheix and Rubinstein, 2001Boucheix C. Rubinstein E. Tetraspanins.Cell Mol Life Sci. 2001; 58: 1189-1205Crossref PubMed Scopus (514) Google Scholar; Hemler, 2001Hemler M.E. Specific tetraspanin functions.J Cell Biol. 2001; 155: 1103-1107Crossref PubMed Scopus (313) Google Scholar; Stipp et al., 2003Stipp C.S. Kolesnikova T.V. Hemler M.E. Functional domains in tetraspanin proteins.Trends Biochem Sci. 2003; 28: 106-112Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). The migration of melanocytes represents a fundamental requirement in a wide variety of physiological and pathological scenarios. Melanocytes migrate from the neural crest to the skin during the first trimester of the embryogenesis. In physiological conditions, melanocytes migrate to display a homogeneous distribution in the epidermis, which have been designated as an “epidermal melanin unit” (Fitzpatrick and Breathnach, 1963Fitzpatrick T. Breathnach A. Das epidermale Melanin-Einheit system.Dermatol Wochenschr. 1963; 147: 481-489PubMed Google Scholar). But insights into the cellular and molecular mechanisms underlying such organization, however, remain unknown (Hoath and Leahy, 2003Hoath S.B. Leahy D.G. The organization of human epidermis: Functional epidermal units and phi proportionality.J Invest Dermatol. 2003; 121: 1440-1446Crossref PubMed Scopus (81) Google Scholar). In adults, melanocytes also migrate during the process of wound healing and to cover the white areas of patients with vitiligo. Although integrins (Morelli et al., 1993Morelli J.G. Yohn J.J. Zekman T. Norris D.A. Melanocyte movement in vitro: Role of matrix proteins and integrin receptors.J Invest Dermatol. 1993; 101: 605-608Abstract Full Text PDF PubMed Google Scholar) and cadherins (Hsu et al., 2000Hsu M.Y. Meier F.E. Nesbit M. Hsu J.Y. van Belle P. Elder D.E. Herlyn M. E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors.Am J Pathol. 2000; 156: 1515-1525Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) are both implicated in adhesion and movement of melanocytes, little is known about other factors that regulate both functions. Tetraspanins have been found on normal epidermis (Okochi et al., 1997Okochi H. Kato M. Nashiro K. Yoshie O. Miyazono K. Furue M. Expression of tetra-spans transmembrane family (CD9, CD37, CD53, CD63, CD81 and CD82) in normal and neoplastic human keratinocytes: An association of CD9 with alpha 3 beta 1 integrin.Br J Dermatol. 1997; 137: 856-863Crossref PubMed Scopus (40) Google Scholar; Sincock et al., 1997Sincock P.M. Mayrhofer G. Ashman L.K. Localization of the transmembrane 4 superfamily (TM4SF) member PETA-3 (CD151) in normal human tissues: Comparison with CD9, CD63, and alpha5beta1 integrin.J Histochem Cytochem. 1997; 45 (225): 515Crossref PubMed Scopus (164) Google Scholar), expressed by keratinocytes (Jones et al., 1996Jones P.H. Bishop L.A. Watt F.M. Functional significance of CD9 association with beta 1 integrins in human epidermal keratinocytes.Cell Adhes Commun. 1996; 4: 297-305Crossref PubMed Scopus (88) Google Scholar; Okochi et al., 1997Okochi H. Kato M. Nashiro K. Yoshie O. Miyazono K. Furue M. Expression of tetra-spans transmembrane family (CD9, CD37, CD53, CD63, CD81 and CD82) in normal and neoplastic human keratinocytes: An association of CD9 with alpha 3 beta 1 integrin.Br J Dermatol. 1997; 137: 856-863Crossref PubMed Scopus (40) Google Scholar; Peñas et al., 2000Peñas P.F. García-Diez A. Sánchez-Madrid F. Yañez-Mó M. Tetraspanins are localized at motility-related structures and involved in normal human keratinocyte wound healing migration.J Invest Dermatol. 2000; 114: 1126-1135Crossref PubMed Scopus (89) Google Scholar). We have previously demonstrated that tetraspanin molecules have an important role in keratinocyte motility (Peñas et al., 2000Peñas P.F. García-Diez A. Sánchez-Madrid F. Yañez-Mó M. Tetraspanins are localized at motility-related structures and involved in normal human keratinocyte wound healing migration.J Invest Dermatol. 2000; 114: 1126-1135Crossref PubMed Scopus (89) Google Scholar). On the other hand, CD9 has been functionally associated with tumor cell motility (Ikeyama et al., 1993Ikeyama S. Koyama M. Yamaoko M. Sasada R. Miyake M. Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA.J Exp Med. 1993; 177: 1231-1237Crossref PubMed Scopus (269) Google Scholar) and melanoma invasion (Longo et al., 2001Longo N. Yanez-Mo M. Mittelbrunn M. de la Rosa G. Munoz M.L. Sanchez-Madrid F. Sanchez-Mateos P. Regulatory role of tetraspanin CD9 in tumor-endothelial cell interaction during transendothelial invasion of melanoma cells.Blood. 2001; 98: 3717-3726Crossref PubMed Scopus (92) Google Scholar). Nevertheless, no formal study of tetraspanins has been performed in human primary melanocytes. In this study, we describe the subcellular localization of CD9 and CD151 tetraspanin molecules and their functional role in melanocyte motility. We analyzed the expression of tetraspanins CD9, CD63, CD81, and CD151, and integrins αvβ3, β1, α2, and α3 by flow cytometry in normal human melanocytes. These primary cells express high levels of β1 integrin and CD9, and lower levels of α2 and α3 chains of integrins and tetraspanins CD81, CD151, and CD63 (Figure 1). Immunofluorescence studies showed that CD9 and CD151 are distributed throughout the cell body and the dendrites of the melanocyte, their clustering at points of intercellular contact and at the tips of dendrites being remarkable, where they preferentially colocalized with β1 integrins (Figure 2a and b). The colocalization percentage obtained considering the overall surface of the melanocytes, however, is rather average (ranging from 14% to 28%), and not all contacts and tips contained accumulation of both molecular types (data not shown). CD81 exhibits a staining pattern similar to that of CD9 and CD151, but no clear CD63 membrane expression was found, although it was highly represented in intracellular vesicles as it has been found in other cell types such as endothelial cells and keratinocytes (data not shown) (Yáñez-Mó et al., 1998Yáñez-Mó M. Alfranca A. Cabañas C. et al.Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions.J Cell Biol. 1998; 141: 791-804Crossref PubMed Scopus (233) Google Scholar; Peñas et al., 2000Peñas P.F. García-Diez A. Sánchez-Madrid F. Yañez-Mó M. Tetraspanins are localized at motility-related structures and involved in normal human keratinocyte wound healing migration.J Invest Dermatol. 2000; 114: 1126-1135Crossref PubMed Scopus (89) Google Scholar). We found footprints in the immunofluorescence studies with CD9 but almost no staining with CD151 (data not shown). These footprints are made of rests of plasma membrane containing receptors for extracellular matrix proteins that remain attached to the ligands as the cell moves. We and other authors have described this phenomenon, also termed ripping, in keratinocytes (Peñas et al., 2000Peñas P.F. García-Diez A. Sánchez-Madrid F. Yañez-Mó M. Tetraspanins are localized at motility-related structures and involved in normal human keratinocyte wound healing migration.J Invest Dermatol. 2000; 114: 1126-1135Crossref PubMed Scopus (89) Google Scholar) and other cell types (Palecek et al., 1996Palecek S.P. Schmidt C.E. Lauffenburger D.A. Horwitz A.F. Integrin dynamics on the tail region of migrating fibroblasts.J Cell Sci. 1996; 109: 941-952PubMed Google Scholar). As these findings suggested an implication of tetraspanins in adhesion to the extracellular matrix, we performed adhesion experiments on collagen. We used TS2/16 (an anti-CD29 that activates integrins β1) and VJ1/14 (an anti-CD29 that inhibits integrins β1) monoclonal antibodies (mAb) as positive and negative controls of adhesion, respectively. Differences between both TS2/16 (enhancing adhesion) and VJ1/14 (inhibiting adhesion) were significant (p<0.005) but moderate (±15% from the control). When melanocytes were incubated with anti-tetraspanin mAb, we also found a reproducible but very mild modification of adhesion (≤15%) compared with control cells without antibody treatment, suggesting the lack of implication of CD9 and CD151 in adhesion to collagen (data not shown) as described for other cell types (Yáñez-Mó et al., 1998Yáñez-Mó M. Alfranca A. Cabañas C. et al.Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions.J Cell Biol. 1998; 141: 791-804Crossref PubMed Scopus (233) Google Scholar). To ascertain the kinetics of the localization of tetraspanins in intercellular homotypic contacts of melanocytes, we erformed time-lapse fluorescence confocal microscopy with CD9–GFP (green fluorescent protein) and CD151–GFP-tagged proteins. Using CD9–GFP we found that this molecule showed a diffuse pattern of accumulation at transient cell–cell contact sites (Figure 3a, Fig S1). CD151–GFP was rapidly and highly concentrated as soon as dendrites of different melanocytes come into contact (Figure 3b, Fig S2). Both findings suggest that CD9 and CD151 transiently cluster at the homotypic intercellular contacts during the scanning of the melanocyte cell surface.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI3NDZmMDg2YjY3ZTdhZmEwYTUxZDc3MTA2OWRjYTJkMiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM0NDg4NTg4fQ.YYI3HjzIb0cPQbDKxz26JNzb-KyHT3A4224Odiao-nj4D7UQg1cdL5BB7iWq_pws8G_2a7DzYdsL2KjIiYMVehu27aTHuSqUOsYNH6Yt28HQ1ss9xSFa_TAVsJsK9VsUTUOO8JK-6kiuog3p1Hw3VSq7iZAZ29Nz3mzJg9akjuoac_YmIloCbA92Uh76eENaKq53u6IWtbSZ3tY5ZAolN9tfnS5pHX0keM5ICf_-Ju35C-MzSUtQympUghys5YROpW510vK2gTyk9shKM1jjjFiYI32jGoKx5QXtZ-fyq26M-l0iAoSdoUbctZmvzb94yZWPbwj7457nmMYzFdDf0Q Download .mp4 (0.37 MB) Help with .mp4 files Figure S1Video S1 (from Fig 3a). Primary human melanocytes transiently transfected with CD9-GFP were seeded on a coverslip for 4 h. Time-lapse fluorescence confocal microscopy was performed after the incubation period. Series of optical sections distanced 0.4 µm on the z-axis were acquired every 10 min for a total time of 7 h. A field containing three melanocytes in contact (two of them transfected) was selected for recording the videosequence. CD9-GFP showed a diffuse pattern of accumulation at the transient cell-cell contact sites.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJiNTBhZTM0MGU5YjlhMDcwYTQ0MTEzYTIxZjJkNzQ1ZCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM0NDg4NTg4fQ.M6oeNU3CuaWJbPNSeE6qB2KM19zkGhgZxHcFf0KldEfwJacMbZWJnDQ1cfcdbX1ceCvXjaDyRl_qDp0K9o_rkcKHx9FOn4mOu8qblvy2Z7B1GrCzRrNRJQsUsdA0oVPgWUfZ9Ov4O2QH7sVLn-qyJ4tBk-S8EImg5Op_EcQo1ZaKJDsOGWuMRBr92741ydeG5zeRjELYUWa5QY6eF5tAYGX-SfRG4At54xVIVnSmYFLyhE_bvH47RHFRGYHmd_yG7kt1HGx1IEMhU0rZcQI_rA-OKRgPeSGpPj1ndW6rsv3LyhtmQ8u2euc9hs_ohG8edO5Rj1G8SDhEU_5AVbloFw Download .mp4 (0.12 MB) Help with .mp4 files Figure S2Video S1 (from Fig 3b).Primary human melanocytes transiently transfected with CD151-GFP were seeded on a coverslip for 4 h. Time-lapse fluorescence confocal microscopy was performed after the incubation period. Series of optical sections distanced 0.4 µm on the z-axis were acquired every 15 min for a total time of 7 h. A field containing two transfected melanocytes was selected for recording the videosequence. CD151-GFP was rapidly and highly concentrated as soon as the dendrites of both melanocytes contacted. On the other hand, another specific area of accumulation was the tips of the dendrites, which are enriched in ruffles. We found enhanced expression of CD9–GFP (Figure 4a, Figure S3) and CD151–GFP (Figure 4b, Fig S4) at the tip of dendrites when melanocytes explore the surrounding milieu. This localization was dynamic and was displayed by all dendrites; those that directed the movement of the melanocyte and those that were retracting.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI3NDEyODQ2ZDg2NTA2YjhjMWM3MTFhMjAyMTdiYjVhOCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM0NDg4NTg4fQ.GOsfH93V3vs46tAYMX864Rtv8pge-RGLw2yLw4WgyntnXodCZghLUR7WK2Bk6V-OeqGbkb0AhEhe2c7qWFCZ_Mn_UhspDRH66jXm9RrpaOoyL_JZXy7fTk0VuF_L5QRydniDZ2yBHVyYdLk-NtXQYfBsYg7mmi9Xh6dlVPrsBAappjiGGKOBLpUrvXvK0U_cLWOIzJBBX2KkMvQJpeJzWgXmZjJK23mPEvAYhtEpNItFULZnQaKVUkhJ5WYHnrdmrT73wVDx-cv4RjDQOJ2TYQmohC-65mvglZxH2GFnD6-2TT4N4TKFq5w0WdePvOWyPvutMwkHxrkdmYRGBYkDew Download .mp4 (0.41 MB) Help with .mp4 files Figure S3Video S1 (from Fig 4a). Primary human melanocytes transiently transfected with CD9-GFP were seeded on a coverslip for 4 h. Time-lapse fluorescence confocal microscopy was performed after the incubation period. Series of optical sections distanced 0.4 µm on the z-axis were acquired every 15 min for a total time of 7 h. A field containing a migrating melanocyte was selected for recording the videosequence. CD9-GFP was accumulated at the tip of the dendrites as the melanocyte moved exploring the surrounding milieu. This localization was dynamic and displayed by all dendrites, those directing the movement of the melanocyte and the one that was retracting.eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJkOGIxZmQ4NzRmNjlhYTIwNmRmNDE5MTg1ZjYwYjczZCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM0NDg4NTg4fQ.Vnd50Bx-z9Niud9MoUKBHgp5-ekpUIwCn9u-rCV1OgY7Y_YvS0Y34He16MMbqLfcxFnnOyYhKxd1NyvWVBI0ngdudVBev5CwZsZFpGsBFHJy52q3GTtnx4blemDJ4f_ZmYxwwxXgkGeOZWIJaG2ZlFjRvhPWX5WJosn5YTfoDokhYd-vARE63YGGiIbdF_UXlFquNy5Q3GebBGAhZS6z3KRlOhC6_lWTW_HpwL1fM4-LohNF_uXjie7rnkA0gkafudvMjkAWk32e2MWxtoESxpMt_qPLSgjyx3KozAMFYFIXEyp5cQCzhtLMY5ezcPDaMQwjKnrobrN3mP59zetf9A Download .mp4 (1.59 MB) Help with .mp4 files Figure S4Video S1 (from Fig 4b). Primary human melanocytes transiently transfected with CD151-GFP were seeded on a coverslip for 4 h. Time-lapse fluorescence confocal microscopy was performed after the incubation period. Series of optical sections distanced 0.4 µm on the z-axis were acquired every 5 min for a total time of 7 h. A field containing a migrating melanocyte was selected for recording the videosequence. CD151-GFP was accumulated at the tip of the dendrites as the melanocyte moved exploring the surrounding milieu. This localization was dynamic and displayed by both dendrites. As these results suggested that CD9 and CD151 may be involved in the movement of melanocytes, they prompted us to formally explore the functional involvement of tetraspanins in melanocyte motility using Transwell chambers with fibronectin in the lower chamber as a stimulus for melanocyte motility. Blocking monoclonal antibodies against CD9 and CD151, as determined in other cellular types (Yáñez-Mó et al., 1998Yáñez-Mó M. Alfranca A. Cabañas C. et al.Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions.J Cell Biol. 1998; 141: 791-804Crossref PubMed Scopus (233) Google Scholar; Peñas et al., 2000Peñas P.F. García-Diez A. Sánchez-Madrid F. Yañez-Mó M. Tetraspanins are localized at motility-related structures and involved in normal human keratinocyte wound healing migration.J Invest Dermatol. 2000; 114: 1126-1135Crossref PubMed Scopus (89) Google Scholar), enhanced melanocyte motility, whereas anti-αvβ3 integrin inhibited the migration of melanocytes. The activatory anti-β1 integrin TS2/16 decreased the migration of melanocytes, whereas the inhibitory anti-β1 integrin VJ1/14 increased it (Figure 5a). To evaluate the influence of β1 integrins in CD9 and CD151-mediated effect on melanocyte migration, we combined the use of TS2/16 and VJ1/14 with anti-CD9 and anti-CD151 (Figure 5b). We found that the enhanced effect of CD9 and CD151 on melanocyte migration was more pronounced when combined with VJ1/14, and was partially reversed by TS2/16. To further assess the functional role of tetraspanins in melanocyte motility, tetraspanin expression was knocked down in melanocytes using small interference RNA (siRNA) against CD9 and CD151. We obtained a significant inhibition in the expression of CD9 and CD151 using specific siRNA (Figure 6a), without affecting the morphological characteristics of siRNA-treated melanocytes (data not shown). We performed similar migration assays using fibronectin as a stimulus of melanocyte motility with these interfered cells and found that siRNA against CD9 and CD151 significantly (p<0.05) stimulates melanocyte migration (Figure 6b). Altogether these data underscore the involvement of tetraspanin in melanocyte migration through regulation of β1 integrin function. The role of tetraspanins in keratinocytes has been explored previously (Peñas et al., 2000Peñas P.F. García-Diez A. Sánchez-Madrid F. Yañez-Mó M. Tetraspanins are localized at motility-related structures and involved in normal human keratinocyte wound healing migration.J Invest Dermatol. 2000; 114: 1126-1135Crossref PubMed Scopus (89) Google Scholar). Tetraspanins are implicated in keratinocyte motility, and a possible implication of these molecules in inter-keratinocyte contacts has been suggested. Although CD9, CD81, and CD151 have been described in melanoma cells (Radford et al., 1996Radford K.J. Thorne R.F. Hersey P. CD63 associates with transmembrane 4 superfamily members, CD9 and CD81, and with beta 1 integrins in human melanoma.Biochem Biophys Res Commun. 1996; 222: 13-18Crossref PubMed Scopus (82) Google Scholar; Longo et al., 2001Longo N. Yanez-Mo M. Mittelbrunn M. de la Rosa G. Munoz M.L. Sanchez-Madrid F. Sanchez-Mateos P. Regulatory role of tetraspanin CD9 in tumor-endothelial cell interaction during transendothelial invasion of melanoma cells.Blood. 2001; 98: 3717-3726Crossref PubMed Scopus (92) Google Scholar), no description of the expression or functional implications of tetraspanins in normal human melanocytes has been reported before. This work addresses the expression, subcellular distribution, and functional role in cell adhesion and motility of the tetraspanins CD9 and CD151 in primary human melanocytes. The accumulation of both tetraspanins at the tips of the melanocyte dendrites and in the intercellular contacts with other melanocytes was remarkable. Moreover, some ripping was found. These findings suggested the involvement of CD9 and CD151 in melanocyte adhesion to the extracellular matrix, in the exploration of the environment and in homotypic interactions. CD63, a component of melanosomes also known as lysosome-associated membrane protein (LAMP)-3, has been described in melanocytes and melanoma, and implicated in melanoma progression (Ota et al., 1998Ota A. Park J.S. Jimbow K. Functional regulation of tyrosinase and LAMP gene family of melanogenesis and cell death in immortal murine melanocytes after repeated exposure to ultraviolet B.Br J Dermatol. 1998; 139: 207-215Crossref PubMed Scopus (7) Google Scholar). As expected, CD63 showed low plasma membrane and high intracellular expression in primary melanocytes. The role of tetraspanins in adhesion to the ECM varies among different cell types, and it has been suggested that tetraspanins are not involved in cellular adhesion in most cells (Berditchevski, 2001Berditchevski F. Complexes of tetraspanins with integrins: More than meets the eye.J Cell Sci. 2001; 114: 4143-4151Crossref PubMed Google Scholar). In this regard, CD9 did not affect MDA-MB 231 (Sugiura and Berditchevski, 1999Sugiura T. Berditchevski F. Function of alpha3beta1-tetraspanin protein complexes in tumor cell invasion. Evidence for the role of the complexes in production of matrix metalloproteinase 2 (MMP-2).J Cell Biol. 1999; 146: 1375-1389Crossref PubMed Scopus (178) Google Scholar), human B cells (Shaw et al., 1995Shaw A.R. Domanska A. Mak A. et al.Ectopic expression of human and feline CD9 in a human B cell line confers beta 1 integrin-dependent motility on fibronectin and laminin substrates and enhanced tyrosine phosphorylation.J Biol Chem. 1995; 270: 24092-24099Crossref PubMed Scopus (120) Google Scholar) or hepatic cells (Mazzocca et al., 2002Mazzocca A. Carloni V. Sciammetta S. et al.Expression of transmembrane 4 superfamily (TM4SF) proteins and their role in hepatic stellate cell motility and wound healing migration.J Hepatol. 2002; 37: 322-330Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) adhesion. Accordingly, our data show that tetraspanins slightly modulate the adhesion of melanocytes to collagen. But CD151 increased adhesion to collagen and fibronectin of the erytroleukemia cell line HEL (Fitter et al., 1999Fitter S. Sincock P.M. Jolliffe C.N. Ashman L.K. Transmembrane 4 superfamily protein CD151 (PETA-3) associates with beta 1 and alpha IIb beta 3 integrins in haemopoietic cell lines and modulates cell–cell adhesion.Biochem J. 1999; 338: 61-70PubMed Google Scholar) but did not affect many other cell lines and human primary cells (Yauch et al., 1998Yauch R.L. Berditchevski F. Harler M.B. Reichner J. Hemler M.E. Highly stoichiometric, stable, and specific association of integrin alpha3beta1 with CD151 provides a major link to phosphatidylinositol 4- kinase, and may regulate cell migration.Mol Biol Cell. 1998; 9: 2751-2765Crossref PubMed Scopus (262) Google Scholar; Fitter et al., 1999Fitter S. Sincock P.M. Jolliffe C.N. Ashman L.K. Transmembrane 4 superfamily protein CD151 (PETA-3) associates with beta 1 and alpha IIb beta 3 integrins in haemopoietic cell lines and modulates cell–cell adhesion.Biochem J. 1999; 338: 61-70PubMed Google Scholar; Sugiura and Berditchevski, 1999Sugiura T. Berditchevski F. Function of alpha3beta1-tetraspanin protein complexes in tumor cell invasion. Evidence for the role of the complexes in production of matrix metalloproteinase 2 (MMP-2).J Cell Biol. 1999; 146: 1375-1389Crossref PubMed Scopus (178) Google Scholar; Mazzocca et al., 2002Mazzocca A. Carloni V. Sciammetta S. et al.Expression of transmembrane 4 superfamily (TM4SF) proteins and their role in hepatic stellate cell motility and wound healing migration.J Hepatol. 2002; 37: 322-330Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). It is remarkable that a defective CD151 protein has been implicated as the cause of epidermolysis bullosa in two patients, suggesting that this tetraspanin could play a role in keratinocyte adhesion (Karamatic Crew et al., 2004Karamatic Crew V. Burton N. Kagan A. et al.CD151, the first member of the tetraspanin (TM4) superfamily detected on erythrocytes, is essential for the correct assembly of human basement membranes in kidney and skin.Blood. 2004; 104: 2217-2223Crossref PubMed Scopus (190) Google Scholar). Tetraspanins are important in inte" @default.
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