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• W2016367573 abstract "The axon-associated protein F11 is a GPI-anchored member of the immunoglobulin superfamily that promotes axon outgrowth and that shows a complex binding pattern toward multiple cell surface and extracellular matrix proteins including tenascin-R and tenascin-C. In this study, we demonstrate that tenascin-R and tenascin-C differentially modulate cell adhesion and neurite outgrowth of tectal cells on F11. While soluble tenascin-R increases the number of attached cells and the percentage of cells with neurites on immobilized F11, tenascin-C stimulates cell attachment to a similar extent but decreases neurite outgrowth. The cellular receptor interacting with F11 has been previously identified as NrCAM; however, in the presence of tenascin-R or tenascin-C cell attachment and neurite extension are independent of NrCAM. Antibody perturbation experiments indicate that β1 integrins instead of NrCAM function as receptor for neurite outgrowth of tectal cells on an F11·TN-R complex. Cellular binding assays support the possibility that the interaction of F11 to NrCAM is blocked in the presence of tenascin-R and tenascin-C. Furthermore, a sandwich binding assay demonstrates that tenascin-R and tenascin-C are able to form larger molecular complexes and to link F11 polypeptides by forming a molecular bridge.These results suggest that the molecular interactions of F11 might be regulated by the presence of tenascin-R and tenascin-C. The axon-associated protein F11 is a GPI-anchored member of the immunoglobulin superfamily that promotes axon outgrowth and that shows a complex binding pattern toward multiple cell surface and extracellular matrix proteins including tenascin-R and tenascin-C. In this study, we demonstrate that tenascin-R and tenascin-C differentially modulate cell adhesion and neurite outgrowth of tectal cells on F11. While soluble tenascin-R increases the number of attached cells and the percentage of cells with neurites on immobilized F11, tenascin-C stimulates cell attachment to a similar extent but decreases neurite outgrowth. The cellular receptor interacting with F11 has been previously identified as NrCAM; however, in the presence of tenascin-R or tenascin-C cell attachment and neurite extension are independent of NrCAM. Antibody perturbation experiments indicate that β1 integrins instead of NrCAM function as receptor for neurite outgrowth of tectal cells on an F11·TN-R complex. Cellular binding assays support the possibility that the interaction of F11 to NrCAM is blocked in the presence of tenascin-R and tenascin-C. Furthermore, a sandwich binding assay demonstrates that tenascin-R and tenascin-C are able to form larger molecular complexes and to link F11 polypeptides by forming a molecular bridge. These results suggest that the molecular interactions of F11 might be regulated by the presence of tenascin-R and tenascin-C. cell adhesion molecule neural cell adhesion molecule immunoglobulin superfamily extracellular matrix fibronectin type III domain tenascin-R tenascin-C monoclonal antibody Cell adhesion molecules (CAMs)1 of the immunoglobulin superfamily (IgSF) act in concert with other cell surface molecules and extracellular matrix (ECM) proteins to regulate cell migration, axonal growth, and guidance during development of the nervous system. IgSF members coexist on many extending axons and show a transient expression pattern during early stages of development. The multidomain nature of glycoproteins of the IgSF suggest that they regulate axonal pathfinding by multiple complex interactions with other axonal and ECM molecules (1Brümmendorf T. Rathjen F.G. Curr. Opin. Neurobiol. 1996; 6: 584-593Crossref PubMed Scopus (153) Google Scholar). The axon-associated F11 glycoprotein is composed of six N-terminal Ig domains followed by four fibronectin type III (FNIII) domains and a glycosylphosphatidylinositol anchor and has been implicated in axonal growth and fasciculation (2Brümmendorf T. Wolff J.M. Frank R. Rathjen F.G. Neuron. 1989; 2: 1351-1361Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 3Chang S. Rathjen F.G. Raper J.A. J. Cell Biol. 1987; 104: 355-362Crossref PubMed Scopus (239) Google Scholar, 4Rathjen F.G. Wolff J.M. Frank R. Bonhoeffer F. Rutishauser U. J. Cell Biol. 1987; 104: 343-353Crossref PubMed Scopus (282) Google Scholar, 5Gennarini G. Cibelli G. Rougon G. Mattei M.G. Goridis C. J. Cell Biol. 1989; 109: 775-788Crossref PubMed Scopus (224) Google Scholar, 6Gennarini G. Durbec P. Boned A. Rougon G. Goridis C. Neuron. 1991; 6: 595-606Abstract Full Text PDF PubMed Scopus (142) Google Scholar). As found for other axonal members of the IgSF, the F11 polypeptide shows a broad binding activity. Interactions with the cell surface proteins NgCAM, NrCAM, neurofascin, Caspr, and RPTPβ/ζ and the ECM glycoproteins tenascin-R (TN-R) and tenascin-C (TN-C) have been revealed by in vitro assays (7Volkmer H. Zacharias U. Nörenberg U. Rathjen F.G. J. Cell Biol. 1998; 142: 1083-1093Crossref PubMed Scopus (93) Google Scholar, 8Brümmendorf T. Hubert M. Treubert U. Leuschner R. Tárnok A. Rathjen F.G. Neuron. 1993; 10: 711-727Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 9Zisch A.H. D'Alessandri L. Ranscht B. Falchetto R. Winterhalter K.H. Vaughan L. J. Cell Biol. 1992; 119: 203-213Crossref PubMed Scopus (154) Google Scholar, 10Morales G. Hubert M. Brümmendorf T. Treubert U. Tárnok A. Schwarz U. Rathjen F.G. Neuron. 1993; 11: 1113-1122Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 11Peles E. Nativ M. Campbell P.L. Sakurai T. Martinez R. Lev S. Clary D.O. Schilling J. Barnea G. Plowman G.D. Grumet M. Schlessinger J. Cell. 1995; 82: 251-260Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 12Peles E. Nativ M. Lustig M. Grumet M. Schilling J. Martinez R. Plowman G.D. Schlessinger J. EMBO. J. 1997; 16: 978-988Crossref PubMed Scopus (358) Google Scholar, 13Sakurai T. Lustig M. Nativ M. Hemperly J.J. Schlessinger J. Peles E. Grumet M. J. Cell Biol. 1997; 136: 907-918Crossref PubMed Scopus (156) Google Scholar, 14Pesheva P. Gennarini G. Goridis C. Schachner M. Neuron. 1993; 10: 69-82Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 15Nörenberg U. Hubert M. Brümmendorf T. Tárnok A. Rathjen F.G. J. Cell Biol. 1995; 130: 473-484Crossref PubMed Scopus (56) Google Scholar, 16Rathjen F.G. Wolff J.M. Chiquet Ehrismann R. Development. 1991; 113: 151-164Crossref PubMed Google Scholar). The N-terminal Ig domains 1–4 of the F11 polypeptide are sufficient for interactions with NgCAM, NrCAM, TN-R, and TN-C, although binding assays with domain-specific anti-F11 monoclonal antibodies and with F11 domain deletion mutants suggest that individual domains of the four N-terminal domains might be more important for specific bindings (8Brümmendorf T. Hubert M. Treubert U. Leuschner R. Tárnok A. Rathjen F.G. Neuron. 1993; 10: 711-727Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 9Zisch A.H. D'Alessandri L. Ranscht B. Falchetto R. Winterhalter K.H. Vaughan L. J. Cell Biol. 1992; 119: 203-213Crossref PubMed Scopus (154) Google Scholar, 10Morales G. Hubert M. Brümmendorf T. Treubert U. Tárnok A. Schwarz U. Rathjen F.G. Neuron. 1993; 11: 1113-1122Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 15Nörenberg U. Hubert M. Brümmendorf T. Tárnok A. Rathjen F.G. J. Cell Biol. 1995; 130: 473-484Crossref PubMed Scopus (56) Google Scholar). The interaction between immobilized F11 and neuronal NrCAM induces neurite outgrowth of tectal cells (10Morales G. Hubert M. Brümmendorf T. Treubert U. Tárnok A. Schwarz U. Rathjen F.G. Neuron. 1993; 11: 1113-1122Abstract Full Text PDF PubMed Scopus (108) Google Scholar). TN-R and TN-C are two major members of the tenascin family of ECM glycoproteins. These multidomain proteins are composed of a cysteine-rich segment, epidermal growth factor-like repeats, FNIII-like domains, and a segment similar to the β- and γ-chains of fibrinogen (for a review, see Ref. 17Chiquet Ehrismann R. Hagios C. Matsumoto K. Perspect. Dev. Neurobiol. 1994; 2: 3-7PubMed Google Scholar). TN-R and TN-C show striking functional analogies, but within the nervous system TN-R has a more restricted localization than TN-C, has a different developmental time course, and is synthesized by oligodendrocytes and a subpopulation of neurons rather than predominantly by astroglia (18Faissner A. Cell Tissue Res. 1997; 290: 331-341Crossref PubMed Scopus (132) Google Scholar). TN-R and TN-C form oligomeric structures as revealed by rotatory shadowing electron microscopy (19Nörenberg U. Wille H. Wolff J.M. Frank R. Rathjen F.G. Neuron. 1992; 8: 849-863Abstract Full Text PDF PubMed Scopus (134) Google Scholar, 20Erickson H.P. Inglesias J.L. Nature. 1984; 311: 267-269Crossref PubMed Scopus (199) Google Scholar). Multiple ligands have been described for TN-R and TN-C including cell surface proteins such as F11, axonin-1, CALEB, RPTPβ/ζ, integrins, and ECM glycoproteins and proteoglycans such as neurocan, phosphacan, versican, brevican, heparin, and fibronectin (21Milev P. Meyer-Puttlitz B. Margolis R.K. Margolis R.U. J. Biol. Chem. 1995; 270: 24650-24653Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 22Milev P. Chiba A. Haring M. Rauvala H. Schachner M. Ranscht B. Margolis R.K. Margolis R.U. J. Biol. Chem. 1998; 273: 6998-7005Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 23Schumacher S. Volkmer H. Buck F. Otto A. Tárnok A. Roth S. Rathjen F.G. J. Cell Biol. 1997; 136: 895-906Crossref PubMed Scopus (51) Google Scholar, 24Weber P. Ferber P. Fischer R. Winterhalter K.H. Vaughan L. FEBS Lett. 1996; 389: 304-308Crossref PubMed Scopus (17) Google Scholar, 25Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (216) Google Scholar, 26Varnum-Finney B. Venstrom K. Muller U. Kypta R. Backus C. Chiquet M. Reichardt L.F. Neuron. 1995; 14: 1213-1222Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 27Aspberg A. Miura R. Bourdoulous S. Shimonaka M. Heinegard D. Schachner M. Ruoslahti E. Yamaguchi Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10116-10121Crossref PubMed Scopus (241) Google Scholar, 28Chung C.Y. Zardi L. Erickson H.P. J. Biol. Chem. 1995; 270: 29012-29017Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 29Sriramarao P. Mendler M. Bourdon M.A. J. Cell Sci. 1993; 105: 1001-1012Crossref PubMed Google Scholar). Interactions between TN-R or TN-C and cell surface molecules affect cell adhesion and neurite growth. The responses can be either stimulatory or inhibitory, depending on the specific neuronal cell type studied, the assay design (choise situation on patterned substrates or homogeneous substrate), and they are probably mediated by separate domains. Neurite outgrowth-promoting, cell-binding, antiadhesive, and nonpermissive regions have been identified in TN-R and TN-C (15Nörenberg U. Hubert M. Brümmendorf T. Tárnok A. Rathjen F.G. J. Cell Biol. 1995; 130: 473-484Crossref PubMed Scopus (56) Google Scholar,30Prieto A.L. Andersson-Fisone C. Crossin K.L. J. Cell Biol. 1992; 119: 663-678Crossref PubMed Scopus (147) Google Scholar, 31Xiao Z.C. Taylor J. Montag D. Rougon G. Schachner M. Eur. J. Neurosci. 1996; 8: 766-782Crossref PubMed Scopus (89) Google Scholar, 32Yokosaki Y. Matsuura N. Higashiyama S. Murakami I. Obara M. Yamakido M. Shigeto N. Chen J. Sheppard D. J. Biol. Chem. 1998; 273: 11423-11428Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 33Gotz B. Scholze A. Clement A. Joester A. Schutte K. Wigger F. Frank R. Spiess E. Ekblom P. Faissner A. J. Cell Biol. 1996; 132: 681-699Crossref PubMed Scopus (141) Google Scholar, 34Fischer D. Brown Ludi M. Schulthess T. Chiquet Ehrismann R. J. Cell Sci. 1997; 110: 1513-1522Crossref PubMed Google Scholar, 35Spring J. Beck K. Chiquet Ehrismann R. Cell. 1989; 59: 325-334Abstract Full Text PDF PubMed Scopus (322) Google Scholar, 36Wehrle Haller B. Chiquet M. J. Cell Sci. 1993; 106: 597-610Crossref PubMed Google Scholar). These observations could also reflect the differential expression of receptor complexes on the responding cells or of downstream effector mechanisms that control the growth cone. The short term attachment site for retinal cells within TN-R was allocated to FNIII domain 8, while the site interacting with F11 has been mapped to the FNIII domains 2 and 3. Furthermore, TN-R FNIII domain 2 has been shown to mediate homophilic interaction (15Nörenberg U. Hubert M. Brümmendorf T. Tárnok A. Rathjen F.G. J. Cell Biol. 1995; 130: 473-484Crossref PubMed Scopus (56) Google Scholar). Cell attachment sites within TN-C have been identified in FNIII domains 3 and 6–8 and in the fibrinogen-like globe (25Prieto A.L. Edelman G.M. Crossin K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10154-10158Crossref PubMed Scopus (216) Google Scholar, 26Varnum-Finney B. Venstrom K. Muller U. Kypta R. Backus C. Chiquet M. Reichardt L.F. Neuron. 1995; 14: 1213-1222Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 29Sriramarao P. Mendler M. Bourdon M.A. J. Cell Sci. 1993; 105: 1001-1012Crossref PubMed Google Scholar, 35Spring J. Beck K. Chiquet Ehrismann R. Cell. 1989; 59: 325-334Abstract Full Text PDF PubMed Scopus (322) Google Scholar, 37Aukhil I. Joshi P. Yan Y. Erickson H.P. J. Biol. Chem. 1993; 268: 2542-2553Abstract Full Text PDF PubMed Google Scholar), whereas F11 binds to FNIII domains 5 and 6 (24Weber P. Ferber P. Fischer R. Winterhalter K.H. Vaughan L. FEBS Lett. 1996; 389: 304-308Crossref PubMed Scopus (17) Google Scholar). The multidomain and oligomeric structure of ECM glycoproteins like TN-R and TN-C, together with their elastic properties (38Oberhauser A.F. Marszalek P.E. Erickson H.P. Fernandez J.M. Nature. 1998; 393: 181-185Crossref PubMed Scopus (748) Google Scholar), suggests that they may serve to link cell surface molecules between different cells and to the ECM network. Immunohistochemistry and in situ hybridization reveal that F11 shows a significant overlap in its expression pattern with TN-R or TN-C, but there are also spatial and temporal differences (14Pesheva P. Gennarini G. Goridis C. Schachner M. Neuron. 1993; 10: 69-82Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 16Rathjen F.G. Wolff J.M. Chiquet Ehrismann R. Development. 1991; 113: 151-164Crossref PubMed Google Scholar, 39Fuss B. Wintergerst E.S. Bartsch U. Schachner M. J. Cell Biol. 1993; 120: 1237-1249Crossref PubMed Scopus (156) Google Scholar). This is consistent with the possibility of interactions between these proteins as well as competition between them for ligands. Although multiple interactions between CAMs and ECM molecules have been described, the question remains which interactions are of functional importance and how these interactions are regulated during complex physiological processes like axon guidance and neural cell migration. Different mechanisms like alternative splicing, posttranslational modifications, and complex formation with other proteins have been proposed in addition to spatial and temporal regulation of protein expression (7Volkmer H. Zacharias U. Nörenberg U. Rathjen F.G. J. Cell Biol. 1998; 142: 1083-1093Crossref PubMed Scopus (93) Google Scholar). Alternative splicing of the VASE exon has been shown to regulate NCAM-mediated neurite outgrowth (40Liu L. Haines S. Shew R. Akeson R.A. J. Neurosci. Res. 1993; 35: 327-345Crossref PubMed Scopus (39) Google Scholar, 41Doherty P. Moolenaar C.E. Ashton S.V. Michalides R.J. Walsh F.S. Nature. 1992; 356: 791-793Crossref PubMed Scopus (127) Google Scholar), and neurite outgrowth induced by TN-C is modulated by alternative splicing of the FNIII domains (33Gotz B. Scholze A. Clement A. Joester A. Schutte K. Wigger F. Frank R. Spiess E. Ekblom P. Faissner A. J. Cell Biol. 1996; 132: 681-699Crossref PubMed Scopus (141) Google Scholar, 42Meiners S. Geller H.M. Mol. Cell. Neurosci. 1997; 10: 100-116Crossref PubMed Scopus (45) Google Scholar). Varying the carbohydrate groups attached to cell surface proteins is another possibility of regulating cell interactions, as it has been described for polysialylation of NCAM (43Tang J. Rutishauser U. Landmesser L. Neuron. 1994; 13: 405-414Abstract Full Text PDF PubMed Scopus (238) Google Scholar,44Rutishauser U. Landmesser L. Trends Neurosci. 1991; 14: 528-532Abstract Full Text PDF PubMed Scopus (94) Google Scholar). The adhesion strength of CAMs can be modulated by intracellular signaling. T cell receptor engagement increases the affinity of the integrin leukocyte function-associated antigen-1 for its ligand, intercellular adhesion molecule-1, probably by a conformational change transmitted across the plasma membrane (45Dustin M.L. Springer T.A. Nature. 1989; 341: 619-624Crossref PubMed Scopus (1286) Google Scholar). Similarly, neurofascin-dependent cell aggregation is regulated by tyrosine phosphorylation of its cytoplasmic domain (46Tuvia S. Garver T.D. Bennett V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12957-12962Crossref PubMed Scopus (112) Google Scholar). Furthermore, complex formation may occlude binding sites on the ligand and/or receptor, thereby regulating cell-cell and cell-matrix interactions. The multiple potential interactions of F11 already identified make it an interesting candidate to evaluate whether different ligands can interact simultaneously or compete with each other for binding to F11. Such information for F11 and other CAMs together with expression patterns would provide a basis for evaluating functions for various interactions in vivo. Here we investigate whether the ECM glycoproteins TN-R and TN-C may regulate the interactions of F11 with its ligands. Competition and sandwich binding assays have been used to establish that TN-R or TN-C compete with NrCAM or NgCAM for binding to the F11 polypeptide. Cellular assays demonstrate that TN-R and TN-C modulate neurite outgrowth of tectal cells on immobilized F11 differentially and that in the presence of TN-R neurite extension is mediated by β1 integrins instead of NrCAM as cellular receptor. NgCAM, NrCAM, TN-R, and TN-C were purified from detergent (CAMs) and urea (TNs) extracts, respectively; F11 and axonin-1 were purified from phosphatidylinositol-specific phospholipase C-treated extracts of plasma membrane preparations of adult chicken brains followed by immunoaffinity chromatography as described previously (4Rathjen F.G. Wolff J.M. Frank R. Bonhoeffer F. Rutishauser U. J. Cell Biol. 1987; 104: 343-353Crossref PubMed Scopus (282) Google Scholar, 8Brümmendorf T. Hubert M. Treubert U. Leuschner R. Tárnok A. Rathjen F.G. Neuron. 1993; 10: 711-727Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 10Morales G. Hubert M. Brümmendorf T. Treubert U. Tárnok A. Schwarz U. Rathjen F.G. Neuron. 1993; 11: 1113-1122Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 16Rathjen F.G. Wolff J.M. Chiquet Ehrismann R. Development. 1991; 113: 151-164Crossref PubMed Google Scholar, 47Rathjen F.G. Wolff J.M. Chang S. Raper J. Acta Histochem. Suppl. 1990; 38: 59-63PubMed Google Scholar, 48Rader C. Kunz B. Lierheimer R. Giger R.J. Berger P. Tittmann P. Gross H. Sonderegger P. EMBO J. 1996; 15: 2056-2068Crossref PubMed Scopus (55) Google Scholar). The purity of isolates was analyzed by SDS-PAGE. Isolation of monoclonal antibodies and generation of Fab fragments of polyclonal antibodies to these antigens are detailed elsewhere (2Brümmendorf T. Wolff J.M. Frank R. Rathjen F.G. Neuron. 1989; 2: 1351-1361Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 10Morales G. Hubert M. Brümmendorf T. Treubert U. Tárnok A. Schwarz U. Rathjen F.G. Neuron. 1993; 11: 1113-1122Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 49Rathjen F.G. Wolff J.M. Chang S. Bonhoeffer F. Raper J.A. Cell. 1987; 51: 841-849Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 50Wolff J.M. Rathjen F.G. Frank R. Roth S. Eur. J. Biochem. 1987; 168: 551-561Crossref PubMed Scopus (40) Google Scholar). TN-R fragments were expressed as glutathione S-transferase fusion proteins and purified as described (15Nörenberg U. Hubert M. Brümmendorf T. Tárnok A. Rathjen F.G. J. Cell Biol. 1995; 130: 473-484Crossref PubMed Scopus (56) Google Scholar). Monoclonal anti-β1 integrin antibody W1B10 was purchased from Sigma (Deisenhofen, Germany), and JG22 was purified from the supernatant of hybridomas that were obtained from the Developmental Studies Hybridoma Bank (John Hopkins University School of Medicine, Baltimore, MD). Immunoaffinity-purified F11 and NrCAM were conjugated to red fluorescent microspheres of 0.5 μm in diameter according to the manufacturer's protocol (Bioclean, Duke Scientific Corp., Palo Alto, CA), and residual binding sites were blocked by bovine serum albumin. COS7 cells were transiently transfected with F11- or NgCAM-encoding plasmids using the DEAE-dextran method as described previously (8Brümmendorf T. Hubert M. Treubert U. Leuschner R. Tárnok A. Rathjen F.G. Neuron. 1993; 10: 711-727Abstract Full Text PDF PubMed Scopus (172) Google Scholar, 51Buchstaller A. Kunz S. Berger P. Kunz B. Ziegler U. Rader C. Sonderegger P. J. Cell Biol. 1996; 135: 1593-1607Crossref PubMed Scopus (106) Google Scholar). After 24 h, transfected cells were transferred to poly-l-lysine (100 μg/ml)-coated eight-well multitest slides (ICN, Costa Mesa, CA), grown overnight, and then incubated with 30 μl of Dulbecco's modified Eagle's medium, 10% fetal calf serum containing 0.3 μl of microsphere solution. For competition binding assays, NrCAM-coated microspheres were allowed to bind to F11-expressing COS7 cells in the absence or presence of TN-R (10 μg/ml), TN-C (20 μg/ml), NgCAM (20 μg/ml), NrCAM (20 μg/ml), TN-R FNIII 2 and 3 (50 μg/ml), or TN-R FNIII 4 to A (50 μg/ml). In parallel, NgCAM-expressing COS7 cells were incubated with F11-coated microspheres in the absence or presence of soluble proteins as described above. For sandwich binding assays, F11-coated microspheres were incubated with F11-expressing COS7 cells in the absence or presence of TN-R (10 μg/ml), TN-C (20 μg/ml), NgCAM (20 μg/ml), TN-R FNIII 2 and 3 (50 μg/ml), or TN-R FNIII 4 to A (50 μg/ml). After incubation for 1 h at 37 °C, cells were washed, fixed in 4% formaldehyde/phosphate-buffered saline, and stained for F11 expression by indirect immunofluorescence using polyclonal antibodies to F11 and fluorescein isothiocyanate-conjugated anti-rabbit polyclonal antibodies (Dianova, Hamburg, FRG). Images were analyzed for red fluorescence, indicating microsphere binding to the cell surface, and for green fluorescence, indicating F11 or NgCAM expression. For quantification, double fluorescence detection was performed with a confocal microscope (MRC 1024; Bio-Rad). Digital images were analyzed using the public domain NIH IMAGE program (developed at the National Institutes of Health and available on the Internet) as detailed previously (52Volkmer H. Leuschner R. Zacharias U. Rathjen F.G. J. Cell Biol. 1996; 135: 1059-1069Crossref PubMed Scopus (81) Google Scholar). F11- or NgCAM-expressing cells were marked, and the mean pixel intensity of fluorescing cells was determined as a measure for F11 or NgCAM expression. The red channel analysis was overlaid with the same frames, and mean pixel intensity was measured to quantify microsphere binding. Microsphere binding to about 20 cells/experiment was normalized to F11 or NgCAM expression, and background values measured over cells that did not express F11 or NgCAM were subtracted. Culture dishes (Petriperm; Bachhofer, Reutlingen, Germany) were coated with 100 μl of affinity-purified F11 (2 μg/ml), NgCAM (10 μg/ml), axonin-1 (10 μg/ml), laminin (10 μg/ml), and bovine serum albumin (10 μg/ml) that was spread over 1 cm2 delineated by a silicon fitting at 4 °C overnight. Note that in previously published experiments, 10 μl of a F11 solution at a concentration of 100 μg/ml was used for coating (10Morales G. Hubert M. Brümmendorf T. Treubert U. Tárnok A. Schwarz U. Rathjen F.G. Neuron. 1993; 11: 1113-1122Abstract Full Text PDF PubMed Scopus (108) Google Scholar, 15Nörenberg U. Hubert M. Brümmendorf T. Tárnok A. Rathjen F.G. J. Cell Biol. 1995; 130: 473-484Crossref PubMed Scopus (56) Google Scholar). Residual binding sites were blocked by washing and incubating with Dulbecco's modified Eagle's medium/10% fetal calf serum for 30 min at 37 °C. Single cell suspensions were obtained by dissociation of chick tecta of embryonic day 6 in a trypsin solution (1 mg/ml, 20 min at 37 °C) and subsequent trituration. After resuspension in Dulbecco's modified Eagle's medium/10% fetal calf serum, 15,000 cells/well were plated on immobilized F11 and grown in the absence or presence of TN-R (0.6–50 μg/ml), TN-C (1.2–20 μg/ml), NrCAM (20 μg/ml), F11 (20 μg/ml), or axonin-1 (20 μg/ml). Monoclonal antibodies to β1 integrin W1B10 and JG22 were added at the time of plating at a final concentration of 20 μg/ml each. All other monoclonal antibodies and Fab fragments of polyclonal antibodies to different proteins were used at a final concentration of 10 and 200 μg/ml, respectively. For preincubation studies, immobilized F11 was incubated with TN-R (10 μg/ml) or TN-C (20 μg/ml) after blocking for 1 h at 37 °C. Unbound TN-R and TN-C were removed by extensive washing with Dulbecco's modified Eagle's medium, 10% fetal calf serum prior to the addition of cells. After cultivation for 40 h, cells were fixed in 4% formaldehyde/phosphate-buffered saline and stained by indirect immunofluorescence using monoclonal antibody A2B5 and Cy3-conjugated anti-mouse polyclonal antibodies (Dianova; Hamburg, FRG). The number of attached cells and the number of extending neurites were quantified with Genias imaging software (Image Works; Teltow, Germany (52Volkmer H. Leuschner R. Zacharias U. Rathjen F.G. J. Cell Biol. 1996; 135: 1059-1069Crossref PubMed Scopus (81) Google Scholar)) and calculated as the percentage of control cultures. An interesting feature of axonal IgSF members and ECM glycoproteins is their complex binding pattern. The binding sites for NrCAM, NgCAM, TN-R, and TN-C have been mapped to a similar region of the F11 polypeptide comprising Ig domains 1–4. This makes it important to evaluate whether these proteins can bind simultaneously to F11 or compete with each other for identical or overlapping regions within the F11 polypeptide. To address this question, we established a competitive cellular binding assay and analyzed the interaction between F11 and NrCAM and between F11 and NgCAM in the presence or absence of soluble TN-R, TN-C, NgCAM, or NrCAM (Fig. 1). Binding of microspheres was quantified and related to the expression of F11 using a confocal microscope as described previously (Fig.1 G) (52Volkmer H. Leuschner R. Zacharias U. Rathjen F.G. J. Cell Biol. 1996; 135: 1059-1069Crossref PubMed Scopus (81) Google Scholar). No direct binding of NrCAM or of NgCAM to TN-R or to TN-C, respectively, has been detected so far in different binding assays (8Brümmendorf T. Hubert M. Treubert U. Leuschner R. Tárnok A. Rathjen F.G. Neuron. 1993; 10: 711-727Abstract Full Text PDF PubMed Scopus (172) Google Scholar). As described previously, NrCAM-coated beads bound to F11-expressing COS7 cells but not to untransfected cells within the same culture (Fig. 1 A) (10Morales G. Hubert M. Brümmendorf T. Treubert U. Tárnok A. Schwarz U. Rathjen F.G. Neuron. 1993; 11: 1113-1122Abstract Full Text PDF PubMed Scopus (108) Google Scholar). The presence of soluble TN-R or TN-C led to a significant reduction in NrCAM bead binding to F11-transfected COS7 cells as depicted in Fig. 1, B, D, and G. This inhibitory effect of TN-R and TN-C was dose-dependent and could be specifically abolished by the addition of Fab fragments of polyclonal antibodies to TN-R or TN-C, respectively (data not shown). In contrast, soluble NgCAM (which is known to bind to F11 (8Brümmendorf T. Hubert M. Treubert U. Leuschner R. Tárnok A. Rathjen F.G. Neuron. 1993; 10: 711-727Abstract Full Text PDF PubMed Scopus (172) Google Scholar)) did not have a similar inhibitory effect on the binding of NrCAM beads to F11-expressing COS7 cells (Fig. 1, C and G). The addition of the recombinant TN-R fragment FNIII 2 and 3 comprising the F11 binding site within TN-R (15Nörenberg U. Hubert M. Brümmendorf T. Tárnok A. Rathjen F.G. J. Cell Biol. 1995; 130: 473-484Crossref PubMed Scopus (56) Google Scholar) did not suppress NrCAM bead binding to F11-expressing COS7 cells (Fig.1, E and G), suggesting that the inhibitory effect of intact TN-R on NrCAM binding is probably caused by steric hindrance. The interaction between F11 and NgCAM was analyzed by quantifying the binding of F11-coated microspheres to NgCAM expressing COS7 cells (Fig.1 I). Similar to the NrCAM-F11 interaction described above, the addition of soluble TN-R and TN-C resulted in a significant reduction of F11 coated beads to bind to NgCAM expressing COS7 cells (Fig. 1 I). However, in this assay system the recombinant TN-R fragment FNIII 2–3 was able to mimic the inhibitory effect of intact TN-R, which was not observed for the TN-R fragments FNIII 4 to A, indicating further specificity. As expected, the addition of soluble F11 could compete for bead binding in the F11-NrCAM and in the F11-NgCAM binding assay (data not shown). Taken together, these results suggest that the ECM glycoprotein TN-R or TN-C can block binding of the IgSF members NrCAM or NgCAM to F11 probably by competing for overlapping binding sites and/or by steric hindrance as illustrated in Fig. 1, H and J. TN-R and TN-C might therefore regulate functional interactions of the F11 polypeptide. Previous studies by us have shown that tectal cells extend long neurites on immobilized F11, and the cellular receptor mediating neurite extension has been identified as NrCAM (10Morales G. Hubert M. Brümmendorf T. Treubert U. Tárnok A. Schwarz U. Rathjen F.G. Neuron. 1993; 11: 1113-1122Abstract" @default.
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• W2016367573 title "Functional Interactions of the Immunoglobulin Superfamily Member F11 Are Differentially Regulated by the Extracellular Matrix Proteins Tenascin-R and Tenascin-C" @default.
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