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Interaction between 5′-nucleotidase and extracellular matrix
BioL Cell (1991), 73
EXPRESSION OF EMBRYONIC NEURAL CELL ADHESION MOLECULE (E-N-CAM) AND DYSTROPHIN IN REGENERATING TAIL SPINAL CORD OF AMPHIBIAN URODELES. Xavier CAUBIT (1), Jean.Pierre ARSANTO (1), Monique DIANO (1), Genevieve ROUGON (1), Dominique FIGARELLA (1), Nathalie AUGIER (2), Dominique MORNET (2) and Yves THOUVENY (t). (1) Laboratoire de Biologie de la diff6rendation Cellulaire, L.A. C.N.R.S. N" 179, Facult6 des Sciences de Marseille-Luminy, 13288 MARSEILLE CEDEX 9, FRANCE, and (2) Pathologie Mol6culaire du Muscle, INSERM U.300, Facultd de Pharmacie,Av. Ch. Flahault, 34060 MONTPELLIER, FRANCE. Expression pattems of the highly sialylated, emb~]onic form, of the Neural Cell Adhesion Molecule (E-N-CAM) and of the cytoskeletal protein Dystrophin were investigated by indirect immunolluorescence and western blotting during post-amputation regeneration of the tail spinal cord (TSC) of newts Pleurndeles waltlii and Notophthalmus viridescens. A moneclonai antibody to group B meninoococcus polysaccharides distinguishing E-N-CAM from adult N-CAM, a monoclonal antibody DyH 5A3 directed against the Cderminal region (residues 3357-3660), and polyclonal and monecional (C-4G10) antibodies raised against the speclrin-like domain (residues 1173-1728)of the chicken skeletal muscle Dystrophin were used. The level of E-N-CAM expression is very low in the normal TSC of the adult animals whereas it is high in the regenerating as well as in the embryonic developing TSC. E-N-CAM expression in the regeneraling TSC is maximal 4 to 6 weeks following amputation, and then gradually decreases. E-N-CAM immunoreactivity observed along the ependymal cell surfaces in regenerating TSC supports the view thai the capacities of 1"80 plasticity in urodeles are correlated with reexprassion of E-N-CAM in ependyma. Furthermore, E.N-CAM expression in axonal compartments of the regenerating TSC suggests that the molecule could be implicated in neurile growth. On the other hand, the sequential accumulation of dystrophin in the zonula adherens, sepia of K611ikerand pia maner as TSC regeneration pro0resses, su0gests thai this cyloskeletal protein, thought to play a crucial role in the stabilization of myoliber plasma membrane, may be also involved in the maintenance of spinal cord organization.
FUNCTIONALSTUDIES AND LOCAUZATIONOF F3 NEURONALCELL SURFACE PROTEIN. P. Durbec, G. Gennarini °, C. Faivre-Sarrailh, O. Gorldis* and G. Rougon. CNRS URA 179, case 901 and *CIML, case 906; Pare Sclentifique et Technologique de Luminy. 13288 Marseille cedex 9. We previously Identified, clone¢l and sequenced a glypiated molecule F3 belonging to a subclass of proteins of the immunoglobulin superfamily with preferential localization on neurites in neuronal cell cultures (Gennarini el el. 1989 a, b). We probed the function of F3 using a eDNA Iransfection approach. Mouse F3 eDNA was Iransfected into a line of CHO cells. Biochemical analysis of the transfected cells established that F3 eDNA codes for a GPI-anchored protein that exists both under membrane bound and soluble form released into the culture medium. F3 expressing transfectants exhibited enhanced adhesive properties aggregating with faster kinetics and forming larger aggregates than F3-negative control cells. However, the protein appears to mediate low affinity Interactions suggesting that F3 may be involved in weak adhesion between cells. When used as culture substrata for sensory neurons, F3transfected cells showed a markedly enhanced ability to promote neurite outgrowth compared with nontransfected cells (Gennarini et al. 1991). We investigated the distribution of F3 In the developing cerebellum by immunocytochemistry at the light and electron microscopic levels, F3 was confined to subsets of neuronal types, In developing cerebellum, the granular cell axons strongly express F3 as soon as they begin to grow, consistent with a functional role in promoting directional outgrowth of neuronal processes. In adult cerebellum, F3 was found at only one side of the synaptic sites never at both. This suggest that F3 might also play a role in the formation and maintenance of synapses and that, if F3 functions as an adhesion molecule, It is likely to mediate heterophilic interactions. Gennarinl el el. 1989a. J. Neurosc. Res. ~2, 1.22. Gennarini et el. 1989b. J. Cell Blol. 109, 775-788. Gennarini el 81. 1991, Neuron, 6, 595-606,
INTERACTION BETWEEN 5'-NUCLEOTIDASE AND EXTRACELLULAR MATRIX. Bruno HEHUL, Mich~le AUBER¥ and Partite CODOGNO. Unite 180 INSERM, 45 rue des Salnts-P~res, 75006 Paris, France. 5'-nucleotidase (5'-N) isolated from chicken gizzard smooth muscle was found to be, in vltro, a extracellular matrix ligand for flbronectln (FN) and laminln (LN) (1). In addition, several assays using poly- and monoclonal antibodies directed against this glycoprotein have shown that this ectomembrane enzyme is Involved in the spreading of chicken embryo flbroblasts and myoblasts (2, 3). Recently, we have demonstrated that the specific activity of 5'-N and its presence on the surface of myoblasts in culture were more enhanced on a support coated with LN than on those coated with FN or gelatin. The colocalizatlon observed, ~n vivo, between 5'-N and LM, during development and the positioning of striated muscle tissues, suggested a for a physiological r61e for 5'-N as ligand of LN. In parallel, we have investigated the relationship that might exist between 5'-N and other LM-blnding proteins (68 kDa (4}, CBG 72 (5) and Integrlns ). 5'-N differs from other receptors by its antigenic proprieties (6) and more partlculary with integrln by its mode of interaction with the matrix (7). However, 5'-N and integrln were found colocalized in specialized structures: Myotendinous junctions and adult striated muscle sarcolemmas.
(1) Stochaj, U. et al., Biochim. Biophys. Acta 992, 385-392 (1989). (2) Codogno, P., et al., Exp. Cell Res. 174, 344-354 (1988). (3) Mehul, B., et al., Cell Biol. Iter. Rep. 14, 155-164 (1990). (4) Lesot, H., et al., Eur. /4ol. Biol. Organ. J. 2, 861-865 (1983}. (5) Moutsita, R. et al., Exp. Cell Res. 192, 236-242 (1991). (6) Gundula, R. et al., Biochim. Biophys. Acta, 994, 258-263 (1989}. (7) Stochaj, U. et al., Eur. J. Cell Biol. 30, 167-176 (1990).
2.
EXPRESSION OF EMBRYONIC NEURAL CELL ADHESION MOLECULE (E-N-CAM) AND DYSTROPHIN IN REGENERATING TAIL SPINAL CORD OF AMPHIBIAN URODELES. Xavier CAUBIT (1), Jean.Pierre ARSANTO (1), Monique DIANO (1), Genevieve ROUGON (1), Dominique FIGARELLA (1), Nathalie AUGIER (2), Dominique MORNET (2) and Yves THOUVENY (t). (1) Laboratoire de Biologie de la diff6rendation Cellulaire, L.A. C.N.R.S. N" 179, Facult6 des Sciences de Marseille-Luminy, 13288 MARSEILLE CEDEX 9, FRANCE, and (2) Pathologie Mol6culaire du Muscle, INSERM U.300, Facultd de Pharmacie,Av. Ch. Flahault, 34060 MONTPELLIER, FRANCE. Expression pattems of the highly sialylated, emb~]onic form, of the Neural Cell Adhesion Molecule (E-N-CAM) and of the cytoskeletal protein Dystrophin were investigated by indirect immunolluorescence and western blotting during post-amputation regeneration of the tail spinal cord (TSC) of newts Pleurndeles waltlii and Notophthalmus viridescens. A moneclonai antibody to group B meninoococcus polysaccharides distinguishing E-N-CAM from adult N-CAM, a monoclonal antibody DyH 5A3 directed against the Cderminal region (residues 3357-3660), and polyclonal and monecional (C-4G10) antibodies raised against the speclrin-like domain (residues 1173-1728)of the chicken skeletal muscle Dystrophin were used. The level of E-N-CAM expression is very low in the normal TSC of the adult animals whereas it is high in the regenerating as well as in the embryonic developing TSC. E-N-CAM expression in the regeneraling TSC is maximal 4 to 6 weeks following amputation, and then gradually decreases. E-N-CAM immunoreactivity observed along the ependymal cell surfaces in regenerating TSC supports the view thai the capacities of 1"80 plasticity in urodeles are correlated with reexprassion of E-N-CAM in ependyma. Furthermore, E.N-CAM expression in axonal compartments of the regenerating TSC suggests that the molecule could be implicated in neurile growth. On the other hand, the sequential accumulation of dystrophin in the zonula adherens, sepia of K611ikerand pia maner as TSC regeneration pro0resses, su0gests thai this cyloskeletal protein, thought to play a crucial role in the stabilization of myoliber plasma membrane, may be also involved in the maintenance of spinal cord organization.
FUNCTIONALSTUDIES AND LOCAUZATIONOF F3 NEURONALCELL SURFACE PROTEIN. P. Durbec, G. Gennarini °, C. Faivre-Sarrailh, O. Gorldis* and G. Rougon. CNRS URA 179, case 901 and *CIML, case 906; Pare Sclentifique et Technologique de Luminy. 13288 Marseille cedex 9. We previously Identified, clone¢l and sequenced a glypiated molecule F3 belonging to a subclass of proteins of the immunoglobulin superfamily with preferential localization on neurites in neuronal cell cultures (Gennarini el el. 1989 a, b). We probed the function of F3 using a eDNA Iransfection approach. Mouse F3 eDNA was Iransfected into a line of CHO cells. Biochemical analysis of the transfected cells established that F3 eDNA codes for a GPI-anchored protein that exists both under membrane bound and soluble form released into the culture medium. F3 expressing transfectants exhibited enhanced adhesive properties aggregating with faster kinetics and forming larger aggregates than F3-negative control cells. However, the protein appears to mediate low affinity Interactions suggesting that F3 may be involved in weak adhesion between cells. When used as culture substrata for sensory neurons, F3transfected cells showed a markedly enhanced ability to promote neurite outgrowth compared with nontransfected cells (Gennarini et al. 1991). We investigated the distribution of F3 In the developing cerebellum by immunocytochemistry at the light and electron microscopic levels, F3 was confined to subsets of neuronal types, In developing cerebellum, the granular cell axons strongly express F3 as soon as they begin to grow, consistent with a functional role in promoting directional outgrowth of neuronal processes. In adult cerebellum, F3 was found at only one side of the synaptic sites never at both. This suggest that F3 might also play a role in the formation and maintenance of synapses and that, if F3 functions as an adhesion molecule, It is likely to mediate heterophilic interactions. Gennarinl el el. 1989a. J. Neurosc. Res. ~2, 1.22. Gennarini et el. 1989b. J. Cell Blol. 109, 775-788. Gennarini el 81. 1991, Neuron, 6, 595-606,
INTERACTION BETWEEN 5'-NUCLEOTIDASE AND EXTRACELLULAR MATRIX. Bruno HEHUL, Mich~le AUBER¥ and Partite CODOGNO. Unite 180 INSERM, 45 rue des Salnts-P~res, 75006 Paris, France. 5'-nucleotidase (5'-N) isolated from chicken gizzard smooth muscle was found to be, in vltro, a extracellular matrix ligand for flbronectln (FN) and laminln (LN) (1). In addition, several assays using poly- and monoclonal antibodies directed against this glycoprotein have shown that this ectomembrane enzyme is Involved in the spreading of chicken embryo flbroblasts and myoblasts (2, 3). Recently, we have demonstrated that the specific activity of 5'-N and its presence on the surface of myoblasts in culture were more enhanced on a support coated with LN than on those coated with FN or gelatin. The colocalizatlon observed, ~n vivo, between 5'-N and LM, during development and the positioning of striated muscle tissues, suggested a for a physiological r61e for 5'-N as ligand of LN. In parallel, we have investigated the relationship that might exist between 5'-N and other LM-blnding proteins (68 kDa (4}, CBG 72 (5) and Integrlns ). 5'-N differs from other receptors by its antigenic proprieties (6) and more partlculary with integrln by its mode of interaction with the matrix (7). However, 5'-N and integrln were found colocalized in specialized structures: Myotendinous junctions and adult striated muscle sarcolemmas.
(1) Stochaj, U. et al., Biochim. Biophys. Acta 992, 385-392 (1989). (2) Codogno, P., et al., Exp. Cell Res. 174, 344-354 (1988). (3) Mehul, B., et al., Cell Biol. Iter. Rep. 14, 155-164 (1990). (4) Lesot, H., et al., Eur. /4ol. Biol. Organ. J. 2, 861-865 (1983}. (5) Moutsita, R. et al., Exp. Cell Res. 192, 236-242 (1991). (6) Gundula, R. et al., Biochim. Biophys. Acta, 994, 258-263 (1989}. (7) Stochaj, U. et al., Eur. J. Cell Biol. 30, 167-176 (1990).
2.