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Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule.

Kunz S, Spirig M, Ginsburg C, Buchstaller A, Berger P, Lanz R, Rader C, Vogt L, Kunz B, Sonderegger P - J. Cell Biol. (1998)

Bottom Line: In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding.These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM.The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.

ABSTRACT
Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

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Binding of NgCAM-microspheres to cells expressing  axonin-1, NgCAM or coexpressing  axonin-1 and NgCAM. COS cells  were transfected with axonin-1 (a– c), NgCAM (d–f) and cotransfected  with axonin-1 and NgCAM (g–i).  The cells were preincubated with  anti-axonin-1, anti-NgCAM, and  control Fab as indicated. Binding of  TRITC-conjugated NgCAM Covaspheres was assessed. Axonin-1 expressing cells in a–c were identified  by indirect immunofluorescence  staining using a polyclonal anti-axonin-1 antibody and a FITC-labeled  secondary antibody. An identical  staining protocol was applied for  the detection of NgCAM in d–f, using a polyclonal anti-NgCAM antibody. Axonin-1–NgCAM coexpressing cells were detected in g and  h by the application of goat polyclonal anti-NgCAM antibody  (green fluorescence due to the  FITC labeled secondary antibody)  combined with rabbit anti-axonin-1  antibody (red fluorescence due to  the Texas red–labeled secondary antibody). In i, axonin-1 was detected by a goat polyclonal antibody, combined with a the FITC-labeled secondary antibody and NgCAM by a rabbit anti-NgCAM antibody using a the Texas red–labeled secondary antibody. Axonin-1–NgCAM coexpressing cells appear in yellow as a Texas red/FITC double filter device was used. TRITC-conjugated NgCAM  Covaspheres can be easily recognized on coexpressing cells. Bar, 20 μm.
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Figure 7: Binding of NgCAM-microspheres to cells expressing axonin-1, NgCAM or coexpressing axonin-1 and NgCAM. COS cells were transfected with axonin-1 (a– c), NgCAM (d–f) and cotransfected with axonin-1 and NgCAM (g–i). The cells were preincubated with anti-axonin-1, anti-NgCAM, and control Fab as indicated. Binding of TRITC-conjugated NgCAM Covaspheres was assessed. Axonin-1 expressing cells in a–c were identified by indirect immunofluorescence staining using a polyclonal anti-axonin-1 antibody and a FITC-labeled secondary antibody. An identical staining protocol was applied for the detection of NgCAM in d–f, using a polyclonal anti-NgCAM antibody. Axonin-1–NgCAM coexpressing cells were detected in g and h by the application of goat polyclonal anti-NgCAM antibody (green fluorescence due to the FITC labeled secondary antibody) combined with rabbit anti-axonin-1 antibody (red fluorescence due to the Texas red–labeled secondary antibody). In i, axonin-1 was detected by a goat polyclonal antibody, combined with a the FITC-labeled secondary antibody and NgCAM by a rabbit anti-NgCAM antibody using a the Texas red–labeled secondary antibody. Axonin-1–NgCAM coexpressing cells appear in yellow as a Texas red/FITC double filter device was used. TRITC-conjugated NgCAM Covaspheres can be easily recognized on coexpressing cells. Bar, 20 μm.

Mentions: The results of the domain mapping studies described above indicate an overlap of the domains of NgCAM involved in axonin-1 binding, Ig2-4 and Fn3, and the domains involved in the homophilic NgCAM interaction, Ig1-4. Likewise, localization of the binding sites on axonin-1 had revealed that the axonin-1 domains Ig1-4, which mediate NgCAM binding, overlap with those identified recently to be involved in axonin-1 homophilic binding, Ig1 and Fn4 (Kunz, B., R. Lierheimer, C. Rader, U. Ziegler, L. Vogt, M. Spirig, and P. Sonderegger, manuscript submitted for publication). The participation of some domains of both axonin-1 and NgCAM in homophilic and heterophilic interaction rises the possibility of a competition between the axonin-1–NgCAM binding and the homophilic interactions of each of the two molecules. To address this question, we investigated the effect of a coexpression of axonin-1 with NgCAM on the homophilic interactions of axonin-1 and NgCAM. For this purpose, the expression constructs pSCTaxonin-1 (Rader et al., 1993) and pSCTNgCAM were introduced individually or together into COS7 cells by electroporation, resulting in the expression of either axonin-1 or NgCAM alone or the coexpression of axonin-1 and NgCAM. 48 h after transfection the cells were preincubated with either control Fab (prepared from preimmune serum), anti-axonin-1, or anti-NgCAM Fab, washed extensively, and subsequently incubated with microspheres conjugated to NgCAM or axonin-1. The expression of axonin-1 and NgCAM was detected by immunofluorescence staining using goat anti-axonin-1 and rabbit anti-NgCAM antibodies and the corresponding secondary antibodies conjugated to FITC or Texas red, respectively. The coexpressing cells were clearly identified by their yellow staining using a narrow band-pass double filter for analysis. For quantification, we selected cells with comparable intensity of the individual immunofluorescence staining of axonin-1 and NgCAM. The results of this study are partially presented in Fig. 7. A summary of all results is represented by Table I and a quantitative analysis is shown in Fig. 8.


Neurite fasciculation mediated by complexes of axonin-1 and Ng cell adhesion molecule.

Kunz S, Spirig M, Ginsburg C, Buchstaller A, Berger P, Lanz R, Rader C, Vogt L, Kunz B, Sonderegger P - J. Cell Biol. (1998)

Binding of NgCAM-microspheres to cells expressing  axonin-1, NgCAM or coexpressing  axonin-1 and NgCAM. COS cells  were transfected with axonin-1 (a– c), NgCAM (d–f) and cotransfected  with axonin-1 and NgCAM (g–i).  The cells were preincubated with  anti-axonin-1, anti-NgCAM, and  control Fab as indicated. Binding of  TRITC-conjugated NgCAM Covaspheres was assessed. Axonin-1 expressing cells in a–c were identified  by indirect immunofluorescence  staining using a polyclonal anti-axonin-1 antibody and a FITC-labeled  secondary antibody. An identical  staining protocol was applied for  the detection of NgCAM in d–f, using a polyclonal anti-NgCAM antibody. Axonin-1–NgCAM coexpressing cells were detected in g and  h by the application of goat polyclonal anti-NgCAM antibody  (green fluorescence due to the  FITC labeled secondary antibody)  combined with rabbit anti-axonin-1  antibody (red fluorescence due to  the Texas red–labeled secondary antibody). In i, axonin-1 was detected by a goat polyclonal antibody, combined with a the FITC-labeled secondary antibody and NgCAM by a rabbit anti-NgCAM antibody using a the Texas red–labeled secondary antibody. Axonin-1–NgCAM coexpressing cells appear in yellow as a Texas red/FITC double filter device was used. TRITC-conjugated NgCAM  Covaspheres can be easily recognized on coexpressing cells. Bar, 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132982&req=5

Figure 7: Binding of NgCAM-microspheres to cells expressing axonin-1, NgCAM or coexpressing axonin-1 and NgCAM. COS cells were transfected with axonin-1 (a– c), NgCAM (d–f) and cotransfected with axonin-1 and NgCAM (g–i). The cells were preincubated with anti-axonin-1, anti-NgCAM, and control Fab as indicated. Binding of TRITC-conjugated NgCAM Covaspheres was assessed. Axonin-1 expressing cells in a–c were identified by indirect immunofluorescence staining using a polyclonal anti-axonin-1 antibody and a FITC-labeled secondary antibody. An identical staining protocol was applied for the detection of NgCAM in d–f, using a polyclonal anti-NgCAM antibody. Axonin-1–NgCAM coexpressing cells were detected in g and h by the application of goat polyclonal anti-NgCAM antibody (green fluorescence due to the FITC labeled secondary antibody) combined with rabbit anti-axonin-1 antibody (red fluorescence due to the Texas red–labeled secondary antibody). In i, axonin-1 was detected by a goat polyclonal antibody, combined with a the FITC-labeled secondary antibody and NgCAM by a rabbit anti-NgCAM antibody using a the Texas red–labeled secondary antibody. Axonin-1–NgCAM coexpressing cells appear in yellow as a Texas red/FITC double filter device was used. TRITC-conjugated NgCAM Covaspheres can be easily recognized on coexpressing cells. Bar, 20 μm.
Mentions: The results of the domain mapping studies described above indicate an overlap of the domains of NgCAM involved in axonin-1 binding, Ig2-4 and Fn3, and the domains involved in the homophilic NgCAM interaction, Ig1-4. Likewise, localization of the binding sites on axonin-1 had revealed that the axonin-1 domains Ig1-4, which mediate NgCAM binding, overlap with those identified recently to be involved in axonin-1 homophilic binding, Ig1 and Fn4 (Kunz, B., R. Lierheimer, C. Rader, U. Ziegler, L. Vogt, M. Spirig, and P. Sonderegger, manuscript submitted for publication). The participation of some domains of both axonin-1 and NgCAM in homophilic and heterophilic interaction rises the possibility of a competition between the axonin-1–NgCAM binding and the homophilic interactions of each of the two molecules. To address this question, we investigated the effect of a coexpression of axonin-1 with NgCAM on the homophilic interactions of axonin-1 and NgCAM. For this purpose, the expression constructs pSCTaxonin-1 (Rader et al., 1993) and pSCTNgCAM were introduced individually or together into COS7 cells by electroporation, resulting in the expression of either axonin-1 or NgCAM alone or the coexpression of axonin-1 and NgCAM. 48 h after transfection the cells were preincubated with either control Fab (prepared from preimmune serum), anti-axonin-1, or anti-NgCAM Fab, washed extensively, and subsequently incubated with microspheres conjugated to NgCAM or axonin-1. The expression of axonin-1 and NgCAM was detected by immunofluorescence staining using goat anti-axonin-1 and rabbit anti-NgCAM antibodies and the corresponding secondary antibodies conjugated to FITC or Texas red, respectively. The coexpressing cells were clearly identified by their yellow staining using a narrow band-pass double filter for analysis. For quantification, we selected cells with comparable intensity of the individual immunofluorescence staining of axonin-1 and NgCAM. The results of this study are partially presented in Fig. 7. A summary of all results is represented by Table I and a quantitative analysis is shown in Fig. 8.

Bottom Line: In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding.These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM.The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.

ABSTRACT
Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.

Show MeSH
Related in: MedlinePlus