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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 and axonin-1-microspheres to cells  that express axonin-1, NgCAM or coexpressed axonin-1 and NgCAM: Quantitative analysis of the binding of NgCAM (A) and  axonin-1 (B) Covaspheres to COS cells expressing axonin-1 (ax-1),  NgCAM (NgCAM) and coexpressing axonin-1 and NgCAM  (ax-1/NgCAM). Cells were preincubated with control- (filled  bars), anti-NgCAM- (shaded bars), and anti-axonin-1- (empty  bars) Fab. In case of the single axonin-1 or NgCAM transfections, green fluorescing cells that bound four or more red fluorescing Covaspheres were scored as positive cells. In axonin-1–NgCAM cotransfection cells of comparable fluorescence for  axonin-1 and NgCAM staining were selected by examination  with the corresponding single-pass filters. Again, cells that bound  four or more red fluorescing Covaspheres were scored as positive. Cells that did not express NgCAM or axonin-1 were used as  internal negative controls and were found to bind on the average  0.1 axonin-1 or NgCAM Covaspheres. Each column corresponds  to four independent experiments (± SD). The capability of NgCAM to bind axonin-1 microspheres is only partially reduced in  axonin-1–NgCAM coexpressing cells. This may be due to a stoichiometric excess of NgCAM versus axonin-1 in the coexpressing cells we selected for quantification. Combined analysis using  immunofluorescence microscopy and quantitative immunoblotting, performed on axonin-1–NgCAM coexpressing cells indeed  revealed a comparably weaker immunostaining of NgCAM on  cells with similar amounts of axonin-1 and NgCAM protein (data  not shown).
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Figure 8: Binding of NgCAM and axonin-1-microspheres to cells that express axonin-1, NgCAM or coexpressed axonin-1 and NgCAM: Quantitative analysis of the binding of NgCAM (A) and axonin-1 (B) Covaspheres to COS cells expressing axonin-1 (ax-1), NgCAM (NgCAM) and coexpressing axonin-1 and NgCAM (ax-1/NgCAM). Cells were preincubated with control- (filled bars), anti-NgCAM- (shaded bars), and anti-axonin-1- (empty bars) Fab. In case of the single axonin-1 or NgCAM transfections, green fluorescing cells that bound four or more red fluorescing Covaspheres were scored as positive cells. In axonin-1–NgCAM cotransfection cells of comparable fluorescence for axonin-1 and NgCAM staining were selected by examination with the corresponding single-pass filters. Again, cells that bound four or more red fluorescing Covaspheres were scored as positive. Cells that did not express NgCAM or axonin-1 were used as internal negative controls and were found to bind on the average 0.1 axonin-1 or NgCAM Covaspheres. Each column corresponds to four independent experiments (± SD). The capability of NgCAM to bind axonin-1 microspheres is only partially reduced in axonin-1–NgCAM coexpressing cells. This may be due to a stoichiometric excess of NgCAM versus axonin-1 in the coexpressing cells we selected for quantification. Combined analysis using immunofluorescence microscopy and quantitative immunoblotting, performed on axonin-1–NgCAM coexpressing cells indeed revealed a comparably weaker immunostaining of NgCAM on cells with similar amounts of axonin-1 and NgCAM protein (data not shown).

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 and axonin-1-microspheres to cells  that express axonin-1, NgCAM or coexpressed axonin-1 and NgCAM: Quantitative analysis of the binding of NgCAM (A) and  axonin-1 (B) Covaspheres to COS cells expressing axonin-1 (ax-1),  NgCAM (NgCAM) and coexpressing axonin-1 and NgCAM  (ax-1/NgCAM). Cells were preincubated with control- (filled  bars), anti-NgCAM- (shaded bars), and anti-axonin-1- (empty  bars) Fab. In case of the single axonin-1 or NgCAM transfections, green fluorescing cells that bound four or more red fluorescing Covaspheres were scored as positive cells. In axonin-1–NgCAM cotransfection cells of comparable fluorescence for  axonin-1 and NgCAM staining were selected by examination  with the corresponding single-pass filters. Again, cells that bound  four or more red fluorescing Covaspheres were scored as positive. Cells that did not express NgCAM or axonin-1 were used as  internal negative controls and were found to bind on the average  0.1 axonin-1 or NgCAM Covaspheres. Each column corresponds  to four independent experiments (± SD). The capability of NgCAM to bind axonin-1 microspheres is only partially reduced in  axonin-1–NgCAM coexpressing cells. This may be due to a stoichiometric excess of NgCAM versus axonin-1 in the coexpressing cells we selected for quantification. Combined analysis using  immunofluorescence microscopy and quantitative immunoblotting, performed on axonin-1–NgCAM coexpressing cells indeed  revealed a comparably weaker immunostaining of NgCAM on  cells with similar amounts of axonin-1 and NgCAM protein (data  not shown).
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Related In: Results  -  Collection

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Figure 8: Binding of NgCAM and axonin-1-microspheres to cells that express axonin-1, NgCAM or coexpressed axonin-1 and NgCAM: Quantitative analysis of the binding of NgCAM (A) and axonin-1 (B) Covaspheres to COS cells expressing axonin-1 (ax-1), NgCAM (NgCAM) and coexpressing axonin-1 and NgCAM (ax-1/NgCAM). Cells were preincubated with control- (filled bars), anti-NgCAM- (shaded bars), and anti-axonin-1- (empty bars) Fab. In case of the single axonin-1 or NgCAM transfections, green fluorescing cells that bound four or more red fluorescing Covaspheres were scored as positive cells. In axonin-1–NgCAM cotransfection cells of comparable fluorescence for axonin-1 and NgCAM staining were selected by examination with the corresponding single-pass filters. Again, cells that bound four or more red fluorescing Covaspheres were scored as positive. Cells that did not express NgCAM or axonin-1 were used as internal negative controls and were found to bind on the average 0.1 axonin-1 or NgCAM Covaspheres. Each column corresponds to four independent experiments (± SD). The capability of NgCAM to bind axonin-1 microspheres is only partially reduced in axonin-1–NgCAM coexpressing cells. This may be due to a stoichiometric excess of NgCAM versus axonin-1 in the coexpressing cells we selected for quantification. Combined analysis using immunofluorescence microscopy and quantitative immunoblotting, performed on axonin-1–NgCAM coexpressing cells indeed revealed a comparably weaker immunostaining of NgCAM on cells with similar amounts of axonin-1 and NgCAM protein (data not shown).
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