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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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Quantitative analysis of the distribution of axonin-1 and NgCAM in stably  transfected CV-1 cells: The  distributions of axonin-1  (filled bars) and NgCAM  (empty bars) were quantified  by analysis of the fluorescence intensity of the axonin-1 and NgCAM immunofluorescence staining in axonin-1 single transfectants (Ax),  NgCAM single transfectants (Ng), axonin-1–NgCAM cotransfectants (Ax-/Ng) and mixtures of axonin-1 and NgCAM single  transfectants (Ax-1+Ng). The combinations of antibodies used  correspond to those in Fig. 9. To obtain cell layers with comparable geometry, identical numbers of confocal sections were integrated for quantitative analysis of the distribution of axonin-1  and NgCAM. For quantification distribution coefficients were  calculated. In case of cultures consisting of only one type of cell  the distribution coefficient was calculated as: q = 2 × Icell-contact/ Icell1 + Icell2 and in cases of contacts formed by different cell types  (e.g., axonin-1 and NgCAM expressing cells) q* = Icell-contact/ Iexpressing cell. The term Icell-contact represents the fluorescence intensity per area at the cell contact and the terms Icell1, Icell2, and  Iexpressing cell represent the fluorescence intensity per area at the  surface of the cells forming the contacts. Bars represent means ±  SD, n = 30 for NgCAM and n = 24 for axonin-1 single transfectants, n = 24 for axonin-1–NgCAM double transfectants, and n =  24 and for the mixtures of axonin-1 and NgCAM single transfectants.
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Figure 10: Quantitative analysis of the distribution of axonin-1 and NgCAM in stably transfected CV-1 cells: The distributions of axonin-1 (filled bars) and NgCAM (empty bars) were quantified by analysis of the fluorescence intensity of the axonin-1 and NgCAM immunofluorescence staining in axonin-1 single transfectants (Ax), NgCAM single transfectants (Ng), axonin-1–NgCAM cotransfectants (Ax-/Ng) and mixtures of axonin-1 and NgCAM single transfectants (Ax-1+Ng). The combinations of antibodies used correspond to those in Fig. 9. To obtain cell layers with comparable geometry, identical numbers of confocal sections were integrated for quantitative analysis of the distribution of axonin-1 and NgCAM. For quantification distribution coefficients were calculated. In case of cultures consisting of only one type of cell the distribution coefficient was calculated as: q = 2 × Icell-contact/ Icell1 + Icell2 and in cases of contacts formed by different cell types (e.g., axonin-1 and NgCAM expressing cells) q* = Icell-contact/ Iexpressing cell. The term Icell-contact represents the fluorescence intensity per area at the cell contact and the terms Icell1, Icell2, and Iexpressing cell represent the fluorescence intensity per area at the surface of the cells forming the contacts. Bars represent means ± SD, n = 30 for NgCAM and n = 24 for axonin-1 single transfectants, n = 24 for axonin-1–NgCAM double transfectants, and n = 24 and for the mixtures of axonin-1 and NgCAM single transfectants.

Mentions: To investigate the molecular interactions underlying the formation of complexes of axonin-1 and NgCAM at cell contacts we used CV-1 cells stably transfected with full-length cDNAs of axonin-1 or NgCAM alone or stably cotransfected with both cDNAs. Cultures of single transfectants, double transfectants and mixtures of single transfectants were kept for 24 h to allow the formation of extended cell contacts. Subsequently, the cells were fixed using 2% (wt/vol) formaldehyde and 0.1% (wt/vol) glutaraldehyde to obtain complete immobilization of membrane proteins without permeabilization of the cells (Dubreuil et al., 1996). Axonin-1 and NgCAM were localized 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 distribution of axonin-1 and NgCAM was quantitatively analyzed by confocal laser scanning microscopy. To obtain cell layers of comparable thickness we integrated identical numbers of confocal sections for image generation and quantitative analysis (Figs. 9 and 10). The accumulation of axonin-1 and NgCAM in the different situations is reflected by the distribution coefficient that represents the ratio of protein detected at the cell contact versus protein at the surface of the adjacent cells (Fig. 10). These distribution coefficients represent underestimations of the actual amount of protein accumulated at the cell contact region due to a limited accessibility of the molecules localized in closely apposed membranes (for details see Materials and Methods).


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)

Quantitative analysis of the distribution of axonin-1 and NgCAM in stably  transfected CV-1 cells: The  distributions of axonin-1  (filled bars) and NgCAM  (empty bars) were quantified  by analysis of the fluorescence intensity of the axonin-1 and NgCAM immunofluorescence staining in axonin-1 single transfectants (Ax),  NgCAM single transfectants (Ng), axonin-1–NgCAM cotransfectants (Ax-/Ng) and mixtures of axonin-1 and NgCAM single  transfectants (Ax-1+Ng). The combinations of antibodies used  correspond to those in Fig. 9. To obtain cell layers with comparable geometry, identical numbers of confocal sections were integrated for quantitative analysis of the distribution of axonin-1  and NgCAM. For quantification distribution coefficients were  calculated. In case of cultures consisting of only one type of cell  the distribution coefficient was calculated as: q = 2 × Icell-contact/ Icell1 + Icell2 and in cases of contacts formed by different cell types  (e.g., axonin-1 and NgCAM expressing cells) q* = Icell-contact/ Iexpressing cell. The term Icell-contact represents the fluorescence intensity per area at the cell contact and the terms Icell1, Icell2, and  Iexpressing cell represent the fluorescence intensity per area at the  surface of the cells forming the contacts. Bars represent means ±  SD, n = 30 for NgCAM and n = 24 for axonin-1 single transfectants, n = 24 for axonin-1–NgCAM double transfectants, and n =  24 and for the mixtures of axonin-1 and NgCAM single transfectants.
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Figure 10: Quantitative analysis of the distribution of axonin-1 and NgCAM in stably transfected CV-1 cells: The distributions of axonin-1 (filled bars) and NgCAM (empty bars) were quantified by analysis of the fluorescence intensity of the axonin-1 and NgCAM immunofluorescence staining in axonin-1 single transfectants (Ax), NgCAM single transfectants (Ng), axonin-1–NgCAM cotransfectants (Ax-/Ng) and mixtures of axonin-1 and NgCAM single transfectants (Ax-1+Ng). The combinations of antibodies used correspond to those in Fig. 9. To obtain cell layers with comparable geometry, identical numbers of confocal sections were integrated for quantitative analysis of the distribution of axonin-1 and NgCAM. For quantification distribution coefficients were calculated. In case of cultures consisting of only one type of cell the distribution coefficient was calculated as: q = 2 × Icell-contact/ Icell1 + Icell2 and in cases of contacts formed by different cell types (e.g., axonin-1 and NgCAM expressing cells) q* = Icell-contact/ Iexpressing cell. The term Icell-contact represents the fluorescence intensity per area at the cell contact and the terms Icell1, Icell2, and Iexpressing cell represent the fluorescence intensity per area at the surface of the cells forming the contacts. Bars represent means ± SD, n = 30 for NgCAM and n = 24 for axonin-1 single transfectants, n = 24 for axonin-1–NgCAM double transfectants, and n = 24 and for the mixtures of axonin-1 and NgCAM single transfectants.
Mentions: To investigate the molecular interactions underlying the formation of complexes of axonin-1 and NgCAM at cell contacts we used CV-1 cells stably transfected with full-length cDNAs of axonin-1 or NgCAM alone or stably cotransfected with both cDNAs. Cultures of single transfectants, double transfectants and mixtures of single transfectants were kept for 24 h to allow the formation of extended cell contacts. Subsequently, the cells were fixed using 2% (wt/vol) formaldehyde and 0.1% (wt/vol) glutaraldehyde to obtain complete immobilization of membrane proteins without permeabilization of the cells (Dubreuil et al., 1996). Axonin-1 and NgCAM were localized 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 distribution of axonin-1 and NgCAM was quantitatively analyzed by confocal laser scanning microscopy. To obtain cell layers of comparable thickness we integrated identical numbers of confocal sections for image generation and quantitative analysis (Figs. 9 and 10). The accumulation of axonin-1 and NgCAM in the different situations is reflected by the distribution coefficient that represents the ratio of protein detected at the cell contact versus protein at the surface of the adjacent cells (Fig. 10). These distribution coefficients represent underestimations of the actual amount of protein accumulated at the cell contact region due to a limited accessibility of the molecules localized in closely apposed membranes (for details see Materials and Methods).

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