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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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Localization of axonin-1 and NgCAM at cell contacts  of stably transfected CV-1 cells: CV-1 cells were stably transfected with NgCAM (a) and axonin-1 (b) or stably cotransfected  with axonin-1 and NgCAM (c and d). e and f represent mixtures  of single axonin-1 and NgCAM transfectants. Axonin-1 and NgCAM were stained using a rabbit polyclonal anti-axonin-1 antibody (axonin-1 staining) combined with a Texas red–labeled secondary antibody and a goat anti-NgCAM antibody (NgCAM  staining) combined with a FITC-labeled secondary antibody. Images were generated by confocal laser-scanning microscopy. To  examine cell layers of equal thickness, equal numbers of confocal  sections were integrated for image generation. Bar, 20 μm.
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Figure 9: Localization of axonin-1 and NgCAM at cell contacts of stably transfected CV-1 cells: CV-1 cells were stably transfected with NgCAM (a) and axonin-1 (b) or stably cotransfected with axonin-1 and NgCAM (c and d). e and f represent mixtures of single axonin-1 and NgCAM transfectants. Axonin-1 and NgCAM were stained using a rabbit polyclonal anti-axonin-1 antibody (axonin-1 staining) combined with a Texas red–labeled secondary antibody and a goat anti-NgCAM antibody (NgCAM staining) combined with a FITC-labeled secondary antibody. Images were generated by confocal laser-scanning microscopy. To examine cell layers of equal thickness, equal numbers of confocal sections were integrated for image generation. Bar, 20 μm.

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)

Localization of axonin-1 and NgCAM at cell contacts  of stably transfected CV-1 cells: CV-1 cells were stably transfected with NgCAM (a) and axonin-1 (b) or stably cotransfected  with axonin-1 and NgCAM (c and d). e and f represent mixtures  of single axonin-1 and NgCAM transfectants. Axonin-1 and NgCAM were stained using a rabbit polyclonal anti-axonin-1 antibody (axonin-1 staining) combined with a Texas red–labeled secondary antibody and a goat anti-NgCAM antibody (NgCAM  staining) combined with a FITC-labeled secondary antibody. Images were generated by confocal laser-scanning microscopy. To  examine cell layers of equal thickness, equal numbers of confocal  sections were integrated for image generation. Bar, 20 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132982&req=5

Figure 9: Localization of axonin-1 and NgCAM at cell contacts of stably transfected CV-1 cells: CV-1 cells were stably transfected with NgCAM (a) and axonin-1 (b) or stably cotransfected with axonin-1 and NgCAM (c and d). e and f represent mixtures of single axonin-1 and NgCAM transfectants. Axonin-1 and NgCAM were stained using a rabbit polyclonal anti-axonin-1 antibody (axonin-1 staining) combined with a Texas red–labeled secondary antibody and a goat anti-NgCAM antibody (NgCAM staining) combined with a FITC-labeled secondary antibody. Images were generated by confocal laser-scanning microscopy. To examine cell layers of equal thickness, equal numbers of confocal sections were integrated for image generation. Bar, 20 μm.
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.

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Related in: MedlinePlus