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Dissection of complex molecular interactions of neurofascin with axonin-1, F11, and tenascin-R, which promote attachment and neurite formation of tectal cells.

Volkmer H, Zacharias U, Nörenberg U, Rathjen FG - J. Cell Biol. (1998)

Bottom Line: In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11.Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding.In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück-Centrum für Molekulare Medizin, D-13122 Berlin, Germany.

ABSTRACT
Neurofascin is a member of the L1 subgroup of the Ig superfamily that promotes axon outgrowth by interactions with neuronal NgCAM-related cell adhesion molecule (NrCAM). We used a combination of cellular binding assays and neurite outgrowth experiments to investigate mechanisms that might modulate the interactions of neurofascin. In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11. Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding. While interactions of neurofascin with F11 are only slightly modulated, binding to axonin-1 and TN-R is strongly regulated by alternatively spliced stretches located in the NH2-terminal half, and by the proline-alanine-threonine-rich segment. In vitro neurite outgrowth and cell attachment assays on a neurofascin-Fc substrate reveal a shift of cellular receptor usage from NrCAM to axonin-1, F11, and at least one additional protein in the presence of TN-R, presumably due to competition of the neurofascin- NrCAM interaction. Thereby, F11 binds to TN-R of the neurofascin/TN-R complex, but not to neurofascin, whereas axonin-1 is not able to bind directly to the neurofascin/TN-R complex as shown by competition binding assays. In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

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Receptor switch from NrCAM to axonin-1 in the presence of TN-R. (A–D) Representive micrographs of cultures of  tectal cells on a neurofascin-Fc substrate in the absence (A and  B) or in the presence of TN-R (C and D). Anti-NrCAM antibodies block neurite outgrowth on a neurofascin substrate in the absence (B) but not in the presence of TN-R (D). Bar, 100 μm. (E)  Quantification of neurite extension on neurofascin-Fc (FcNF15)  in the presence or absence of TN-R, and Fab fragments of polyclonal antibodies to F11, axonin-1, or NgCAM. The broken line  indicates that tectal cells do not extend neurites on immobilized  TN-R. The lengths of individual neurites were measured with the  GENIAS imaging software, and are plotted as introduced by  Chang et al. (1987). The percentage of neurons (vertical axes)  with neurites longer than or equal to 20 μm (horizontal axis) is  shown. For each experimental condition, 80–120 neurites were  measured. Neurite length of tectal cells is not affected in the presence of TN-R. Only Fab fragments of polyclonal antibodies to axonin-1 and not to NgCAM or F11 reduce neurite length on a  combined neurofascin/TN-R substrate (P values of the t test:  TN-R vs. TN-R+anti-F11, 0.98; TN-R vs. TN-R+anti-NgCAM,  0.81; TN-R vs. TN-R+anti-Ax-1; 0.0002).
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Figure 4: Receptor switch from NrCAM to axonin-1 in the presence of TN-R. (A–D) Representive micrographs of cultures of tectal cells on a neurofascin-Fc substrate in the absence (A and B) or in the presence of TN-R (C and D). Anti-NrCAM antibodies block neurite outgrowth on a neurofascin substrate in the absence (B) but not in the presence of TN-R (D). Bar, 100 μm. (E) Quantification of neurite extension on neurofascin-Fc (FcNF15) in the presence or absence of TN-R, and Fab fragments of polyclonal antibodies to F11, axonin-1, or NgCAM. The broken line indicates that tectal cells do not extend neurites on immobilized TN-R. The lengths of individual neurites were measured with the GENIAS imaging software, and are plotted as introduced by Chang et al. (1987). The percentage of neurons (vertical axes) with neurites longer than or equal to 20 μm (horizontal axis) is shown. For each experimental condition, 80–120 neurites were measured. Neurite length of tectal cells is not affected in the presence of TN-R. Only Fab fragments of polyclonal antibodies to axonin-1 and not to NgCAM or F11 reduce neurite length on a combined neurofascin/TN-R substrate (P values of the t test: TN-R vs. TN-R+anti-F11, 0.98; TN-R vs. TN-R+anti-NgCAM, 0.81; TN-R vs. TN-R+anti-Ax-1; 0.0002).

Mentions: In addition to alternative splicing, the activities of neurofascin may be regulated by the presence of specific binding proteins. Therefore, to analyze the function of the interaction between neurofascin and axonin-1, between neurofascin and F11, and between neurofascin and TN-R, we used in vitro neurite outgrowth and cell attachment assays in which tectal cells were cultivated on neurofascin-Fc immobilized on culture dishes in the presence of blocking antibodies or of purified soluble proteins. Antibodies to F11, axonin-1, or TN-R were not found to affect neurite extension on immobilized neurofascin-Fc, which is known to be mediated by neuritic NrCAM (see Fig. 4 B; Volkmer et al., 1996), suggesting that F11, axonin-1, or TN-R are not important for neurite extension in this experimental system. We therefore incubated tectal cells on immobilized neurofascin-Fc in the presence or absence of soluble F11, axonin-1, or TN-R to study their influence on neurite outgrowth and long-term cell attachment. A neurofascin isoform was chosen that binds to TN-R and to the other ligands, and is composed of the extracellular region of NF15 fused to the Fc portion of human IgG1. It therefore contains the short exons at the NH2 terminus, and between the Ig- and FNIII-like domains; however, it lacks the PAT segment of neurofascin (see Fig. 2 D for a schematic representation of NF15). Among the different combinations that we analyzed in these assays, the interaction between neurofascin and TN-R appeared to be of particular importance since it was found to modulate the behavior of tectal cells to neurofascin as described below.


Dissection of complex molecular interactions of neurofascin with axonin-1, F11, and tenascin-R, which promote attachment and neurite formation of tectal cells.

Volkmer H, Zacharias U, Nörenberg U, Rathjen FG - J. Cell Biol. (1998)

Receptor switch from NrCAM to axonin-1 in the presence of TN-R. (A–D) Representive micrographs of cultures of  tectal cells on a neurofascin-Fc substrate in the absence (A and  B) or in the presence of TN-R (C and D). Anti-NrCAM antibodies block neurite outgrowth on a neurofascin substrate in the absence (B) but not in the presence of TN-R (D). Bar, 100 μm. (E)  Quantification of neurite extension on neurofascin-Fc (FcNF15)  in the presence or absence of TN-R, and Fab fragments of polyclonal antibodies to F11, axonin-1, or NgCAM. The broken line  indicates that tectal cells do not extend neurites on immobilized  TN-R. The lengths of individual neurites were measured with the  GENIAS imaging software, and are plotted as introduced by  Chang et al. (1987). The percentage of neurons (vertical axes)  with neurites longer than or equal to 20 μm (horizontal axis) is  shown. For each experimental condition, 80–120 neurites were  measured. Neurite length of tectal cells is not affected in the presence of TN-R. Only Fab fragments of polyclonal antibodies to axonin-1 and not to NgCAM or F11 reduce neurite length on a  combined neurofascin/TN-R substrate (P values of the t test:  TN-R vs. TN-R+anti-F11, 0.98; TN-R vs. TN-R+anti-NgCAM,  0.81; TN-R vs. TN-R+anti-Ax-1; 0.0002).
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Figure 4: Receptor switch from NrCAM to axonin-1 in the presence of TN-R. (A–D) Representive micrographs of cultures of tectal cells on a neurofascin-Fc substrate in the absence (A and B) or in the presence of TN-R (C and D). Anti-NrCAM antibodies block neurite outgrowth on a neurofascin substrate in the absence (B) but not in the presence of TN-R (D). Bar, 100 μm. (E) Quantification of neurite extension on neurofascin-Fc (FcNF15) in the presence or absence of TN-R, and Fab fragments of polyclonal antibodies to F11, axonin-1, or NgCAM. The broken line indicates that tectal cells do not extend neurites on immobilized TN-R. The lengths of individual neurites were measured with the GENIAS imaging software, and are plotted as introduced by Chang et al. (1987). The percentage of neurons (vertical axes) with neurites longer than or equal to 20 μm (horizontal axis) is shown. For each experimental condition, 80–120 neurites were measured. Neurite length of tectal cells is not affected in the presence of TN-R. Only Fab fragments of polyclonal antibodies to axonin-1 and not to NgCAM or F11 reduce neurite length on a combined neurofascin/TN-R substrate (P values of the t test: TN-R vs. TN-R+anti-F11, 0.98; TN-R vs. TN-R+anti-NgCAM, 0.81; TN-R vs. TN-R+anti-Ax-1; 0.0002).
Mentions: In addition to alternative splicing, the activities of neurofascin may be regulated by the presence of specific binding proteins. Therefore, to analyze the function of the interaction between neurofascin and axonin-1, between neurofascin and F11, and between neurofascin and TN-R, we used in vitro neurite outgrowth and cell attachment assays in which tectal cells were cultivated on neurofascin-Fc immobilized on culture dishes in the presence of blocking antibodies or of purified soluble proteins. Antibodies to F11, axonin-1, or TN-R were not found to affect neurite extension on immobilized neurofascin-Fc, which is known to be mediated by neuritic NrCAM (see Fig. 4 B; Volkmer et al., 1996), suggesting that F11, axonin-1, or TN-R are not important for neurite extension in this experimental system. We therefore incubated tectal cells on immobilized neurofascin-Fc in the presence or absence of soluble F11, axonin-1, or TN-R to study their influence on neurite outgrowth and long-term cell attachment. A neurofascin isoform was chosen that binds to TN-R and to the other ligands, and is composed of the extracellular region of NF15 fused to the Fc portion of human IgG1. It therefore contains the short exons at the NH2 terminus, and between the Ig- and FNIII-like domains; however, it lacks the PAT segment of neurofascin (see Fig. 2 D for a schematic representation of NF15). Among the different combinations that we analyzed in these assays, the interaction between neurofascin and TN-R appeared to be of particular importance since it was found to modulate the behavior of tectal cells to neurofascin as described below.

Bottom Line: In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11.Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding.In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

View Article: PubMed Central - PubMed

Affiliation: Max-Delbrück-Centrum für Molekulare Medizin, D-13122 Berlin, Germany.

ABSTRACT
Neurofascin is a member of the L1 subgroup of the Ig superfamily that promotes axon outgrowth by interactions with neuronal NgCAM-related cell adhesion molecule (NrCAM). We used a combination of cellular binding assays and neurite outgrowth experiments to investigate mechanisms that might modulate the interactions of neurofascin. In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11. Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding. While interactions of neurofascin with F11 are only slightly modulated, binding to axonin-1 and TN-R is strongly regulated by alternatively spliced stretches located in the NH2-terminal half, and by the proline-alanine-threonine-rich segment. In vitro neurite outgrowth and cell attachment assays on a neurofascin-Fc substrate reveal a shift of cellular receptor usage from NrCAM to axonin-1, F11, and at least one additional protein in the presence of TN-R, presumably due to competition of the neurofascin- NrCAM interaction. Thereby, F11 binds to TN-R of the neurofascin/TN-R complex, but not to neurofascin, whereas axonin-1 is not able to bind directly to the neurofascin/TN-R complex as shown by competition binding assays. In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

Show MeSH
Related in: MedlinePlus