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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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(A–C) The adherence of tectal cells to a neurofascin-Fc  substrate is enhanced in the presence of TN-R. Neurofascin fusion protein with the Fc portion of human IgG1 (FcNF15), the Fc  portion without neurofascin sequences (Fc), F11, BSA, or collagen were immobilized on petriperm dishes, and dissociated tectal cells were incubated in the presence or absence of TN-R (A  and C) or in the presence of F11 or axonin-1 (B). The immobilized proteins, the protein in solution, or the applied antibody is  given at the left of each panel. (A) 10 μg/ml or (C) 50 μg/ml of  TN-R was used to monitor the effects of the different antibodies.  The concentration of soluble F11 or axonin-1 was 20 μg/ml in B.  Long-term attachment (after 22 h) of cells was quantified with  the GENIAS imaging software, and the adherence of tectal cells  to neurofascin (FcNF15) was calculated to 1. Error bars indicate  SEM. TN-R was either provided in the supernatant of a neurofascin substrate during the whole culture period (FcNF15 + TN-R)  or washed out after a 1 h incubation period before adding the  cells (FcNF15 + TN-Rprec). Cells were also incubated in the  presence of Fab fragments of polyclonal antibodies specific for  neurofascin (antiNF), NrCAM (antiNr), F11 (antiF11), NgCAM  (antiNg), axonin-1 (antiAx-1), or TN-R (antiTN-R) at a concentration of 100 μg/ml.
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Figure 3: (A–C) The adherence of tectal cells to a neurofascin-Fc substrate is enhanced in the presence of TN-R. Neurofascin fusion protein with the Fc portion of human IgG1 (FcNF15), the Fc portion without neurofascin sequences (Fc), F11, BSA, or collagen were immobilized on petriperm dishes, and dissociated tectal cells were incubated in the presence or absence of TN-R (A and C) or in the presence of F11 or axonin-1 (B). The immobilized proteins, the protein in solution, or the applied antibody is given at the left of each panel. (A) 10 μg/ml or (C) 50 μg/ml of TN-R was used to monitor the effects of the different antibodies. The concentration of soluble F11 or axonin-1 was 20 μg/ml in B. Long-term attachment (after 22 h) of cells was quantified with the GENIAS imaging software, and the adherence of tectal cells to neurofascin (FcNF15) was calculated to 1. Error bars indicate SEM. TN-R was either provided in the supernatant of a neurofascin substrate during the whole culture period (FcNF15 + TN-R) or washed out after a 1 h incubation period before adding the cells (FcNF15 + TN-Rprec). Cells were also incubated in the presence of Fab fragments of polyclonal antibodies specific for neurofascin (antiNF), NrCAM (antiNr), F11 (antiF11), NgCAM (antiNg), axonin-1 (antiAx-1), or TN-R (antiTN-R) at a concentration of 100 μg/ml.

Mentions: For analyzing neurite extension, 100 μl of FcNF15, collagen, BSA, or the Fc portion of human IgG1 (DiFc) were immobilized at a concentration of 10 μg/ml or of F11 at a concentration of 100 μg/ml per cm2 culture dish (Petriperm; Bachhofer GmbH, Reutlingen, Germany). Residual binding sites were blocked by washing and incubating with DMEM/10% FCS. 10,000 chick embryonic day 6 tectal cells per cm2 delineated by a silicon fitting were plated in a volume of 100 μl DMEM/10%FCS and incubated for 22 h. TN-R or TN-C was applied at a final concentration of 10 or 50 μg/ml during the cultivation period. The different concentrations used gave qualitatively the same results; however, the percentage of adhering cells increased in the presence of the higher TN-R concentration (Fig. 3, A and C). Seven independent experiments were performed, two of these with 50 μg/ml of TN-R. In cases of preincubation with TN-R, immobilized neurofascin-Fc was incubated with TN-R at a concentration of 10 μg/ml after blocking for 1 h. Unbound TN-R was removed by extensive washing with DMEM/FCS before adding cells. Soluble axonin-1 or F11 was used at 20 μg/ml. Fab fragments of polyclonal antibodies to the different cell surface proteins were added at a final concentration of 100 μg/ml, and monoclonal antibodies were added at a final concentration of 10 μg/ml of culture medium. After a 22-h cultivation, cells were fixed and stained by indirect immunofluorescence using mAb A2B5 followed by Cy3-conjugated rabbit anti–mouse as secondary antibody (Dianova, Hamburg, Germany). The cultures were analyzed using the GENIAS imaging software to measure the number of attached cells and the length of neurites (Image works, Teltow, Germany).


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)

(A–C) The adherence of tectal cells to a neurofascin-Fc  substrate is enhanced in the presence of TN-R. Neurofascin fusion protein with the Fc portion of human IgG1 (FcNF15), the Fc  portion without neurofascin sequences (Fc), F11, BSA, or collagen were immobilized on petriperm dishes, and dissociated tectal cells were incubated in the presence or absence of TN-R (A  and C) or in the presence of F11 or axonin-1 (B). The immobilized proteins, the protein in solution, or the applied antibody is  given at the left of each panel. (A) 10 μg/ml or (C) 50 μg/ml of  TN-R was used to monitor the effects of the different antibodies.  The concentration of soluble F11 or axonin-1 was 20 μg/ml in B.  Long-term attachment (after 22 h) of cells was quantified with  the GENIAS imaging software, and the adherence of tectal cells  to neurofascin (FcNF15) was calculated to 1. Error bars indicate  SEM. TN-R was either provided in the supernatant of a neurofascin substrate during the whole culture period (FcNF15 + TN-R)  or washed out after a 1 h incubation period before adding the  cells (FcNF15 + TN-Rprec). Cells were also incubated in the  presence of Fab fragments of polyclonal antibodies specific for  neurofascin (antiNF), NrCAM (antiNr), F11 (antiF11), NgCAM  (antiNg), axonin-1 (antiAx-1), or TN-R (antiTN-R) at a concentration of 100 μg/ml.
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Figure 3: (A–C) The adherence of tectal cells to a neurofascin-Fc substrate is enhanced in the presence of TN-R. Neurofascin fusion protein with the Fc portion of human IgG1 (FcNF15), the Fc portion without neurofascin sequences (Fc), F11, BSA, or collagen were immobilized on petriperm dishes, and dissociated tectal cells were incubated in the presence or absence of TN-R (A and C) or in the presence of F11 or axonin-1 (B). The immobilized proteins, the protein in solution, or the applied antibody is given at the left of each panel. (A) 10 μg/ml or (C) 50 μg/ml of TN-R was used to monitor the effects of the different antibodies. The concentration of soluble F11 or axonin-1 was 20 μg/ml in B. Long-term attachment (after 22 h) of cells was quantified with the GENIAS imaging software, and the adherence of tectal cells to neurofascin (FcNF15) was calculated to 1. Error bars indicate SEM. TN-R was either provided in the supernatant of a neurofascin substrate during the whole culture period (FcNF15 + TN-R) or washed out after a 1 h incubation period before adding the cells (FcNF15 + TN-Rprec). Cells were also incubated in the presence of Fab fragments of polyclonal antibodies specific for neurofascin (antiNF), NrCAM (antiNr), F11 (antiF11), NgCAM (antiNg), axonin-1 (antiAx-1), or TN-R (antiTN-R) at a concentration of 100 μg/ml.
Mentions: For analyzing neurite extension, 100 μl of FcNF15, collagen, BSA, or the Fc portion of human IgG1 (DiFc) were immobilized at a concentration of 10 μg/ml or of F11 at a concentration of 100 μg/ml per cm2 culture dish (Petriperm; Bachhofer GmbH, Reutlingen, Germany). Residual binding sites were blocked by washing and incubating with DMEM/10% FCS. 10,000 chick embryonic day 6 tectal cells per cm2 delineated by a silicon fitting were plated in a volume of 100 μl DMEM/10%FCS and incubated for 22 h. TN-R or TN-C was applied at a final concentration of 10 or 50 μg/ml during the cultivation period. The different concentrations used gave qualitatively the same results; however, the percentage of adhering cells increased in the presence of the higher TN-R concentration (Fig. 3, A and C). Seven independent experiments were performed, two of these with 50 μg/ml of TN-R. In cases of preincubation with TN-R, immobilized neurofascin-Fc was incubated with TN-R at a concentration of 10 μg/ml after blocking for 1 h. Unbound TN-R was removed by extensive washing with DMEM/FCS before adding cells. Soluble axonin-1 or F11 was used at 20 μg/ml. Fab fragments of polyclonal antibodies to the different cell surface proteins were added at a final concentration of 100 μg/ml, and monoclonal antibodies were added at a final concentration of 10 μg/ml of culture medium. After a 22-h cultivation, cells were fixed and stained by indirect immunofluorescence using mAb A2B5 followed by Cy3-conjugated rabbit anti–mouse as secondary antibody (Dianova, Hamburg, Germany). The cultures were analyzed using the GENIAS imaging software to measure the number of attached cells and the length of neurites (Image works, Teltow, Germany).

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