Ultrastructural co-localization of calmodulin and B-50/growth-associated protein-43 at the plasma membrane of proximal unmyelinated axon shafts studied in the model of the regenerating rat sciatic nerve
Section snippets
Animals and surgical procedures
Crush lesions of the right sciatic nerve of adult male Wistar rats (TNO, Zeist, The Netherlands; body weight 200 to 250 g) were made as described by De Koning et al.[17]All experiments were carried out under Hypnorm anaesthesia (0.5 ml/100 g body weight, Janssen Pharmaceutica, Tilburg, The Netherlands), in accordance with protocols approved by the Animal Welfare Officer of the University of Utrecht. After seven days, the animals were killed with an overdose of Hypnorm (0.5 ml/100 g body weight) and
Double immunofluorescence in axon shafts of the regenerating sciatic nerve
In the double label immunofluorescence experiments (Fig. 2), calmodulin was detected throughout structures of the proximal part of the regenerating nerve. Compact myelin did not reveal any significant immunolabelling for calmodulin. Although the immunofluorescence detection was not quantitative, it appeared that the labelling in unmyelinated axons was equal or somewhat higher than in myelinated axons (see Fig. 2B). This observation was confirmed qualitatively at the ultrastructural level, using
B-50/GAP-43 and calmodulin are localized at the same site of the plasma membrane
The starting rationale for this study in this specific model was our demonstration by immunoelectron microscopy that the density of B-50/GAP-43 is increased at the plasma membrane of unmyelinated axons in the nerve segment proximal to the site of previous injury.[62]In these unmyelinated axon shafts, 77% of the total B-50/GAP-43 immunolabelling was associated with the plasma membrane. Moreover, immunofluorescence light microscopy experiments on frozen sections of regenerating sciatic nerve
Conclusion
In conclusion, this ultrastructural study shows that in the proximal axon shafts of regenerating unmyelinated axons of the rat sciatic nerve, B-50/GAP-43, which in de-phosphorylated form binds calmodulin at low calcium concentrations, is co-localized with calmodulin at the axolemma in situ. This finding suggests a physiological interaction between B-50/GAP-43 and calmodulin, serving to sequester calmodulin at the plasma membrane, and thus preventing both B-50/GAP-43 and calmodulin to
Acknowledgements
The authors are grateful to Dr Karina F. Meiri (SUNY Health Science Center, Rochester, NY, U.S.A.) for providing the monoclonal B-50/GAP-43 antibodies 2G12/c7 and 10E8/E7, Arnoud Marquart for the characterization of the calmodulin antibodies, Leo van Halewijn for his skilful assistance in the surgical procedures, and Cas Kruitwagen for his assistance with the analysis of the theoretical model for co-localization.
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Present address: Cell Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69012 Heidelberg, Germany.