Elsevier

Neuroscience

Volume 79, Issue 4, 6 June 1997, Pages 1207-1218
Neuroscience

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

https://doi.org/10.1016/S0306-4522(97)00041-9Get rights and content

Abstract

Calmodulin and de-phosphorylated B-50/growth-associated protein-43 (GAP-43) have been shown to bind in vitro in a molecular complex, but evidence for an in situ association in the nervous system does not exist.

Previously, we have reported that, in the model of the regenerating rat sciatic nerve, the B-50/GAP-43 immunoreactivity is increased and concentrated at the axolemma of unmyelinated axons located proximal to the site of injury and axon outgrowth. To explore a putative function of B-50/GAP-43, namely, the capacity of binding calmodulin to the plasma membrane, we examined the ultrastructural distribution of calmodulin in the proximal unmyelinated axon shafts of this model, using double immunolabelling and detection by fluorescent or gold probes conjugated to second antibodies. Immunofluorescence showed that seven days post-sciatic nerve crush the calmodulin immunoreactivity, similar to B-50/GAP-43 immunoreactivity, was intense in unmyelinated axon shafts located proximal to the site of injury of the regenerating nerve. Ultrastructurally, calmodulin was located at the axolemma of these regenerating unmyelinated axon shafts and inside the axoplasm, where it was associated with vesicles and microtubules. The plasma membrane labelling (approximately 69%) was significantly higher than the axoplasmic labelling. Over 60% of the plasma membrane-associated calmodulin co-localized with B-50/GAP-43 in a non-random distribution.

Since normally calmodulin is largely present in the cytoplasm, these data suggest that calmodulin has been concentrated at the plasma membrane of unmyelinated axons, most probably by B-50/GAP-43. If the concentrating effect is due to B-50/GAP-43, then there is a possibility that these proteins may be present as a molecular complex in situ. The physiological significance could be that this association regulates the local availability of both B-50/GAP-43 and calmodulin for other interactions.

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.

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