ULTRASTRUCTURAL COLOCALIZATION 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/

Calmodulin and de-phosphorylated B-50/growth-associated protein-43 (GA P-43) have been shown to bind in vitro in a molecular complex, but evi dence for an in situ association in the nervous system does not exist. Previously, we have reported that, in the model of the regenerating r at sciatic nerve, the B-50/GAP-43 immunoreactivity is increased and co ncentrated at the axolemma of unmyelinated axons located proximal to t he site of injury and axon outgrowth. To explore a putative function o f B-50/GAP-43, namely, the capacity of binding calmodulin to the plasm a 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/G AP-43 immunoreactivity, was intense in unmyelinated axon shafts locate d proximal to the site of injury of the regenerating nerve. Ultrastruc turally, calmodulin was located at the axolemma of these regenerating unmyelinated axon shafts and inside the axoplasm, where it was associa ted with vesicles and microtubules. The plasma membrane labelling (app roximately 69%) was significantly higher than the axoplasmic labelling . Over 60% of the plasma membrane-associated calmodulin co-localized w ith B-50/GAP-43 in a non-random distribution. Since normally calmoduli n is largely present in the cytoplasm, these data suggest that calmodu lin 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 pr esent as a molecular complex in situ. The physiological significance c ould be that this association regulates the local availability of both B-50/GAP-43 and calmodulin for other interactions. (C) 1997 IBRO. Pub lished by Elsevier Science Ltd.