Rabphilin-3A is transported with fast anterograde axonal transport and associated with synaptic vesicles

Abstract

Rab3a has been proposed to be involved in the process of exocytosis. It undergoes a membrane association/dissociation cycle which is dependent upon alteration in its GTP-GDP state. Rabphilin-3A is one of the most attractive candidates for a Rab3a effector molecule in synaptic vesicle exocytosis. It binds selectively to the GTP-bound form of Rab3a as well as of Rab3c. Fast axonal transport was studied by crushing spinal roots and sciatic nerves, resulting in accumulations of transported, organelle-bound substances. Rabphilin-3A was transported with fast anterograde transport, but < 10% of the proximal accumulation was detected in the recycling vesicle population. The pattern for accumulation of rabphilin-3A was similar to that of Rab3a accumulation, including the poor recycling. However, synaptophysin and SV2, two transmembrane synaptic vesicle proteins, accumulated in large amounts in many axons distal to the crushes, and the amounts of recycling synaptophysin and SV2, in percentages of anterograde accumulations, were about 70% and 60%, respectively. Double-labelling showed a high degree of colocalization between Rab3a and rabphilin-3A in spinal roots and sciatic nerves. The two proteins essentially colocalized in the nerve terminals of the spinal cord and in motor endplates. Immuno-EM studies demonstrated that rabphilin-3A was present on clear small synaptic vesicles in the proximal segment, but was absent from vesicles in the distal segment. Rabphilin-3A also associated with the synaptic vesicles in the presynaptic compartment. Results indicate that rabphilin-3A is associated with the membrane of synaptic vesicles during anterograde transport. Furthermore, as it colocalized in most, but not all, structures with Rab3a, results support the hypothesis that rabphilin-3A is interacting with Rab3a.