Preferential localization of annexin V to the axon terminal

To examine the participation of annexin V, a member of Ca(2+)-dependent phospholipid-binding proteins, in the process of synaptic vesicle exocytosis, rat central nervous tissue was analysed using biochemical and morphological techniques. By both fluorescence and confocal laser scanning microscopy, immunoreactivity for annexin V was predominantly localized around neuronal somata and dendrites, and the reactivity was mostly co-labeled with that for synaptophysin. The annexin V immunoreactivity was also detectable, but less intensely, in neuronal perikarya, glial cells and endothelial cells. Both immunoblot and immunoelectron microscopic analyses with intact tissues, synaptosomes and purified synaptic vesicles showed that annexin V was expressed in neurons, preferentially concentrated in axon terminals and associated with synaptic vesicles. Purified synaptic vesicles were relatively homogeneously distributed in the medium where Ca2+ was removed and thus the amount of annexin V was reduced drastically. The vesicles tended to be clustered in the fraction where endogenous annexin V is maintained, and the clusters were more conspicuous when purified human annexin V was added. Synaptic vesicles forming the clusters were not directly fused with each other but separated by a 10-15 nm gap that corresponded well with the size of single annexin V molecules. In axon terminals, globular structures 12-13 nm in diameter, similar in dimension to annexin V molecules, were distinctly found to be attached to the cytoplasmic surface of both vesicle membranes when the two vesicles were close to each other. These results suggest that annexin V belongs to the group of synaptic vesicle-associated proteins. Although its localization and significance in non-neuronal cells were not analysed here, at least in the axon terminal annexin V may participate in the cluster formation of synaptic vesicles by linking with the cytoplasmic surface of the vesicles in a Ca(2+)-dependent manner.