Limited intermixing of synaptic vesicle components upon vesicle recycling

Synaptic vesicles recycle repeatedly in order to maintain synaptic transmission. We have previously proposed that upon exocytosis the vesicle components persist as clusters, which would be endocytosed as whole units. It has also been proposed that the vesicle components diffuse into the plasma membrane and are then randomly gathered into new vesicles. We found here that while strong stimulation (releasing the entire recycling pool) causes the diffusion of the vesicle marker synaptotagmin out of synaptic boutons, moderate stimulation (releasing approximately 19% of all vesicles) is followed by no measurable diffusion. In agreement with this observation, synaptotagmin molecules labeled with different fluorescently tagged antibodies did not appear to mix upon vesicle recycling, when investigated by subdiffraction resolution stimulated emission depletion (STED) microscopy. Finally, as protein diffusion from vesicles has been mainly observed using molecules tagged with pH-sensitive green fluorescent protein (pHluorin), we have also investigated the membrane patterning of several native and pHluorin-tagged proteins. While the native proteins had a clustered distribution, the GFP-tagged ones were diffused in the plasma membrane. We conclude that synaptic vesicle components intermix little, at least under moderate stimulation, possibly because of the formation of clusters in the plasma membrane. We suggest that several pHluorin-tagged vesicle proteins are less well integrated in clusters.