Intermediates in synaptic vesicle recycling revealed by optical imaging of Drosophila neuromuscular junctions

We show that uptake and release of the styryl dye FM-143 may be used to monitor synaptic vesicle exocytosis and recycling at Drosophila larval neuromuscular junctions. At Drosophila nerve terminals, FM1–43 specifically labels subsynaptic domains enriched in synaptotagmin, in a manner that requires Ca 2+, membrane depolarization, and shibire ( shi) function. Endocytosis rates, very low in unstimulated synapses, are induced severalfold by the exocytosis of synaptic vesicles. Using shi ts1 mutant synapses to separate synaptic vesicle fusion and recycling temporally, we show that recycling events subsequent to the shi block do not require extracellular Ca 2+. We suggest that two distinct intermediate stages in vesicle recycling may be trapped and analyzed at Drosophila neuromuscular junctions.