Live imaging of bulk endocytosis in frog motor nerve terminals using FM dyes

We observed endocytosis in real time in stimulated frog motor nerve terminals by imaging the growth of large membrane infoldings labeled with a low concentration of FM dye. The spatial and temporal information made available by these experiments allowed us to image several new aspects of this synaptic vesicle recycling pathway. Membrane infoldings appeared near synaptic vesicle clusters and grew rapidly during long-duration, high-frequency stimulation. In some cases, we observed large, elongated infoldings growing laterally into the terminal. We used these observations to calculate infolding growth rates. A decrease in stimulation frequency caused a decrease in growth rates, but the overall length of these structures was unaffected by frequency changes. Attempts to wash the dye from these infoldings after stimulation were unsuccessful, demonstrating that the fluorescent structures had been endocytosed. We also used this technique to trigger and image infoldings during repeated, short trains. We found that membrane uptake occurred repeatedly at individual endocytosis sites, but only during a portion of the total number of trains delivered to the terminal. Finally, we showed that phosphatidylinositol 3-kinase, but not actin, was involved in this endocytosis pathway. The ability to monitor many individual bulk endocytosis sites in real time should allow for new types of endocytosis measurements and could reveal novel and unexpected mechanisms for coordinating membrane recovery during synaptic activity.