Synaptic vesicles are responsible for releasing neurotransmitters and are thus essential to brain function. The classical mode of vesicle recycling includes full collapse of the vesicle into the plasma membrane and clathrin-mediated regeneration of a new vesicle. In contrast, a nonclassical mode known as "kiss-and-run" features fusion by a transient fusion pore without complete loss of vesicle identity and offers possible advantages for increasing the throughput of neurotransmission. Studies of vesicular traffic have benefited greatly from fluorescent probes like FM dyes and synaptopHluorin. However, intrinsic properties of these probes limit their ability to provide a simple and precise distinction between classical and nonclassical modes. Here we report a novel optical probe specific to full collapse fusion, capitalizing on the size and superior photo-properties of photoluminescent quantum dots (Qdots). Qdots with exposed carboxyl groups were readily taken up by synaptic vesicles in an activity-, Ca2+-, and clathrin-dependent manner. Electron microscopy showed that Qdots were harbored within individual vesicles in a 1:1 ratio. The release of Qdots was activity- and Ca2+-dependent, similar to FM dyes. As artificial cargo, ≈15 nm in diameter, Qdots will not escape vesicles during kiss-and-run but only with full collapse fusion. Strikingly, Qdots unloaded with kinetics substantially slower than destaining of FM dye, indicating that full-collapse fusion contributed only a fraction of all fusion events. As a full-collapse-fusion- responsive reporter, Qdots will likely promote better understanding of vesicle recycling at small CNS nerve terminals.
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Nov 6 2007|
- Electron microscopy
- Fusion pore
- Nerve terminal
- Quantal release