Research & Publications
Neurotransmitters and hormones are released when a cargo-filled vesicle fuses with the plasma membrane. This requires trafficking and docking of the vesicle to the release site, priming of the vesicle to acquire fusion-competence, and a trigger signal in the form of an increase in calcium concentration (elicited by calcium influx through voltage-gated calcium channels that open in response to a depolarization). The fusion step is driven by formation of a complex between vesicular v-SNARE proteins and target membrane t-SNAREs. Other proteins are also essential. Synaptotagmin couples calcium entry to fusion with astonishing speed, complexin regulates both spontaneous and evoked release, Mun13 primes vesicles, Munc18 chaperones SNARE assembly, and other proteins dissociate post-fusion SNARE complexes for recycling.
The initial, nanometer-sized pore that opens is also subject to regulation. High resolution electrophysiological and electrochemical measurements found that the pore may fluctuate in size, and can flicker open and shut multiple times. Cargo release and vesicle recycling depend on the fate of the pore, which may reseal or dilate irreversibly. Mechanisms governing pore dynamics are not understood.
We have developed novel in vitro assays with biochemically defined components to study the exocytotic fusion process with single pore, or even single molecule sensitivity, and sub-ms to ~10 ms time resolution. We found pores are more stable than previously appreciated, and pore lifetimes are very sensitive to mutations in the SNARE transmembrane domains. We also found only a few SNARE complexes are required for opening a pore, but the subsequent pore dilation requires many more SNAREs. We are currently exploring how synaptotagmin couples calcium binding to fusion.
Specialized Terms: Membrane fusion; Exocytosis; Secretory vesicle dynamics; Fluorescence microscopy; Image analysis; Microfluidics; Supported bilayers; Proteoliposomes
Exocytosis; Liposomes; Membrane Fusion; Microscopy, Fluorescence; Molecular Biology; Physiology; Secretory Vesicles; Microfluidics