Membrane fusion is a key process in cell biology from intracellular transport to release of neurotransmitters and hormones and viral infection. This release occurs from the interior of the secretory vesicle to the outside of the cell via formation of a fusion pore. In neurosecretory cells release is stimulated by calcium entry, which induces rapid release of primed vesicles that form a readily releasable pool. The SNARE (Soluble NSF Attachment REceptor) complex, which in mammalian neurons and neuroendocrine cells is composed of the proteins synaptobrevin-2, syntaxin-1, and SNAP-25, plays a key role in vesicle fusion. One example for the medical relevance of SNARE complex function is the BoTox treatment, which inhibits transmitter release by specific cleavage of the SNARE protein SNAP-25. Although the components of the SNARE complex and the identity of several accessory proteins are known, it is still unclear how SNARE complex assembly induces priming of vesicles, fusion pore opening and dilation, and how many SNARE complexes participate in these steps. One method to probe conformational changes in a protein complex utilizes Fluorescence Resonance Energy Transfer (FRET). In this approach, a FRET donor and a FRET acceptor are incorporated into the protein. The fluorescent proteins CFP and YFP (and their derivatives with similar spectra) form a suitable FRET pair and have been incorporated at the N terminal ends of the SNAP-25 SNARE domains. We recently found that this SNARE COmplex REporter (SCORE) shows a rapid transient FRET change specifically associated with fusion events. This research uses FRET probes of SNAP-25 as well as syntaxin-1 based FRET constructs that report transitions between the open and closed state of syntaxin to elucidate the nanomechanical molecular steps and rearrangements associated with vesicle priming and fusion.
Neurotransmitters and hormones are stored at high concentration in secretory vesicles from which they are released by fusion of vesicle and plasma membrane in response to stimulation. The proteins SNAP-25 and syntaxin 1 play a key role in the fusion mechanism. This project is aimed at determining their mechanistic nanomechanical function in vesicle fusion and transmitter release.