Membrane fusion is a key process in cell biology from intracellular transport to release of neurotransmitters and hormones and viral infection. Transmitter release from neurons and neuroendocrine cells in response to specific stimuli occurs from the interior of the secretory vesicle to the outside of the cell via formation of a fuson pore. The formation of such a fusion pore may be followed by rapid full fusion of the vesicle membrane with the plasma membrane or by delayed fusion pore formation dilation. 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 this process. One example for the medical relevance of SNARE complex function is the BoTox treatment of spasms and for cosmetic purpose, which inhibits transmitter release by specific cleavage of the SNARE protein SNAP-25. One component of SNARE complex, Syntaxin 1, is anchored in the plasma membrane by a single transmembrane helix and forms nanodomains in the membrane that contain ~70 copies of syntaxin. The function of these nanodomains is unknown. SNAP-25 is another plasma membrane component of the SNARE complex that is lipid-anchored. A small fraction of these molecules also form clusters, possibly overlapping with the syntaxin 1 clusters. Based on recent evidence that rapid full fusion occurs at sites with more SNAP-25 while fusion pore dilation is delayed at sites with less SNAP-25 (Zhao et al PNAS 2013), this research will test the hypothesis that rapid full fusion occurs at sites with syntaxin 1 t-SNARE clusters whereas at sites without such a cluster, fusion pores are formed that dilate with a delay. To investigate the relation of syntaxin 1 cluster dynamics to fusion pore formation and expansion, a novel innovative time-superresolution imaging method named Event COrrelation Microscopy (ECOM) will be applied. ECOM beats the time resolution limit of imaging frames by taking advantage of the high time resolution of amperometric detection of single fusion events. The method is potentially applicable in a wide range of other experiments and will be further developed in this project. Using a recently developed syntaxin 1 Fluorescence Resonance Transfer (FRET) probe (Greitzer-Antes JCS 2013), it will be determined if the transition between the closed and open conformation of syntaxin 1 is specifically related to brief stimuli and/or to individual fusion events. If successful, this reseach will provide a powerful tool to determine the specific function of the supramolecular assemblies formed by syntaxin 1 in the plasma membrane. It will advance understanding of medical problems from deficiencies in release and their treatment and lead to better understanding of the mechanisms of drugs and behavior.
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 molecular machine that performs this function is organized in nanodomains the function of which is unknown. This project is aimed at understanding the role of these molecular nanodomains in the release mechanism to improve understanding of medical problems from deficiencies in release and their treatment and to better understand the mechanisms of drugs and behavior.
|Sharma, Satyan; Lindau, Manfred (2016) The mystery of the fusion pore. Nat Struct Mol Biol 23:5-6|