Time superresolution microscopy to study of the function of syntaxin clusters

描述（申请人提供）：膜融合是细胞生物学中从细胞内转运到释放神经递质和激素以及病毒感染的关键过程。神经元和神经内分泌细胞的递质释放是对特定刺激的反应，从分泌囊的内部通过融合孔的形成向细胞外释放。这种融合孔的形成可伴随囊泡膜与质膜的快速完全融合或延迟融合孔形成扩张。在哺乳动物神经元和神经内分泌细胞中，由synaptobrein -2、syntaxin-1和SNAP-25蛋白组成的SNARE（可溶性NSF附着受体）复合物在这一过程中起关键作用。SNARE复合物功能的医学相关性的一个例子是用于治疗痉挛和美容目的的肉毒杆菌毒素，它通过特异性切割SNARE蛋白SNAP-25来抑制递质释放。SNARE复合体的一个组成部分Syntaxin 1通过一个单跨膜螺旋锚定在质膜上，并在膜上形成纳米结构域，其中包含约70个Syntaxin拷贝。这些纳米结构域的功能尚不清楚。SNAP-25是SNARE复合体的另一种质膜成分，是脂质锚定的。这些分子的一小部分也形成簇，可能与syntaxin 1簇重叠。根据最近的证据，快速的完全融合发生在SNAP-25较多的位点，而融合孔扩张在SNAP-25较少的位点被延迟（Zhao et al PNAS 2013），本研究将验证这样的假设，即快速的完全融合发生在具有syntaxin 1 t-SNARE簇的位点，而在没有这种簇的位点，融合孔形成并延迟扩张。为了研究syntaxin 1簇动力学与融合孔形成和扩展的关系，将采用一种新颖的时间超分辨率成像方法——事件相关显微镜（ECOM）。ECOM利用安培检测单个融合事件的高时间分辨率，突破了成像帧的时间分辨率限制。该方法可能适用于广泛的其他实验，并将在本项目中进一步发展。使用最近开发的syntaxin 1荧光共振转移（FRET）探针（Greitzer-Antes JCS 2013），将确定syntaxin 1的封闭和开放构象之间的转变是否与短暂刺激和/或单个融合事件特别相关。如果成功，本研究将为确定syntaxin 1在质膜中形成的超分子组装体的具体功能提供有力的工具。它将促进对释放缺陷及其治疗的医学问题的理解，并导致对药物和行为机制的更好理解。

DESCRIPTION (provided by applicant): 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.