Time superresolution microscopy to study of the function of syntaxin clusters

时间超分辨率显微镜研究突触蛋白簇的功能

• 批准号: 8748044
• 负责人: Manfred LINDAU
• 金额: $ 24.15万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8748044
• 关键词: Back; Behavior; Botox; C-terminal; Cell membrane; Cells; Cellular biology; Color; Complex; Cosmetics; Cytoplasmic Granules; Detection; Event; Exocytosis; Fluorescence; Glass; Hormones; Image; Individual; Intracellular Transport; Laboratories; Lead; Link; Lipids; Mediating; Medical; Medicine; Membrane; Membrane Fusion; Methods; Microscopy; Modeling; Molecular; Molecular Conformation; Molecular Machines; N-terminal; Names; Neuroendocrine Cell; Neurons; Neurotransmitters; Nobel Prize; PC12 Cells; Pattern; Play; Process; Property; Protein Isoforms; Proteins; Regulation; Reporting; Research; Resolution; Role; Running; S-nitro-N-acetylpenicillamine; SNAP receptor; Secretory Vesicles; Site; Spasm; Stimulus; Testing; Time; Transmembrane Domain; VAMP-2; Vesicle; Virus Diseases; base; detector; drug mechanism; fluorescence imaging; imaging modality; improved; innovation; neurotransmitter release; novel; public health relevance; receptor; research study; response; syntaxin; syntaxin 1; syntaxin 1A; syntaxin 1B; target SNARE proteins; tool

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.

描述（由申请人提供）：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响应特定刺激而释放递质，通过形成融合孔从分泌囊泡内部释放到细胞外部。这样的融合孔的形成之后可以是囊泡膜与质膜的快速完全融合或延迟的融合孔形成扩张。SNARE（可溶性NSF附着受体）复合物在哺乳动物神经元和神经内分泌细胞中由小突触泡蛋白-2、突触融合蛋白-1和SNAP-25蛋白组成，在该过程中起关键作用。SNARE复合物功能的医学相关性的一个例子是痉挛的BoTox治疗和美容目的，其通过特异性切割SNARE蛋白SNAP-25来抑制递质释放。SNARE复合物的一种组分，突触融合蛋白1，通过单个跨膜螺旋锚定在质膜上，并在膜上形成含有约70个突触融合蛋白拷贝的纳米结构域。这些纳米畴的功能是未知的。SNAP-25是脂质锚定的SNARE复合物的另一种质膜组分。这些分子的一小部分也形成簇，可能与突触融合蛋白1簇重叠。基于最近的证据，即快速完全融合发生在具有更多SNAP-25的位点，而融合孔扩张在具有较少SNAP-25的位点被延迟（Zhao et al PNAS 2013），本研究将检验快速完全融合发生在具有突触融合蛋白1 t-SNARE簇的位点，而在没有这样的簇的位点，融合孔形成为延迟扩张。为了研究突触融合蛋白1簇动力学与融合孔形成和扩张的关系，将应用一种新的创新时间超分辨率成像方法，称为事件相关显微镜（ECOM）。ECOM利用电流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。该方法具有广泛的应用前景，并将在本项目中得到进一步发展。使用最近开发的突触融合蛋白1荧光共振转移（FRET）探针（Greitzer-Antes JCS 2013），将确定突触融合蛋白1的闭合和开放构象之间的转变是否与短暂刺激和/或个体融合事件特异性相关。如果成功的话，这一研究将为确定syntaxin 1在质膜上形成的超分子组装体的功能提供一个强有力的工具。它将促进对释放缺陷及其治疗的医学问题的理解，并导致更好地理解药物和行为的机制。