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的关键过程，从细胞内转运到释放神经递质和激素以及病毒感染。从神经元和神经内分泌细胞中释放出对特定刺激的递质释放，是通过梭形孔的形成从分泌囊泡的内部到细胞外部的。这种融合孔的形成可以在囊泡膜与质膜或延迟融合孔的形成扩张之后快速完全融合。在哺乳动物神经元和神经内分泌细胞中，该蛋白质触发蛋白突触oftinaptobrevin-2，Syntaxin-1和SNAP-25在哺乳动物神经元和神经内分泌细胞中，该复合物（可溶性NSF附着受体）复合物在此过程中起关键作用。 SNARE复合功能的医学相关性的一个例子是痉挛的痉挛处理和化妆品，它通过SNARE蛋白SNAP-25的特定切割抑制了发射机释放。 SNARE复合物的一种组成部分语法1由单个跨膜螺旋锚定在质膜中，并在膜上形成纳米构域，其中包含〜70份语法素的副本。这些纳米域的功能尚不清楚。 SNAP-25是SNARE复合物的另一个质膜成分，是脂质锚定的。这些分子中的一小部分也形成簇，可能与句法1簇重叠。 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.为了研究语法1簇动力学与融合孔的形成和膨胀的关系，将应用一种新型创新的时间渗透性成像方法，称为事件相关显微镜（ECOM）。 ECOM通过利用单个融合事件的安培检测的高时间分辨率来击败成像框架的时间分辨率限制。该方法可能适用于广泛的其他实验，并将在该项目中进一步开发。使用最近开发的语法1荧光共振转移（FRET）探针（Greitzer-Antes JCS 2013），将确定语法1的闭合和开放构象之间的过渡是否特别与短暂刺激和/或与个体融合事件有关。如果成功的话，此研究将提供强大的工具来确定由语法1在质膜中形成的超分子组件的特定功能。它将从释放中的缺陷及其治疗中了解医学问题，并可以更好地理解对药物和行为的机制。