Development of a high resolution assay to characterize exocytotic vesicle fusion
开发高分辨率测定法来表征胞吐囊泡融合
基本信息
- 批准号:10041876
- 负责人:
- 金额:$ 9.55万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-06-01 至 2021-10-01
- 项目状态:已结题
- 来源:
- 关键词:Alzheimer&aposs DiseaseAmino AcidsBiological AssayC-terminalCattleCell membraneCellsChromaffin CellsChromaffin granuleCollaborationsComplexDementiaDense Core VesicleDepositionDevelopmentDockingDrug TargetingDrug usageEnsureEnvironmentEventFluorescenceFluorescence Recovery After PhotobleachingFluorescence Resonance Energy TransferHormonesImageIndividualLabelLengthLipidsMeasurementMeasuresMediatingMembraneMembrane FluidityMethodsMicroscopyMolecularMolecular ConformationMolecular MachinesMolecular StructureNamesNatureNeurodegenerative DisordersNeuronsOxidesParkinson DiseasePatternPharmaceutical PreparationsProcessProtein ConformationProteinsProtocols documentationQuartzRecombinantsRegulationResolutionSNAP receptorSecretory VesiclesSiliconSiteStructureSurfaceSystemTechnologyTestingTimeTransmembrane DomainUniversitiesVAMP-2ValidationVenusVesicleVirginiaWorkdetectorexperimental studyfluorescence imagingfluorophorehigh riskinnovative technologiesmillisecondmolecular dynamicsmolecular rearrangementmonolayerneurotransmitter releasenovelprotein functionreceptorreconstitutionsingle moleculesyntaxin 1technology developmenttime use
项目摘要
Transmitter release is mediated by fusion of neurosecretory vesicles with the plasma membrane. While it is
known that Soluble NSF Attachment REceptor (SNARE) proteins form the core complex of the molecular fusion
machine, the precise molecular rearrangements leading to fusion pore formation are still unknown. We will
develop a highly innovative technology that will enable experiments to achieve a precise mechanistic
understanding of structural molecular rearrangements associated with the fusion of neurosecretory vesicles at
the plasma membrane. The approach combines electrochemical detector (ECD) arrays, with reconstituted
supported membranes to study fusion of isolated chromaffin granules simultaneously by amperometry and total
internal reflection fluorescence (TIRF) imaging. We have previously performed combined ECD and TIRF
experiments using intact chromaffin cells and discovered a rapid conformational change in SNAP25 associated
with fusion events. However, these measurements were performed using a FRET construct incorporating
CFP/Venus and the actual nature of the structural change remains unknown. Proceeding to the reconstituted
system will make it possible to incorporate small labels at arbitrary sites in the SNARE proteins or other
accessory proteins, a technology that will make it possible to identify precisely which amino acids in the SNARE
complex and accessory proteins move and change distance at specific times during the fusion process. The
amperometric recordings can be performed with a time resolution of a millisecond or less and by averaging
fluorescence changes from multiple fusion events, the time of such fluorescence changes relative to the fusion
event can be determined with very high precision, not limited by the exposure time used in the fluorescence
image acquisition. This has become possible with the time super-resolution approach named Event Correlation
Microscopy (ECOM), developed in the Lindau Lab. The technology we propose to develop is high risk because
we need to establish a protocol to form the supported bilayers on top of the ECD arrays and explore how a
sufficiently high rate of fusion events at a given ECD array can be achieved to perform the required averaging of
large numbers of fusion events (as we did in the cells). It is known, that when supported bilayers incorporating
SNARE proteins are formed on a quartz or silicon oxide surface, fusion of dense core vesicles does occur. The
project will be performed in collaboration between Dr. Lindau at Cornell who has developed the ECD and ECOM
methods and Dr. Kiessling who has pioneered the study of SNARE protein conformations in the supported bilayer
system. If successful, this technology will enable the experimental identification of the detailed molecular steps
in vesicle fusion and to test the predictions from structural work as well as molecular dynamics simulations.
递质释放是由神经分泌囊泡与质膜融合介导的。当它是
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Manfred LINDAU其他文献
Manfred LINDAU的其他文献
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{{ truncateString('Manfred LINDAU', 18)}}的其他基金
Molecular mechanisms of exocytotic vesicle fusion and release.
胞吐囊泡融合和释放的分子机制。
- 批准号:
10529686 - 财政年份:2021
- 资助金额:
$ 9.55万 - 项目类别:
Molecular mechanisms of exocytotic vesicle fusion and release.
胞吐囊泡融合和释放的分子机制。
- 批准号:
10311492 - 财政年份:2021
- 资助金额:
$ 9.55万 - 项目类别:
Molecular mechanisms of exocytotic vesicle fusion and release.
胞吐囊泡融合和释放的分子机制。
- 批准号:
10553597 - 财政年份:2021
- 资助金额:
$ 9.55万 - 项目类别:
Development of a high resolution assay to characterize exocytotic vesicle fusion.
开发高分辨率测定法来表征胞吐囊泡融合。
- 批准号:
10528722 - 财政年份:2020
- 资助金额:
$ 9.55万 - 项目类别:
Scalable amperometric microchip array for high-throughput screening of small molecules, peptides or genetic perturbations for modulation of quantal transmitter release
可扩展的电流微芯片阵列,用于小分子、肽或遗传扰动的高通量筛选,以调节量子递质释放
- 批准号:
9201261 - 财政年份:2016
- 资助金额:
$ 9.55万 - 项目类别:
Scalable amperometric microchip array for high-throughput screening of small molecules, peptides or genetic perturbations for modulation of quantal transmitter release
可扩展的电流微芯片阵列,用于小分子、肽或遗传扰动的高通量筛选,以调节量子递质释放
- 批准号:
9334939 - 财政年份:2016
- 资助金额:
$ 9.55万 - 项目类别:
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
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8748044 - 财政年份:2014
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A Scalable N x N Electrochemical Detector Array Platform for Analysis of Quantal
用于量子分析的可扩展 N x N 电化学检测器阵列平台
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8322641 - 财政年份:2011
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$ 9.55万 - 项目类别:
A Scalable N x N Electrochemical Detector Array Platform for Analysis of Quantal
用于量子分析的可扩展 N x N 电化学检测器阵列平台
- 批准号:
8660337 - 财政年份:2011
- 资助金额:
$ 9.55万 - 项目类别:
Scalable sensor array platform for analysis of quantal transmitter release events
用于分析量子发射器释放事件的可扩展传感器阵列平台
- 批准号:
8460585 - 财政年份:2011
- 资助金额:
$ 9.55万 - 项目类别:
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