Direct single-molecule observation ofregulated SNARE assembly
调节 SNARE 组装的直接单分子观察
基本信息
- 批准号:9256820
- 负责人:
- 金额:$ 2.87万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2017
- 资助国家:美国
- 起止时间:2017-01-01 至 2018-12-31
- 项目状态:已结题
- 来源:
- 关键词:Action PotentialsAddressAffectAtaxiaBindingBiological AssayBiological ProcessC-terminalCalciumCis TestsClosure by clampComplexDiseaseEpilepsyEventExocytosisHormonesHumanImpairmentIntellectual functioning disabilityKineticsLengthLightLipidsMeasurementMeasuresMechanicsMediatingMembraneMembrane FusionMental disordersMethodsModelingMolecularMolecular ConformationMutationN-terminalNervous System PhysiologyNeurologicNeuronsNeuropathyNeurotransmittersNon-Insulin-Dependent Diabetes MellitusParkinson DiseasePathway interactionsPositioning AttributeProcessProteinsPsyche structureRegulationResearchResolutionRoleS-nitro-N-acetylpenicillamineSNAP receptorSchizophreniaStructureSumSynapsesSynaptic TransmissionSynaptic VesiclesTestingTransmembrane DomainVesiclebasecontrolled releasedisabilitydisease-causing mutationexperimental studyfallsinsightinsulin secretioninterestlaser tweezermembrane reconstitutionmillisecondnanodisknanometernervous system disorderneurotransmitter releasenew therapeutic targetreceptorreconstitutionresponsesingle moleculesynaptotagminvesicular release
项目摘要
Project Summary – Aleksander A. Rebane
Imbalances in Ca2+-triggered exocytosis of hormones and neurotransmitters cause severe diseases, including
type 2 diabetes and various neurological disorders such as Parkinson’s disease, schizophrenia, and epilepsy.
Yet it remains poorly understood how Ca2+ triggers exocytosis. Of particular interest is synaptic exocytosis of
neurotransmitters, which occurs in response to the local influx of Ca2+, triggered by the arrival of an action
potential at the axonal terminal. The core machinery responsible for membrane fusion in regulated exocytosis
consists of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), complexin, and
synaptotagmin. SNAREs form a broad class of molecular fusion machines that associate by forcefully
zippering into a coiled-coil four-helix structure, thus drawing opposing membranes into close proximity for
fusion. Additional components are required to turn fusion on and off to regulate synaptic transmission. This
task is achieved by complexin and synaptotagmin, which suppress unwanted spontaneous synaptic exocytosis
by suspending SNARE zippering halfway as a clamp. Controlled release occurs when calcium removes the
clamp to resume SNARE zippering and induce neurotransmitter release. However, the exact constituents of
the clamp and the mechanism of clamping and de-clamping are poorly understood. It has been difficult to
observe the molecular events along the regulated SNARE assembly pathway using traditional ensemble-based
methods. These events are inherently transient and occur solely in the presence of the membrane’s repulsive
force. We address these difficulties by using optical tweezers to apply precisely known pulling forces on single
SNARE complex molecules to mimic membrane repulsion and to stabilize the partially assembled
intermediates, while using molecular extension measurements to determine the structures on millisecond
timescale and at nanometer resolution. We analyze these measurements with state-of-the-art methods to
derive the conformations, energies, and kinetics of SNARE complex assembly intermediates. In Aim 1, we will
analyze the effect of two mutations in the SNARE complex, SNAP-25 I67T and I67N, which cause severe
neuropathy, including ataxia and intellectual disability. We will use optical tweezers to measure how these
mutations change the energetics, kinetics, and pathways of SNARE zippering. We will then employ
reconstituted membrane-fusion assays to compare how the observed changes in SNARE zippering affect
SNARE-mediated membrane fusion. In Aim 2, we will directly observe the effect of complexin, synaptotagmin,
and calcium on SNARE assembly. We will investigate by what mechanism SNARE assembly is clamped by
complexin, and how synaptotagmin and Ca2+ release this clamp. Our research will reveal how SNARE
mutations may cause neuropathy and how complexin and synaptotagmin regulate Ca2+-dependent release.
Our research will provide concrete molecular mechanisms to act as new drug targets for neurological disease.
项目总结- Aleksander A. Rebane
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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