Identifying the Mechanisms and Localization of Activity-Dependent CaMKII Synthesis
确定活性依赖性 CaMKII 合成的机制和定位
基本信息
- 批准号:10750472
- 负责人:
- 金额:$ 3.86万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2023
- 资助国家:美国
- 起止时间:2023-07-16 至 2026-07-15
- 项目状态:未结题
- 来源:
- 关键词:3&apos Untranslated RegionsAdultAffectAffinity ChromatographyAllelesAlzheimer&aposs DiseaseAxonBindingBinding ProteinsBinding SitesBiological ModelsBrain DiseasesCRISPR/Cas technologyCa(2+)-Calmodulin Dependent Protein KinaseCatalytic DomainCellsChemosensitizationCommunicationCycloheximideDataDendritesDrosophila genusElementsExcisionFrequenciesFunctional disorderGenesGeneticGenetic RecombinationGoalsGrowthImage AnalysisImaging DeviceIntellectual functioning disabilityInvertebratesLearningLinkMammalsMemoryMicroRNAsModelingModificationMolecularMotor NeuronsMovementMuscleNeuromuscular JunctionNeuronsPhysiologic pulsePotassiumProcessProtein BiosynthesisProtein translocationProteinsProtocols documentationRNARNA-Binding ProteinsReporterResearchResearch DesignResearch ProposalsRoleSideSpatial DistributionStatistical Data InterpretationStimulusStructureSynapsesSystemTrainingTrans-ActivatorsTranslationsUntranslated Regionscalmodulin-dependent protein kinase IIexperimental studyflyinhibitorinsightnanopostsynapticpresynapticprotein transportskillssuperresolution imagingsuperresolution microscopysynaptic functiontool
项目摘要
Project Summary
At the Drosophila neuromuscular junction (NMJ), repetitive stimulation has been shown to induce various
forms of protein synthesis-dependent plasticity, including growth of immature “ghost” boutons and potentiation
of miniature excitatory junction potential (mEJP) frequency. There is a concurrent increase in
calcium/calmodulin-dependent protein kinase II (CaMKII) protein abundance, which is required for plasticity.
The molecular mechanisms of this increase in CaMKII are not well understood, and previous studies
examining this effect did not differentiate between the presynaptic and postsynaptic sides of the NMJ. The
Drosophila NMJ has a wide variety of genetic tools to separate the motoneuron and muscle, making it an ideal
model for separating presynaptic and postsynaptic mechanisms. Preliminary data indicate that after spaced
depolarization with a high potassium solution, CaMKII levels increase in both the presynaptic and postsynaptic
sides of the NMJ. Additionally, this increase was blocked by application of cycloheximide, a translation
inhibitor, indicating that CaMKII accumulation is dependent on protein synthesis and is unlikely to be due to
protein translocation or transport. The goal of this research proposal is to identify the molecular mechanisms
and localization of CaMKII synthesis on both sides of the synapse. In the first part of Aim 1, GFP reporter
constructs will be used to determine the sequences in the CaMKII 3’UTR that are required for activity-
dependent translation. In the second part of Aim 1, the trans-acting proteins that bind to the UTR will be
identified using RNA affinity purification. The results from these experiments will elucidate the specific cis- and
trans-acting factors that are required for activity-dependent CaMKII protein synthesis, and determine whether
similar mechanisms act pre- and post-synaptically. In Aim 2, Flp-Frt recombination will be used to delete either
the CaMKII catalytic domain or the 3’UTR and the effect on potentiation of mEJP frequency will be determined.
The excision of the domains will be done exclusively in the presynaptic or postsynaptic compartment, so that
the contribution of either the motoneuron or muscle can be clearly separated. In Aim 3, the subcellular
localization of newly-synthesized CaMKII will be compared to that of pre-existing CaMKII. The function of this
newly-synthesized CaMKII pool is unknown, and this experiment will determine whether the newly-synthesized
CaMKII is targeted to a distinct subcellular nanodomain. Ultimately, this research plan will clarify how
compartment-specific CaMKII synthesis may be contributing to long-term functional changes in the synapse
and may identify new avenues of research into CaMKII-dependent plasticity. While conducting this research,
in-depth training will be provided in a variety of crucial skills, including research design, statistical analysis,
communication skills, super-resolution microscopy, and image analysis.
项目总结
项目成果
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