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

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.
项目摘要 在果蝇神经肌肉接头(NMJ),重复刺激被证明能诱导出不同的 依赖蛋白质合成的可塑性的形式,包括未成熟的“幽灵”突起的生长和增强 微型兴奋连接电位(MEJP)频率。同时还增加了 钙/钙调蛋白依赖的蛋白激酶II(CaMKII)蛋白丰富,这是可塑性所必需的。 CaMKII增加的分子机制还不是很清楚,以前的研究 检查这一效应并没有区分NMJ的突触前和突触后。这个 果蝇NMJ有各种各样的遗传工具来分离运动神经元和肌肉,这使它成为理想的 分离突触前和突触后机制的模型。初步数据显示,在间隔后 高钾溶液去极化,突触前和突触后CaMKII水平升高 NMJ的两边。此外,这种增加被环己亚胺的应用所阻止。 抑制剂,表明CaMKII的积累依赖于蛋白质的合成,不太可能是由于 蛋白质的移位或运输。这项研究计划的目标是确定分子机制。 CaMKII合成在突触两侧的定位。在目标1的第一部分,GFP记者 构建物将用于确定CaMKII 3‘非编码区中活性所需的序列- 从属翻译。在目标1的第二部分中,与UTR结合的反式作用蛋白将是 采用RNA亲和纯化技术进行鉴定。这些实验的结果将阐明特定的顺式-和 活性依赖的CaMKII蛋白合成所需的反式作用因子,并决定是否 类似的机制在突触前和突触后起作用。在目标2中,将使用FLP-FRT重组来删除 将确定CaMKII催化结构域或3‘非编码区以及对mEJP频率增强的影响。 区域的切除将仅在突触前或突触后间隔进行,以便 运动神经元和肌肉的作用可以清楚地区分开来。在《目标3》中,亚细胞 新合成的CaMKII的本地化将与现有的CaMKII进行比较。它的功能是 新合成的CaMKII池未知,本次实验将确定新合成的 CaMKII针对的是一个独特的亚细胞纳米结构域。最终,这项研究计划将阐明 脑室特异性CaMKII的合成可能有助于突触的长期功能变化 并可能确定研究CaMKII依赖的可塑性的新途径。在进行这项研究时, 将提供各种关键技能方面的深入培训,包括研究设计、统计分析、 沟通技巧、超分辨率显微镜和图像分析。

项目成果

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