Identifying the Mechanisms and Localization of Activity-Dependent CaMKII Synthesis

项目摘要 在果蝇神经肌肉接头（NMJ），重复刺激已被证明可诱导各种 蛋白质合成依赖的可塑性形式，包括未成熟的"幽灵"钮的生长和增强 微型兴奋性接点电位（mEJP）频率。与此同时， 钙/钙调蛋白依赖性蛋白激酶II（CaMKII）蛋白丰度，这是可塑性所需的。 CaMKII增加的分子机制还不清楚，以前的研究 对这种效应的检查没有区分NMJ的突触前和突触后侧。的 果蝇NMJ具有多种遗传工具来分离运动神经元和肌肉，使其成为理想的 分离突触前和突触后机制的模型。初步数据显示， 高钾溶液去极化，突触前和突触后CaMK Ⅱ水平均增加， NMJ的侧面。此外，这种增加被放线菌酮的应用所阻断，放线菌酮是一种翻译物， 抑制剂，表明CaMKII积累依赖于蛋白质合成，不太可能是由于 蛋白质移位或转运。这项研究计划的目标是确定分子机制 和突触两侧CaMK Ⅱ合成的定位。在目标1的第一部分，GFP报告基因 构建体将用于确定活性所需的CaMK II 3'UTR中的序列- 依赖翻译在目标1的第二部分中，结合到UTR的反式作用蛋白将是 使用RNA亲和纯化鉴定。这些实验的结果将阐明特定的顺式和 活性依赖性CaMKII蛋白合成所需的反式作用因子，并确定是否 类似的机制在突触前和突触后起作用。在目的2中，Flp-Frt重组将用于缺失 将确定CaMK II催化结构域或3'UTR以及对mEJP频率增强的影响。 结构域的切除将仅在突触前或突触后区室中进行，使得 运动神经元或肌肉的贡献可以清楚地分开。在目标3中，亚细胞 将新合成的CaMKII的定位与预先存在的CaMKII的定位进行比较。这个的功能 新合成的CaMKII库未知，本实验将确定新合成的CaMKII库是否 CaMKII靶向不同的亚细胞纳米结构域。最终，这项研究计划将阐明如何 隔室特异性CaMKII合成可能有助于突触的长期功能变化 并可能发现研究CaMKII依赖性可塑性的新途径。在进行这项研究时， 将提供各种关键技能的深入培训，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。

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.