REGULATION OF THE NEUROMUSCULAR JUNCTION BY THE CYTOSKELETON-ASSOCIATED PROTEIN, ADDUCIN

神经肌肉接头(NMJ)包括运动神经元终末、突触间隙和突触后肌。我的研究项目以果蝇(果蝇)为模型，试图了解NMJ的突触后区域是如何构建的，以及突触活动是如何修改的。我的研究计划的长期目标是了解突触后的细胞骨架如何调节NMJ的形态和功能，因为它是一个蛋白质晶格，关键的突触蛋白质，如支架分子和神经递质受体，在其上整合。我们发现细胞骨架蛋白adducin(ADD；在果蝇中称为HTS：ADD/HTS)是一种肌动蛋白-幽灵蛋白结合蛋白，调节着NMJ的突触结构。在果蝇中，我们发现突触后ADD/HTS的过度表达导致NMJ突触过度生长和NMJ支架分子Discs Large(DLG)的磷酸化，DLG是一种与SAP-97同源的膜相关鸟氨酸激酶。我们有数据表明ADD/HTS也可能从突触移位到细胞核，这表明ADD/HTS是一种突触核蛋白。*我们的短期目标包括：*目标1：检查ADD/HTS相对于包括DLG在内的其他蛋白质在NMJ突触的分布：我们假设，当ADD/HTS在突触激活后被磷酸化时，它从突触移位到细胞核，在那里它改变了几种激酶(例如，钙/钙调素依赖的蛋白激酶II；CaMKII)的核转录输出，从而磷酸化DLG，导致DLG在突触上的去定位。这些作用将破坏突触的稳定性，使突触发生生长或收缩。*目标2：确定ADD/HTS在细胞核中的作用：我们将确定ADD/HTS与其他核蛋白和RNA物种之间的分子相互作用，如CaMKII转录本。*目标3：探索ADD/HTS作为核输出调节因子的作用：我们将确定ADD/HTS改变突触中特定蛋白激酶水平的机制，包括CaMKII和分割缺陷-1/微管亲和力调节激酶，两者都是DLG的磷酸化，以及细胞核中的ADD/HTS是否有助于这些蛋白激酶转录本的输出。*这项工作是原创性的，也是重要的，因为我们以前的和初步的数据指向了细胞骨架相关蛋白如何修改突触功能的新视角。我们现在认为，突触中的信号信息并不停留在突触中，而是在细胞内广泛传播，包括通过ADD/HTS本身以及其他分子传播到细胞核。建议的实验提供了一个相对简单、易处理的模型，说明如何在完整的动物身上确定ADD/HTS的功能，并导致了解它如何调节NMJ的突触形式和功能。

The neuromuscular junction (NMJ) includes the motoneuron terminal, the synaptic gap and post-synaptic muscle. My research program seeks to understand how the post-synaptic region of the NMJ is constructed and is modified by synaptic activity, using the fruit fly (Drosophila) as a model. The long-term goal of my research program is focused on understanding how the cytoskeleton of the post synapse regulates the morphology and function of the NMJ as it is a protein lattice upon which critical synaptic proteins such as scaffolding molecules and neurotransmitter receptors are integrated. We have found that the cytoskeletal protein adducin (Add; known as Hts in Drosophila: Add/Hts), an actin-spectrin binding protein, regulates synapse structure at the NMJ. In Drosophila, we found that post-synaptic Add/Hts over-expression causes NMJ synaptic overgrowth and the phosphorylation of the NMJ scaffolding molecule Discs large (Dlg), a membrane-associated guanylate kinase with homology to SAP-97. We have data that Add/Hts also translocates to the nucleus, likely from the synapse, suggesting that Add/Hts is a synapto-nuclear protein.******Our short-term objectives include: ***Aim 1: To examine Add/Hts distribution at the NMJ synapse in relation to other proteins including Dlg: We hypothesize that when Add/Hts is phosphorylated following synaptic activation it translocates away from the synapse to the cell nucleus where it alters nuclear export of transcripts for several kinases (e.g. Ca2+/calmodulin-dependent protein kinase II; CaMKII) which phosphorylate Dlg leading to a delocalization of Dlg at the synapse. These actions will ‘de-stabilize' the synapse allowing growth or retraction to occur.***Aim 2: To determine the role of Add/Hts in the cell nucleus: We will identify molecular interactions between Add/Hts and other nuclear proteins and RNA species, such as CaMKII transcripts. ***Aim 3: To explore the role of Add/Hts as a regulator of export from the nucleus: We will determine the mechanism by which Add/Hts alters the levels of specific protein kinases in the synapse including CaMKII and partitioning defective-1/ microtubule affinity-regulating kinase, both of which phosphorylate Dlg, and whether Add/Hts actions in the cell nucleus assists in the export of transcripts for these protein kinases.******This work is original and important as our previous and preliminary data point to a novel view of how cytoskeletal-associated proteins modify synaptic function. We now think that signaling information in the synapse does not stay in the synapse, but is disseminated widely in the cell, including to the nucleus, likely by Add/Hts itself, as well as other molecules. The proposed experiments provide a relatively simple, tractable model of how the functions of Add/Hts can be determined in an intact animal and lead to an understanding of how it can regulate synaptic form and function at the NMJ.