Single-Molecule Imaging of Ubiquitination Dynamics in Neurons

泛素化的亚细胞动力学在基因的许多生物学过程中起着重要的作用。 突触功能和损伤的表达可能直接导致和/或促成神经疾病。 虽然过去在固定细胞上使用生化分离或显微镜的研究表明， 泛素化-蛋白酶体系统(UPS)在突触蛋白稳态和神经元功能中的作用 使用不允许亚细胞定位和泛素化动力学的直接可视化。这个 泛素化产物的瞬变性质阻碍了它们的检测，并阻止了对比率和 突触可塑性和/或神经学改变如何影响蛋白质泛素化的速度 疾病。我们最近开发了一种新的单分子方法，单分子泛素化 介导的荧光互补(SM-UbFC)，使用裂解金星为基础的荧光记者 可视化和量化活神经元泛素化的亚细胞动力学。此方法将应用于 利用培养的小鼠皮质阐明突触和RNA生物学在健康和疾病中的新机制 神经元和人类IPSC来源的神经元。AIM-1验证可塑性诱导刺激调节的假说 突触支架蛋白和谷氨酸受体亚基直接在树突棘中泛素化， 在脆性X综合征中会发生改变。我们将进一步开发、优化和应用SM-UbFC来进行动态检测 小鼠和人iPSC来源神经元突触蛋白泛素化的变化 可塑性诱导刺激。AIM-2验证了CDH1-APC E3连接酶复合体调节RNA的假设 通过泛素化多个RNA结合蛋白在突触上的颗粒，专注于我们已有的候选 最近在对CDH1相互作用组的质谱分析中被鉴定出来。其中包括FMRP、FXR1P和 卡普林-1。我们将进一步开发、优化和应用SM-UbFC来检测数据中的动态变化 利用小鼠皮质神经元和人IPSC泛素化RNA颗粒中的这些RNA结合蛋白 衍生的神经元。我们将进一步验证在FMR1 KO中RNA颗粒动力学改变的假设 神经细胞或与疾病相关的Caprin-1突变。在Aims1+2中选择的型号系统将揭示新的 泛素化动力学调节突触后密度和谷氨酸受体的机制(AIM-1) 定位于突触的RNA颗粒(AIM-2)。SM-UbFC方法有望在未来的应用中发挥作用 研究阐明泛素化动力学的调节机制并了解它们可能出错的原因 在导致突触蛋白稳态失调的神经系统疾病中。这种敏感的方法 提供了一种潜在的工具来评估纠正泛素化缺陷的治疗策略的有效性 神经疾病的动力学。

The subcellular dynamics of ubiquitination has important roles in many biological processes from gene expression to synaptic function and impairments may directly result and/or contribute to neurological disease. While past studies using biochemical fractionation or microscopy on fixed cells suggest the importance of the ubiquitination-proteasome system (UPS) in synaptic protein homeostasis and neuronal function, the methods used do not allow for the direct visualization of subcellular localization and dynamics of ubiquitination. The transient nature of the ubiquitinated products hinders their detection and prevents the analysis of the rate and how the rate of protein ubiquitination may be affected by synaptic plasticity and/or altered in neurological diseases. We have recently developed a new single molecule method, Single Molecule Ubiquitination Mediated Fluorescence Complementation (SM-UbFC), using split-Venus-based fluorescent reporters to visualize and quantify the subcellular dynamics of ubiquitination in live neurons. This method will be applied to elucidate new mechanisms of synapse and RNA biology in health and disease using cultured mouse cortical neurons and human iPSC derived neurons. Aim-1 tests the hypothesis that plasticity-inducing stimuli regulate ubiquitination of synaptic scaffolding proteins and glutamate receptor subunits directly in dendritic spines, which is altered in fragile x syndrome. We will further develop, optimize and apply SM-UbFC to detect dynamic changes in the ubiquitination of synaptic proteins in mouse and human iPSC derived neurons in response to plasticity inducing stimuli. Aim- 2 tests the hypothesis that the Cdh1-APC E3 ligase complex regulates RNA granules at synapses by ubiquitination of multiple RNA binding proteins, focusing on candidates we have recently identified in a mass spectrometry analysis of the Cdh1 interactome. These include FMRP, FXR1P and Caprin-1. We will further develop, optimize and apply SM-UbFC to detect dynamic changes in the ubiquitination of these RNA binding proteins in RNA granules using mouse cortical neurons and human iPSC derived neurons. We will further test the hypothesis that RNA granule dynamics are altered in FMR1 KO neurons or by disease linked mutations in Caprin-1. The model systems selected in Aims1+2 will uncover new mechanisms of ubiquitination dynamics regulating the postsynaptic density and glutamate receptors (Aim-1) and RNA granules localized to synapses (Aim-2). The SM-UbFC method is anticipated to be useful for future studies to elucidate mechanisms regulating ubiquitination dynamics and understanding how they may go awry in neurological diseases leading to dysregulation of synaptic protein homeostasis. This sensitive method provides a potential tool to assess efficacy of therapeutic strategies to correct defects in ubiquitination dynamics in neurological disease.