Department of Cell and Developmental Biology, State University of New York, Upstate Medical University, Weiskotten Hall, 766 Irving Avenue, Syracuse, NY 13210, USA; Department of Cell Biology, Emory University School of Medicine, 615 Michael St., Atlanta, GA 30322, USA.
Department of Cell Biology, Emory University School of Medicine, 615 Michael St., Atlanta, GA 30322, USA.
Cell Rep. 2022 Nov 15;41(7):111658. doi: 10.1016/j.celrep.2022.111658.
The ubiquitination/proteasome system is important for the spatiotemporal control of protein synthesis and degradation at synapses, while dysregulation may underlie autism spectrum disorders (ASDs). However, methods allowing direct visualization of the subcellular localization and temporal dynamics of protein ubiquitination are lacking. Here we report the development of Single-Molecule Ubiquitin Mediated Fluorescence Complementation (SM-UbFC) as a method to visualize and quantify the dynamics of protein ubiquitination in dendrites of live neurons in culture. Using SM-UbFC, we demonstrate that the rate of PSD-95 ubiquitination is elevated in dendrites of FMR1 KO neurons compared with wild-type controls. We further demonstrate the rapid ubiquitination of the fragile X messenger ribonucleoprotein, FMRP, and the AMPA receptor subunit, GluA1, which are known to be key events in the regulation of synaptic protein synthesis and plasticity. SM-UbFC will be useful for future studies on the regulation of synaptic protein homeostasis.
泛素/蛋白酶体系统对于突触处蛋白质合成和降解的时空控制非常重要，而其失调可能是自闭症谱系障碍（ASD）的基础。然而，目前还缺乏能够直接观察蛋白质泛素化的亚细胞定位和时间动态的方法。在这里，我们报告了单分子泛素介导的荧光互补（SM-UbFC）的发展，这是一种在培养的活神经元树突中可视化和定量蛋白质泛素化动力学的方法。使用 SM-UbFC，我们证明与野生型对照相比，FMR1 KO 神经元树突中的 PSD-95 泛素化速率升高。我们进一步证明了脆性 X 信使核糖核蛋白 FMRP 和 AMPA 受体亚基 GluA1 的快速泛素化，这是调节突触蛋白合成和可塑性的关键事件。SM-UbFC 将有助于未来研究突触蛋白动态平衡的调节。
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