Lorenz-Guertin Joshua M, Wilcox Madeleine R, Zhang Ming, Larsen Mads B, Pilli Jyotsna, Schmidt Brigitte F, Bruchez Marcel P, Johnson Jon W, Waggoner Alan S, Watkins Simon C, Jacob Tija C
Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA.
J Cell Sci. 2017 Nov 15;130(22):3933-3945. doi: 10.1242/jcs.205286. Epub 2017 Oct 12.
Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic γ-aminobutyric acid (GABA) type A receptor (GABAR) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABAR γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2FAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2FAP GABARs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2FAP GABARs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2FAP-transfected neurons and MG dyes show time-dependent receptor accumulation into intracellular vesicles, revealing constitutive endosomal and lysosomal trafficking. Simultaneous analysis of synaptic, surface and lysosomal receptors using the γ2FAP-MG dye approach reveals enhanced GABAR turnover following a bicucculine-induced seizure paradigm, a finding not detected by standard surface receptor measurements. To our knowledge, this is the first application of the FAP-MG dye system in neurons, demonstrating the versatility to study nearly all phases of GABAR trafficking.
活细胞成像方法能够提供关键的实时受体运输测量数据。在此，我们描述一种光学工具，通过适应性荧光追踪能力来研究突触γ-氨基丁酸（GABA）A型受体（GABAR）的动力学。一种荧光团激活肽（FAP）被基因插入到标记有pH敏感绿色荧光蛋白的GABARγ2亚基中（γ2FAP）。FAP选择性地结合并激活孔雀石绿（MG）染料，而这些染料在溶液中原本是无荧光的。γ2FAP GABARs在转染的皮层神经元的细胞表面表达，形成突触簇，并且不会干扰神经元发育。电生理研究表明γ2FAP GABARs对GABA有反应，并且在用苯二氮䓬类药物地西泮刺激时表现出正向调节作用。使用γ2FAP转染神经元和MG染料的成像研究显示，受体随时间积累到细胞内囊泡中，揭示了组成型内体和溶酶体运输。使用γ2FAP-MG染料方法对突触、表面和溶酶体受体进行同步分析，发现在荷包牡丹碱诱导的癫痫发作模式后GABAR周转增强，这一发现通过标准表面受体测量未检测到。据我们所知，这是FAP-MG染料系统在神经元中的首次应用，证明了其在研究GABAR运输几乎所有阶段的通用性。
