Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA.
Neuron. 2013 Jun 19;78(6):971-85. doi: 10.1016/j.neuron.2013.04.017.
The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed Fibronectin intrabodies generated with mRNA display (FingRs), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and that, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices, FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo.
在活神经元中可视化内源性蛋白质的能力为研究神经元结构和功能提供了一种强大的手段。在这里，我们生成了重组抗体样蛋白，称为使用 mRNA 展示生成的纤维连接蛋白内含子（FingR），它们与内源性神经元蛋白 PSD-95 和 Gephyrin 具有高亲和力，并且当与 GFP 融合时，可以在活神经元中可视化兴奋性和抑制性突触。FingR 的设计包含一个转录调控系统，该系统将 FingR 的表达与靶标水平联系起来，并降低背景荧光。在分离的神经元和脑片中，针对 PSD-95 和 Gephyrin 生成的 FingR 不会影响其内源性靶蛋白的表达模式或突触的数量或强度。总之，我们的数据表明 PSD-95 和 Gephyrin FingR 可以报告活神经元中内源性突触蛋白的定位和数量，因此可用于研究体内突触强度的变化。
