Single-cell quantification of the concentrations and dissociation constants of endogenous proteins
Single-cell quantification of the concentrations and dissociation constants of endogenous proteins
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DOI：
10.1074/jbc.ra119.007685
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发表时间：
2019-04-12
影响因子：
4.8
通讯作者：
Aoki, Kazuhiro
中科院分区：
文献类型：
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作者：
Komatsubara, Akira T.;Goto, Yuhei;Aoki, Kazuhiro
动力学模拟是一种阐明复杂细胞信号系统的有效方法。活细胞动力学建模所需的数值模拟关键需要诸如蛋白质浓度和解离常数（K-d）等参数。然而，只有有限数量的参数在活细胞中被实验测量。在这里，我们描述了一种利用CRISPR/ cas9介导的敲入和荧光相互关联光谱在单细胞水平上定量内源蛋白浓度和K-d的方法。首先，通过同源性定向修复或微同源性介导的末端连接，将mEGFP基因敲入丝裂原活化蛋白激酶1 （MAPK1）基因末端，该基因编码细胞外信号调节激酶2 （ERK2）。接下来，在核糖体S6激酶2 （RSK2）基因末端敲入HaloTag基因。然后，我们使用荧光相关光谱测量活细胞中内源性ERK2-mEGFP和RSK2-HaloTag融合构建物的蛋白浓度，揭示了大量的异质性。此外，荧光互相关光谱分析揭示了ERK2-mEGFP和RSK2-HaloTag结合的表观K-d值在表皮生长因子刺激下的时间变化。我们在这里提出的方法提供了一种强大而有效的方法来量化活细胞中的内源性蛋白质浓度和解离常数。
Kinetic simulation is a useful approach for elucidating complex cell-signaling systems. The numerical simulations required for kinetic modeling in live cells critically require parameters such as protein concentrations and dissociation constants (K-d). However, only a limited number of parameters have been measured experimentally in living cells. Here we describe an approach for quantifying the concentration and K-d of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in and fluorescence cross-correlation spectroscopy. First, the mEGFP gene was knocked in at the end of the mitogen-activated protein kinase 1 (MAPK1) gene, encoding extracellular signal-regulated kinase 2 (ERK2), through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was knocked in at the end of the ribosomal S6 kinase 2 (RSK2) gene. We then used fluorescence correlation spectroscopy to measure the protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag fusion constructs in living cells, revealing substantial heterogeneities. Moreover, fluorescence cross-correlation spectroscopy analyses revealed temporal changes in the apparent K-d values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to epidermal growth factor stimulation. Our approach presented here provides a robust and efficient method for quantifying endogenous protein concentrations and dissociation constants in living cells.