AbstractKinetic simulation is a useful approach for the elucidation of complex cell-signaling systems. To perform the numerical simulations required for kinetic modeling, parameters such as the protein concentration and dissociation constant (Kd) are essential. However, only a limited number of parameters have been measured experimentally in living cells. Here, we describe a method for quantifying the concentration and Kd of endogenous proteins at the single-cell level with CRISPR/Cas9-mediated knock-in and fluorescence cross-correlation spectroscopy (FCCS). First, the mEGFP gene was knocked-in at the end of the MAPK1 gene, which encoded ERK2, through homology-directed repair or microhomology-mediated end joining. Next, the HaloTag gene was further knocked-in at the end of the RSK2 gene. Protein concentrations of endogenous ERK2-mEGFP and RSK2-HaloTag were quantified in living cells by fluorescence correlation spectroscopy (FCS), revealing substantial cellular heterogeneities. Interestingly, the levels of ERK2-mEGFP and RSK2-HaloTag were strongly positively correlated, suggesting a global mechanism underlying their expressions. In addition, FCCS measurement revealed temporal changes in the apparent Kd values of the binding between ERK2-mEGFP and RSK2-HaloTag in response to EGF stimulation. Our method provides a new approach for quantification of the endogenous protein concentration and dissociation constant in living cells.
Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies