Biology subjects

Aoki, K.

Publications and source records attributed to Aoki, K..

2 recordsLinked to original sources

bioRxiv2017

Single-cell quantification of the concentration and dissociation constant of endogenous proteins

Kinetic 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.

biochemistry↗

bioRxiv2017

Cross-inhibition of JNK by p38 visualized by multiplexed fluorescence imaging

AbstractThe stress activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK) and p38, are important players in cell fate decisions in response to environmental stress signals. Crosstalk signaling between JNK and p38 is emerging as an important regulatory mechanism in the inflammatory and stress responses. However, it is still unknown how this crosstalk affects signaling dynamics, cell-to-cell variation, and cellular responses at the single-cell level. To address these questions, we established a multiplexed live-cell imaging system based on kinase translocation reporters to simultaneously monitor JNK and p38 activities with high specificity and sensitivity at single-cell resolution. Various stresses, such as anisomycin, osmotic stress, and UV irradiation, and pro-inflammatory cytokines activated JNK and p38 with various dynamics. In all cases, however, p38 suppressed JNK activity in a cross-inhibitory manner. We demonstrate that p38 antagonizes JNK through both transcriptional and post-translational mechanisms. This cross-inhibition of JNK appears to generate cellular heterogeneity in JNK activity after stress exposure. Our data indicate that this heterogeneity in JNK activity plays a role in fractional killing in response to UV stress. Our highly sensitive multiplexed imaging system enables detailed investigation into the p38-JNK interplay in single cells.\n\nOne Sentence SummaryCross-inhibition by p38 generates cell-to-cell variability in JNK activity.

cell biology↗