Visualizing cellular heterogeneity by quantifying the dynamics of MAPK activity in live mammalian cells with synthetic fluorescent biosensors

Min Ma,Pino Bordignon,Gian-Paolo Dotto,Serge Pelet

doi:10.1016/j.heliyon.2020.e05574

Abstract

Mitogen-Activated Protein Kinases (MAPKs) control a wide array of cellular functions by transducing extracellular information into defined biological responses. In order to understand how these pathways are regulated, dynamic single cell measurements are highly needed. Fluorescence microscopy is well suited to perform these measurements. However, more dynamic and sensitive biosensors that allow the quantification of signaling activity in living mammalian cells are required. We have engineered a synthetic fluorescent substrate for human MAPKs (ERK, JNK and p38) that relocates from the nucleus to the cytoplasm when phosphorylated by the kinases. We demonstrate that this reporter displays an improved response compared to other relocation biosensors. This assay allows to monitor the heterogeneity in the MAPK response in a population of isogenic cells, revealing pulses of ERK activity upon a physiological EGFR stimulation. We show applicability of this approach to the analysis of multiple cancer cell lines and primary cells as well as its application in vivo to developing tumors. Using this ERK biosensor, dynamic single cell measurements with high temporal resolution can be obtained. These MAPK reporters can be widely applied to the analysis of molecular mechanisms of MAPK signaling in healthy and diseased state, in cell culture assays or in vivo.

Highlights

Given that cells live in changing environments, they have developed complex biochemical networks to sense and transmit extracellular information
Nuclear Localization Signals (NLSs) are small amino acid sequences that confer on proteins the ability to be transported in the nucleus
These domains are highly conserved throughout eukaryotes, and for instance the same SV40 NLS motif is commonly used in yeast as well as in mammalian cells

Summary

Introduction

Given that cells live in changing environments, they have developed complex biochemical networks to sense and transmit extracellular information. Intensive biological research has allowed the identification of multiple signal transduction pathways that relay the presence of nutrients, stress or hormones in the cell's surroundings. To fully grasp the complex regulation of these biochemical signals, quantitative and dynamic measurements have to be performed. Population-averaged measurements can prevent the identification of important signaling patterns such as oscillations or bimodality [2, 3]. Microscopy and flow cytometry, as well as novel techniques such as mass cytometry or single cell sequencing, have gained in importance to unravel the biochemical signals taking place in single cells. More tools are required to enable the quantification of signaling activity in single cells in order to uncover novel biochemical regulations in signal transduction cascades

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