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

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 that relocates from the nucleus to the cytoplasm when phosphorylated by the kinase. We demonstrate that this reporter provides a better sensitivity relative to other similar biosensors and has allowed the monitoring of ERK MAPK activity pulses upon a single physiological EGF stimulation. In addition, we display its applicability to other MAPKs and in multiple cancer cell lines or primary cells as well as its application in vivo in developing tumors. Using our newly developed biosensors, dynamic single cell measurements with high temporal resolution can be obtained. These 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.

Real-time sensing of MAPK signaling in medulloblastoma cells reveals cellular evasion mechanism counteracting dasatinib blockade of ERK activation during invasion

“…Nuclear translocation of ERK1/2 is necessary for controlling nuclear substrates and is associated with oncogenesis (reviewed in [11] ). To determine nuclear translocation dynamics of activated ERK1/2 in real time in living MB cells, we established the SKARS [16] biosensor for ERK1/2 in DAOY MB cells (DAOY-ESKARS, Fig. S3 ).…”

“…Serum-free medium (SFM) is IMEM, supplemented with penicillin/streptomycin (1%). The pMM55_pLV-CMV-MEK2-2NLS-mClover plasmid [16] for CMV-driven ERK Synthetic Kinase Activation Relocation Sensor (ESKARS) expression was provided by M. Ma. Infectious lentiviral particles of rLV.EF1.mCherry-Nuc-9 for nuclear expression of mCherry were purchased from Takara Bio Inc. (Kusatsu, Japan).…”

“…With new tools for real-time visualization of nuclear ERK1/2 activities established, including ERK1/2-nKTR, a genetically encoded biosensor for extracellular regulated kinase [15] , ERK1/2 activation patterns can now be tracked and correlated with cellular functions. This sensor was further developed and adapted to the mammalian system for quantifying MAPK dynamics in living mammalian cells [16] .…”