Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters

Maryu, Gembu; Miura, Hideaki; Uda, Youichi; Komatsubara, Akira T.; Matsuda, Minoru; Aoki, Kazuhiro

doi:10.1247/csf.18003

Cited by 24 publications

(18 citation statements)

References 142 publications

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“…Different classes of enzymes and transcriptional factors are subjected to such regulation. To date, numerous genetically encoded probes have been used for visualizing PK activity; however, most of them utilize FRET technology, and only few are based on cpFPs [211]. This design based on cpFPs was first implemented in sinphoses, which represent a family of probes for insulin-induced protein phosphorylation [212].…”

Section: Classification Of Cpfp-based Gefis By Analyte Measured Anmentioning

confidence: 99%

Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification

Kostyuk

Demidovich

Kotova

et al. 2019

IJMS

104

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Genetically encoded biosensors based on fluorescent proteins (FPs) are a reliable tool for studying the various biological processes in living systems. The circular permutation of single FPs led to the development of an extensive class of biosensors that allow the monitoring of many intracellular events. In circularly permuted FPs (cpFPs), the original N- and C-termini are fused using a peptide linker, while new termini are formed near the chromophore. Such a structure imparts greater mobility to the FP than that of the native variant, allowing greater lability of the spectral characteristics. One of the common principles of creating genetically encoded biosensors is based on the integration of a cpFP into a flexible region of a sensory domain or between two interacting domains, which are selected according to certain characteristics. Conformational rearrangements of the sensory domain associated with ligand interaction or changes in the cellular parameter are transferred to the cpFP, changing the chromophore environment. In this review, we highlight the basic principles of such sensors, the history of their creation, and a complete classification of the available biosensors.

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Section: Classification Of Cpfp-based Gefis By Analyte Measured Anmentioning

confidence: 99%

Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification

Kostyuk

Demidovich

Kotova

et al. 2019

IJMS

104

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“…Both groups used SH2 domain for the detection of phosphotyrosine of the substrate domain in the FRET biosensors. Thereafter, a large number of FRET biosensors for protein kinases have been reported to cover all seven classes of protein kinases, with essentially the same backbones (Oldach and Zhang, 2014;Maryu et al, 2018).…”

Section: Protein Conformationmentioning

confidence: 99%

“…radiative process and that "fluorescence resonance energy transfer" may be an inappropriate term. Because the principles and the pitfalls of FRET technology based on fluorescent proteins have already been described in previous review papers (Pietraszewska-Bogiel and Gadella, 2011;Vogel et al, 2012;Lindenburg and Merkx, 2014;Maryu et al, 2018), here we will limit ourselves to briefly introducing the keywords referred to in this article.…”

Section: Introductionmentioning

confidence: 99%

Two Decades of Genetically Encoded Biosensors Based on Förster Resonance Energy Transfer

Terai

Imanishi

2019

Cell Struct. Funct.

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Two decades have passed since the development of the first calcium indicator based on the green fluorescent protein (GFP) and the principle of Förster resonance energy transfer (FRET). During this period, researchers have advanced many novel ideas for the improvement of such genetically encoded FRET biosensors, which have allowed them to expand their targets from small molecules to signaling proteins and physicochemical properties. Although the merits of "genetically encoded" FRET biosensors became clear once various cell lines were established and several transgenic organisms were generated, the road to these developments was not necessarily a smooth one. Moreover, even today the development of new FRET biosensors remains a very laborintensive, trial-and-error process. Therefore, at this junction, it may be worthwhile to summarize the progress of the FRET biosensor and discuss the future direction of its development and application.

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“…We decided to use kinase-translocation reporters (KTRs) due to the flexibility in the choice of the fluorophore and the fact that they use a single channel [10]. Among other, there exist KTRs suitable for monitoring of MAPKs and Akt [11,12]. Since aberrant behavior of ERK and Akt is found across cancer types, these kinases are possibly the most investigated as potential therapeutic targets [13].…”

Section: Introductionmentioning

confidence: 99%

Heterogeneity and dynamics of ERK and Akt activation by G protein-coupled receptors depend on the activated heterotrimeric G proteins

Chavez-Abiega

Grönloh

Gadella

et al. 2021

Preprint

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Kinases are fundamental regulators of cellular functions and play key roles in GPCR-mediated signaling pathways. Kinase activities are generally inferred from cell lysates, hiding the heterogeneity of the individual cellular responses to extracellular stimuli. Here, we study the dynamics and heterogeneity of ERK and Akt in genetically identical cells in response to activation of endogenously expressed GPCRs. We use kinase translocation reporters, high-content imaging, automated segmentation and clustering methods to assess cell-to-cell signaling heterogeneity. We observed ligand-concentration dependent response kinetics to histamine, α2-adrenergic, and S1P receptor stimulation that varied between cells. By using G protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to α2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single cell level, especially for ERK. In all cases, the cellular heterogeneity was not explained by distinct pre-stimulation levels or saturation of the measured response. Cluster analysis of time-series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. The single-cell ERK responses to histamine and UK showed remarkably similar dynamics, despite the activation of different heterotrimeric G proteins. In contrast, the ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs.

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Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters

Cited by 24 publications

References 142 publications

Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification

Circularly Permuted Fluorescent Protein-Based Indicators: History, Principles, and Classification

Two Decades of Genetically Encoded Biosensors Based on Förster Resonance Energy Transfer

Heterogeneity and dynamics of ERK and Akt activation by G protein-coupled receptors depend on the activated heterotrimeric G proteins

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