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Nat Cell Biol. 2018 Oct;20(10):1215-1225. doi: 10.1038/s41556-018-0200-6. Epub 2018 Sep 24.

Single-fluorophore biosensors for sensitive and multiplexed detection of signalling activities.

Author information

1
Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA. sohum@ucsd.edu.
2
Department of Neurobiology, School of Basic Medical Sciences and Neuroscience Research Institute; Key Lab for Neuroscience, Ministry of Education of China and National Health Commission of the P.R. China; IDG/McGovern Institute for Brain Research at PKU, Peking University, Beijing, China.
3
The Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
4
The Kavli Neuroscience Discovery Institute, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
5
Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA.
6
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
7
The Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD, USA. rhuganir@jhmi.edu.
8
The Kavli Neuroscience Discovery Institute, The Johns Hopkins University School of Medicine, Baltimore, MD, USA. rhuganir@jhmi.edu.
9
Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA. jzhang32@ucsd.edu.
10
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA. jzhang32@ucsd.edu.

Abstract

Unravelling the dynamic molecular interplay behind complex physiological processes such as neuronal plasticity requires the ability to both detect minute changes in biochemical states in response to physiological signals and track multiple signalling activities simultaneously. Fluorescent protein-based biosensors have enabled the real-time monitoring of dynamic signalling processes within the native context of living cells, yet most commonly used biosensors exhibit poor sensitivity (for example, due to low dynamic range) and are limited to imaging signalling activities in isolation. Here, we address this challenge by developing a suite of excitation ratiometric kinase activity biosensors that offer the highest reported dynamic range and enable the detection of subtle changes in signalling activity that could not be reliably detected previously, as well as a suite of single-fluorophore biosensors that enable the simultaneous tracking of as many as six distinct signalling activities in single living cells.

PMID:
30250062
PMCID:
PMC6258557
DOI:
10.1038/s41556-018-0200-6
[Indexed for MEDLINE]
Free PMC Article

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