Single-color, ratiometric biosensors for detecting signaling activities in live cells
- University of California, San Diego, United States
- University of Maryland, United States
Abstract
Genetically encoded fluorescent biosensors have revolutionized the study of signal transduction by enabling the real-time tracking of signaling activities in live cells. Investigating the interaction between signaling networks has become increasingly important to understanding complex cellular phenomena, necessitating an update of the biosensor toolkit to allow monitoring and perturbing multiple activities simultaneously in the same cell. We therefore developed a new class of fluorescent biosensors based on homo-FRET, deemed FLuorescence Anisotropy REporters (FLAREs), which combine the multiplexing ability of single-color sensors with a quantitative, ratiometric readout. Using an array of color variants, we were able to demonstrate multiplexed imaging of three activity reporters simultaneously in the same cell. We further demonstrate the compatibility of FLAREs for use with optogenetic tools as well as intravital two-photon imaging.
Data availability
Source data have been provided for Figures 1 to 4.
Article and author information
Author details
Funding
National Institutes of Health (R35 CA197622)
- Jin Zhang
National Institutes of Health (R01 DK073368)
- Jin Zhang
National Institutes of Health (R01 MH111516)
- Jin Zhang
National Institutes of Health (R01 DK077140)
- Megan A Rizzo
National Institutes of Health (R01 HL122827)
- Megan A Rizzo
National Institutes of Health (R01 MH111527)
- Megan A Rizzo
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All work involving mice was performed in accordance and recommendations of the NIH's Guide for the Care and Use of Laboratory Animals. Work was performed under protocols approved by the University of Maryland, Baltimore's Institutional Animal Care and Use Committee (protocol # 1213012). Procedures were performed under isoflurane anesthesia to minimize suffering.
Copyright
© 2018, Ross et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Single-color, ratiometric biosensors for detecting signaling activities in live cells
eLife 7:e35458.
https://doi.org/10.7554/eLife.35458
Further reading
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- Biochemistry and Chemical Biology
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Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.
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- Biochemistry and Chemical Biology
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