Journal
BIOSENSORS & BIOELECTRONICS
Volume 31, Issue 1, Pages 292-298Publisher
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2011.10.034
Keywords
Ratiometric fluorescent imaging; Fluorescent protein; Biosensor; mVenus; mKO kappa; Grx1-roGFP2
Categories
Funding
- National Major Scientific Research Program of China [2011CB910401]
- National Natural Science Foundation of China [81172153, 30911120489, 30800339, 30800208]
- Program for New Century Excellent Talents in University of China [NCET-08-0220]
Ask authors/readers for more resources
Genetically coded fluorescent protein (FP)-based biosensors are powerful tools for the non-invasive tracking of molecular events in living cells. Although a variety of FP biosensors are available, the simultaneous imaging of multiple biosensors (multi-parameter imaging) in single living cells remains a challenge and is far from routinely used to elucidate the intricate networks of molecular events. In this study, we established a novel combination of FP biosensors for dual-parameter ratiometric imaging, consisting of a new fluorescence resonance energy transfer (FRET) pair mVenus (yellow FP)/mKO kappa (orange FP)-based (abbreviated as YO)biosensor and a single FP-based biosensor Grx1-roGFP2. Under our imaging condition, 1.4 +/- 0.05% of Grx1-roGFP2 signal contributes to the mVenus channel and 5.2 +/- 0.12% of the mVenus signal contributes to the Grx1-roGFP2 channel. We demonstrate that such low degree of cross-talk causes negligible distortion of the ratiometric signal of the YO-based FRET biosensor and Grx1-roGFP2. By using this dual-parameter ratiometric imaging approach, we achieved simultaneous imaging of Src/Ca2+ signaling and glutathione (GSH) redox potential in a single cell, which was previously unattainable. Furthermore, we provided direct evidence that epidermal growth factor (EGF)-induced Src signaling was negatively regulated by H2O2 via its effect on GSH-based redox system, demonstrating the power of this dual-parameter imaging approach for elucidating new connections between different molecular events that occur in a single cell. More importantly, the dual-parameter imaging approach described in this study is highly extendable. (C) 2011 Elsevier B.V. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available