The suboptimal features of the spectral properties of the leading fluorescence resonance energy transfer (FRET) pair, cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP), limit the full promise of FRET imaging. To overcome the drawbacks, we developed FRET-based, intra-molecular biosensors consisting of CFP/discomona sp red fluorescent protein (DsRed) or green fluorescent protein (GFP)/DsRed as donor/acceptor fluorophores.
The biosensors were expressed in NIH3T3 cells. In vitro fluorescence spectroscopy and Rho GTPase activation assays were used to confirm that Rac1 or Cdc42 was activated in serum-starved cells following stimulation with insulin or bradykinin. The transient changes of the amount, location, and translocation of activated Rac1 or Cdc42 in living cells were tracked with confocal imaging.
The increase of FRET efficiency was achieved in the cells expressing the biosensors and was proportional to the levels of activated Rac1 or Cdc42. The localized, transitional, and transient FRET signals were directly and quantitatively imaged with high spatial and temporal resolution. The biosensors were used to analyze and judge the GEF or GAP activities of putative regulatory proteins for Rac1 or Cdc42.
DsRed is a more suitable acceptor in FRET pair with CFP than with GFP in terms of the spectral overlap between the donor and acceptor. The approach can also be applied to many other types of protein behavior in living cells.
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Fluorescence resonance energy transfer
Cyan fluorescent protein
Green fluorescent protein
Yellow fluorescent protein
Guanine nucleotide exchange factors
Neural Wiskott-Aldrich syndrome protein
GTPase binding domain
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We thank Dr. Chunyi Tong for the help in assays of fluorescence spectroscopy and Mr. Ju Peng for the work in confocal microscopy. This work was supported by the grants from National Natural Science Foundation of China (30470887, 30871269), Hunan Provincial Natural Science Foundation of China (98JJY2004), and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry of China.
Conflict of Interest
The authors declare that they have no conflict of interest.
This manuscript describes the FRET-based intra-molecular biosensors using DsRed as an acceptor in FRET pair with CFP or GFP that can be used to image the induced activation of Rac1, Cdc42 in living cells, and to analyze and judge the GEF or GAP activities of putative regulatory proteins for Rac1 or Cdc42.
Rushi Liu and Daoquan Ren contributed equally to this work.
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Liu, R., Ren, D., Liu, Y. et al. Biosensors of DsRed as FRET Partner with CFP or GFP for Quantitatively Imaging Induced Activation of Rac, Cdc42 in Living Cells. Mol Imaging Biol 13, 424–431 (2011). https://doi.org/10.1007/s11307-010-0381-2
- FRET pair
- Induced activation