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Biosensors of DsRed as FRET Partner with CFP or GFP for Quantitatively Imaging Induced Activation of Rac, Cdc42 in Living Cells

Abstract

Purpose

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.

Procedures

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.

Results

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.

Conclusion

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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Abbreviations

FRET:

Fluorescence resonance energy transfer

CFP:

Cyan fluorescent protein

GFP:

Green fluorescent protein

YFP:

Yellow fluorescent protein

GEF:

Guanine nucleotide exchange factors

GAP:

GTPase-activating proteins

PAK1:

p21-activated kinase1

N-WASP:

Neural Wiskott-Aldrich syndrome protein

GBD:

GTPase binding domain

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Acknowledgements

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.

Author information

Correspondence to Xiangrong Guo.

Additional information

Significance:

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.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1
figure8

Fluorescence emission spectra analysis in cell lysates expressing the biosensors of GFP/DsRed upon excitation at 488 nm after stimulation for 5 min. Two emission peaks at 488 nm (CFP) and 583 nm (DsRed) were observed (JPEG 50 kb)

Fig. S2
figure9

FRET measurements for two individual cells depicted the changing fluorescence intensity of FRET signals, representing the changing Rac1 activation levels in vivo in process of the induced activation (JPEG 53 kb)

Fig. S3
figure10

FRET measurements for two individual cells revealed the changes of Cdc42 activation levels in vivo in process of the induced activation (JPEG 46 kb)

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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

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Key words

  • Biosensors
  • FRET pair
  • Induced activation
  • Rac1
  • Cdc42