Biosensors of DsRed as FRET partner with CFP or GFP for quantitatively imaging induced activation of Rac, Cdc42 in living cells.
OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
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.
Liu
Rushi
R
Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha, 410081, China.
Ren
Daoquan
D
Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha, 410081, China.
Liu
Yizhou
Y
Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha, 410081, China.
Deng
Yuting
Y
Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha, 410081, China.
Sun
Bin
B
Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha, 410081, China.
Zhang
Qingyan
Q
Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha, 410081, China.
Guo
Xiangrong
X
Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hunan Normal University, Changsha, 410081, China. xiangrongguo@hotmail.com.
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D001920:Bradykinin / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D020690:GTPase-Activating Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D057054:Molecular Imaging; D041681:NIH 3T3 Cells; D021381:Protein Transport / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000378:metabolism*
C485182:Cyan Fluorescent Protein; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D008164:Luminescent Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D001920:Bradykinin