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High Dynamic‐Range LRET Biosensors of Rac1
Author(s) -
Pham Ha Thi,
Chen Ting,
Miller Lawrence
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.767.20
Subject(s) - förster resonance energy transfer , biosensor , linker , chemistry , rac1 , biophysics , fluorescence , biochemistry , biology , signal transduction , computer science , physics , operating system , quantum mechanics
Rac1, a member of Rho GTPase family, is well recognized for controlling a diverse range of cellular process, including cytoskeletal dynamics, cell migration and transcription. The diverse functions of Rac1 require a complex regulation of its subcellular location and interactions with multiple downstream targets. Over the past 10 years, rapid progress was made in understanding Rac1 signaling, in which FRET‐based biosensors using fluorescent proteins (FPs) play a vital role in quantifying the spatio‐temporal dynamics of the Rac1 nucleotide state in living cells. While proven, valuable research tools, FP biosensors of Rac1 and in general often have low signal‐to‐noise and/or dynamic range, which makes it very difficult to quantify small changes in activity. This problem can be overcome with lanthanide‐based FRET (LRET) biosensors that incorporate Tb(III) or Eu(III) complexes as energy transfer donors and FPs as acceptors. Lanthanide and lanthanide‐to‐FP sensitized emission signals have ms‐scale lifetimes that permit the use of time‐gated detection to eliminate non‐specific, ns‐scale autofluorescence and directly excited acceptor fluorescence. We prepared stably transformed NIH 3T3 fibroblast lines that express a single polypeptide consisting of the following elements ( N to C ): 1) EGFP; 2) p21‐binding domain (PBD); 3) a 30 nm α‐helix linker; 4) Escherichia coli dihydrofolate reductase (eDHFR); and 5) Rac1. The eDHFR moiety binds to a trimethorprim (TMP)‐Tb(III) complex heterodimer, completing the sensor. The presence of a rigid α‐helix linker keeps each end of the inactive sensor far apart, so there is a low baseline LRET signal. The single‐chain LRET sensors permit straightforward, two‐color ratiometric imaging of Rac1 activity. Moreover, a large difference in LRET signal (up to 25‐fold) between positive and negative controls was observed directly in sensor‐expressing fibroblasts grown in 96‐well plates. Single‐chain LRET affinity biosensors will permit both dynamic microscopic imaging and medium‐high throughput analysis of Rac1 or other protein activities in living cells. Support or Funding Information Funding provided by the National Institutes of Health, R01 GM081030.