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Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors

Nature Protocols volume 4, pages 1623–1631 (2009)Cite this article

Abstract

Small GTPases act as molecular switches that regulate a variety of cellular functions, such as proliferation, cell movement and vesicle trafficking. Genetically encoded biosensors based on the principle of fluorescence resonance energy transfer (FRET) can visualize a spatio-temporal activity of small GTPases in living cells, thereby helping us to understand the role of small GTPases intuitively and vividly. Here we describe protocols of live cell imaging with the FRET biosensors. There are several types of FRET biosensors; this protocol focuses on intramolecular or unimolecular FRET biosensors of small GTPases that are made up of donor and acceptor fluorescence proteins, a small GTPase, its binding partner, and, if necessary, a subcellular localization signal. These FRET biosensors uncover the spatio-temporal activity of the small GTPases in living cells, which could not be obtained by conventional biochemical methods. Preparation of FRET biosensors and cell culture takes 6 d. Imaging and processing take 3–4 d to complete.

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Figure 1: Schematic representation of intramolecular fluorescence resonance energy transfer (FRET) biosensors of small GTPases.
Figure 2: Layout of the experimental setup.
Figure 3: Fluorescence resonance energy transfer (FRET) imaging of epidermal growth factor (EGF)-induced activation of Ras.
Figure 4: Fluorescence resonance energy transfer (FRET) imaging of Rac1 and Cdc42 in randomly migrating HT-1,080 cells.

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Acknowledgements

We thank M. Kitano and K. Kunida for providing the data of FRET imaging, and A. Nishiyama-Abe and Y. Kasakawa for their technical assistance. We are grateful to the members of the Matsuda laboratory for helpful discussions. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, Japan, Sagawa Cancer Research Grant, and by the Kyoto University Global COE program Center for Frontier Medicine.

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Authors and Affiliations

  1. Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan

    Kazuhiro Aoki & Michiyuki Matsuda

  2. Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Michiyuki Matsuda

Authors
  1. Kazuhiro Aoki
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  2. Michiyuki Matsuda
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Contributions

K.A. assembled and analyzed data. K.A. and M.M. wrote the paper.

Corresponding author

Correspondence to Michiyuki Matsuda.

Supplementary information

Supplementary method 1

Journals for the use of Metamorph software (ZIP 67 kb)

Supplementary video 1

EGF-induced Ras activation. Cos7 cells expressing Raichu-Ras were serum starved for 6 h, and stimulated with 50 ng ml-1 EGF. FRET (upper left), CFP (upper right), and phase contrast images (PH, lower right) were acquired every 2 min. The FRET/CFP ratio image (lower left) was generated with images of CFP and YFP. (MOV 1191 kb)

Supplementary video 2

Rac1 activity in a stochastically migrating HT-1080 cell. HT-1080 cells expressing Raichu-Rac1 were replated onto collagen-coated glass bottom dishes. FRET, CFP, and phase images (PH, right) were acquired every 2 min. The FRET/CFP ratio image (left) was generated with images of CFP and FRET. (MOV 1028 kb)

Supplementary video 3

Cdc42 activity in a motile HT-1080 cell. HT-1080 cells expressing Raichu-Cdc42 were replated onto collagen-coated glass bottom dishes. FRET, CFP, and phase images (PH, right) were acquired every 2 min. The FRET/VFP image (left) was generated with images of CFP and FRET. (MOV 1402 kb)

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Aoki, K., Matsuda, M. Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nat Protoc 4, 1623–1631 (2009). https://doi.org/10.1038/nprot.2009.175

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