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Multiphoton excitation–evoked chromophore-assisted laser inactivation using green fluorescent protein

Nature Methods volume 2, pages 503–505 (2005)Cite this article

Abstract

Noninvasive, straightforward methods to inactivate selected proteins in living cells with high spatiotemporal resolution are needed. Chromophore-assisted laser inactivation (CALI) can be used to photochemically inactivate proteins, but it has several drawbacks, such as procedural complexity and nonspecific photodamage. Here we show that by application of multiphoton excitation to CALI, enhanced green fluorescent protein (EGFP) is an effective chromophore for inactivation of a protein's function without nonspecific photodamage in living mammalian cells.

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Figure 1: MP-CALI of Cx43 tagged with EGFP.
Figure 2: MP-CALI inactivates aurora B tagged with EGFP.

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Acknowledgements

This work was supported by funds for Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation (JST) and by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We wish to thank R.Y. Tsien (University of California at San Diego) for providing the gene encoding mRFP1, Y.L. Wang (University of Massachusetts Medical School) for providing the gene encoding aurora B-EGFP, and R. Weingart (University of Bern, Switzerland) for helpful suggestions, discussions and critical review of this manuscript.

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Authors and Affiliations

  1. Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, 465 Kajii-cho Hirokoji Kawaramachi, Kamigyo-ku, Kyoto, 602-8566, Japan

    Takuji Tanabe, Masahito Oyamada, Ping Dai, Hideo Tanaka & Tetsuro Takamatsu

  2. Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Katsumasa Fujita

Authors
  1. Takuji Tanabe
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  2. Masahito Oyamada
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  3. Katsumasa Fujita
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  4. Ping Dai
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  5. Hideo Tanaka
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  6. Tetsuro Takamatsu
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Correspondence to Tetsuro Takamatsu.

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Supplementary information

Supplementary Fig. 1

MP-CALI does not influence the integrity of the plasma membrane. (PDF 3820 kb)

Supplementary Fig. 2

Recovery of Cx43-EGFP signals after MP-CALI. (PDF 2914 kb)

Supplementary Fig. 3

Proteins in the proximity of the EGFP fusion proteins undergoing MP-CALI are not destroyed via bystander effect. (PDF 96 kb)

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Tanabe, T., Oyamada, M., Fujita, K. et al. Multiphoton excitation–evoked chromophore-assisted laser inactivation using green fluorescent protein. Nat Methods 2, 503–505 (2005). https://doi.org/10.1038/nmeth770

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