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Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein

Nature Methods volume 5, pages 339–345 (2008)Cite this article

Abstract

All biological reactions depend on the diffusion and re-localization of biomolecules. Our understanding of biological processes requires accurate measurement of biomolecule mobility in living cells. Currently, approaches for investigating the mobility of biomolecules are generally restricted to measuring either fast or slow diffusion kinetics. We describe the development and application of a photoconvertible fluorescent protein, Phamret, that can be highlighted by UV light stimulation inducing a change in fluorescence emission from cyan fluorescent protein (CFP) to photoactivated GFP (PA-GFP). Phamret can be monitored by single excitation-dual emission mode for visualization of molecular dynamics for a broad range of kinetics. We also devised a microscopy-based method to measure the diffusion coefficient from the fluorescence decay after photostimulation of Phamret, enabling analysis of diffusion kinetics ranging from less than 0.1 μm2/s up to ∼100 μm2/s, and found significant changes in free protein movement during cell-cycle progression.

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Figure 1: Structure and spectral properties of Phamret.
Figure 2: Photoconversion of Phamret fusion protein in living cells.
Figure 3: Visualization of rapid protein dynamics using Phamret.
Figure 4: Determination of diffusion coefficient of Phamret by FDAP.

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Acknowledgements

We thank H. Kimura and Y. Yoneda (Osaka University) for providing the cDNA encoding H2B-GFP and PP2Cγ, and fibrillalin, respectively. We also thank D. Goto and I. Kotera for helpful comments. We also thank F. Inagaki and N. Noda for help with gel filtration analysis. This work was partially supported by grants from Scientific Research on Advanced Medical Technology of the Ministry of Labor, Health and Welfare of Japan, Precursory Research for Embryonic Science and Technology of the Japan Science and Technology Agency, and the Japanese Ministry of Education, Science and Technology.

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Authors and Affiliations

  1. Laboratory for Nanosystems Physiology, Research Institute for Electronic Science, Hokkaido University, Kita-12 Nishi-6 Kita-ku, Sapporo, 060-0812, Hokkaido, Japan

    Tomoki Matsuda & Takeharu Nagai

  2. Laboratory for Cell Function and Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, 351-0198, Saitama, Japan

    Atsushi Miyawaki

Authors
  1. Tomoki Matsuda
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  2. Atsushi Miyawaki
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  3. Takeharu Nagai
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Contributions

T.M. performed experiments, analyzed data and prepared the manuscript; A.M. contributed to data analysis; T.N. contributed to the conceptual development and experimental design and performed experiments, analyzed data and prepared the manuscript.

Corresponding author

Correspondence to Takeharu Nagai.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Methods (PDF 496 kb)

Supplementary Video 1

Tracking of mitochondria fusion with mitochondria-Phamret in HeLa cells. (MOV 3037 kb)

Supplementary Video 2

Tracking distribution of activated H2B-Phamret throughout mitosis in HeLa cells. (MOV 2172 kb)

Supplementary Video 3

Tracking rapid diffusion of PP2Cγ-Phamret within nucleus in HeLa Cells. (MOV 20357 kb)

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Matsuda, T., Miyawaki, A. & Nagai, T. Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein. Nat Methods 5, 339–345 (2008). https://doi.org/10.1038/nmeth.1193

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