Spatio-temporal images of growth-factor-induced activation of Ras and Rap1 (original) (raw)

Nature volume 411, pages 1065–1068 (2001)Cite this article

Abstract

G proteins of the Ras family function as molecular switches in many signalling cascades1,2,3; however, little is known about where they become activated in living cells. Here we use FRET (fluorescent resonance energy transfer)-based sensors to report on the spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Epidermal growth factor activated Ras at the peripheral plasma membrane and Rap1 at the intracellular perinuclear region of COS-1 cells. In PC12 cells, nerve growth factor-induced activation of Ras was initiated at the plasma membrane and transmitted to the whole cell body. After three hours, high Ras activity was observed at the extending neurites. By using the FRAP (fluorescence recovery after photobleaching) technique, we found that Ras at the neurites turned over rapidly; therefore, the sustained Ras activity at neurites was due to high GTP/GDP exchange rate and/or low GTPase activity, but not to the retention of the active Ras. These observations may resolve long-standing questions as to how Ras and Rap1 induce different cellular responses4 and how the signals for differentiation and survival are distinguished by neuronal cells5.

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Acknowledgements

We thank D. Bowtell, K. Kaibuchi, J. Miyazaki, N. Minato and N. Nomura for plasmids; H. Mizuno, F. Ohba, N. Otsuka, K. Kimura and K. Okuda for technical assistance; and S. Hattori, M. Dutta and A. Dutta for the critical reading of the manuscript. Supported by grants from the Ministry of Health and Welfare, from the Ministry of Education, Science, Sports and Culture, Mitsui Life Social Welfare Foundation, the Princess Takamatsu Cancer Research Fund and from the Japan Health Science Foundation.

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Authors and Affiliations

  1. Department of Structural Analysis, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita-shi, 565-8565, Osaka, Japan
    Naoki Mochizuki
  2. Department of Pathology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, 162-8655, Tokyo, Japan
    Shigeko Yamashita
  3. Department of Tumor Virology, Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, 565-0871, Osaka, Japan
    Kazuo Kurokawa, Yusuke Ohba & Michiyuki Matsuda
  4. Laboratory for Cell Function and Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-shi, 351-0198, Saitama, Japan
    Takeharu Nagai & Atsushi Miyawaki

Authors

  1. Naoki Mochizuki
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  2. Shigeko Yamashita
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  3. Kazuo Kurokawa
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  4. Yusuke Ohba
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  5. Takeharu Nagai
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  6. Atsushi Miyawaki
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  7. Michiyuki Matsuda
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Supplementary information

Supplementary Information

Movie 1

EGF-induced Ras activation. COS-1 cells expressing Raichu-Ras were serum-starved and stimulated with EGF. Phase contrast (upper left), CFP (lower left), and YFP images (lower right) were obtained every 20 sec. The FRET image (upper right) was created from images of CFP and YFP. A series of images were saved as a stack file, which was converted to a video file.

Movie 2

EGF-induced activation of Ras and Rap1. See legend to Figure 2a. COS-1 cells transfected with pRaichu-Ras or pRaichu-Rap1 were serum-starved and stimulated with EGF. FRET image was obtained every 20 sec and a series of images were saved as a stack file, which was converted to a video file.

Movie 3

Effect of the dominant-negative H-Ras mutant and the active Rap1 mutant on EGF-induced Ras activation. COS-1 cells expressing Raichu-Ras with H-RasN17 (left) or Rap1V12 (right) were serum-starved and stimulated with EGF. FRET image was obtained every 20 sec and a series of images were saved as a stack file, which was converted to a video file.

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Mochizuki, N., Yamashita, S., Kurokawa, K. et al. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1.Nature 411, 1065–1068 (2001). https://doi.org/10.1038/35082594

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