Multiplexed Fluorescence Imaging of ERK and Akt Activities and Cell-cycle Progression

• Maryu Gembu

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

• Matsuda Michiyuki

  Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University

• Aoki Kazuhiro

  Imaging Platform for Spatio-Temporal Information, Graduate School of Medicine, Kyoto University Division of Quantitative Biology, Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences Department of Basic Biology, Faculty of Life Science, SOKENDAI (Graduate University for Advanced Studies)

Abstract

The Ras-ERK pathway controls cell proliferation and differentiation, whereas the PI3K-Akt pathway plays a role in the process of cell-cycle progression and cell survival. Both pathways are activated by many stimuli such as epidermal growth factor (EGF), and coordinately regulate each other through cross-talk. However, it remains unclear how cells accommodate the dynamics and interplay between the Ras-ERK and PI3K-Akt pathways to regulate cell-fate decisions, mainly because of the lack of good tools to visualize ERK and Akt activities simultaneously in live cells. Here, we developed a multiplexed fluorescence system for imaging ERK and Akt signaling and the cell-cycle status at the single cell level. Based on the principle of the kinase translocation reporter (KTR), we created Akt-FoxO3a-KTR, which shuttled between nucleus and cytoplasm in a manner regulated by Akt phosphorylation. To simultaneously measure ERK, Akt and the cell-cycle status, we generated a polycistronic vector expressing ERK-KTR, Akt-FoxO3a-KTR, a cell-cycle reporter and a nuclear reporter, and applied linear unmixing to these four images to remove spectral overlap among fluorescent proteins. The specificity and sensitivity of ERK-KTR and Akt-FoxO3a-KTR were characterized quantitatively. We examined the cellular heterogeneity of relationship between ERK and Akt activities under a basal or EGF-stimulated condition, and found that ERK and Akt were regulated in a highly cooperative and cell-cycle-dependent manner. Our study provides a useful tool for quantifying the dynamics among ERK and Akt activities and the cell cycle in a live cell, and for addressing the mechanisms underlying intrinsic resistance to molecularly targeted drugs.

Journal

• Cell Structure and Function

  Cell Structure and Function 41 (2), 81-92, 2016

  Japan Society for Cell Biology

Keywords

• ERK
• Akt
• Fluorescence imaging

Details

• CRID: 1390001204695850368
• NII Article ID: 130005165901
• NII Book ID: AA0060007X
• DOI: 10.1247/csf.16007
• ISSN: 13473700
• ISSN: 03867196
• NDL BIB ID: 028100148
• PubMed: 27247077
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I028100148
• Web Site: https://www.jstage.jst.go.jp/article/csf/41/2/41_16007/_pdf
• Web Site: https://search.jamas.or.jp/link/ui/2017381376
• Text Lang: en
• Data Source:
  • JaLC
  • NDL
  • Crossref
  • PubMed
  • CiNii Articles
  • KAKEN
• Abstract License Flag: Disallowed