Miura, Haruko Matsuda, Michiyuki Aoki, Kazuhiro
Laboratory of Bio-imaging and Cell Signalling, Graduate School of Bio studies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, 2 Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, 3 Imaging Platform for Spatio-Temporal Information, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
The serine/threonine kinase Akt plays a critical role in cell proliferation, survival, and tumorigenesis. As a central kinase in the phosphatidylinositol 3-kinase pathway, its activation mechanism at the plasma membrane has been well characterized. However, the subcellular Akt activity in living cells is still largely unknown. Fluorescence resonance energy transfer (FRET)-based biosensors have emerged as indispensable tools to visualize the subcellular activities of signalling molecules. In this study, we developed a highly specific FRET biosensor for Akt based on the Eevee backbone, called Eevee-iAkt. Using inhibitors targeting kinases upstream and downstream of Akt, we showed that Eevee-iAkt specifically monitors Akt activity in living cells. To visualize Akt activity at different subcellular compartments, we targeted Eevee-iAkt to raft and non-raft regions of the plasma membrane, mitochondria, and nucleus in HeLa and Cos7 cells. Interestingly, we revealed substantial differences in Akt activation between HeLa and Cos7 cells upon epidermal growth factor (EGF) stimulation: Akt was transiently activated in HeLa cells with comparable levels at the plasma membrane, cytosol, and mitochondria. In contrast, sustained and spatially localized Akt activation was observed in EGF-stimulated Cos7 cells. We found high Akt activity at the plasma membrane, low activity in the cytosol, and no detectable activity at the mitochondria and nucleus in Cos7 cells. The Eevee-iAkt biosensor was shown to be a valuable tool to study the functional relationship between subcellular Akt activation and its anti-apoptotic role in living cells.
… “camera (Roper Scientific, Tucson, AZ), a CoolLED precisExcite LED illumination system (Molecular Devices, Sunnyvale, CA)”…
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pE-100: A range of compact, simple-to-use, single wavelength illumination systems for screening fluorescence.
Cell Structure and Function
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