論文 - 詳細
| RRC ID | 19643 |
|---|---|
| 著者 | Adachi E, Kazoe Y, Sato Y, Suzuki Y, Urano T, Ueyama T, Saito N, Nikolaev VO, Lohse MJ, Tominaga M, Mogami H. |
| タイトル | A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy. |
| ジャーナル | Pflugers Arch |
| Abstract |
Physiological phenomena are regulated by multiple signal pathways upon receptor stimulation. Here, we have introduced a new technique with a combination of prism-based total internal reflection fluorescence microscopy (PBTIRFM) and epifluorescence microscopy (EPI) to simultaneously monitor multiple signal pathways. This instrumentation allows us to visualize three signal pathways, Ca2+, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), and diacylglycerol (DAG)/protein kinase C (PKC) signals in living cells. Three fluorescent indicators were employed for this purpose: (1) Fura-2 AM as a calcium sensor; (2) Epac1-camp, a cyan fluorescent protein-yellow fluorescent protein fluorescence resonance energy transfer-based cAMP indicator, as a cAMP sensor; and (3) C1-tagged monomeric red fluorescent protein, a tandem DAG-binding domain of PKC gamma, as a DAG sensor or myristoylated alanine-rich C kinase substrate-tagged DsRed for the PKC activation pathway. The DAG signal was monitored by PBTIRFM, whereas the Ca2+ and cAMP signals were monitored by EPI. Adenosine trisphosphate resulted in generation of all three second messengers in triple probe-loaded Cos-7 cells. The spectral overlap between these signal probes was evaluated by means of linear unmixing. Forskolin also evoked Ca2+, cAMP/PKA, and DAG/PKC signals whereas acetylcholine activated Ca2+ and DAG/PKC signals as well as inhibiting cAMP generation in triple probe-loaded insulin-secreting cells. Thus, the optical observation system combining PBTIRFM and EPI offers a great advance in analyzing interplay of multiple signaling pathways, such as these second messengers, upon G-protein-coupled receptor stimulation in living cells. |
| 巻・号 | 459(1) |
| ページ | 227-34 |
| 公開日 | 2009-11-1 |
| DOI | 10.1007/s00424-009-0705-8 |
| PMID | 19680684 |
| MeSH | Animals COS Cells Calcium Signaling / physiology Chlorocebus aethiops Cyclic AMP / physiology Cyclic AMP-Dependent Protein Kinases / physiology Diacylglycerol Kinase / physiology Fluorescent Dyes Microscopy, Fluorescence / instrumentation Microscopy, Fluorescence / methods* Microscopy, Interference / instrumentation Microscopy, Interference / methods* Protein Kinase C / physiology Signal Transduction / physiology* |
| IF | 3.158 |
| 引用数 | 3 |
| WOS 分野 | PHYSIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 6 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| ヒト・動物細胞 | COS-7(RCB0539) |