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
|
リソース情報 |
ヒト・動物細胞 |
COS-7(RCB0539) |