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) Epacl-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 γ, 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 Ca 2+ 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-proteincoupled receptor stimulation in living cells.
| Original language | English |
|---|---|
| Pages (from-to) | 227-234 |
| Number of pages | 8 |
| Journal | Pflugers Archiv European Journal of Physiology |
| Volume | 459 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2009 Nov |
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Keywords
- G-protein-coupled receptor
- Live cell imaging
- Prism-based total internal reflection fluorescence microscopy linear unmixing
- Second messenger
ASJC Scopus subject areas
- Physiology
- Clinical Biochemistry
- Physiology (medical)
- Medicine(all)
Cite this
A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy. / Adachi, Eisuke; Kazoe, Yutaka; Sato, Yohei; Suzuki, Yuko; Urano, Tetsumei; Ueyama, Takehiko; Saito, Naoaki; Nikolaev, Viacheslav O.; Lohse, Martin J.; Tominaga, Makoto; Mogami, Hideo.
In: Pflugers Archiv European Journal of Physiology, Vol. 459, No. 1, 11.2009, p. 227-234.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy
AU - Adachi, Eisuke
AU - Kazoe, Yutaka
AU - Sato, Yohei
AU - Suzuki, Yuko
AU - Urano, Tetsumei
AU - Ueyama, Takehiko
AU - Saito, Naoaki
AU - Nikolaev, Viacheslav O.
AU - Lohse, Martin J.
AU - Tominaga, Makoto
AU - Mogami, Hideo
PY - 2009/11
Y1 - 2009/11
N2 - 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) Epacl-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 γ, 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 Ca 2+ 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-proteincoupled receptor stimulation in living cells.
AB - 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) Epacl-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 γ, 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 Ca 2+ 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-proteincoupled receptor stimulation in living cells.
KW - G-protein-coupled receptor
KW - Live cell imaging
KW - Prism-based total internal reflection fluorescence microscopy linear unmixing
KW - Second messenger
UR - http://www.scopus.com/inward/record.url?scp=73949155996&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=73949155996&partnerID=8YFLogxK
U2 - 10.1007/s00424-009-0705-8
DO - 10.1007/s00424-009-0705-8
M3 - Article
C2 - 19680684
AN - SCOPUS:73949155996
VL - 459
SP - 227
EP - 234
JO - Pflugers Archiv European Journal of Physiology
JF - Pflugers Archiv European Journal of Physiology
SN - 0031-6768
IS - 1
ER -