A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy

Eisuke Adachi, Yutaka Kazoe, Yohei Sato, Yuko Suzuki, Tetsumei Urano, Takehiko Ueyama, Naoaki Saito, Viacheslav O. Nikolaev, Martin J. Lohse, Makoto Tominaga, Hideo Mogami

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 languageEnglish
Pages (from-to)227-234
Number of pages8
JournalPflugers Archiv European Journal of Physiology
Volume459
Issue number1
DOIs
Publication statusPublished - 2009 Nov

Fingerprint

Fluorescence microscopy
Diglycerides
Prisms
Fluorescence Microscopy
Cyclic AMP
Protein Kinase C
Microscopy
Diacylglycerol Kinase
Microscopic examination
Monitoring
Signal Transduction
Second Messenger Systems
Cyclic AMP-Dependent Protein Kinases
Physiological Phenomena
Sensors
Optical Devices
Fluorescence Resonance Energy Transfer
Cells
Fura-2
Insulin-Secreting Cells

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 journalArticle

Adachi, Eisuke ; Kazoe, Yutaka ; Sato, Yohei ; Suzuki, Yuko ; Urano, Tetsumei ; Ueyama, Takehiko ; Saito, Naoaki ; Nikolaev, Viacheslav O. ; Lohse, Martin J. ; Tominaga, Makoto ; Mogami, Hideo. / A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy. In: Pflugers Archiv European Journal of Physiology. 2009 ; Vol. 459, No. 1. pp. 227-234.
@article{976188d7524b4f269b0d744ae83bfc94,
title = "A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy",
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.",
keywords = "G-protein-coupled receptor, Live cell imaging, Prism-based total internal reflection fluorescence microscopy linear unmixing, Second messenger",
author = "Eisuke Adachi and Yutaka Kazoe and Yohei Sato and Yuko Suzuki and Tetsumei Urano and Takehiko Ueyama and Naoaki Saito and Nikolaev, {Viacheslav O.} and Lohse, {Martin J.} and Makoto Tominaga and Hideo Mogami",
year = "2009",
month = "11",
doi = "10.1007/s00424-009-0705-8",
language = "English",
volume = "459",
pages = "227--234",
journal = "Pflugers Archiv European Journal of Physiology",
issn = "0031-6768",
publisher = "Springer Verlag",
number = "1",

}

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 -