A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy.
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.
Adachi
Eisuke
E
Department of Physiology, Hamamatsu University School of Medicine, Handa-yama 1-20-1, Higashi-ku, Hamamatsu, Shizuoka, 431-3192, Japan.
Kazoe
Yutaka
Y
Sato
Yohei
Y
Suzuki
Yuko
Y
Urano
Tetsumei
T
Ueyama
Takehiko
T
Saito
Naoaki
N
Nikolaev
Viacheslav O
VO
Lohse
Martin J
MJ
Tominaga
Makoto
M
Mogami
Hideo
H
D000818:Animals; D019556:COS Cells; D020013:Calcium Signaling / Q000502:physiology; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D019852:Diacylglycerol Kinase / Q000502:physiology; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008857:Microscopy, Interference / Q000295:instrumentation / Q000379:methods*; D011493:Protein Kinase C / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*
D005456:Fluorescent Dyes; D000242:Cyclic AMP; D019852:Diacylglycerol Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C
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