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.
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