%0 Journal Article %1 Adachi2009 %A Adachi, E. %A Kazoe, Y. %A Sato, Y. %A Suzuki, Y. %A Urano, T. %A Ueyama, T. %A Saito, N. %A Nikolaev, V. O. %A Lohse, M. J. %A Tominaga, M. %A Mogami, H. %D 2009 %J Pflugers Arch %K AMP-Dependent AMP/physiology Animals C/physiology COS Calcium Cercopithecus Cyclic Diacylglycerol Dyes/diagnostic Fluorescence/instrumentation/*methods Fluorescent Interference/instrumentation/*methods Kinase Kinase/physiology Kinases/physiology Microscopy, Protein Signal Signaling/physiology Transduction/*physiology aethiops use Cell %N 1 %P 227-34 %T A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19680684 %V 459 %X 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.

@article{Adachi2009, 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.}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Adachi, E. and Kazoe, Y. and Sato, Y. and Suzuki, Y. and Urano, T. and Ueyama, T. and Saito, N. and Nikolaev, V. O. and Lohse, M. J. and Tominaga, M. and Mogami, H.}, biburl = {https://www.bibsonomy.org/bibtex/2c133ecafe109d9ce9b0b72daa8b6a942/pharmawuerz}, endnotereftype = {Journal Article}, groups = {private}, interhash = {fbda68cf704b0fb8b48e104dd4d49524}, intrahash = {c133ecafe109d9ce9b0b72daa8b6a942}, issn = {1432-2013 (Electronic) 1432-2013 (Linking)}, journal = {Pflugers Arch}, keywords = {AMP-Dependent AMP/physiology Animals C/physiology COS Calcium Cercopithecus Cyclic Diacylglycerol Dyes/diagnostic Fluorescence/instrumentation/*methods Fluorescent Interference/instrumentation/*methods Kinase Kinase/physiology Kinases/physiology Microscopy, Protein Signal Signaling/physiology Transduction/*physiology aethiops use Cell}, month = Nov, note = {Adachi, Eisuke Kazoe, Yutaka Sato, Yohei Suzuki, Yuko Urano, Tetsumei Ueyama, Takehiko Saito, Naoaki Nikolaev, Viacheslav O Lohse, Martin J Tominaga, Makoto Mogami, Hideo Research Support, Non-U.S. Gov't Germany Pflugers Archiv : European journal of physiology Pflugers Arch. 2009 Nov;459(1):227-34. Epub 2009 Aug 13.}, number = 1, pages = {227-34}, shorttitle = {A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy}, timestamp = {2010-12-14T18:20:38.000+0100}, title = {A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19680684}, volume = 459, year = 2009 }