Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems.

European Molecular Biology Laboratory Cell Biology and Biophysics Unit Meyerhofstraße 1 69117 Heidelberg Germany

Institut Interdisciplinaire de Neurosciences CNRS UMR 5297 Université Bordeaux 2 146 rue Léo Saignat 33077 Bordeaux France

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Flemingovo náměstí 2 16610 Prague 6 Czech Republic

Max Planck Institute of Molecular Cell Biology and Genetics Pfotenhauerstraße 108 01307 Dresden Germany

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Purvis J. E. & Lahav G. Encoding and decoding cellular information through signaling dynamics. Cell 152, 945–956 (2013) . PubMed PMC

Marks F., Klingmueller U. & Mueller-Decker K. Cellular Signal Processing: an Introduction to the Molecular Mechanisms of Signal Transduction Garland Science (2009) .

Ouyang X. & Chen J. K. Synthetic strategies for studying embryonic development. ChemBiol. 17, 590–606 (2010) . PubMed PMC

Putyrski M. & Schultz C. Protein translocation as a tool: The current rapamycin story. FEBS Lett. 586, 2097–2105 (2012) . PubMed

Rutkowska A. & Schultz C. Protein tango: the toolbox to capture interacting partners. Angew. Chem. Int. Ed. Engl. 51, 8166–8176 (2012) . PubMed

Fegan A., White B., Carlson J. C. T. & Wagner C. R. Chemically controlled protein assembly: techniques and applications. Chem. Rev. 110, 3315–3336 (2010) . PubMed

Pastrana E. Optogenetics: controlling cell function with light. Nat. Methods 8, 24–25 (2011) .

Kramer R. H., Mourot A. & Adesnik H. Optogenetic pharmacology for control of native neuronal signaling proteins. Nat. Neurosci. 16, 816–823 (2013) . PubMed PMC

Kao J. P. Caged molecules: principles and practical considerations. Curr. Protoc. Neurosci Chapter 6, Unit 6 20 (2006) . PubMed

Gautier A. et al.. How to control proteins with light in living systems. Nat. Chem. Biol. 10, 533–541 (2014) . PubMed

Nadler A. et al.. The fatty acid composition of diacylglycerols determines local signaling patterns. Angew. Chem. Int. Ed. Engl. 52, 6330–6334 (2013) . PubMed

Artamonov M. V. et al.. Agonist-induced Ca2+ sensitization in smooth muscle: redundancy of Rho guanine nucleotide exchange factors (RhoGEFs) and response kinetics, a caged compound study. J. Biol. Chem. 288, 34030–34040 (2013) . PubMed PMC

Ellis-Davies G. C. A practical guide to the synthesis of dinitroindolinyl-caged neurotransmitters. Nat. Protoc. 6, 314–326 (2011) . PubMed PMC

Hoglinger D., Nadler A. & Schultz C. Caged lipids as tools for investigating cellular signaling. Biophys. Biochim. Acta 1841, 1085–1096 (2014) . PubMed

Huang X. P., Sreekumar R., Patel J. R. & Walker J. W. Response of cardiac myocytes to a ramp increase of diacylglycerol generated by photolysis of a novel caged diacylglycerol. Biophys. J. 70, 2448–2457 (1996) . PubMed PMC

Thompson S. M. et al.. Flashy science: controlling neural function with light. J. Neurosci. 25, 10358–10365 (2005) . PubMed PMC

Neveu P. et al.. A caged retinoic acid for one- and two-photon excitation in zebrafish embryos. Angew. Chem. Int. Ed. Engl. 47, 3744–3746 (2008) . PubMed

Feng S. H. et al.. A rapidly reversible chemical dimerizer system to study lipid signaling in living cells. Angew. Chem. Int. Ed. Engl. 53, 6720–6723 (2014) . PubMed

Ahmed S., Xie J., Horne D. & Williams J. C. Photocleavable dimerizer for the rapid reversal of molecular trap antagonists. J. Biol. Chem. 289, 4546–4552 (2014) . PubMed PMC

Zimmermann M. et al.. Cell-permeant and photocleavable chemical inducer of dimerization. Angew. Chem. Int. Ed. Engl. 53, 4717–4720 (2014) . PubMed PMC

Liu P. et al.. A bioorthogonal small-molecule-switch system for controlling protein function in live cells. Angew. Chem. Int. Ed. Engl. 53, 10049–10055 (2014) . PubMed

Tischer D. & Weiner O. D. Illuminating cell signalling with optogenetic tools. Nat. Rev. Mol. Cell Biol. 15, 551–558 (2014) . PubMed PMC

Matsuzaki M. & Kasai H. Two-photon uncaging microscopy. Cold Spring Harb. Protoc. 2011, pdb prot5620 (2011) . PubMed

Olson J. P. et al.. Optically selective two-photon uncaging of glutamate at 900 nm. J. Am. Chem. Soc. 135, 5954–5957 (2013) . PubMed PMC

Fournier L. et al.. Coumarinylmethyl caging groups with redshifted absorption. Chemistry 19, 17494–17507 (2013) . PubMed

Brieke C., Rohrbach F., Gottschalk A., Mayer G. & Heckel A. Light-controlled tools. Angew. Chem. Int. Ed. Engl. 51, 8446–8476 (2012) . PubMed

Banala S., Maurel D., Manley S. & Johnsson K. A caged, localizable rhodamine derivative for superresolution microscopy. ACS Chem. Biol. 7, 288–292 (2012) . PubMed

Brash A. R. Arachidonic acid as a bioactive molecule. J. Clin. Invest. 107, 1339–1345 (2001) . PubMed PMC

Jacobson D. A., Weber C. R., Bao S. Z., Turk J. & Philipson L. H. Modulation of the pancreatic islet beta-cell-delayed rectifier potassium channel Kv2.1 by the polyunsaturated fatty acid arachidonate. J. Biol. Chem. 282, 7442–7449 (2007) . PubMed PMC

Carta M. et al.. Membrane lipids tune synaptic transmission by direct modulation of presynaptic potassium channels. Neuron 81, 787–799 (2014) . PubMed

Alexander L. D. & Hamzeh M. T. Arachidonic acid-induced apoptosis in human renal proximal tubular epithelial cells. FASEB J. 27, 727.12 (2013) .

Meves H. Arachidonic acid and ion channels: an update. Br. J. Pharmacol. 155, 4–16 (2008) . PubMed PMC

Oliver D. et al.. Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids. Science 304, 265–270 (2004) . PubMed

Connell E. et al.. Mechanism of arachidonic acid action on syntaxin-Munc18. EMBO. Rep. 8, 414–419 (2007) . PubMed PMC

Itoh Y. et al.. Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40. Nature 422, 173–176 (2003) . PubMed

O'Flaherty J. T., Chadwell B. A., Kearns M. W., Sergeant S. & Daniel L. W. Protein kinases C translocation responses to low concentrations of arachidonic acid. J. Biol. Chem. 276, 24743–24750 (2001) . PubMed

Bennett W. F. D. & Tieleman D. P. Molecular simulation of rapid translocation of cholesterol, diacylglycerol, and ceramide in model raft and nonraft membranes. J. Lipid Res. 53, 421–429 (2012) . PubMed PMC

Ramanadham S., Gross R. & Turk J. Arachidonic-acid induces an increase in the cytosolic calcium-concentration in single pancreatic-islet beta-cells. Biochem. Biophys. Res. Commun. 184, 647–653 (1992) . PubMed

Yeung-Yam-Wah V., Lee A. K. & Tse A. Arachidonic acid mobilizes Ca2+ from the endoplasmic reticulum and an acidic store in rat pancreatic beta cells. Cell Calcium 51, 140–148 (2012) . PubMed

Ramanadham S., Gross R. W., Han X. L. & Turk J. Inhibition of arachidonate release by secretagogue-stimulated pancreatic-islets suppresses both insulin-secretion and the rise in beta-cell cytosolic calcium-ion concentration. Biochemistry 32, 337–346 (1993) . PubMed

Ishihara H. et al.. Pancreatic beta-cell line min6 exhibits characteristics of glucose-metabolism and glucose-stimulated insulin-secretion similar to those of normal islets. Diabetologia 36, 1139–1145 (1993) . PubMed

Zhao Y. et al.. An expanded palette of genetically encoded Ca(2)(+) indicators. Science 333, 1888–1891 (2011) . PubMed PMC

Hu H. et al.. A novel class of antagonists for the FFAs receptor GPR40. Biochem. Biophys. Res. Commun. 390, 557–563 (2009) . PubMed

Wu J. et al.. Inhibition of GPR40 protects MIN6 beta cells from palmitate-induced ER stress and apoptosis. J. Cell. Biochem. 113, 1152–1158 (2012) . PubMed

Briscoe C. P. et al.. Pharmacological regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules. Br. J. Pharmacol. 148, 619–628 (2006) . PubMed PMC

Lipina C., Rastedt W., Irving A. J. & Hundal H. S. Endocannabinoids in obesity:brewing up the perfect metabolicstorm? WIREs Membr. Transp. Signal 2, 49–63 (2013) .

Eckardt K. et al.. Cannabinoid type 1 receptors in human skeletal muscle cells participate in the negative crosstalk between fat and muscle. Diabetologia 52, 664–674 (2009) . PubMed

Alle H., Kubota H. & Geiger J. R. P. Sparse but highly efficient K(v)3 outpace BKCa channels in action potential repolarization at hippocampal mossy fiber boutons. J. Neurosci. 31, 8001–8012 (2011) . PubMed PMC

Geiger J. R. P. & Jonas P. Dynamic control of presynaptic ca2+ inflow by fast-inactivating K+ channels in hippocampal mossy fiber boutons. Neuron 28, 927–939 (2000) . PubMed

Horinouchi T., Nakagawa H., Suzuki T., Fukuhara K. & Miyata N. A novel mitochondria-localizing nitrobenzene derivative as a donor for photo-uncaging of nitric oxide. Bioorg. Med. Chem. Lett. 21, 2000–2002 (2011) . PubMed

Leonidova A. et al.. Photo-induced uncaging of a specific Re(I) organometallic complex in living cells. Chem. Sci. 5, 4044–4056 (2014) .

Riesgo E. C., Jin X. & Thummel R. P. Introduction of benzo[h]quinoline and 1,10-phenanthroline subunits by friedlander methodology. J. Org. Chem. 61, 3017–3022 (1996) . PubMed

Ito K. & Nakajima K. Selenium dioxide oxidation of alkylcoumarins and related methyl-substituted heteroaromatics. J. Heterocycl. Chem. 25, 511–515 (1988) .

Hagen V. et al.. Coumarinylmethyl esters for ultrafast release of high concentrations of cyclic nucleotides upon one- and two-photon photolysis. Angew. Chem. Int. Ed. Engl. 44, 7887–7891 (2005) . PubMed

Jones G., Jackson W. R., Choi C. & Bergmark W. R. Solvent effects on emission yield and lifetime for coumarin laser-dyes - requirements for a rotatory decay mechanism. J. Phys. Chem. 89, 294–300 (1985) .

Miyazaki J. I. et al.. Establishment of a pancreatic beta-cell line that retains glucose-inducible insulin-secretion - special reference to expression of glucose transporter isoforms. Endocrinology 127, 126–132 (1990) . PubMed

Stein F., Kress M., Reither S., Piljic A. & Schultz C. FluoQ: a tool for rapid analysis of multiparameter fluorescence imaging data applied to oscillatory events. ACS Chem. Biol. 8, 1862–1868 (2013) . PubMed

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