Abstract
Photo-activatable (caged) probes are powerful research tools for biological investigation. The superb maneuverability of a light beam allows researchers to activate caged probes with pinpoint accuracy. Recent developments in caging chemistry and two-photon excitation technique further enhance our capability to perform photo-uncaging with even higher spatial and temporal resolution, offering new photonic approaches to study cell signaling dynamics in greater detail. Here we present a sample method that combines the techniques of photo-activation and digital fluorescence microscopy to assay an important class of intracellular receptors for the second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3, or IP3). The imaging assay is performed in fully intact living cells using a caged and cell membrane permeable ester derivative of IP3, cm-IP3/PM.
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References
Kaplan, J. H., Forbush, B., 3rd, and Hoffman, J. F. (1978) Rapid photolytic release of adenosine 5'-triphosphate from a protected analogue: utilization by the Na:K pump of human red blood cell ghosts. Biochemistry 17, 1929–35.
Marriott, G. (Ed.) (1998) Caged Compounds, Methods Enzymol, vol 291, Academic Press, New York.
Goeldner, M., and Givens, R. S. (Eds.) (2005) Dynamic Studies in Biology: Phototriggers, Photoswitches and Caged Biomolecules, Wiley-VCH Verlag GmbH, Weinheim.
Papageorgiou, G., Ogden, D. C., Barth, A., and Corrie, J. E. T. (1999) Photorelease of carboxylic acids from 1-acyl-7-nitroindolines in aqueous solution: Rapid and efficient photorelease of L-glutamate. J Am Chem Soc 121, 6503–4.
Walker, J. W., Feeney, J., and Trentham, D. R. (1989) Photolabile precursors of inositol phosphates – preparation and properties of 1-(2-nitrophenyl)ethyl esters of myo-inositol 1,4,5-trisphosphate. Biochemistry 28, 3272–80.
Li, W. H., Llopis, J., Whitney, M., Zlokarnik, G., and Tsien, R. Y. (1998) Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature 392, 936–41.
Aarhus, R., Gee, K., and Lee, H. C. (1995) Caged cyclic ADP-ribose. Synthesis and use. J Biol Chem 270, 7745–9.
Dakin, K., and Li, W. H. (2007) Cell membrane permeable esters of D-myo-inositol 1,4,5-trisphosphate. Cell Calcium 42, 291–301.
Zipfel, W. R., Williams, R. M., and Webb, W. W. (2003) Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21, 1369–77.
Haugland, R. P. (2005) The Handbook: A Guide to Fluorescent Probes and Labeling Technologies, Invitrogen Corporation, Eugene, OR, Ch. 19, pp. 879–906.
Palmer, A. E., and Tsien, R. Y. (2006) Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc 1, 1057–65.
West, D. J., and Williams, A. J. (2007) Pharmacological regulators of intracellular calcium release channels. Curr Pharm Des 13, 2428–42.
Furuta, T., Wang, S. S., Dantzker, J. L., Dore, T. M., Bybee, W. J., Callaway, E. M., Denk, W., and Tsien, R. Y. (1999) Brominated 7-hydroxycoumarin-4-ylmethyls: photolabile protecting groups with biologically useful cross-sections for two photon photolysis. Proc Natl Acad Sci U S A 96, 1193–200.
Zhao, Y., Zheng, Q., Dakin, K., Xu, K., Martinez, M. L., and Li, W. H. (2004) New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications. J Am Chem Soc 126, 4653–63.
Dakin, K., and Li, W. H. (2006) Infrared-LAMP: two-photon uncaging and imaging of gap junctional communication in three dimensions. Nat Methods 3, 959.
Zhu, Y., Pavlos, C. M., Toscano, J. P., and Dore, T. M. (2006) 8-Bromo-7-hydroxyquinoline as a photoremovable protecting group for physiological use: mechanism and scope. J Am Chem Soc 128, 4267–76.
Momotake, A., Lindegger, N., Niggli, E., Barsotti, R. J., and Ellis-Davies, G. C. (2006) The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells. Nat Methods 3, 35–40.
Gug, S., Bolze, F., Specht, A., Bourgogne, C., Goeldner, M., and Nicoud, J. F. (2008) Molecular engineering of photoremovable protecting groups for two-photon uncaging. Angew Chem Int Ed Engl 47, 9525–9.
Brown, E. B., Shear, J. B., Adams, S. R., Tsien, R. Y., and Webb, W. W. (1999) Photolysis of caged calcium in femtoliter volumes using two-photon excitation. Biophys J 76, 489–99.
Newton, C. L., Mignery, G. A., and Sudhof, T. C. (1994) Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3. J Biol Chem 269, 28613–9.
Yoneshima, H., Miyawaki, A., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1997) Ca2+ differentially regulates the ligand-affinity states of type 1 and type 3 inositol 1,4,5-trisphosphate receptors. Biochem J 322 (Pt 2), 591–6.
Patterson, G. H., and Piston, D. W. (2000) Photobleaching in two-photon excitation microscopy. Biophys J 78, 2159–62.
Kiskin, N. I., Chillingworth, R., McCray, J. A., Piston, D., and Ogden, D. (2002) The efficiency of two-photon photolysis of a “caged” fluorophore, o-1-(2-nitrophenyl)ethylpyranine, in relation to photodamage of synaptic terminals. Eur Biophys J 30, 588–604.
Hatchard, C. G., and Parker, C. A. (1956) A new sensitive chemical actinometer. II. Potassium ferrioxalate as a standard chemical actinometer. Proc R Acad London A 235, 518–36.
Bernardinelli, Y., Haeberli, C., and Chatton, J. Y. (2005) Flash photolysis using a light emitting diode: an efficient, compact, and affordable solution. Cell Calcium 37, 565–72.
Rapp, G. (1998) Flash lamp-based irradiation of caged compounds. Methods Enzymol 291, 202–22.
Yang, S., and Li, W. H. (2009) Assaying dynamic cell-cell junctional communication using noninvasive and quantitative fluorescence imaging techniques: LAMP and infrared-LAMP. Nat Protoc 4, 94–101.
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Li, WH. (2010). Photo-Activatable Probes for the Analysis of Receptor Function in Living Cells. In: Papkovsky, D. (eds) Live Cell Imaging. Methods in Molecular Biology, vol 591. Humana Press. https://doi.org/10.1007/978-1-60761-404-3_6
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DOI: https://doi.org/10.1007/978-1-60761-404-3_6
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