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Visualization of the Orai1 Homodimer and the Functional Coupling of Orai1-STIM1 by Live-Cell Fluorescence Lifetime Imaging

Published online by Cambridge University Press:  09 April 2010

Ping-Chun Huang ,
Tai-Yu Chiu ,
Li-Chun Wang ,
Hsiao-Chuan Teng ,
Fu-Jen Kao  and
De-Ming Yang
Ping-Chun Huang
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan, Republic of China Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan, Republic of China
Tai-Yu Chiu
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan, Republic of China Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan, Republic of China
Li-Chun Wang
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan, Republic of China
Hsiao-Chuan Teng
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan, Republic of China
Fu-Jen Kao
Affiliation:
Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan, Republic of China
De-Ming Yang*
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan, Republic of China Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan, Republic of China
*
Corresponding author. E-mail: yang.deming@gmail.com
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Abstract

The Orai1-STIM1 constructed store-operated Ca2+ channels (SOCs) have been found to exert several essential Ca2+ entry/signaling cascades, e.g., the generation of immune response in T lymphocytes. Although biochemical and novel imaging evidence appear to indicate that Orai1 and STIM1 interact with each other to achieve store-operated Ca2+ entry (SOCE), the detailed mechanism of functional SOCE in situ has yet to be fully understood. In this study, green fluorescence protein (EGFP as donor) targeted to either the N- or C-terminal of Orai1 (wild type or ▵1-90+▵267-301 double deletion type) and mOrange (as acceptor) tagged STIM1 were used to comprise a fluorescence resonance energy transfer (FRET) pair within living PC12 cells. The fluorescence lifetime map and histogram/distribution of each single cell, determined by one-photon excitation fluorescence lifetime imaging microscopy (FLIM), was used to visualize FRET and show the Orai1 homodimer and Orai1-STIM1 binding. Both the color-coded lifetime map and the distribution of EGFP-tagged Orai1 significantly changed after the administration of thapsigargin, the SOCE stimulating agent. The FRET efficiency from each experimental set was also calculated and compared using double exponential analysis. In summary, we show the detailed interactions Orai1-Orai1 and Orai1-STIM1 within intact living cells by using the FLIM-FRET technique.

Keywords

fluorescence resonance energy transfer (FRET)fluorescence lifetime imaging microscopy (FLIM)time-correlated single-photon counting (TCSPC)green fluorescent proteins (GFPs)store-operated calcium channels (SOCs)
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 16 , Issue 3 , June 2010 , pp. 313 - 326
Copyright
Copyright © Microscopy Society of America 2010

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References

REFERENCES

Barr, V.A., Bernot, K.M., Srikanth, S., Gwack, Y., Balagopalan, L., Regan, C.K., Helman, D.J., Sommers, C.L., Oh-Hora, M., Rao, A. & Samelson, L.E. (2008). Dynamic movement of the calcium sensor STIM1 and the calcium channel Orai1 in activated T-cells: Puncta and distal caps. Mol Biol Cell 19, 2802–2817.Google Scholar
Birnbaumer, L. (2009). The TRPC class of ion channels: A critical review of their roles in slow, sustained increases in intracellular Ca2+ concentrations. Annu Rev Pharmacol Toxicol 49, 395–426.CrossRefGoogle Scholar
Brymora, A., Valova, V.A. & Robinson, P.J. (2004). Protein-protein interactions identified by pull-down experiments and mass spectrometry. Curr Protoc Cell Biol 22, 17.5.1–17.5.51 (Chapter 17, Unit 17.5).Google Scholar
Calloway, N., Vig, M., Kinet, J.P., Holowka, D. & Baird, B. (2009). Molecular clustering of STIM1 with Orai1/CRACM1 at the plasma membrane depends dynamically on depletion of Ca2+ stores and on electrostatic interactions. Mol Biol Cell 20, 389–399.CrossRefGoogle ScholarPubMed
Chang, Y.F., Teng, H.C., Cheng, S.Y., Wang, C.T., Chiou, S.H., Kao, L.S., Kao, F.J., Chiou, A. & Yang, D.M. (2008). Orai1-STIM1 formed store-operated Ca2+ channels (SOCs) as the molecular components needed for Pb2+ entry in living cells. Toxicol Appl Pharmacol 227, 430–439.Google Scholar
Chiu, T.Y., Teng, H.C., Huang, P.C., Kao, F.J. & Yang, D.M. (2009). Dominant role of Orai1 with STIM1 on the cytosolic entry and cytotoxicity of lead ions. Toxicol Sci 110, 353–362.Google Scholar
Ciruela, F. (2008). Fluorescence-based methods in the study of protein-protein interactions in living cells. Curr Opin Biotech 19, 338–343.CrossRefGoogle Scholar
Dai, S., Hall, D.D. & Hell, J.W. (2009). Supramolecular assemblies and localized regulation of voltage-gated ion channels. Physiol Rev 89, 411–452.Google Scholar
Fahrner, M., Muik, M., Derler, I., Schindl, R., Fritsch, R., Frischauf, I. & Romanin, C. (2009). Mechanistic view on domains mediating STIM1-Orai coupling. Immunol Rev 231, 99–112.Google Scholar
Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S.H., Tanasa, B., Hogan, P.G., Lewis, R.S., Daly, M. & Rao, A. (2006). A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 441, 179–185.CrossRefGoogle ScholarPubMed
Förster, T. (1948). Intermolecular energy migration and fluorescence. Ann Phys 2, 55–75.Google Scholar
Gandía, J., Lluís, C., Ferre, S., Franco, R. & Ciruela, F. (2007). Light resonance energy transfer-based methods into the study of G protein-coupled receptor oligomerization. BioEssays 30, 82–89.Google Scholar
Goedhart, J., Vermeer, J.E., Adjobo-Hermans, M.J., van Weeren, L. & Gadella, T.W. Jr. (2007). Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples. PlosOne 10, e-011.Google Scholar
Huang, G.N., Zeng, W., Kim, J.Y., Yuan, J.P., Han, L., Muallem, S. & Worley, P.F. (2006). STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels. Nat Cell Biol 8, 1003–1010.CrossRefGoogle ScholarPubMed
Ji, W., Xu, P., Li, Z., Lu, J., Liu, L., Zhan, Y., Chen, Y., Hille, B., Xu, T. & Chen, L. (2008). Functional stoichiometry of the unitary calcium-release-activated calcium channel. Proc Natl Acad Sci USA 105, 13668–13673.CrossRefGoogle ScholarPubMed
Koch, H.P., Kurokawa, T., Okochi, Y., Sasaki, M., Okamura, Y. & Larsson, H.P. (2008). Multimeric nature of voltage-gated proton channels. Proc Natl Acad Sci USA 105, 9111–9116.Google Scholar
Lee, J.D., Chang, Y.F., Kao, F.J., Kao, L.S., Lin, C.C., Lu, A.C., Shyyu, B.C., Chiou, S.H. & Yang, D.M. (2008a). Detection of the interaction between SNAP25 and rabphilin in neuroendocrine PC12 cells using the FLIM/FRET technique. Microsco Res Tech 71, 26–34.CrossRefGoogle ScholarPubMed
Lee, J.D., Huang, P.C., Lin, Y.C., Kao, L.S., Huang, C.C., Kao, F.J., Lin, C.C. & Yang, D.M. (2008b). In-depth fluorescence lifetime imaging analysis revealing SNAP25A-rabphilin 3A interactions. Micro Microanal 14, 507–518.Google Scholar
Lee, S.Y., Letts, J.A. & MacKinnon, R. (2008c). Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1. Proc Natl Acad Sci USA 105, 7692–7695.CrossRefGoogle ScholarPubMed
Levitt, J.A., Matthews, D.R., Ameer-Beg, S.M. & Suhling, K. (2009). Fluorescence lifetime and polarization-resolved imaging in cell biology. Curr Opin Biotechnol 20, 28–36.Google Scholar
Li, Z., Lu, J., Xu, P., Xie, X., Chen, L. & Xu, T. (2007). Mapping the interacting domains of STIM1 and Orai1 in Ca2+ release-activated Ca2+ channel activation. J Biol Chem 282, 29448–29456.CrossRefGoogle ScholarPubMed
Liou, J., Kim, M.L., Heo, W.D., Jones, J.T., Myers, J.W., Ferrell, J.E. Jr. & Meyer, T. (2005). STIM is a Ca2+ sensor essential for Ca2+-store depletion-triggered Ca2+ influx. Curr Biol 15, 1235–1241.Google Scholar
Luik, R.M., Wu, M.M., Buchanan, J. & Lewis, R.S. (2006). The elementary unit of store-operated Ca2+ entry: Local activation of CRAC channels by STIM1 at ER-plasma membrane junctions. J Cell Biol 174, 815–825.Google Scholar
Maruyama, Y., Ogura, T., Mio, K., Kato, K., Kaneko, T., Kiyonaka, S., Mori, Y. & Sato, C. (2009). Tetrameric Orai1 is a teardrop-shaped molecule with a long, tapered cytoplasmic domain. J Biol Chem 284, 13676–13685.CrossRefGoogle ScholarPubMed
Mignen, O., Thompson, J.L. & Shuttleworth, T.J. (2008). Orai1 subunit stoichiometry of the mammalian CRAC channel pore. J Physiol 586 (2), 419–425.Google Scholar
Muik, M., Frischauf, I., Derler, I., Fahrner, M., Bergsmann, J., Eder, P., Schindl, R., Hesch, C., Polzinger, B., Fritsch, R., Kahr, H., Madl, J., Gruber, H., Groschner, K. & Romanin, C. (2008). Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation. J Biol Chem 283, 8014–8022.Google Scholar
Navarro-Borelly, L., Somasundaram, A., Yamashita, M., Ren, D., Miller, R.J. & Prakriya, M. (2008). STIM1-Orai1 interactions and Orai1 conformational changes revealed by live-cell FRET microscopy. J Physiol 586, 5383–5401.Google Scholar
Padilla-Parra, S., Audugé, N., Coppey-Moisanand, M. & Tramier, M. (2008). Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells. Biophys J 95, 2976–2988.Google Scholar
Park, C.Y., Hoover, P.J., Mullins, F.M., Bachhawat, P., Covington, E.D., Raunser, S., Walz, T., Garcia, K.C., Dolmetsch, R.E. & Lewis, R.S. (2009). STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell 136, 876–890.Google Scholar
Penna, A., Demuro, A., Yeromin, A.V., Zhang, S.L., Safrina, O., Parker, I. & Cahalan, M.D. (2008). The CRAC channel consists of a tetramer formed by Stim-induced dimerzation of Orai1 dimers. Nature 456, 116–120CrossRefGoogle Scholar
Periasamy, A., Wallrabe, H., Chen, Y. & Barroso, M. (2008). Chapter 22: Quantitation of protein-protein interactions: Confocal FRET microscopy. Methods Cell Biol 89, 569–98.CrossRefGoogle ScholarPubMed
Peter, M., Ameer-Beg, S.M., Hughes, M.K.Y., Keppler, M.D., Prag, S., Marsh, M., Vojnovic, B. & Ng, T. (2005). Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions. Biophy J 88, 1224–1237.CrossRefGoogle ScholarPubMed
Putney, J.W. Jr. (1986). A model for receptor-regulated calcium entry. Cell Calcium 7, 1–12.Google Scholar
Roda, A., Guardigli, M., Michelini, E. & Mirasoli, M. (2009). Nanobioanalytical lumenscence: Förster-type energy transfer methods. Anal Bioanal Chem 393, 109–123.Google Scholar
Shyu, Y.J., Suarez, C.D. & Hu, C.-D. (2008). Visualization of AP-1–NF-kB ternary complexes in living cells by using a BiFC-based FRET. Proc Natl Acad Sci USA 105, 151–156.Google Scholar
Suter, B., Kittanakom, S. & Stagljar, I. (2008). Two-hybrid techniques in proteomics research. Curr Opin Biotech 19, 316–323.Google Scholar
Tregidgo, C., Levitt, J.A. & Suhling, K. (2008). Effect of refractive index on the fluorescence lifetime of green fluorescent protein. J Biomed Opt 13, 031218-1–031218-8.CrossRefGoogle ScholarPubMed
Várnai, P., Tóth, B., Tóth, D.J., Hunyady, L. & Balla, T. (2007). Visualization and manipulation of plasma membrane-endoplasmic reticulum contact sites indicates the presence of additional molecular components within the STIM1-Orai1 Complex. J Biol Chem 282, 29678–29690.CrossRefGoogle ScholarPubMed
Vig, M., Peinelt, C., Beck, A., Koomoa, D.L., Rabah, D., Koblan-Huberson, M., Kraft, S., Turner, H., Fleig, A., Penner, R. & Kinet, J.P. (2006). CRACM1is a plasma membrane protein essential for store-operated Ca2+ entry. Science 312, 1220–1223.Google Scholar
Vogel, S.S., Thaler, C. & Koushik, S.V. (2006). Fanciful FRET. Sci STKE 331, re2 PMID: 16622184.Google Scholar
Wang, Y., Deng, X., Zhou, Y., Hendron, E., Mancarella, S., Ritchie, M.F., Tang, X.D., Baba, Y., Kurosaki, T., Mori, Y., Soboloff, J. & Gill, D.L. (2009). STIM protein coupling in the activation of Orai channels. Proc Natl Acad Sci USA 106, 7391–7396.Google Scholar