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Green fluorescent protein based pH indicators for in vivo use: a review

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Abstract

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community.

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References

  1. Srivastava J, Barber DL, Jacobson MP (2007) Physiology (Bethesda) 22:30–39

    CAS  Google Scholar 

  2. Wang H, Singh D, Fliegel L (1997) J Biol Chem 272:26545–26549

    Article  CAS  Google Scholar 

  3. Putney LK, Barber DL (2003) J Biol Chem 278:44645–44649

    Article  CAS  Google Scholar 

  4. Fliegel L (2005) Int J Biochem Cell Biol 37:33–37

    Article  CAS  Google Scholar 

  5. Hunte C, Screpanti E, Venturi M, Rimon A, Padan E, Michel H (2005) Nature 435:1197–1202

    Article  CAS  Google Scholar 

  6. Wakabayashi I, Poteser M, Groschner K (2006) J Vasc Res 43:238–250

    Article  Google Scholar 

  7. Sun HY, Wang NP, Halkos ME, Kerendi F, Kin H, Wang RX, Guyton RA, Zhao ZQ (2004) Eur J Pharmacol 486:121–131

    Article  CAS  Google Scholar 

  8. Denker SP, Barber DL (2002) J Cell Biol 159:1087–1096

    Article  CAS  Google Scholar 

  9. Kapus A, Szaszi K, Sun J, Rizoli S, Rotstein OD (1999) J Biol Chem 274:8093–8102

    Article  CAS  Google Scholar 

  10. Mellman I, Warren G (2000) Cell 100:99–112

    Article  CAS  Google Scholar 

  11. Rybak SL, Lanni F, Murphy RF (1997) Biophys J 73:674–687

    Article  CAS  Google Scholar 

  12. Jahn R, Lang T, Sudhof TC (2003) Cell 112:519–533

    Article  CAS  Google Scholar 

  13. Abad MFC, Di Benedetto G, Magalhaes PJ, Filippin L, Pozzan T (2004) J Biol Chem 279:11521–11529

    Article  CAS  Google Scholar 

  14. Carnell L, Moore HP (1994) J Cell Biol 127:693–705

    Article  CAS  Google Scholar 

  15. Chanat E, Huttner WB (1991) J Cell Biol 115:1505–1519

    Article  CAS  Google Scholar 

  16. Puri S, Bachert C, Fimmel CJ, Linstedt AD (2002) Traffic 3:641–653

    Article  CAS  Google Scholar 

  17. Kellokumpu S, Sormunen R, Kellokumpu I (2002) FEBS Lett 516:217–224

    Article  CAS  Google Scholar 

  18. Lakowicz JR (1999) Principles of fluorescence spectroscopy. Plenum, Oakland

    Google Scholar 

  19. Rink TJ, Tsien RY, Pozzan T (1982) J Cell Biol 95:189–196

    Article  CAS  Google Scholar 

  20. Tsien RY (1989) Methods Cell Biol 30:127–156

    Article  CAS  Google Scholar 

  21. Lin HJ, Herman P, Lakowicz JR (2003) Cytometry Part A 52A:77–89

    Article  Google Scholar 

  22. Shimomura O, Johnson FH, Saiga Y (1962) J Cell Comp Physiol 59:223–239

    Article  CAS  Google Scholar 

  23. Tsien RY (1998) Annu Rev Biochem 67:509–544

    Article  CAS  Google Scholar 

  24. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Gene 111:229–233

    Article  CAS  Google Scholar 

  25. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Science 263:802–805

    Article  CAS  Google Scholar 

  26. Chudakov DM, Lukyanov S, Lukyanov KA (2005) Trends Biotechnol 23:605–613

    Article  CAS  Google Scholar 

  27. Miyawaki A (2005) Neuron 48:189–199

    Article  CAS  Google Scholar 

  28. Shaner NC, Steinbach PA, Tsien RY (2005) Nat Methods 2:905–909

    Article  CAS  Google Scholar 

  29. Zhang J, Campbell RE, Ting AY, Tsien RY (2002) Nat Rev Mol Cell Biol 3:906–918

    Article  CAS  Google Scholar 

  30. Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Science 273:1392–1395

    Article  CAS  Google Scholar 

  31. Wachter RM (2007) Acc Chem Res 40:120–127

    Article  CAS  Google Scholar 

  32. Niwa H, Inouye S, Hirano T, Matsuno T, Kojima S, Kubota M, Ohashi M, Tsuji FI (1996) Proc Natl Acad Sci USA 93:13617–13622

    Article  CAS  Google Scholar 

  33. Webber NM, Litvinenko KL, Meech SR (2001) J Phys Chem B 105:8036–8039

    Article  CAS  Google Scholar 

  34. Ward WW, Bokman SH (1982) Biochemistry 21:4535–4540

    Article  CAS  Google Scholar 

  35. Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormo M, Remington SJ (1997) Proc Natl Acad Sci USA 94:2306–2311

    Article  CAS  Google Scholar 

  36. Kummer AD, Wiehler J, Rehaber H, Kompa C, Steipe B, Michel-Beyerle ME (2000) J Phys Chem B 104:4791–4798

    Article  CAS  Google Scholar 

  37. Wachter RM, Yarbrough D, Kallio K, Remington SJ (2000) J Mol Biol 301:157–171

    Article  CAS  Google Scholar 

  38. Sniegowski JA, Lappe JW, Patel HN, Huffman HA, Wachter RM (2005) J Biol Chem 280:26248–26255

    Article  CAS  Google Scholar 

  39. Zhang L, Patel HN, Lappe JW, Wachter RM (2006) J Am Chem Soc 128:4766–4772

    Article  CAS  Google Scholar 

  40. Bell AF, He X, Wachter RM, Tonge PJ (2000) Biochemistry 39:4423–4431

    Article  CAS  Google Scholar 

  41. Dong J, Solntsev KM, Tolbert LM (2006) J Am Chem Soc 128:12038–12039

    Article  CAS  Google Scholar 

  42. Elsliger MA, Wachter RM, Hanson GT, Kallio K, Remington SJ (1999) Biochemistry 38:5296–5301

    Article  CAS  Google Scholar 

  43. Bizzarri R, Nifosi R, Abbruzzetti S, Rocchia W, Guidi S, Arosio D, Garau G, Campanini B, Grandi E, Ricci F, Viappiani C, Beltram F (2007) Biochemistry 46:5494–5504

    Article  CAS  Google Scholar 

  44. Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY (1998) Proc Natl Acad Sci USA 95:6803–6808

    Article  CAS  Google Scholar 

  45. Voityuk AA, Michel-Beyerle ME, Rosch N (1998) Chem Phys 231:13–25

    Article  CAS  Google Scholar 

  46. Chattoraj M, King BA, Bublitz GU, Boxer SG (1996) Proc Natl Acad Sci USA 93:8362–8367

    Article  CAS  Google Scholar 

  47. McAnaney TB, Park ES, Hanson GT, Remington SJ, Boxer SG (2002) Biochemistry 41:15489–15494

    Article  CAS  Google Scholar 

  48. Bonsma S, Purchase R, Jezowski S, Gallus J, Konz F, Volker S (2005) Chemphyschem 6:838–849

    Article  CAS  Google Scholar 

  49. Creemers TM, Lock AJ, Subramaniam V, Jovin TM, Volker S (1999) Nat Struct Biol 6:557–560

    Article  CAS  Google Scholar 

  50. Wiehler J, Jung G, Seebacher C, Zumbusch A, Steipe B (2003) Chembiochem 4:1164–1171

    Article  CAS  Google Scholar 

  51. Kennis JT, Larsen DS, van Stokkum IH, Vengris M, van Thor JJ, van Grondelle R (2004) Proc Natl Acad Sci USA 101:17988–17993

    Article  CAS  Google Scholar 

  52. Lukyanov KA, Chudakov DM, Fradkov AF, Labas YA, Matz MV, Lukyanov S (2006) Methods Biochem Anal 47:121–138

    Article  Google Scholar 

  53. Kneen M, Farinas J, Li Y, Verkman AS (1998) Biophys J 74:1591–1599

    Article  CAS  Google Scholar 

  54. Abbruzzetti S, Grandi E, Viappiani C, Bologna S, Campanini B, Raboni S, Bettati S, Mozzarelli A (2005) J Am Chem Soc 127:626–635

    Article  CAS  Google Scholar 

  55. Hanson GT, McAnaney TB, Park ES, Rendell ME, Yarbrough DK, Chu S, Xi L, Boxer SG, Montrose MH, Remington SJ (2002) Biochemistry 41:15477–15488

    Article  CAS  Google Scholar 

  56. Ullmann GM (2003) J Phys Chem B 107:1263–1271

    Article  CAS  Google Scholar 

  57. Grynkiewicz G, Poenie M, Tsien RY (1985) J Biol Chem 260:3440–3450

    CAS  Google Scholar 

  58. Sonnleitner A, Schutz GJ, Schmidt T (1999) Biophys J 77:2638–2642

    Article  CAS  Google Scholar 

  59. Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E (2005) Biophys J 89:1317–1327

    Article  CAS  Google Scholar 

  60. Cardarelli F, Serresi M, Bizzarri R, Beltram F (2008) Traffic 9:528–539

    Article  CAS  Google Scholar 

  61. Cardarelli F, Serresi M, Bizzarri R, Giacca M, Beltram F (2007) Molec Ther 15:1313–1322

    Article  CAS  Google Scholar 

  62. Hell SW (2003) Nat Biotechnol 21:1347–1355

    Article  CAS  Google Scholar 

  63. Haupts U, Maiti S, Schwille P, Webb WW (1998) Proc Natl Acad Sci USA 95:13573–13578

    Article  CAS  Google Scholar 

  64. Bosisio C, Quercioli V, Collini M, D’Alfonso L, Baldini G, Bettati S, Campanini B, Raboni S, Chirico G (2008) J Phys Chem B 112:8806–8814

    Article  CAS  Google Scholar 

  65. Widengren J, Terry B, Rigler R (1999) Chem Phys 249:259–271

    Article  CAS  Google Scholar 

  66. Charier S, Meglio A, Alcor D, Cogne-Laage E, Allemand JF, Jullien L, Lemarchand A (2005) J Am Chem Soc 127:15491–15505

    Article  CAS  Google Scholar 

  67. Sankaranarayanan S, De Angelis D, Rothman JE, Ryan TA (2000) Biophys J 79:2199–2208

    Article  CAS  Google Scholar 

  68. Nakabayashi T, Wang HP, Kinjo M, Ohta N (2008) Photochem Photobiol Sci 7:668–670

    Article  CAS  Google Scholar 

  69. Miesenbock G, De Angelis DA, Rothman JE (1998) Nature 394:192–195

    Article  CAS  Google Scholar 

  70. Hess ST, Heikal AA, Webb WW (2004) J Phys Chem B 108:10138–10148

    Article  CAS  Google Scholar 

  71. Wachter RM, Elsliger MA, Kallio K, Hanson GT, Remington SJ (1998) Structure 6:1267–1277

    Article  CAS  Google Scholar 

  72. Patterson G, Day RN, Piston D (2001) J Cell Sci 114:837–838

    CAS  Google Scholar 

  73. Jayaraman S, Haggie P, Wachter RM, Remington SJ, Verkman AS (2000) J Biol Chem 275:6047–6050

    Article  CAS  Google Scholar 

  74. Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY (2001) J Biol Chem 276:29188–29194

    Article  CAS  Google Scholar 

  75. Karagiannis J, Young PG (2001) J Cell Sci 114:2929–2941

    CAS  Google Scholar 

  76. Jankowski A, Kim JH, Collins RF, Daneman R, Walton P, Grinstein S (2001) J Biol Chem 276:48748–48753

    Article  CAS  Google Scholar 

  77. Machen TE, Leigh MJ, Taylor C, Kimura T, Asano S, Moore HP (2003) Am J Physiol 285:C205–214

    CAS  Google Scholar 

  78. Schulte A, Lorenzen I, Bottcher M, Plieth C (2006) Plant Methods 2:7

    Article  CAS  Google Scholar 

  79. Shu X, Leiderman P, Gepshtein R, Smith NR, Kallio K, Huppert D, Remington SJ (2007) Protein Sci 16:2703–2710

    Article  CAS  Google Scholar 

  80. Shu X, Kallio K, Shi X, Abbyad P, Kanchanawong P, Childs W, Boxer SG, Remington SJ (2007) Biochemistry 46:12005–12013

    Article  CAS  Google Scholar 

  81. Cody SH, Dubbin PN, Beischer AD, Duncan ND, Hill JS, Kaye AH, Williams DA (1993) Micron 24:573–580

    Article  Google Scholar 

  82. Dubbin PN, Cody SH, Williams DA (1993) Micron 24:581–586

    Article  Google Scholar 

  83. Bizzarri R, Arcangeli C, Arosio D, Ricci F, Faraci P, Cardarelli F, Beltram F (2006) Biophys J 90:3300–3314

    Article  CAS  Google Scholar 

  84. Marcello A, Lusic M, Pegoraro G, Pellegrini V, Beltram F, Giacca M (2004) Gene 326:1–11

    Article  CAS  Google Scholar 

  85. Arosio D, Garau G, Ricci F, Marchetti L, Bizzarri R, Nifosi R, Beltram F (2007) Biophys J 93:232–244

    Article  CAS  Google Scholar 

  86. Serresi M, Bizzarri R, Cardarelli F, Beltram F (2009) Anal Bioanal Chem. doi:10.1007/s00216-008-2489-7

  87. Awaji T, Hirasawa A, Shirakawa H, Tsujimoto G, Miyazaki S (2001) Biochem Biophys Res Commun 289:457–462

    Article  CAS  Google Scholar 

  88. Chan FK, Siegel RM, Zacharias D, Swofford R, Holmes KL, Tsien RY, Lenardo MJ (2001) Cytometry 44:361–368

    Article  CAS  Google Scholar 

  89. Wlodarczyk J, Woehler A, Kobe F, Ponimaskin E, Zeug A, Neher E (2008) Biophys J 94:986–1000

    Article  CAS  Google Scholar 

  90. Urra J, Sandoval M, Cornejo I, Barros LF, Sepúlveda FV, Cid LP (2008) Pflugers Arch 457:233–242

    Article  CAS  Google Scholar 

  91. Markova O, Mukhtarov M, Real E, Jacob Y, Bregestovski P (2008) J Neurosci Methods 170:67–76

    Article  CAS  Google Scholar 

  92. Tsien RY (2003) Nat Rev Mol Cell Biol Suppl S16–21

  93. Paddock S (2008) Biotechniques 44:643–644 646, 648

    Article  CAS  Google Scholar 

  94. Diaspro A, Bianchini P, Vicidomini G, Faretta M, Ramoino P, Usai C (2006) Biomed Eng online 5:36

    Article  Google Scholar 

  95. Digman MA, Dalal R, Horwitz AF, Gratton E (2008) Biophys J 94:2320–2332

    Article  CAS  Google Scholar 

  96. Zipfel WR, Williams RM, Webb WW (2003) Nat Biotechnol 21:1369–1377

    Article  CAS  Google Scholar 

  97. Svoboda K, Yasuda R (2006) Neuron 50:823–839

    Article  CAS  Google Scholar 

  98. Chirico G, Diaspro A, Cannone F, Collini M, Bologna S, Pellegrini V, Beltram F (2005) Chemphyschem 6:328–335

    Article  CAS  Google Scholar 

  99. Chirico G, Cannone F, Diaspro A, Bologna S, Pellegrini V, Nifosi R, Beltram F (2004) Phys Rev E 70:030901

    Article  CAS  Google Scholar 

  100. Albota MA, Xu C, Webb WW (1998) Appl Opt 37:7352–7356

    Article  CAS  Google Scholar 

  101. Heikal A, Hess S, Webb WW (2001) Chem Phys 274:37–55

    Article  CAS  Google Scholar 

  102. Stoner-Ma D, Jaye AA, Ronayne KL, Nappa J, Meech SR, Tonge PJ (2008) J Am Chem Soc 130:1227–1235

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Riccardo Nifosì (Scuola Normale Superiore) for stimulating discussions. We also gratefully acknowledge the partial financial support of the Italian Ministry for University and Research (FIRB no. RBLA03ER38).

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Authors and Affiliations

  1. NEST, Scuola Normale Superiore and Italian Institute of Technology, Piazza San Silvestro 12, 56124, Pisa, Italy

    Ranieri Bizzarri, Michela Serresi & Fabio Beltram

  2. NEST, Scuola Normale Superiore and CNR-INFM, Piazza San Silvestro 12, 56124, Pisa, Italy

    Ranieri Bizzarri, Stefano Luin & Fabio Beltram

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  1. Ranieri Bizzarri
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  2. Michela Serresi
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Correspondence to Ranieri Bizzarri.

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Bizzarri, R., Serresi, M., Luin, S. et al. Green fluorescent protein based pH indicators for in vivo use: a review. Anal Bioanal Chem 393, 1107–1122 (2009). https://doi.org/10.1007/s00216-008-2515-9

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