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Two-Photon Permeabilization and Calcium Measurements in Cellular Organelles

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Live Cell Imaging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 591))

Abstract

Inositol trisphosphate and cyclic ADP-ribose, main intracellular Ca2+ messengers, induce release from the intracellular Ca2+ stores via inositol trisphosphate and ryanodine receptors, respectively. Recently, studies using novel messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releasing Ca2+ from calcium stores in organelles other than endoplasmic reticulum (ER) have been conducted. However, technical difficulties of Ca2+ measurements in relatively small Ca2+ stores prompted us to develop a new, more sensitive, and less damaging two-photon permeabilization technique. Applied to pancreatic acinar cells, this technique allowed us to show that all three messengers – IP3, cADPR, and NAADP – release Ca2+ from two intracellular stores: the endoplasmic reticulum and an acidic store in the granular region. This chapter describes a detailed procedure of using this technique with pancreatic acinar cells.

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References

  1. Nielsen, S. P. and Petersen, O. H. (1972). Transport of calcium in the perfused submandibular gland of the cat. J. Physiol. 223, 685–697.

    CAS  PubMed  Google Scholar 

  2. Streb, H., Irvine, R. F., Berridge, M. J. and Schulz, I. (1983). Release of Ca2+ from a nonmitochondrial intracellular store in pancreatic acinar cells by inositol-1,4,5-trisphosphate. Nature 306, 67–69.

    Article  CAS  PubMed  Google Scholar 

  3. Berridge, M. J. (1984). Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J. 220, 345–360.

    CAS  PubMed  Google Scholar 

  4. Berridge, M. J. (1993). Inositol trisphosphate and calcium signalling. Nature 361, 315–325.

    Article  CAS  PubMed  Google Scholar 

  5. Berridge, M. J., Bootman, M. D. and Roderick, H. L. (2003). Calcium signalling: dynamics, homeostasis and remodelling. Nature Rev. Mol. Cell. Biol. 14, 517–529.

    Article  Google Scholar 

  6. Meldolesi, J. and Pozzan, T. (1998). The endoplasmic reticulum Ca2+ store: a view from the lumen. Trends. BioChem. Sci. 23, 10–14.

    Article  CAS  PubMed  Google Scholar 

  7. Petersen, O. H., Petersen, C. C. H., and Kasai, H. (1994). Calcium and hormone action. Annu. Rev. Physiol. 56, 297–319.

    Article  CAS  PubMed  Google Scholar 

  8. Pozzan, T., Rizzuto, R., Volpe, P. and Meldolesi, J. (1994). Molecular and cellular physiology of intracellular calcium stores. Physiol. Rev. 74, 595–636.

    CAS  PubMed  Google Scholar 

  9. Ashby, M. C. and Tepikin, A. V. (2002) Polarized calcium and calmodulin signaling in secretory epithelia. Physiol. Rev. 82, 701–734.

    CAS  PubMed  Google Scholar 

  10. Rizzuto, R., Brini, M., Murgia, M. and Pozzan, T. (1993). Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science 262, 744–747.

    Article  CAS  PubMed  Google Scholar 

  11. Hajnoczky, G., Robb-Gaspers, L. D., Seitz, M. B. and Thomas, A. P. (1995). Decoding of cytosolic calcium oscillations in the mitochondria. Cell 82, 415–424.

    Article  CAS  PubMed  Google Scholar 

  12. Tinel, H., Cancela, J. M., Mogami, H., Gerasimenko, J. V., Gerasimenko, O. V., Tepikin, A. V. and Petersen, O. H. (1999). Active mitochondria surrounding the pancreatic acinar granule region prevent spreading of inositol trisphosphate-evoked local cytosolic Ca2+ signals. EMBO J. 18, 4999–5008.

    Article  CAS  PubMed  Google Scholar 

  13. Pozzan, T., Magalhaes, P. and Rizzuto, R. (2000). The comeback of mitochondria to calcium signaling. Cell. Calcium 28, 279–283.

    Article  CAS  PubMed  Google Scholar 

  14. Nicotera, P., Orrenius, S., Nilsson, T. and Berggren, P. O. (1990). An inositol 1,4,5 trisphosphate-sensitive Ca2+ pool in liver nuclei. Proc. Natl. Acad. Sci. USA 87, 6858–6862.

    Article  CAS  PubMed  Google Scholar 

  15. Malviya, A. N., Rogue, P. and Vincendon, G. (1990). Stereospecific inositol 1,4,5[32P] trisphosphate binding to isolated rat liver nuclei: evidence for inositol trisphosphate receptor-mediated calcium release from the nucleus. Proc. Natl. Acad. Sci. USA 87, 9270–9274.

    Article  CAS  PubMed  Google Scholar 

  16. Gerasimenko, O. V., Gerasimenko, J. V., Tepikin, A. V. and Petersen, O. H. (1995). ATP dependent accumulation and inositol trisphosphate- or cyclic ADP-ribose-mediated release of Ca2+ from the nuclear envelope. Cell 80, 439–444.

    Article  CAS  PubMed  Google Scholar 

  17. Gerasimenko, O. V., Gerasimenko, J. V., Tepikin, A. V. and Petersen, O. H. (1996a). Calcium transport pathways in the nucleus. Pflugers Arch. 432, 1–6.

    Article  CAS  PubMed  Google Scholar 

  18. Gerasimenko, J. V., Maruyama, Y., Yano, K., Dolman, N. J., Tepikin, A. V., Petersen, O. H. and Gerasimenko, O. V. (2003). NAADP mobilizes Ca2+ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors. J. Cell. Biol. 163, 271–282.

    Article  CAS  PubMed  Google Scholar 

  19. Gerasimenko, O. V. and Gerasimenko, J. V. (2004), New aspects of nuclear calcium signalling. J. Cell. Sci. 117, 3087–3094.

    Article  CAS  PubMed  Google Scholar 

  20. Pinton, P., Pozzan, T. and Rizzuto, R. (1998). The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, with functional properties distinct from those of the endoplasmic reticulum. EMBO J. 17, 5298–5308.

    Article  CAS  PubMed  Google Scholar 

  21. Missiaen, L., Van Acker, K., Van Baelen, K., Raeymaekers, L., Wuytack, F., Parys, J. B., De Smedt, H., Vanoevelen, J., Dode, L., Rizzuto, R. and Callewaert, G. (2004). Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments. Cell. Calcium 36, 479–487.

    Article  CAS  PubMed  Google Scholar 

  22. Dolman, N. J., Gerasimenko, J. V., Gerasimenko, O. V., Voronina, S. G., Petersen, O. H. and Tepikin, A. V. (2005). Stable Golgi-mitochondria complexes and formation of Golgi Ca2+ gradients in pancreatic acinar cells. J. Biol. Chem., 280, 15794–15799.

    Article  CAS  PubMed  Google Scholar 

  23. Fasolato, C., Zottini, M., Clementi, E., Zacchetti, D., Meldolesi, J. and Pozzan, T. (1991). Intracellular Ca2+ pools in PC12 cells. Three intracellular pools are distinguished by their turnover and mechanisms of Ca2+ accumulation, storage, and release. J. Biol. Chem. 266, 20159–20167.

    CAS  PubMed  Google Scholar 

  24. Gerasimenko, O. V., Gerasimenko, J. V., Belan, P. V. and Petersen, O. H. (1996b). Inositol trisphosphate and cyclic ADP-ribose-mediated release of Ca2+ from single isolated pancreatic zymogen granules. Cell 84, 473–480.

    Article  CAS  PubMed  Google Scholar 

  25. Nguyen, T., Chin, W. C., and Verdugo, P. (1998). Role of Ca2+/K+ ion exchange in intracellular storage and release of Ca2+. Nature 395, 908–912.

    Article  CAS  PubMed  Google Scholar 

  26. Quesada, I., Chin, W. C., Steed, J., Campos-Bedolla, P., and Verdugo, P. (2001). Mouse mast cell secretory granules can function as intracellular ionic oscillators. Biophys. J. 80, 2133–2139.

    Article  CAS  PubMed  Google Scholar 

  27. Gerasimenko, J. V., Tepikin, A. V., Petersen, O. H. and Gerasimenko, O. V. (1998). Calcium uptake via endocytosis with rapid release from acidifying endosomes. Curr. Biol. 8, 1335–1338.

    Article  CAS  PubMed  Google Scholar 

  28. Petersen, O. H., Gerasimenko, O. V., Gerasimenko, J. V., Mogami, H. and Tepikin, A. V. (1998). The calcium store in the nuclear envelope. Cell. Calcium 23, 87–90.

    Article  CAS  PubMed  Google Scholar 

  29. Gerasimenko, O. V., Gerasimenko, J. V., Rizzuto, R. R., Treiman, M., Tepikin, A. V. and Petersen, O. H. (2002). The distribution of the endoplasmic reticulum in living pancreatic acinar cells. Cell. Calcium 32, 261–268.

    Article  CAS  PubMed  Google Scholar 

  30. Thorn, P., Lawrie, A. M., Smith, P., Gallacher, D. V. and Petersen, O. H. (1993). Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate. Cell 74, 661–668.

    Article  CAS  PubMed  Google Scholar 

  31. Thorn, P., Gerasimenko, O. and Petersen, O. H. (1994). Cyclic ADP-ribose regulation of ryanodine receptors involved in agonist evoked cytosolic Ca2+ oscillations in pancreatic acinar cells. EMBO J. 13, 2038–2043.

    CAS  PubMed  Google Scholar 

  32. Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N. and Clapper, D. L. (1989). Structural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activity. J. Biol. Chem. 264, 1608–1615.

    CAS  PubMed  Google Scholar 

  33. Chini, E. N., Beers, K. W. and Dousa, T. P. (1995). Nicotinate adenine dinucleotide phosphate (NAADP) triggers a specific calcium release system in sea urchin eggs. J. Biol. Chem. 270, 3216–3223.

    Article  CAS  PubMed  Google Scholar 

  34. Cancela, J. M., Churchill, G. C. and Galione, A. (1999). Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells. Nature 398, 74–76.

    Article  CAS  PubMed  Google Scholar 

  35. Cancela, J. M., Gerasimenko, O. V., Gerasimenko, J. V., Tepikin, A. V. and Petersen, O. H. (2000). Two different but converging messenger pathways to intracellular Ca2+ release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate. EMBO J. 19, 2549–2557.

    Article  CAS  PubMed  Google Scholar 

  36. Hohenegger, M., Suko, J., Gscheidlinger, R., Drobny, H. and Zidar, A. (2002). Nicotinic acid-adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor. Biochem. J. 367, 423–431.

    Article  CAS  PubMed  Google Scholar 

  37. Galione, A., and Petersen, O. H. (2005) The NAADP receptor: new receptors or new regulation? Mol. Interv. 5, 73–79.

    Article  CAS  PubMed  Google Scholar 

  38. Dammermann, W. and Guse, A. H. (2005). Functional ryanodine receptor expression is required for NAADP-mediated local Ca2+ signaling in T-lymphocytes. J. Biol. Chem. 280, 21394–21399.

    Article  CAS  PubMed  Google Scholar 

  39. Malavasi, F., Deaglio, S., Funaro, A., Ferrero, E., Horenstein, A. L., Ortolan, E., Vaisitti, T., Aydin, S. (2008) Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology. Physiol. Rev. 88(3):841–886.

    Article  CAS  PubMed  Google Scholar 

  40. Piston, D. W. (1999). Imaging living cells and tissues by two-photon excitation microscopy. Trends. Cell. Biol. 9, 66–69.

    Article  CAS  PubMed  Google Scholar 

  41. Tirlapur, U. K. and Konig, K. (2002). Targeted transfection by femtosecond laser. Nature 418, 290–291.

    Article  CAS  PubMed  Google Scholar 

  42. Gerasimenko, J. V., Sherwood, M., Tepikin, A. V., Petersen, O. H., Gerasimenko, O. V. (2006a). NAADP, cADPR and IP3 all release Ca2+ from the endoplasmic reticulum and an acidic store in the secretory granule area. J. Cell. Sci. 119(Pt 2):226–238.

    Article  CAS  PubMed  Google Scholar 

  43. Gerasimenko, J. V., Flowerdew, S. E., Voronina, S. G., Sukhomlin, T. K., Tepikin, A. V., Petersen, O. H., Gerasimenko, OV. (2006b). Bile acids induce Ca2+ release from both the endoplasmic reticulum and acidic intracellular calcium stores through activation of inositol trisphosphate receptors and ryanodine receptors. J. Biol. Chem. 281(52): 40154–40163.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Physiology, Biomedical School, University of Liverpool, Liverpool, UK

    Oleg Gerasimenko & Julia Gerasimenko

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  1. Oleg Gerasimenko
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  2. Julia Gerasimenko
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Editors and Affiliations

  1. Dept. Biochemistry, University College Cork, College Road, Cork, Ireland

    Dmitri B. Papkovsky

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Gerasimenko, O., Gerasimenko, J. (2010). Two-Photon Permeabilization and Calcium Measurements in Cellular Organelles. 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_12

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  • DOI: https://doi.org/10.1007/978-1-60761-404-3_12

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-403-6

  • Online ISBN: 978-1-60761-404-3

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