Mast Cells pp 347-363 | Cite as

Real-Time Imaging of Ca2+ Mobilization and Degranulation in Mast Cells

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1220)

Abstract

Mast cells play a key role in allergy and inflammation processes as part of the immune response. The activation of mast cells via antigen binding and cross-linking of IgE receptors initiates the onset of dramatic calcium (Ca2+) mobilization dynamics that promote the release of mediators of inflammation and allergy. Ca2+ signaling in mast cells has been studied extensively using a variety of research tools and techniques. In these studies, a large number of proteins have been identified to participate in various stages of these processes.

Here we describe single-cell imaging as an important approach for examining Ca2+ signaling and exocytosis in mast cells. Single-cell imaging tools have advanced significantly over the last 10 years, in part due to improvements in microscope technology and in part due to the development of a new generation of Ca2+ indicators and genetically encoded Ca2+ sensors. The single-cell imaging techniques described here provide the spatial and temporal resolution required to decipher the signaling events that are critical for mast cell functions.

Key words

Live-cell imaging Calcium (Ca2+) signaling Calcium dynamics Mast cell degranulation 

References

  1. 1.
    Ma HT, Beaven MA (2009) Regulation of Ca2+ signaling with particular focus on mast cells. Crit Rev Immunol 29(2):155–186PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Holowka D, Calloway N, Cohen R, Gadi D, Lee J, Smith NL, Baird B (2012) Roles for ca(2+) mobilization and its regulation in mast cell functions. Front Immun 3:104. doi: 10.3389/fimmu.2012.00104 CrossRefGoogle Scholar
  3. 3.
    Cohen R, Torres A, Ma HT, Holowka D, Baird B (2009) Ca2+ waves initiate antigen-stimulated Ca2+ responses in mast cells. J Immunol 183(10): 6478–6488. doi:10.4049/jimmunol.0901615 PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260(6):3440–3450PubMedGoogle Scholar
  5. 5.
    Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388(6645):882–887. doi:10.1038/42264 PubMedCrossRefGoogle Scholar
  6. 6.
    Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA, Schreiter ER, Bargmann CI, Jayaraman V, Svoboda K, Looger LL (2009) Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods 6(12):875–881. doi:10.1038/nmeth.1398 PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Zhao Y, Araki S, Wu J, Teramoto T, Chang YF, Nakano M, Abdelfattah AS, Fujiwara M, Ishihara T, Nagai T, Campbell RE (2011) An expanded palette of genetically encoded Ca(2)(+) indicators. Science 333(6051):1888–1891. doi:10.1126/science.1208592 PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    McCombs JE, Palmer AE (2008) Measuring calcium dynamics in living cells with genetically encodable calcium indicators. Methods 46(3): 152–159. doi:10.1016/j.ymeth.2008.09.015 PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Kim TD, Eddlestone GT, Mahmoud SF, Kuchtey J, Fewtrell C (1997) Correlating Ca2+ responses and secretion in individual RBL-2H3 mucosal mast cells. J Biol Chem 272(50): 31225–31229PubMedCrossRefGoogle Scholar
  10. 10.
    Crivellato E, Baldini G, Basa M, Fusaroli P (1993) The three-dimensional structure of interdigitating cells. Ital J Anat Embryol 98(4): 243–258PubMedGoogle Scholar
  11. 11.
    Demitsu T, Kiyosawa T, Kakurai M, Murata S, Yaoita H (1999) Local injection of recombinant human stem cell factor promotes human skin mast cell survival and neurofibroma cell proliferation in the transplanted neurofibroma in nude mice. Arch Dermatol Res 291(6): 318–324PubMedCrossRefGoogle Scholar
  12. 12.
    Metcalfe DD, Baram D, Mekori YA (1997) Mast cells. Physiol Rev 77(4):1033–1079PubMedGoogle Scholar
  13. 13.
    Blank U (2011) The mechanisms of exocytosis in mast cells. Adv Exp Med Biol 716:107–122. doi:10.1007/978-1-4419-9533-9_7 PubMedCrossRefGoogle Scholar
  14. 14.
    Chapman, E. R. (2008). “How does synaptotagmin trigger neurotransmitter release?” Annual review of biochemistry 77:615–641PubMedCrossRefGoogle Scholar
  15. 15.
    Naal RM, Tabb J, Holowka D, Baird B (2004) In situ measurement of degranulation as a biosensor based on RBL-2H3 mast cells. Biosens Bioelectron 20(4):791–796. doi:10.1016/j.bios.2004.03.017 PubMedCrossRefGoogle Scholar
  16. 16.
    Gadi D, Wagenknecht-Wiesner A, Holowka D, Baird B (2011) Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca(2+) mobilization and degranulation in mast cells. Mol Biol Cell 22(24): 4908–4917. doi:10.1091/mbc.E11-07-0614 PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Hohman RJ, Dreskin SC (2001) Measuring degranulation of mast cells. In: John E. Coligan et al (eds). Current protocols in immunology. Chapter 7:Unit 7;26. doi:10.1002/0471142735.im0726s08
  18. 18.
    Kawasaki Y, Saitoh T, Okabe T, Kumakura K, Ohara-Imaizumi M (1991) Visualization of exocytotic secretory processes of mast cells by fluorescence techniques. Biochim Biophys Acta 1067(1):71–80PubMedCrossRefGoogle Scholar
  19. 19.
    Williams RM, Webb WW (2000) Single granule pH cycling in antigen-induced mast cell secretion. J Cell Sci 113(Pt 21):3839–3850PubMedGoogle Scholar
  20. 20.
    Jaiswal JK, Fix M, Takano T, Nedergaard M, Simon SM (2007) Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes. Proc Natl Acad Sci U S A 104(35):14151–14156. doi:10.1073/pnas.0704935104 PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Williams RM, Shear JB, Zipfel WR, Maiti S, Webb WW (1999) Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence. Biophys J 76(4):1835–1846. doi:10.1016/S0006-3495(99)77343-1 PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Barsumian EL, Isersky C, Petrino MG, Siraganian RP (1981) IgE-induced histamine release from rat basophilic leukemia cell lines: isolation of releasing and nonreleasing clones. Eur J Immunol 11(4):317–323. doi:10.1002/eji.1830110410 PubMedCrossRefGoogle Scholar
  23. 23.
    Posner RG, Lee B, Conrad DH, Holowka D, Baird B, Goldstein B (1992) Aggregation of IgE-receptor complexes on rat basophilic leukemia cells does not change the intrinsic affinity but can alter the kinetics of the ligand-IgE interaction. Biochemistry 31(23): 5350–5356PubMedCrossRefGoogle Scholar
  24. 24.
    Collins TJ (2007) ImageJ for microscopy. Biotechniques 43(1 Suppl):25–30PubMedCrossRefGoogle Scholar
  25. 25.
    Bonifacino JS, Yuan L, Sandoval IV (1989) Internalization and recycling to serotonin-containing granules of the 80K integral membrane protein exposed on the surface of secreting rat basophilic leukaemia cells. J Cell Sci 92(Pt 4):701–712PubMedGoogle Scholar
  26. 26.
    Cohen R, Corwith K, Holowka D, Baird B (2012) Spatiotemporal resolution of mast cell granule exocytosis reveals correlation with Ca2+ wave initiation. J Cell Sci 125(Pt 12): 2986–2994. doi:10.1242/jcs.102632 PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Roy Cohen
    • 1
  • David A. Holowka
    • 2
  • Barbara A. Baird
    • 2
  1. 1.Baker Institute for Animal HealthCornell University College of Veterinary MedicineIthacaUSA
  2. 2.Department of Chemistry and Chemical BiologyCornell UniversityIthacaUSA

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