Abstract
The hallmark of mast cell activation is secretion of immune mediators by regulated exocytosis. Measurements of mediator secretion from mast cells that are genetically manipulated by transient transfections provide a powerful tool for deciphering the underlying mechanisms of mast cell exocytosis. However, common methods to study regulated exocytosis in bulk culture of mast cells suffer from the drawback of high signal-to-noise ratio because of their failure to distinguish between the different mast cell populations, that is, genetically modified mast cells versus their non-transfected counterparts. In particular, the low transfection efficiency of mast cells poses a significant limitation on the use of conventional methodologies. To overcome this hurdle, we developed a method, which discriminates and allows detection of regulated exocytosis of transfected cells based on the secretion of a fluorescent secretory reporter. We used a plasmid encoding for Neuropeptide Y (NPY) fused to a monomeric red fluorescent protein (NPY-mRFP), yielding a fluorescent secretory granule-targeted reporter that is co-transfected with a plasmid encoding a gene of interest. Upon cell trigger, NPY-mRFP is released from the cells by regulated exocytosis, alongside the endogenous mediators. Therefore, using NPY-mRFP as a reporter for mast cell exocytosis allows either quantitative, via a fluorimeter assay, or qualitative analysis, via confocal microscopy, of the genetically manipulated mast cells. Moreover, this method may be easily modified to accommodate studies of regulated exocytosis in any other type of cell.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dahlin JS, Hallgren J (2015) Mast cell progenitors: origin, development and migration to tissues. Mol Immunol 63:9–17. https://doi.org/10.1016/j.molimm.2014.01.018
Klein O, Sagi-Eisenberg R (2019) Anaphylactic degranulation of mast cells: focus on compound exocytosis. J Immunol Res 2019:1–12. https://doi.org/10.1155/2019/9542656
Wernersson S, Pejler G (2014) Mast cell secretory granules: armed for battle. Nat Rev Immunol 14:478–494. https://doi.org/10.1038/nri3690
Johansson SGO, Bieber T, Dahl R et al (2004) Revised nomenclature for allergy for global use: report of the nomenclature review Committee of the World Allergy Organization, October 2003. J Allergy Clin Immunol 113:832–836. https://doi.org/10.1016/j.jaci.2003.12.591
Rådinger M, Jensen BM, Swindle E, Gilfillan AM (2015) Assay of mast cell mediators. In: Methods in molecular biology. Humana Press, New Jersey, pp 307–323
Cruse G, Gilfillan AM, Smrz D (2015) Flow cytometry-based monitoring of mast cell activation. In: Methods in molecular biology. Humana Press, New Jersey, pp 365–379
Johnson DA (2006) Human mast cell proteases: activity assays using thiobenzyl ester substrates. In: Mast cells. Humana Press, New Jersey, pp 193–202
Chi DS, Fitzgerald SM, Krishnaswamy G (2006) Mast cell histamine and cytokine assays. In: Mast cells. Humana Press, New Jersey, pp 203–216
Jensen BM, Falkencrone S, Skov PS (2014) Measuring histamine and cytokine release from basophils and mast cells. In: Methods in molecular biology. Humana Press, New Jersey, pp 135–145
Hohman RJ, Dreskin SC (2001) Measuring degranulation of mast cells. In: Current protocols in immunology. John Wiley & Sons, Inc., Hoboken, NJ, USA, p Unit 7.26
Kuehn HS, Radinger M, Gilfillan AM (2010) Measuring mast cell mediator release. In: Current protocols in immunology. John Wiley & Sons, Inc., Hoboken, NJ, USA, p Unit7.38
Demo SD, Masuda E, Rossi AB et al (1999) Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay. Cytometry 36:340–348. https://doi.org/10.1002/(SICI)1097-0320(19990801)36:4<340::AID-CYTO9>3.0.CO;2-C
Naal RMZG, Tabb J, Holowka D, Baird B (2004) In situ measurement of degranulation as a biosensor based on RBL-2H3 mast cells. Biosens Bioelectron 20:791–796. https://doi.org/10.1016/J.BIOS.2004.03.017
Gadi D, Wagenknecht-Wiesner A, Holowka D, Baird B (2011) Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca2+ mobilization and degranulation in mast cells. Mol Biol Cell 22:4908–4917. https://doi.org/10.1091/mbc.e11-07-0614
Cohen R, Holowka DA, Baird BA (2015) Real-time imaging of Ca(2+) mobilization and degranulation in mast cells. In: Methods in molecular biology. Humana Press, New Jersey, pp 347–363. https://doi.org/10.1007/978-1-4939-1568-2_22
Gaudenzio N, Sibilano R, Marichal T et al (2016) Different activation signals induce distinct mast cell degranulation strategies. J Clin Invest 126:3981–3998. https://doi.org/10.1172/JCI85538
Klein O, Roded A, Hirschberg K et al (2018) Imaging FITC-dextran as a reporter for regulated exocytosis. J Vis Exp 136:57936. https://doi.org/10.3791/57936
Williams RM, Webb WW (2000) Single granule pH cycling in antigen-induced mast cell secretion. J Cell Sci 113(Pt 21):3839–3850
Williams RM, Shear JB, Zipfel WR et al (1999) Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence. Biophys J 76:1835–1846. https://doi.org/10.1016/S0006-3495(99)77343-1
Kawasaki Y, Saitoh T, Okabe T et al (1991) Visualization of exocytotic secretory processes of mast cells by fluorescence techniques. Biochim Biophys Acta Biomembr 1067:71–80. https://doi.org/10.1016/0005-2736(91)90027-6
Balseiro-Gomez S, Flores JA, Acosta J et al (2016) Transient fusion ensures granule replenishment to maintain repeated release after IgE-mediated mast cell degranulation. J Cell Sci 129:3989–4000. https://doi.org/10.1242/jcs.194340
Tharp MD, Seelig LL, Tigelaar RE, Bergstresser PR (1985) Conjugated avidin binds to mast cell granules. J Histochem Cytochem 33:27–32. https://doi.org/10.1177/33.1.2578142
Wilson JD, Shelby SA, Holowka D, Baird B (2016) Rab11 regulates the mast cell exocytic response. Traffic 17:1027–1041. https://doi.org/10.1111/tra.12418
Azouz NP, Matsui T, Fukuda M, Sagi-Eisenberg R (2012) Decoding the regulation of mast cell exocytosis by networks of Rab GTPases. J Immunol 189:2169–2180. https://doi.org/10.4049/jimmunol.1200542
Blott EJ, Griffiths GM (2002) Secretory lysosomes. Nat Rev Mol Cell Biol 3:122–131. https://doi.org/10.1038/nrm732
Makhmutova M, Liang T, Gaisano H et al (2017) Confocal imaging of neuropeptide Y-pHluorin: a technique to visualize insulin granule exocytosis in intact murine and human islets. J Vis Exp:e56089. https://doi.org/10.3791/56089
Almaça J, Liang T, Gaisano HY et al (2015) Spatial and temporal coordination of insulin granule exocytosis in intact human pancreatic islets. Diabetologia 58:2810–2818. https://doi.org/10.1007/s00125-015-3747-9
Dominguez N, van Weering JRT, Borges R et al (2017) Dense-core vesicle biogenesis and exocytosis in neurons lacking chromogranins A and B. J Neurochem 144:241–254. https://doi.org/10.1111/jnc.14263
Burrone J, Li Z, Murthy VN (2007) Studying vesicle cycling in presynaptic terminals using the genetically encoded probe synaptopHluorin. Nat Protoc 1:2970–2978. https://doi.org/10.1038/nprot.2006.449
Jacobs DT, Weigert R, Grode KD et al (2009) Myosin Vc is a molecular motor that functions in secretory granule trafficking. Mol Biol Cell 20:4471–4488. https://doi.org/10.1091/mbc.e08-08-0865
Tan CMJ, Green P, Tapoulal N et al (2018) The role of neuropeptide Y in cardiovascular health and disease. Front Physiol 9:1281. https://doi.org/10.3389/fphys.2018.01281
Redegeld FA, Yu Y, Kumari S et al (2018) Non-IgE mediated mast cell activation. Immunol Rev 282:87–113. https://doi.org/10.1111/imr.12629
Campbell RE, Tour O, Palmer AE et al (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci 99:7877–7882. https://doi.org/10.1073/pnas.082243699
Falcone FH, Wan D, Barwary N, Sagi-Eisenberg R (2018) RBL cells as models for in vitro studies of mast cells and basophils. Immunol Rev 282:47–57. https://doi.org/10.1111/imr.12628
Klein O, Krier-Burris RA, Lazki-Hagenbach P et al (2019) Mammalian diaphanous-related formin 1 (mDia1) coordinates mast cell migration and secretion through its actin-nucleating activity. J Allergy Clin Immunol 144(4):1074–1090. https://doi.org/10.1016/j.jaci.2019.06.028
Ljubicic S, Bezzi P, Brajkovic S et al (2013) The GTPase rab37 participates in the control of insulin exocytosis. PLoS One 8:e68255. https://doi.org/10.1371/journal.pone.0068255
Golzio M, Mora MP, Raynaud C et al (1998) Control by osmotic pressure of voltage-induced permeabilization and gene transfer in mammalian cells. Biophys J 74:3015–3022. https://doi.org/10.1016/S0006-3495(98)78009-9
Luft C, Ketteler R (2015) Electroporation knows no boundaries: the use of electrostimulation for siRNA delivery in cells and tissues. J Biomol Screen 20:932–942. https://doi.org/10.1177/1087057115579638
Martin TFJ (2003) Tuning exocytosis for speed: fast and slow modes. Biochim Biophys Acta, Mol Cell Res 1641:157–165. https://doi.org/10.1016/S0167-4889(03)00093-4
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Klein, O., Azouz, N.P., Sagi-Eisenberg, R. (2021). Measurement of Exocytosis in Genetically Manipulated Mast Cells. In: Niedergang, F., Vitale, N., Gasman, S. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 2233. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1044-2_12
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1044-2_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1043-5
Online ISBN: 978-1-0716-1044-2
eBook Packages: Springer Protocols