Abstract
Phagocytosis and phagosome maturation are central to the development of the innate and adaptive immune response. Phagosome maturation is a continuous and dynamic process that occurs rapidly. In this chapter, we describe fluorescence-based live cell imaging methods for the quantitative and temporal analysis of phagosome maturation of latex beads and M. tuberculosis as two phagocytic targets. We also describe two simple protocols for monitoring phagosome maturation: the use of the acidotropic probe LysoTracker and analyzing the recruitment of EGFP-tagged host proteins by phagosomes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fairn GD, Grinstein S (2012) How nascent phagosomes mature to become phagolysosomes. Trends Immunol 33:397–405
Flannagan RS, Jaumouille V, Grinstein S (2012) The cell biology of phagocytosis. Annu Rev Pathol 7:61–98
Haas A (2007) The phagosome: compartment with a license to kill. Traffic 8:311–330
Jutras I, Desjardins M (2005) Phagocytosis: at the crossroads of innate and adaptive immunity. Annu Rev Cell Dev Biol 21:511–527
Flannagan RS, Cosio G, Grinstein S (2009) Antimicrobial mechanisms of phagocytes and bacterial evasion strategies. Nat Rev Microbiol 7:355–366
Kasmapour B, Gronow A, Bleck CK, Hong W, Gutierrez MG (2012) Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34. Proc Natl Acad Sci U S A 109:20485–20490
Stuart LM, Ezekowitz RA (2005) Phagocytosis: elegant complexity. Immunity 22:539–550
Hoffmann E, Marion S, Mishra BB, John M, Kratzke R, Ahmad SF, Holzer D, Anand PK, Weiss DG, Griffiths G et al (2010) Initial receptor-ligand interactions modulate gene expression and phagosomal properties during both early and late stages of phagocytosis. Eur J Cell Biol 89:693–704
Lu N, Zhou Z (2012) Membrane trafficking and phagosome maturation during the clearance of apoptotic cells. Int Rev Cell Mol Biol 293:269–309
Colas C, Menezes S, Gutierrez-Martinez E, Pean CB, Dionne MS, Guermonprez P (2014) An improved flow cytometry assay to monitor phagosome acidification. J Immunol Methods 412:1–13
Rogers LD, Foster LJ (2007) The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum. Proc Natl Acad Sci U S A 104:18520–18525
Trost M, English L, Lemieux S, Courcelles M, Desjardins M, Thibault P (2009) The phagosomal proteome in interferon-gamma-activated macrophages. Immunity 30:143–154
de Souza Carvalho C, Kasmapour B, Gronow A, Rohde M, Rabinovitch M, Gutierrez MG (2011) Internalization, phagolysosomal biogenesis and killing of mycobacteria in enucleated epithelial cells. Cell Microbiol 13:1234–1249
Bronietzki M, Kasmapour B, Gutierrez MG (2014) Study of phagolysosome biogenesis in live macrophages. J Vis Exp 10:85
Pei G, Repnik U, Griffiths G, Gutierrez MG (2014) Identification of an immune-regulated phagosomal Rab cascade in macrophages. J Cell Sci 127:2071–2082
Zhang X, Edwards JP, Mosser DM (2009) The expression of exogenous genes in macrophages: obstacles and opportunities. Methods Mol Biol 531:123–143
Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V (2004) Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119:753–766
Acknowledgments
We thank the Host–Pathogen Interactions in Tuberculosis Laboratory for useful discussions and comments on the manuscript. This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001092), the UK Medical Research Council (FC001092), the Wellcome Trust (FC001092) and by the UK Medical Research Council (MC_UP_12012/11) to MGG.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Schnettger, L., Gutierrez, M.G. (2017). Quantitative Spatiotemporal Analysis of Phagosome Maturation in Live Cells. In: Botelho, R. (eds) Phagocytosis and Phagosomes. Methods in Molecular Biology, vol 1519. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6581-6_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6581-6_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6579-3
Online ISBN: 978-1-4939-6581-6
eBook Packages: Springer Protocols