Abstract
Phagocytosis is a receptor-mediated process whereby professional phagocytes internalize invading pathogens and apoptotic bodies into an intracellular vacuole or phagosome, leading to their degradation. During the formation and maturation of the phagosome, several lipids undergo changes and effector proteins are recruited on the nascent phagosome in a concerted manner. These highly localized, dynamic, and transient processes can only be studied by methods capable of high spatial and temporal resolution. The use of genetically encoded chimeric constructs coupled with fluorescence confocal microscopy enables the continuous, noninvasive analysis of the distribution and metabolism of lipids and effector proteins during phagocytosis. Here, we describe a method where the mouse macrophage cell line, RAW 264.7, and primary macrophages are transiently transfected with fluorescent chimeric probes to analyze and quantify phagocytosis of immunoglobulin-opsonized particles, using confocal microscopy.
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
Hochreiter-Hufford A, Ravichandran KS (2013) Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion. Cold Spring Harb Perspect Biol 5:a008748
Niedergang F, Chavrier P (2004) Signaling and membrane dynamics during phagocytosis: many roads lead to the phagos(R)ome. Curr Opin Cell Biol 16:422–428
Viera OV, Botelho RJ, Grinstein S (2002) Phagosome maturation: aging gracefully. Biochem J 15:689–704
Crowley MT, Costello PS, Fitzer-Attas CJ et al (1997) A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages. J Exp Med 186:1027–1039
Marshall JG, Booth JW, Stambolic V et al (2001) Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis. J Cell Biol 153:1369–1380
Botelho RJ, Teruel M, Dierckman R et al (2000) Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J Cell Biol 151:1353–1368
Vieira OV, Botelho RJ, Rameh L et al (2001) Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation. J Cell Biol 155:19–25
Scott CC, Dobson W, Botelho RJ et al (2005) Phosphatidylinositol-4,5, bisphosphate hydrolysis directs actin remodeling during phagocytosis. J Cell Biol 169:139–149
Ellson CD, Anderson KE, Morgan G et al (2001) Phosphatidylinositol 3-phosphate is generated in phagosomal membranes. Curr Biol 11:1631–1635
Vieira OV, Bucci C, Harrison RE et al (2003) Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase. Mol Cell Biol 23:2501–2514
Swanson JA, Hoppe AD (2004) The coordination of signaling during Fc receptor-mediated phagocytosis. J Leukoc Biol 76:1093–1103
Lee WL, Mason D, Schreiber AD et al (2007) Quantitative analysis of membrane remodeling at the phagocytic cup. Mol Biol Cell 8:2883–2892
Riedl J, Creveenna AH, Kessenbrock K et al (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods 5:605–607
Gillooly DJ, Morrow IC, Lindsay M et al (2000) Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J 19:4577–4588
Raiborg C, Bremnes B, Mehlum A et al (2001) FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes. J Cell Sci 114:2255–2263
Roy A, Levine TP (2004) Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p. J Biol Chem 279:44683–44689
Godi A, Di Campli A, Konstantakopoulos A et al (2004) FAPPs control Golgi-to-cell surface membrane traffic by binding to ARF and PtdIns(4)P. Nat Cell Biol 6:393–404
Hammond GR, Machner MP, Balla T (2014) A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi. J Cell Biol 205:113–126
Stauffer TP, Ahn S, Meyer T (1998) Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells. Curr Biol 8:343–346
Varnai P, Rother K I, Balla T (1999) Phosphatidylinositol 3-kinasedependent membrane association of the Bruton’s tyrosine kinase pleckstrin homology domain visualized in single living cells. J Biol Chem 274:10983-10989
Gu H, Botelho RJ, Yu M et al (2003) Critical role for scaffolding adapter Gab2 in Fc gamma R-mediated phagocytosis. J Cell Biol 161:1151–1161
Servant G, Weiner OD, Herzmark P et al (2000) Polarization of chemoattractant receptor signaling during neutrophil chemotaxis. Science 287:1037–1040
Yeung T, Gilbert GE, Shi J et al (2008) Membrane phosphatidylserine regulates surface charge and protein localization. Science 319:210–213
Chen J, Deng F, Wang QJ (2008) Selective binding of phorbol esters and diacylglycerol by individual C1 domains of the PKD family. Biochem J 411:333–342
Colon-Gonzalez F, Kazanietz MG (2006) C1 domains exposed: from diacylglycerol binding to protein-protein interactions. Biochim Biophys Acta 1761:827–837
Ohno-Iwashita Y, Shimada Y, Waheed AA et al (2004) Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipids rafts. Anaerobe 10:125–134
Acknowledgements
S.M.L. is a recipient of the Alexander Graham Bell Scholarship from the Natural Science and Engineering Research Council of Canada. G.D.F. is a recipient of a New Investigator Award from Canadian Institutes of Health Research (CIHR) and an Early Researcher Award from the Government of Ontario. Original work in the authors’ laboratories is supported by a St. Michael’s Hospital New Investigator Start-up Fund (to G.D.F), and by grants FDN-143202 and MOP-126069, and MOP-133656 awarded by the Canadian Institutes of Health Research (to S.G. and G.D.F., respectively).
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
Lu, S.M., Grinstein, S., Fairn, G.D. (2017). Quantitative Live-Cell Fluorescence Microscopy During Phagocytosis. 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_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6581-6_6
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