RRC ID 65132
Author Morita M, Sawaki K, Kinoshita D, Sakurai C, Hori N, Hatsuzawa K.
Title Quantitative analysis of phagosome formation and maturation using an Escherichia coli probe expressing a tandem fluorescent protein.
Journal J Biochem
Abstract Phagosome formation and maturation are essential innate immune mechanisms to engulf and digest foreign particles. To analyze these processes quantitatively, we established a specific Escherichia coli probe expressing a tandem fluorescent protein, comprising glutathione S-transferase fused with monomeric Cherry (mCherry) and monomeric Venus (mVenus). We demonstrated that mVenus was more susceptible to bleaching in an acidic environment than mCherry, and that the mVenus:mCherry fluorescence intensity ratio can be used to monitor phagosomal pH changes during maturation. Using this probe, we revealed that synaptosomal-associated protein of 23 kDa, a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein, actively regulated phagocytosis of E. coli and subsequent phagosome maturation in macrophages. Our results indicated that this probe has the potential to be a powerful tool in understanding the molecular mechanisms of phagosome formation and maturation.
Volume 162(5)
Pages 309-316
Published 2017-11-1
DOI 10.1093/jb/mvx034
PII 3858071
PMID 28575453
MeSH Animals Cell Line Escherichia coli / physiology* Gene Expression Regulation, Bacterial / physiology Gene Expression Regulation, Enzymologic / physiology Glutathione Transferase / genetics Glutathione Transferase / metabolism Hydrogen-Ion Concentration Luminescent Proteins / genetics Luminescent Proteins / metabolism* Mice Phagocytosis / physiology Phagosomes / physiology* Qb-SNARE Proteins / genetics Qb-SNARE Proteins / metabolism Qc-SNARE Proteins / genetics Qc-SNARE Proteins / metabolism
IF 2.476
Human and Animal Cells J774