Use of granzyme B-based fluorescent protein reporters to monitor granzyme distribution and granule integrity in live cells.
Reporter proteins comprising granzyme B (GrB) fused to eGFP, ecliptic pHluorin or mCherry, were generated and used to study granule (lysosome) distribution and properties in COS-1 cells and natural killer cells. The reporters resembled native GrB in biosynthesis and localization, and accumulated in granules. In live cells both the eGFP and pHluorin reporters were dark in lysosomes, but fluoresced when the granule integrity or pH was perturbed by Leu-Leu methyl ester, hydrogen peroxide, naphthazarin, or sphingosine treatment. By contrast, fluorescence of the mCherry reporter was not pH-dependent. The quenching of eGFP within granules indicates that this commonly-used fluorescent protein is not appropriate as a vital intra-lysosomal marker.
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Melbourne, Australia.
Catherina H
CH
Bird
Alexandra
A
Rizzitelli
Ian
I
Harper
Mark
M
Prescott
Phillip I
PI
Bird
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D053804:Granzymes / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007694:Killer Cells, Natural; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000097573:Red Fluorescent Protein
D004792:Enzyme Precursors; D053804:Granzymes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C485187:PHluorin; C485184:enhanced green fluorescent protein; C509213:GZMB protein, human