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- Title
Use of granzyme B-based fluorescent protein reporters to monitor granzyme distribution and granule integrity in live cells.
- Authors
Bird, Catherina H.; Rizzitelli, Alexandra; Harper, Ian; Prescott, Mark; Bird, Phillip I.
- Abstract
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.
- Subjects
PROTEINS; LYSOSOMES; KILLER cells; BIOSYNTHESIS; HYDROGEN peroxide; NAPHTHAZARIN; SPHINGOSINE
- Publication
Biological Chemistry, 2010, Vol 391, Issue 8, p999
- ISSN
1431-6730
- Publication type
Article
- DOI
10.1515/BC.2010.099