Use of granzyme B-based fluorescent protein reporters to monitor granzyme distribution and granule integrity in live cells

Catherina Hedwig Bird, Alexandra Rizzitelli, Ian Harper, Mark Prescott, Phillip Ian Bird

Research output: Contribution to journalArticleResearchpeer-review

7 Citations (Scopus)

Abstract

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 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.
Original languageEnglish
Pages (from-to)999 - 1004
Number of pages6
JournalBiological Chemistry
Volume391
Issue number8
DOIs
Publication statusPublished - 2010

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