Abstract

Cellular reporters of enzyme activity are based on either fluorescent proteins or small molecules. Such reporters provide information corresponding to wherever inside cells the enzyme is maximally active and preclude minor populations present in subcellular compartments. Here we describe a chemical imaging strategy to selectively interrogate minor, subcellular pools of enzymatic activity. This new technology confines the detection chemistry to a designated organelle, enabling imaging of enzymatic cleavage exclusively within the organelle. We have thus quantitatively mapped disulfide reduction exclusively in endosomes in Caenorhabditis elegans and identified that exchange is mediated by minor populations of the enzymes PDI-3 and TRX-1 resident in endosomes. Impeding intra-endosomal disulfide reduction by knocking down TRX-1 protects nematodes from infection by Corynebacterium diphtheriae, revealing the importance of this minor pool of endosomal TRX-1. TRX-1 also mediates endosomal disulfide reduction in human cells. A range of enzymatic cleavage reactions in organelles are amenable to analysis by this new reporter strategy.

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The data that support the plots within this paper and other finding of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank J. Clardy, J. Kuriyan, S. Modi and A. Lin Chun for valuable comments on this work. The authors thank the Integrated Light Microscopy facility at the University of Chicago, the Caenorhabditis Genetic Center (CGC), J. Fares and M. Edgley for C. elegans strains, M. Glotzer and F. M. Ausubel for RNAi clones and valuable discussions, S. Crosson at the BSL facility for C. diphtheriae work and C. Cui for BMDMs and flow cytometry. This work was supported by the University of Chicago Women’s Board, Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust, C-084 as well as a CBC post-doctoral research grant (PDR-073), Pilot and Feasibility award from an NIDDK Center grant P30DK42086 to the University of Chicago Digestive Diseases Research Core Center and University of Chicago start-up funds to Y.K. Y.K. is a Brain Research Foundation Fellow.

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  1. Department of Chemistry, University of Chicago, Chicago, IL, USA

    • Krishna Dan
    • , Aneesh T. Veetil
    • , Kasturi Chakraborty
    •  & Yamuna Krishnan
  2. Grossman Institute of Neuroscience, Quantitative Biology and Human Behaviour, University of Chicago, Chicago, IL, USA

    • Krishna Dan
    • , Aneesh T. Veetil
    • , Kasturi Chakraborty
    •  & Yamuna Krishnan

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Contributions

K.D. and Y.K. designed the project. K.D. developed tripartite TDX reporters and performed all experiments related to TDX reporter in C. elegans and mammalian cells. A.T.V. prepared the dextran encapsulated icosahedron and in vitro experiments related to icosahedron. K.C. contributed to cathepsin-related experiments. K.D., A.T.V., K.C. and Y.K. analysed the data. K.D., A.T.V. and Y.K. wrote the paper. All authors discussed the results and provided input on the manuscript.

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The authors declare no competing interests.

Corresponding author

Correspondence to Yamuna Krishnan.

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https://doi.org/10.1038/s41565-019-0365-6