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Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas

Nature Chemical Biology volume 10, pages 1034–1042 (2014)Cite this article

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An Erratum to this article was published on 17 February 2015

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Abstract

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano–secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu+ accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu+ became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

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Figure 1: Zn deficiency induces CRR1-dependent Cu hyperaccumulation.
Figure 2: Zn-limited cells also express Cu deficiency markers.
Figure 3: Cu+-sensitive CS3 staining suggests Cu accumulation in intracellular compartments.
Figure 4: Intracellular Cu is traceable to Cu-accumulating compartments.
Figure 5: Changes in abundance of electron-dense bodies and Cu redistribution upon Zn resupply.
Figure 6: Transcriptome response to Zn resupply monitored by RNA-seq analysis.

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  • 06 January 2015

    In the version of this article initially published, the labels in Figure 4b were incorrect. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work is supported, in part, by grants from the US National Institutes of Health (NIH; GM42143 and GM092473 to S.S.M., DK068139 to T.L.S. and GM079465 to C.J.C.), the United States Department of Energy Cooperative Agreement (DE-FC02-02ER63421 to D. Eisenberg for support of J.A.L.) and the German Academic Exchange Service DAAD (D0847579 to A.H.-H. and D1242134 to M.M.). Work at Lawrence Livermore National Laboratory (LLNL) was performed under the auspices of the US Department of Energy at LLNL under contract DE-AC52-07NA27344, with funding provided by the US Department of Energy Genomic Science Program under contract SCW1039. Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL). SSRL is a national user facility operated by Stanford University, and the SSRL Structural Molecular Biology Program is supported by the Department of Energy–Office of Biological and Environmental Research and by the NIH–National Center for Research Resources Biomedical Technology Program. D.B. is supported by the NIH (T32HL120822), and C.J.C. is an investigator with the Howard Hughes Medical Institute. Electron microscopy was performed at the Electron Microscopy Services Center of the University of California–Los Angeles Brain Research Institute. We thank A. Aron and K.M. Ramos-Torres for their help with resynthesis and optical spectroscopy of fresh CS3 and Ctrl-CS3 for control experiments.

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  1. Anne Hong-Hermesdorf and Marcus Miethke: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California–Los Angeles, Los Angeles, California, USA

    Anne Hong-Hermesdorf, Marcus Miethke, Sean D Gallaher, Janette Kropat, Joseph A Loo & Sabeeha S Merchant

  2. Department of Chemistry, University of California–Berkeley, Berkeley, California, USA

    Sheel C Dodani, Jefferson Chan, Dylan W Domaille & Christopher J Chang

  3. Howard Hughes Medical Institute, University of California–Berkeley, Berkeley, California, USA

    Sheel C Dodani, Jefferson Chan, Dylan W Domaille & Christopher J Chang

  4. Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan, USA

    Dulmini Barupala & Timothy L Stemmler

  5. Department of Biological Chemistry, University of California–Los Angeles, Los Angeles, California, USA

    Dyna I Shirasaki & Joseph A Loo

  6. Institute for Genomics and Proteomics, University of California–Los Angeles, Los Angeles, USA

    Joseph A Loo & Sabeeha S Merchant

  7. Chemical Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA

    Peter K Weber & Jennifer Pett-Ridge

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Contributions

S.S.M., A.H.-H. and M.M. designed experiments. A.H.-H., M.M. and J.K. cultured cells and supplied samples for NanoSIMS, X-ray absorption spectroscopy (XAS) and RNA-seq. M.M. and J.K. measured cellular metal contents by ICP-MS. A.H.-H. performed immunoblotting and qRT-PCR for expression analysis. A.H.-H. and M.M. imaged cells by confocal and electron microscopy and analyzed the resulting data. J.P.-R., P.K.W. and M.M. analyzed intracellular metal distribution by NanoSIMS, and D.B. and T.L.S. collected and analyzed XAS data. M.M. isolated Cu-containing compartments and did the Cu isotope labeling experiments in conjunction with LC-ICP-MS analysis. D.I.S. and M.M. performed quantitative MS of protein fractions under the supervision of J.A.L. S.D.G. prepared RNA-seq libraries and analyzed the resulting data. S.C.D., D.W.D. and J.C. synthesized the Cu+-sensitive CS3 dye (and control) under the supervision of C.J.C., M.M. and S.S.M., and A.H.-H. wrote the manuscript with input from J.C. and P.K.W.

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Correspondence to Sabeeha S Merchant.

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Hong-Hermesdorf, A., Miethke, M., Gallaher, S. et al. Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas. Nat Chem Biol 10, 1034–1042 (2014). https://doi.org/10.1038/nchembio.1662

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