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Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

Hong-Hermesdorf A, Miethke M, Gallaher SD, Kropat J, Dodani SC, Chan J, Barupala D, Domaille DW, Shirasaki DI, Loo JA, Weber PK, Pett-Ridge J, Stemmler TL, Chang CJ, Merchant SS - Nat. Chem. Biol. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, USA.

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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Intracellular Cu is traceable to Cu accumulating compartments(a) Transmission electron microscopy (TEM) revealed electron-dense structures in Zn-limited cells (scale bar 2 μm). Manually defined areas of cells and the contained electron-dense structures were measured with Image J. We analyzed the statistical significance of Zn-limited vs Zn-replete cells by Kruskal-Wallis One Way Analysis of Variance on ranks (P = <0.001). Error bar shows SD +/− from three independent experiments. (b) NanoSIMS shows 40Ca+ and 63Cu+ co-localize in Zn-limited cells, coinciding with electron-dense structures in TEM (scale bars 1 μm). (c–d) Relative intracellular Ca (c) and Cu (d) measured during Zn resupply (to −Zn compared to +Zn). Samples from three independent cultures were collected 0, 10, and 24 h after Zn addition, and ten cells per time point were examined by NanoSIMS. Average ion ratio values were plotted based on whole cell area (“cell”) and intracellular areas of Ca and Cu accumulations with their corresponding standard deviations. (e) Ratio of 40Ca+ over 63Cu+ at different time points. All NanoSIMS counts were normalized to 12C+. (f–h) XAS spectra for Cu in a representative Zn-limited C. reinhardtii sample. (f) Cu XANES spectrum with a predominant spectral feature at 8984 eV, which corresponds to a 1s → 4p electronic transition typically seen in centrosymmetric Cu+ samples. (g) Expansion of the Cu pre-edge spectral features (red) offset and compared to the Cu2+SO4 (black) model. (h) Fourier transforms of the raw Cu EXAFS (black) with best fit simulation (green).
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Figure 4: Intracellular Cu is traceable to Cu accumulating compartments(a) Transmission electron microscopy (TEM) revealed electron-dense structures in Zn-limited cells (scale bar 2 μm). Manually defined areas of cells and the contained electron-dense structures were measured with Image J. We analyzed the statistical significance of Zn-limited vs Zn-replete cells by Kruskal-Wallis One Way Analysis of Variance on ranks (P = <0.001). Error bar shows SD +/− from three independent experiments. (b) NanoSIMS shows 40Ca+ and 63Cu+ co-localize in Zn-limited cells, coinciding with electron-dense structures in TEM (scale bars 1 μm). (c–d) Relative intracellular Ca (c) and Cu (d) measured during Zn resupply (to −Zn compared to +Zn). Samples from three independent cultures were collected 0, 10, and 24 h after Zn addition, and ten cells per time point were examined by NanoSIMS. Average ion ratio values were plotted based on whole cell area (“cell”) and intracellular areas of Ca and Cu accumulations with their corresponding standard deviations. (e) Ratio of 40Ca+ over 63Cu+ at different time points. All NanoSIMS counts were normalized to 12C+. (f–h) XAS spectra for Cu in a representative Zn-limited C. reinhardtii sample. (f) Cu XANES spectrum with a predominant spectral feature at 8984 eV, which corresponds to a 1s → 4p electronic transition typically seen in centrosymmetric Cu+ samples. (g) Expansion of the Cu pre-edge spectral features (red) offset and compared to the Cu2+SO4 (black) model. (h) Fourier transforms of the raw Cu EXAFS (black) with best fit simulation (green).

Mentions: With these pilot data suggesting further investigation, we sought to use an unequivocal physical technique to identify the intracellular Cu foci. Direct metal detection techniques such as X-ray fluorescence microscopy have been valuable to support indirect metal imaging obtained using synthetic fluorophores27. In the present study, electron-dense structures were revealed in microtomed thin-sections of fixed cells by transmission electron microscopy (TEM) and these were candidate sites for Cu accumulation. Indeed, the abundance and size of the electron dense structures correlated with Zn nutrition (Figure 4a). Kruskal-Wallis One Way Analysis of Variance on ranks of the electron-dense areas in the electron micrographs in relation to total cell areas found a significant difference between the Zn-limited and Zn-replete sections (p-value P= <0.001) (Figure 4a, right panel). NanoSIMS imaging, which allows simultaneous spatial detection of elements based on their mass, indicated that regions containing calcium (Ca) and Cu correlated with electron-dense structures at the periphery of the cells visible in TEM images of the same sections (Figure 4b). Co-localization of Ca and Cu in Zn-limited cells was confirmed independently by confocal microscopy and dual staining of cells with CS3 and Fluo-4-AM, a Ca2+-specific fluorescent dye (Supplementary Fig. 5).


Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

Hong-Hermesdorf A, Miethke M, Gallaher SD, Kropat J, Dodani SC, Chan J, Barupala D, Domaille DW, Shirasaki DI, Loo JA, Weber PK, Pett-Ridge J, Stemmler TL, Chang CJ, Merchant SS - Nat. Chem. Biol. (2014)

Intracellular Cu is traceable to Cu accumulating compartments(a) Transmission electron microscopy (TEM) revealed electron-dense structures in Zn-limited cells (scale bar 2 μm). Manually defined areas of cells and the contained electron-dense structures were measured with Image J. We analyzed the statistical significance of Zn-limited vs Zn-replete cells by Kruskal-Wallis One Way Analysis of Variance on ranks (P = <0.001). Error bar shows SD +/− from three independent experiments. (b) NanoSIMS shows 40Ca+ and 63Cu+ co-localize in Zn-limited cells, coinciding with electron-dense structures in TEM (scale bars 1 μm). (c–d) Relative intracellular Ca (c) and Cu (d) measured during Zn resupply (to −Zn compared to +Zn). Samples from three independent cultures were collected 0, 10, and 24 h after Zn addition, and ten cells per time point were examined by NanoSIMS. Average ion ratio values were plotted based on whole cell area (“cell”) and intracellular areas of Ca and Cu accumulations with their corresponding standard deviations. (e) Ratio of 40Ca+ over 63Cu+ at different time points. All NanoSIMS counts were normalized to 12C+. (f–h) XAS spectra for Cu in a representative Zn-limited C. reinhardtii sample. (f) Cu XANES spectrum with a predominant spectral feature at 8984 eV, which corresponds to a 1s → 4p electronic transition typically seen in centrosymmetric Cu+ samples. (g) Expansion of the Cu pre-edge spectral features (red) offset and compared to the Cu2+SO4 (black) model. (h) Fourier transforms of the raw Cu EXAFS (black) with best fit simulation (green).
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Figure 4: Intracellular Cu is traceable to Cu accumulating compartments(a) Transmission electron microscopy (TEM) revealed electron-dense structures in Zn-limited cells (scale bar 2 μm). Manually defined areas of cells and the contained electron-dense structures were measured with Image J. We analyzed the statistical significance of Zn-limited vs Zn-replete cells by Kruskal-Wallis One Way Analysis of Variance on ranks (P = <0.001). Error bar shows SD +/− from three independent experiments. (b) NanoSIMS shows 40Ca+ and 63Cu+ co-localize in Zn-limited cells, coinciding with electron-dense structures in TEM (scale bars 1 μm). (c–d) Relative intracellular Ca (c) and Cu (d) measured during Zn resupply (to −Zn compared to +Zn). Samples from three independent cultures were collected 0, 10, and 24 h after Zn addition, and ten cells per time point were examined by NanoSIMS. Average ion ratio values were plotted based on whole cell area (“cell”) and intracellular areas of Ca and Cu accumulations with their corresponding standard deviations. (e) Ratio of 40Ca+ over 63Cu+ at different time points. All NanoSIMS counts were normalized to 12C+. (f–h) XAS spectra for Cu in a representative Zn-limited C. reinhardtii sample. (f) Cu XANES spectrum with a predominant spectral feature at 8984 eV, which corresponds to a 1s → 4p electronic transition typically seen in centrosymmetric Cu+ samples. (g) Expansion of the Cu pre-edge spectral features (red) offset and compared to the Cu2+SO4 (black) model. (h) Fourier transforms of the raw Cu EXAFS (black) with best fit simulation (green).
Mentions: With these pilot data suggesting further investigation, we sought to use an unequivocal physical technique to identify the intracellular Cu foci. Direct metal detection techniques such as X-ray fluorescence microscopy have been valuable to support indirect metal imaging obtained using synthetic fluorophores27. In the present study, electron-dense structures were revealed in microtomed thin-sections of fixed cells by transmission electron microscopy (TEM) and these were candidate sites for Cu accumulation. Indeed, the abundance and size of the electron dense structures correlated with Zn nutrition (Figure 4a). Kruskal-Wallis One Way Analysis of Variance on ranks of the electron-dense areas in the electron micrographs in relation to total cell areas found a significant difference between the Zn-limited and Zn-replete sections (p-value P= <0.001) (Figure 4a, right panel). NanoSIMS imaging, which allows simultaneous spatial detection of elements based on their mass, indicated that regions containing calcium (Ca) and Cu correlated with electron-dense structures at the periphery of the cells visible in TEM images of the same sections (Figure 4b). Co-localization of Ca and Cu in Zn-limited cells was confirmed independently by confocal microscopy and dual staining of cells with CS3 and Fluo-4-AM, a Ca2+-specific fluorescent dye (Supplementary Fig. 5).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, USA.

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.

Show MeSH
Related in: MedlinePlus