Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas

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.

Figure 1. Zn deficiency induces CRR1-dependent Cu hyperaccumulation

ICP-MS analysis of Cu and Zn contents in cells grown under Zn-limited (−Zn) or -replete (+Zn) conditions. (a) Cellular Cu contents in cells from three independent C. reinhardtii cultures (strain CC-4532) that were grown to mid-logarithmic phase in either −Zn or +Zn (containing 2.5 μM ZnCl₂) TAP medium (in first (1) and second (2) round of transfer to indicated medium). Quantifications of Cu (b) and Zn (d) contents in cells grown under a concentration range of supplemented Cu (0, 0.02, 0.05, 0.2, 0.5, 2, 5, 10, 25, and 50 μM) in −Zn or +Zn TAP medium. The inset in b is the rescaled representation of Cu contents in the Zn-replete cells. The difference in total Cu contents between −Zn and +Zn cells is about 20-fold. (c) Cu contents in crr1-2 frameshift mutants (CC-3960) and CRR1 complemented strains (a and b representing two independent isolates). Quantification results from three different experiments per strain and condition are shown as separate data points (a) or as averages with corresponding standard deviations (b–d).

Figure 2. Zn-limited cells also express Cu deficiency markers

(a) Quantitative RT-PCR (qRT-PCR) to test for the expression of genes that react to altered Zn and Cu availabilities, respectively. The tested genes were CYC6 encoding cytochrome c₆ (Cyt c₆) as a marker for the Cu-deficiency regulon and ZRT3/ZRT5 encoding ZIP transporters as markers of the Zn-deficiency regulon. The relative transcript abundances as calculated with the LinRegPCR program are plotted as separate data points for triplicate cultures grown either under replete conditions (+Zn/+Cu), Zn deficiency (−Zn/+Cu), or under Cu deficiency (+Zn/−Cu) in first and second round of limitation, respectively. (b) Immunoblot analyses showed that Cyt c₆ and plastocyanin (PC) were adjusted in Zn deficient cells to levels expected from Cu deficient cells. Shown are immunoblots with dilution series (100, 50, and 25 %) of soluble and insoluble protein extracts that were incubated with antiPC, antiCyt c₆, and an antibody against a Cu-binding subunit of Cyt oxidase (antiCox2b), respectively. This experiment was carried out three times and a representative set of those is shown. Full Coomassie-stained gels of loading controls are shown in Supplementary Figure 18.

Figure 3. Cu(I)-sensitive CS3 staining suggests Cu accumulation in intracellular compartments

(a) Chemical structure of the Cu⁺-binding fluorescent dye CS3 and the non-copper binding analog control-CS3 (Ctrl-CS3), where the four metal binding sulfur atoms (CS3) are replaced by isosteric carbons (Ctrl-CS3). For the detailed synthesis refer to the Supplementary note and Supplementary Figure 2a. (b) Zn-limited and Zn-replete wildtype C. reinhardtii (CC-4532) cells were stained with the cuprous dye CS3 to observe intracellular Cu distribution by confocal microscopy. For control, we stained cells from the same cultures with the control dye Ctrl-CS3. DIC = differential interference contrast; Chl = chlorophyll autofluorescence. The shown scale bar represents 10 μm for all images. We analyzed 55 Zn-deficient cells stained with CS3, 30 Zn-deficient cells treated with Ctrl-CS3, 54 Zn-replete cells stained with CS3, and 64 Zn-replete cells treated with Ctrl-CS3 in total.

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).

Figure 5. Changes in abundance of electron dense bodies and Cu redistribution upon Zn resupply

(a) Electron microscopy of Zn-limited cells during Zn resupply (0, 10, 24, 60 h after addition of ZnCl₂) and Zn-replete cells in early and late logarithmic growth phases (+Zn early and +Zn late). Two representative specimens from three independent cultures are shown for each condition. Arrows point to electron-dense bodies, mainly in the periphery of the cell (scale bars 2 μm). (b) Growth curves of Zn-limited or replete cells, which were inoculated into fresh TAP medium containing ZnCl₂ but no Cu (left panel) or no Cu and 0.1 μM Mn and 0.5 μM Fe (right panel). We show mean values of three independent experiments with corresponding standard deviations. (c–e) ⁶⁵Cu-labelled cells grown under Zn limitation were supplied with ⁶³Cu (10 μM ⁶³CuCl₂) either together with Zn resupply (c) or 5h after Zn resupply (d). Soluble proteins were extracted anaerobically at 0, 10, 24 and 60 h after Zn addition, and analyzed by LC-ICP-MS to monitor the Cu isotope content of the protein species. This experiment was performed twice with ⁶⁵Cu as label and chased with ⁶³Cu and twice with ⁶³Cu as label and chased with ⁶⁵Cu. Shown are the ion chromatograms for Cu isotopes 63 and 65. The emerging Cu isotope peak at 18.2 min corresponds to plastocyanin-associated Cu. The corresponding LC fractions were analyzed by immunoblotting regarding their plastocyanin contents (e). Immunoblot analysis of respective fractions with purified plastocyanin (PC) from C. reinhardtii as standard.

Figure 6. Transcriptome response to Zn resupply monitored by RNAseq analysis

(a) Heatmap showing Z scores (interpreted as a measure of standard deviation away from the mean) for the changes of FPKM (fragments per kilobase of exon per million fragments mapped) of select genes indicated at the right margin. The time points 0–24 h after Zn addition indicate the sampling during Zn resupply to a Zn-limited culture. Columns “+Zn early” and “+Zn late” indicate samples from Zn-replete cultures taken in the stages of early logarithmic and beginning stationary growth phase, respectively. (c–g) The mRNA abundances in FPKM are given for (b) members of the CTR family, (c) CYC6, (d) genes encoding members of the ZIP family, (e) genes encoding members of the P_1B-type ATPase family, (f) CRR1 and Cu chaperones ATX1, PCC1 and COX17, and (g) PCY1 (plastocyanin). The y-axes of the diagrams are log₂ scaled.