Limits...
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.

Show MeSH
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 P1B-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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4232477&req=5

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 P1B-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.

Mentions: To assess the operation of CRR1, we monitored the transcriptome as a function of Zn re-supply (between 0 and 24 h) by RNA-Seq. A heat map showing individual changes in transcript abundance for 17 genes involved in Cu and Zn homeostasis is presented (Figure 6a and with absolute scaling in Supplementary Figure 17a). The P-values indicate the significance of FPKM changes between time points (Supplementary Table 2). CTR transcripts were abundant in Zn-limited cells, supporting the contention that Cu+-hyper-accumulation was driven by high CTR expression (Figure 6b). Transcript abundance for all three CTR genes decreased rapidly (comparable to the behavior of CYC6, a sentinel gene of nutritional Cu signaling) during the first 3–5 h, which is consistent with Cu+ mobilization from the internal compartment (Supplementary Fig. 11) and hence de-activation of CRR116(Figure 6c). Interestingly, as intracellular Cu was depleted for synthesis of cuproproteins during growth and division, CRR1 and hence expression of its target genes were re-activated. RNAs encoding the candidate Zn2+ transporters, members of the ZIP family, were also rapidly reduced upon Zn2+ addition and those genes remained repressed throughout the time course (Figure 6d).


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)

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 P1B-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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232477&req=5

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 P1B-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.
Mentions: To assess the operation of CRR1, we monitored the transcriptome as a function of Zn re-supply (between 0 and 24 h) by RNA-Seq. A heat map showing individual changes in transcript abundance for 17 genes involved in Cu and Zn homeostasis is presented (Figure 6a and with absolute scaling in Supplementary Figure 17a). The P-values indicate the significance of FPKM changes between time points (Supplementary Table 2). CTR transcripts were abundant in Zn-limited cells, supporting the contention that Cu+-hyper-accumulation was driven by high CTR expression (Figure 6b). Transcript abundance for all three CTR genes decreased rapidly (comparable to the behavior of CYC6, a sentinel gene of nutritional Cu signaling) during the first 3–5 h, which is consistent with Cu+ mobilization from the internal compartment (Supplementary Fig. 11) and hence de-activation of CRR116(Figure 6c). Interestingly, as intracellular Cu was depleted for synthesis of cuproproteins during growth and division, CRR1 and hence expression of its target genes were re-activated. RNAs encoding the candidate Zn2+ transporters, members of the ZIP family, were also rapidly reduced upon Zn2+ addition and those genes remained repressed throughout the time course (Figure 6d).

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