CHCA-1 is a copper-regulated CTR1 homolog required for normal development, copper accumulation, and copper-sensing behavior in Caenorhabditis elegans

Abstract

Copper plays key roles in catalytic and regulatory biochemical reactions essential for normal growth, development, and health. Dietary copper deficiencies or mutations in copper homeostasis genes can lead to abnormal musculoskeletal development, cognitive disorders, and poor growth. In yeast and mammals, copper is acquired through the activities of the CTR1 family of high-affinity copper transporters. However, the mechanisms of systemic responses to dietary or tissue-specific copper deficiency remain unclear. Here, taking advantage of the animal model Caenorhabditis elegans for studying whole-body copper homeostasis, we investigated the role of a C. elegans CTR1 homolog, CHCA-1, in copper acquisition and in worm growth, development, and behavior. Using sequence homology searches, we identified 10 potential orthologs to mammalian CTR1 Among these genes, we found that chca-1, which is transcriptionally up-regulated in the intestine and hypodermis of C. elegans during copper deficiency, is required for normal growth, reproduction, and maintenance of systemic copper balance under copper deprivation. The intestinal copper transporter CUA-1 normally traffics to endosomes to sequester excess copper, and we found here that loss of chca-1 caused CUA-1 to mislocalize to the basolateral membrane under copper overload conditions. Moreover, animals lacking chca-1 exhibited significantly reduced copper avoidance behavior in response to toxic copper conditions compared with WT worms. These results establish that CHCA-1-mediated copper acquisition in C. elegans is crucial for normal growth, development, and copper-sensing behavior.

Keywords: Caenorhabditis elegans (C. elegans); copper; copper avoidance; copper transport; intestine; metal homeostasis; CHCA-1.

Figure 1.

Transcriptional regulation of C. elegans CTR1 candidate genes by copper. A, copper-dependent C. elegans growth in axenic media. Total worm numbers were calculated for three independent experiments. Asterisks indicate significant difference from optimal 10 μm CuCl₂ condition (one-way ANOVA, Dunnett post hoc test, **, p < 0.01; ****, p < 0.0001). B and C, qRT-PCR analysis of C. elegans CTR candidate genes under copper-limited (B) and high-copper (C) conditions. Worms were synchronized and supplemented with 100 μm BCS, 10 μm CuCl₂, or 300 μm CuCl₂ from the L1 to L4 stage in axenic media. Individual gene expression was normalized first to pmp-3 and then to its own expression under copper-optimal conditions. Two independent experiments were conducted under each condition. Asterisks indicate significant difference from indicated gene expression levels under optimal conditions. (Two-way ANOVA, Sidak post hoc test, *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.) Error bars, mean ± S.E.

Figure 2.

Requirement of CTR1 candidate genes for growth, reproduction, and copper accumulation in worms. A–C, copper-dependent growth assay under basal (A), high copper (B), and limited copper (C) conditions. Synchronized L1 stage N2 worms were cultured on RNAi plates until the vector-treated worms reached the L4 stage. Worm length (TOF) was quantified using a COPAS BioSort system, and worm length under each RNAi condition was normalized to vector TOF. ∼400 individual animals were analyzed under every condition. Values with asterisk are significantly different from vector (one-way ANOVA, Dunnett post hoc test, *, p < 0.05; **, p < 0.01; ***, p < 0.001). D, representative images of P₀ worms treated with indicated dsRNA-expressing bacteria. E, copper levels in C. elegans were measured by ICP-MS after exposure to 10 μm copper for two generations. Values with asterisk are significantly different from vector with three independent trials (one-way ANOVA, Dunnett post hoc test, *, p < 0.05). F, schematic presentation of copper-pulse assays. C. elegans worms were cultured in axenic media supplemented with 25 μm BCS for 5 days prior to synchronization. Synchronized L1 animals were cultured on 50 μm BCS NGM agar plates expressing indicated dsRNA for 72 h. Animals were then washed, aliquoted, and re-plated on fresh 50 μm BCS or 50 μm CuCl₂ plates for 12 h. Restored copper levels are indicated by normalizing copper-treated worms to BCS-cultured animals. G, restored copper levels under indicated RNAi treatments. Values with asterisk are significantly different from vector (one-way ANOVA, Dunnett post hoc test, ***, p < 0.001; ****, p < 0.0001). H, brood size analysis of F58G6.9 RNAi animals. Error bars indicate mean ± S.E. of five independent experiments. Values with asterisk are significantly different from vector under the same culture condition (two-way ANOVA, Sidak post hoc test, ****, p < 0.0001.) Error bars in this figure represent mean ± S.E.

Figure 3.

Depletion of chca-1 gene by RNAi decreases intestinal copper availability in C. elegans. A, synchronized L1 stage BK015 transgenic worms (P_vha-6::CUA-1.1::GFP::unc-54 3′ UTR; cua-1 (ok904)) were cultured on NGM agar plates seeded with E. coli expressing dsRNA against CHCA-1 or vector. CUA-1.1::GFP localization in L4 worms was examined using confocal microscopy. Scale bar, 15 μm. B, chca-1–depleted worms display increased endogenous CUA-1.1::GFP expression in the hypodermis. BK017 (P_cua-1::CUA-1.1::GFP::unc-54 3′ UTR; cua-1 (ok904)) transgenic animals were maintained on 10 μm CuCl₂ plates prior to synchronization. L1 animals were then re-plated for chca-1 RNAi. After 60 h of culture, CUA-1.1::GFP expression levels in L4 animals were examined using confocal microscopy. Scale bar, 50 μm.

Figure 4.

Characterization of the chca-1 (tm6506) IV strain. A, schematic of human CTR1 and C. elegans chca-1 gene loci, with blue-colored ORFs and gray-colored UTR regions. Note that CHCA-1b1 and CHCA-1b2 isoforms differ at the 5′ UTR region but express identical proteins. The deleted region in the tm6506 allele is indicated by the red bar. Scale bar indicates 100 bp. B and C, growth of tm6506 animals under various CuCl₂- or BCS-supplemented conditions. Worms homozygous for the tm6506 allele and their outcrossing WT brood mates (WT) were cultured from synchronized L1s for 72 h. B, representative images of animals growing under indicated conditions. C, worm growth quantification using a COPAS BioSort system. Error bars indicate mean ± S.E. of around 75 worms. Values with asterisk are significantly different from WT animals under same copper or BCS concentrations (two-way ANOVA, Dunnett post hoc test, *, p < 0.05; **, p < 0.01; ****, p < 0.0001). D, WT and tm6506 mutant worms grown on 50 μm BCS plates or 50 μm BCS plus indicated concentrations of metals. Error bars indicate mean ± S.E. of around 100 individual animals. Means with different letters are significantly different at p = 0.05 (two-way ANOVA, Tukey's post hoc test). E, copper levels of tm6506 and WT animals. Synchronized L1 animals were cultured on indicated concentrations of copper- or BCS-supplemented media for 60 h and then pelleted for ICP-MS. For each condition, three or four samples were analyzed. Values with asterisk are significantly different from those of WT animals (t test for each treatment condition, *, p < 0.05; ***, p < 0.001; ****, p < 0.0001). F, copper-acquisition capacity of tm6506 worms. WT or tm6506 worms pre-treated with 15 μm BCS were washed and separately cultured on fresh 15 μm BCS or 50 μm CuCl₂ NGM plates for 12 h, followed by ICP-MS analysis. Three independent samples were assayed for each condition. Asterisks indicate that copper levels in tm6506 worms post-pulse are significantly different from those in the WT strain (ANCOVA, Bonferroni post hoc test, p = 0.016).

Figure 5.

Intestinal CHCA-1 plays an important role in copper-dependent growth and copper accumulation. A, tissue-specific expression of the chca-1 gene. Transgenic animals expressing GFP driven by the 2.8-kb chca-1 promoter region were cultured on NGM plates containing 10 μm CuCl₂ (panels a and b) or 200 μm BCS (panels c–f, same animal, different focus layers). Arrowhead indicates intestine, and arrow indicates hypodermis cells. Panels a, c, and e, bright field; panels b, d, and f, fluorescence. Scale bar, 50 μm. B and C, copper-dependent growth following chca-1 gene depletion in specific tissues. N2, RNAi-resistant strains (rde-1, WM27) and tissue-specific rde-1-expressing strains (VP303, NR222, and WM118) were used to knock down the chca-1 gene in the indicated tissues (Int, intestine; Hyp, hypodermis; Mus, muscle). Synchronized L1s were cultured to the L4 stage (B, P₀) or re-synchronized and cultured for another generation (C, F₁) on indicated copper-deficient NGM plates prior to quantification. Under each condition, ∼200 P₀ or F₁ worms' TOF was quantified by a COPAS BioSort. Growth of chca-1–depleted worms was normalized to vector for each condition. chca-1 RNAi in N2 and VP303 strains on 100 μm BCS plates exhibited severe defects in P₀ reproduction (U.D., under detection limit). Error bars indicate mean ± S.E. of ∼200 individual animals. Values with asterisk are significantly different from the same strain under basal conditions (two-way ANOVA, Dunnett post hoc test, *, p < 0.05; **, p < 0.01; ***, p < 0.001, ns, not significant). D and E, copper accumulation in N2 and tissue-specific chca-1–depleted animals. Different strains of worms were pre-cultured on 50 μm BCS NGM plates, and then half of the population was separated and treated with copper, as described above in Fig. 2, F and G. D, restored copper levels after normalizing to BCS-cultured samples. Error bars represent mean ± S.E. of four independent experiments. Values with asterisk are significantly different from vector (two-way ANOVA, Sidak post hoc test, ***, p < 0.001; ****, p < 0.0001). Copper levels following BCS pre-culture were not significantly different among strains by Two-way ANOVA (data not shown). E, percentage of copper levels restored by chca-1 RNAi after normalizing to vector animals under the same conditions. Error bars, mean ± S.E. of four independent experiments. Values with asterisk are significantly different from one another (one-way ANOVA, Dunnett post hoc test, *, p < 0.05; **, p < 0.01, ns, not significant).

Figure 6.

Intestinal expression of CHCA-1::GFP. A, intestinal expression of CHCA-1::GFP under basal conditions (P_vha-6::CHCA-1::GFP, unc-54 3′UTR). Scale bar, 50 μm. B, CHCA-1::GFP expression in the intestine under basal, copper-deficient, and high-copper conditions. A DAPI channel was used to observe intestinal autofluorescence from gut granules. Scale bar, 15 μm. C, CHCA-1::GFP signal intensity was quantified under high, low, or replete copper conditions using a COPAS BioSort system. At least 100 synchronized L4 CHCA-1::GFP-expressing worms were used following 2.5 days of copper or BCS-supplemented cultures in each condition (one-way ANOVA, Dunnett post hoc test, ns, not significant). D, intestinal expression of CHCA-1 partially rescued growth of CHCA-1 mutant animals. Transgenic animals expressing CHCA-1::SL2::GFP protein were crossed with tm6506 animals to generate an intestinal CHCA-1 expression animal in a whole-body chca-1 mutant background. These transgenic animals, together with their WT brood mates (WT), as well as tm6506 animals, were quantified by TOF after 60 h of culture from synchronized L1s in the indicated conditions. Error bars indicate mean TOF ± S.E. of ∼150 individuals. Values with asterisk are significantly different from tm6506 worms cultured at the same condition (two-way ANOVA, Tukey's post hoc test, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 7.

Worms lacking CHCA-1 exhibit reduced copper-sensing behavior. A, schematic of copper avoidance assay: copper gradient plates were made by adding a given concentration of CuCl₂ to one side of a rectangular plate. Non-copper–containing plates were used as controls. At least 150 animals were used on each plate with a total of three independent experiments. B, representative results of N2 worm distribution on a non-copper plate (left) or 10 mm copper gradient plate (right). Error bars, mean ± S.E. of two independent experiments. Asterisk indicates the percentage of animals in the low-copper area (sections 1 and 2) is significantly different from the percentage in the high-copper area (sections 4 and 5) (two-way ANOVA, Sidak post hoc test, **, p < 0.01, ns, not significant). C, N2 worm avoidance index on copper gradient plates with varied concentrations of copper. Error bars indicate mean ± S.E. of three independent experiments. Asterisk values are significantly different from the avoidance index on non-copper plates (one-way ANOVA, Dunnett post hoc test, ***, p < 0.001; ****, p < 0.0001). D, avoidance index of CB1033 (che-2 (e1033) X) and N2 vector worms or chca-1 RNAi worms on 8 mm copper gradient plates. Three independent experiments for CB1033 and six independent experiments for N2 were analyzed (one-way ANOVA, Dunnett post hoc test, *, p < 0.05; ***, p < 0.001). E, worms were pre-cultured with 100 μm CuCl₂ or 100 μm BCS for one generation and assayed on 8 mm copper gradient plates. (One-way ANOVA, Dunnett post hoc test, ***, p < 0.001; ****, p < 0.0001.) F, avoidance index of RNAi-hypersensitive worms (TU3335) lacking the egl-3 or egl-21 gene on 10 mm copper gradient plates. Error bars indicate mean ± S.E. of eight independent experiments under each condition. Values with asterisk are significantly different from each other (one-way ANOVA, Dunnett's post hoc test, *, p < 0.05; **, p < 0.01; ****, p < 0.0001). G, PAM genes (pgal-1, pghm-1, and pamn-1) were co-depleted by RNAi for two consecutive generations in TU3335 strain, followed by the avoidance assay on 10 mm copper gradient plates. Error bars indicate mean ± S.E. of two independent experiments. Values with asterisk are significantly different (one-way ANOVA, Dunnett post hoc test, *, p < 0.05; **, p < 0.01).