Figure 1.
Phylogenetic tree of CDF family members.
A dendrogram showing 14 predicted C. elegans CDF family members (red) identified by PSI-BLAST and all predicted CDF proteins from Homo sapiens (blue), Arabidopsis thaliana (green), and the yeast Saccaromyces cerevisiae (yellow). TTM-1 and CDF-2 are indicated by arrows. Notably, all ten human ZnT proteins cluster with highly related C. elegans proteins. For genes that encode multiple protein isoforms, only one isoform is listed.
Figure 2.
Structure of the ttm-1 gene and protein.
(A) The straight line indicates genomic DNA on chromosome III: numbered boxes indicate exons, and arrows indicate transcription start sites. The splicing patterns of ttm-1a and ttm-1b are shown above: Exons 1 and 2 (red) are unique to ttm-1a, exon 3 (blue) is unique to ttm-1b, and exons 4 and 5 (yellow) are common to both transcripts. SL1 indicates trans-spliced leader sequence. The black bar denotes the genomic region deleted in the ok3503 allele. Constructs used for expression pattern analysis are depicted below. Black lines denote promoter regions, and colored boxes denote protein coding sequences. (B) An alignment of the predicted TTM-1A and TTM-1B proteins with C. elegans CDF-2 and human ZnT2. Identical and similar amino acids are highlighted in black and gray, respectively. Blue boxes indicate the predicted six transmembrane motifs (labeled I–VI). The red box indicates the conserved histidine-rich motif, (HX)n, and the red bar indicates the additional histidine-rich motif in TTM-1B. Red triangles flank the amino acids encoded by the region deleted in the ok3503 allele.
Figure 3.
Fluorescence microscope images of live transgenic hermaphrodites expressing GFP under the control of (A) the predicted ttm-1a promoter [Pttm-1a::GFP] and (B) the predicted ttm-1b promoter [Pttm-1b::GFP]. The left panel shows an entire worm, and both promoters are expressed in intestinal cells. Right panels are magnified views of hypodermal and intestinal expression driven by the ttm-1a promoter and head neuron and seam cell expression driven by the ttm-1b promoter. To visualize the relatively weak GFP fluorescence in head neurons, we used a longer exposure time compared to Figure 4B. Scale bars represent 100 µm (left) and 10 µm (right).
Figure 4.
Regulation of ttm-1 expression by zinc.
(A) Wild-type animals were cultured with 0 µM or 200 µM supplemental zinc. RNA was extracted from a synchronized population at L4/adult stages, and mRNA levels of indicated genes were determined by quantitative real-time PCR. The Y-axis represents the fold changes of mRNA levels between 0 µM and 200 µM supplemental zinc, and the bars indicate the average ± SEM of three independent experiments. The mRNA levels at 0 µM supplemental zinc were set equal to 1.0 for each gene. ttm-1b was significantly induced by 200 µM compared to 0 µM supplemental zinc (*p<0.05), similar to the positive control gene cdf-2 (**p<0.01) [28], [30]. ttm-1a was not responsive to zinc, similar to the negative control gene ama-1 [28], [30]. (B) Fluorescence microscope images of Pttm-1b::GFP transgenic animals cultured with 0 µM or 200 µM supplemental zinc. Whole animals are shown in the top panels, and the head and anterior most intestinal cells are shown in the bottom panels (anterior to the right). While GFP expression was induced in intestinal cells by 200 µM supplemental zinc (top panel and brackets in lower panel), GFP expression was not induced in the head neurons (arrow heads). Images in each panel were captured with the identical settings and exposure times. Scale bars represent 100 µm (top) and 20 µm (bottom).
Figure 5.
Intracellular localization of TTM-1 isoforms and CDF-1.
Bright field and fluorescence microscope images of live transgenic animals expressing TTM-1A::GFP [Pttm-1a::TTM-1A::GFP] (A–F) or TTM-1B::GFP [Pttm-1b::TTM-1B::GFP] (G–L). Differential interference contrast (DIC) and fluorescence microscope images of a fixed transgenic animal expressing CDF-1::GFP [Pcdf-1::CDF-1::GFP] that was immunostained with an anti-GFP antibody (M–N). Bright field and DIC show morphology, and green displays GFP. Images show an entire animal (A–B, G–H), intestinal cells (C–D, K–L, M–N), hypodermal cells (E–F), and seam cells (I–J). The arrows indicate the lumen of the intestine (K–N). TTM-1A::GFP displayed diffuse, punctuate staining in intestinal and hypodermal cells. TTM-1B::GFP displayed punctuate staining in seam cells and was localized to the apical plasma membrane of intestinal cells. The diffuse fluorescence signal in panel J derives from GFP that is out of the focal plane. CDF-1::GFP was localized to the basolaternal plasma membrane of intestinal cells (triangles). Scale bars represent 100 µm (A, G) and 10 µm (C, E, I, K, M).
Figure 6.
ttm-1 functions in zinc excretion.
(A) Total zinc content of wild-type and ttm-1(ok3503) animals. Populations consisting of mixed developmental stages were cultured with the indicated concentrations of supplemental zinc and analyzed for total zinc content by ICP-MS. Total zinc content was calculated in parts-per-million (ppm) by dividing the weight of zinc by the weight of dessicated worms (µg/g). Bars indicate average ± SEM of two independent experiments (*p<0.05). (B–C) RT-PCR analysis of mRNA levels of mtl-1 or mtl-2 in wild-type and ttm-1(ok3503) animals cultured with 0 µM or 100 µM supplemental zinc. The Y-axis represents the fold changes of mRNA levels, and the bars indicate the average ± SEM of two independent experiments. Wild-type animals cultured in 0 µM supplemental zinc were set equal to 1.0, and the other samples were relative to that sample (*p<0.05). (D–E) Fluorescence microscope images of live wild-type, cdf-1(n2527), ttm-1(ok3503), and ttm-1(ok3503);cdf-1(n2527) animals stained with Zinpyr-1. Animals were cultured with 0 µM or 100 µM supplemental zinc – a concentration that causes zinc accumulation without causing excessive toxicity. Images show whole animals, and insets show magnified images of the boxed regions. Arrows indicate strong Zinpyr-1 staining in the pseudoceolomic space around the uterus. The scale bars represent 100 µm and 20 µm (insets). To quantify Zinpyr-1 staining, we categorized animals into four groups based on Zinpyr-1 fluorescence intensity: highest (dark red), high (red), medium (pink) and low (white). The Y-axis represents the percent of each group in the population (n≥20). The quantification was repeated twice with similar results, and panel D shows representative data from one experiment.
Figure 7.
Role of ttm-1 in zinc detoxification.
(A,B,C,E,F) Zinc sensitivity was determined by analyzing the length of animals cultured from the first larval (L1) stage with the indicated concentrations of supplemental zinc. The length of worms was analyzed using microscopy and imageJ software (n = 20). The alleles were ttm-1(ok3503), cdf-1(n2527), sur-7(ku119), cdf-2(tm788), glo-1(zu391), and pgp-2(kx48). (D) Analysis of cdf-2 mRNA levels in wild-type and ttm-1(ok3503) animals cultured with the indicated concentrations of supplemental zinc. The Y-axis represents the fold changes of mRNA levels, and the bars indicate the average ± SEM of two independent experiments. The mRNA level in wild-type animals at 0 µM supplemental zinc was set equal to 1.0, and the other samples were relative to that sample. Compared to wild-type animals, cdf-2 mRNA levels were elevated in ttm-1 mutant animals cultured with supplemental zinc in both independent trials, indicating this is a reproducible result. However, the combined data did not reach statistical significance at the level of p<0.05 because the values of the fold changes varied between the experiments. (G–H) Zinc sensitivity of cdf-2(tm788), ttm-1(ok3503);cdf-2(tm788), and transgenic ttm-1(ok3503);cdf-2(tm788) animals expressing TTM-1A::GFP driven by the ttm-1a promoter [Pttm-1a::TTM-1A::GFP] (G), TTM-1B::GFP driven by the ttm-1b promoter [Pttm-1b::TTM-1B::GFP] (G), or TTM-1B::GFP driven by the cdf-2 promoter [Pcdf-2::TTM-1B::GFP] (H) (n = 20).
Figure 8.
Relationships between ttm-1 and cdf-2.
(A) Total zinc content of wild-type, ttm-1(ok3503), cdf-2(tm788), and ttm-1(ok3503);cdf-2(tm788) animals analyzed by ICP-MS. Bars indicate average ± SEM of two independent experiments (ns, p>0.05, *p<0.05, ***p<0.005). The wild type and ttm-1(ok3503) data are the same as Figure 6A. (B) mRNA levels of mtl-1 (left) or mtl-2 (right) in animals cultured with no supplemental zinc determined by quantitative RT-PCR. The bars indicate the average ± SEM of three independent experiments. The mRNA level in WT animals was set equal to 1.0, and other samples were relative to that sample. ttm-1;cdf-2 double mutant animals displayed elevated levels of mtl-1 and mtl-2 mRNA compared to WT and single mutant animals in all three experiments, indicating this is a reproducible result. However, the combined data did not reach statistical significance at the level of p<0.05 because the values of the fold changes varied between the experiments.
Figure 9.
Network of CDF zinc transporters in intestinal cells.
(A) Dietary zinc moves from the intestinal lumen to the cytoplasm of intestinal cells by an undefined mechanism. High levels of cytoplasmic zinc increase transcription of ttm-1b and cdf-2. CDF-1 (green) functions in distribution; it is localized to the basolateral surface of the plasma membrane of intestinal cells and transports cytoplasmic zinc into the pseudocoelum for use by cells such as epithelia, muscles and neurons. TTM-1B (red) functions in excretion; it is localized to the apical surface of the plasma membrane of intestinal cells and transports cytoplasmic zinc into the intestinal lumen. CDF-2 (blue) functions in storage: it is localized to the membrane of gut granules, lysosome-related organelles that acquire a bilobed morphology in response to high zinc, and it transports zinc into the lumen of these organelles [30]. TTM-1A (yellow) is localized to vesicles and may also promote zinc excretion and/or sequestration. (B) A parallel negative feedback circuit promotes zinc homeostasis in intestinal cells. TTM-1B and CDF-2 proteins reduce the level of cytoplasmic zinc and promote zinc detoxification by distinct mechanisms of zinc excretion and zinc sequestration, respectively. ttm-1b and cdf-2 transcripts are both induced by high levels of cytoplasmic zinc. Mutations that reduce the activity of ttm-1 or cdf-2 cause induction of the remaining protein.