Chloride intracellular channel proteins respond to heat stress in Caenorhabditis elegans

Chloride intracellular channel proteins (CLICs) are multi-functional proteins that are expressed in various cell types and differ in their subcellular location. Two CLIC homologs, EXL-1 (excretory canal abnormal like-1) and EXC-4 (excretory canal abnormal± 4), are encoded in the Caenorhabditis elegans genome, providing an excellent model to study the functional diversification of CLIC proteins. EXC-4 functions in excretory canal formation during normal animal development. However, to date, the physiological function of EXL-1 remains largely unknown. In this study, we demonstrate that EXL-1 responds specifically to heat stress and translocates from the cytoplasm to the nucleus in intestinal cells and body wall muscle cells under heat shock. In contrast, we do not observe EXC-4 nuclear translocation under heat shock. Full protein sequence analysis shows that EXL-1 bears a non-classic nuclear localization signal (NLS) that EXC-4 is lacking. All mammalian CLIC members have a nuclear localization signal, with the exception of CLIC3. Our phylogenetic analysis of the CLIC gene families across various animal species demonstrates that the duplication of CLICs in protostomes and deuterostomes occurred independently and that the NLS was subsequently lost in amniotes and nematodes, suggesting convergent evolution. We also observe that EXL-1 nuclear translocation occurs in a timely ordered manner in the intestine, from posterior to anterior regions. Finally, we find that exl-1 loss of function mutants are more susceptible to heat stress than wild-type animals, demonstrating functional relevance of the nuclear translocation. This research provides the first link between CLICs and environmental heat stress. We propose that C. elegans CLICs evolved to achieve different physiological functions through subcellular localization change and spatial separation in response to external or internal signals

Chloride intracellular channel proteins respond to heat stress in <i>Caenorhabditis elegans</i>

<div><p>Chloride intracellular channel proteins (CLICs) are multi-functional proteins that are expressed in various cell types and differ in their subcellular location. Two CLIC homologs, EXL-1 (<u>ex</u>cretory canal abnormal <u>l</u>ike-1) and EXC-4 (<u>exc</u>retory canal abnormal– 4), are encoded in the <i>Caenorhabditis elegans</i> genome, providing an excellent model to study the functional diversification of CLIC proteins. EXC-4 functions in excretory canal formation during normal animal development. However, to date, the physiological function of EXL-1 remains largely unknown. In this study, we demonstrate that EXL-1 responds specifically to heat stress and translocates from the cytoplasm to the nucleus in intestinal cells and body wall muscle cells under heat shock. In contrast, we do not observe EXC-4 nuclear translocation under heat shock. Full protein sequence analysis shows that EXL-1 bears a non-classic nuclear localization signal (NLS) that EXC-4 is lacking. All mammalian CLIC members have a nuclear localization signal, with the exception of CLIC3. Our phylogenetic analysis of the CLIC gene families across various animal species demonstrates that the duplication of CLICs in protostomes and deuterostomes occurred independently and that the NLS was subsequently lost in amniotes and nematodes, suggesting convergent evolution. We also observe that EXL-1 nuclear translocation occurs in a timely ordered manner in the intestine, from posterior to anterior regions. Finally, we find that <i>exl-1</i> loss of function mutants are more susceptible to heat stress than wild-type animals, demonstrating functional relevance of the nuclear translocation. This research provides the first link between CLICs and environmental heat stress. We propose that <i>C</i>. <i>elegans</i> CLICs evolved to achieve different physiological functions through subcellular localization change and spatial separation in response to external or internal signals.</p></div

EXL-1::GFP nuclear translocation in intestine occurs in an ordered manner.

<p>(A1-A3): At 20°C, EXL-1::GFP has basal nuclear accumulation at the most posterior region (arrow in A1). (B1-B4): After heat shock at 35°C for 1 hour, EXL-1::GFP nuclear accumulation still remains in the most posterior region. B4 is enlarged image of the rectangular part of B1. (C1-C4): After 2 hour of heat shock, EXL-1::GFP nuclear translocation mostly occurs between middle region (around vulva) and tail region. C4 is enlarged image of the rectangular part of C1. (D1-D3): After 3 hours of heat shock, EXL-1::GFP nuclear translocation appears in the middle region and posterior region. (E1-E3): Enlarged images show EXL-1::GFP nuclear translocation in the middle region of intestine after 3 hours of heat treatment. (F1-F3): After 4 hours of heat shock, EXL-1::GFP nuclear translocation was observed in the whole intestinal region from anterior to posterior. (G1-G3): Enlarged images of EXL-1::GFP nuclear translocation at anterior region of intestine after 4 hours of heat treatment. Image A1-3, B4, C4, E1-3, G1-3 are taken with 400 magnification power; all the other images are taken with 100 magnification power; (H): Quantification of nuclear to cytoplasmic fluorescence ratio under different heat shock treatments.</p

<i>exl-1</i> loss of function mutants are more susceptible to heat stress than wild type animals.

<p>(A): Animals were synchronized to young adult stage at 20°C and then were subjected to heat shock at 35°C. <i>exl-1</i> loss of function mutants are more sensitive to heat stress than wild type animals. (B): Animals were subjected to heat shock at 32°C. <i>exl-1</i> loss of function mutants lived significantly shorter than wild type animals. (Log-rank test was used to determine statistic power. * p value < 0.01; ** p value < 0.001)</p

EXC-4 does not accumulate into the nucleus upon heat shock.

<p>(A1-A3): At 20°C, EXC-4::GFP is targeted to seam cells (arrow in A1) and intestinal cells. (B1-B3): Upon heat shock at 35°C for 2.5 hours, EXC-4::GFP did not accumulate into the intestinal nuclei (arrow in B2).</p

Sequence analysis of CLICs.

<p>(A): A putative NLS sequence is identified in EXL-1 protein sequence, but not in EXC-4. All human CLICs have a NLS motif except CLIC3 (shown here CLIC2 and CLIC4). EXL-1, CLIC2, and CLIC4 share conserved residues Lys at P2 (second binding position to importin-α) and Lys/Arg at P5. Both EXC-4 and CLIC3 sequences, however, lack NLS. Conserved amino acids are in red. NLS from CLIC are underlined. Two amino acids at P2 and P5 are bolded (B): Phylogenetic relationships among animal CLIC proteins. The tree was built using the maximum likelihood method. Nodes with higher than 70% bootstrap support are indicated with a star.</p

EXL-1 accumulates into the nucleus after heat shock.

<p>(A1-A3): EXL-1::GFP is expressed in intestinal cells at standard culture condition (20°C). The protein is diffuse inside the cells. No exclusion of nuclear expression was observed. (B1-B3): EXL-1::GFP accumulates into the intestinal nuclei (arrows) when animals were subjected to heat shock at 35°C for 2 hours. Inset shows enlarged image of the nucleus. (C1-C3): EXL-1::GFP is expressed in body wall muscle cells. (D1-D3): EXL-1::GFP translocates into the nucleus in body wall muscle cells (arrows) under heat shock at 35°C for 2 hours. Inset shows enlarged image of the nucleus. (E1-E3): DAPI staining in L4 animals shows that EXL-1::GFP signal overlaps with DNA staining inside the nucleus. (F1-F3): EXL-1::GFP does not translocate into the nucleus under 200mM paraquat treatment for 4 hours. Images are taken at L4 stage. 1: GFP fluorescence image; 2: Nomarski image (E2: DAPI staining); 3: merged image.</p