Roles of the ClC chloride channel CLH-1 in food-associated salt chemotaxis behavior of C. elegans
- Department of Biological Sciences, School of Science, The University of Tokyo, Japan
- Laboratory of Physiology, Atmosphere and Ocean Research Institute, The University of Tokyo, Japan
Figures
Figure 1 with 3 supplements
Two missense mutations in clh-1 give rise to food-associated salt chemotaxis disorder.
(a) Salt concentration chemotaxis of wild type. Adult hermaphrodites were cultivated at 0 mM or 100 mM of NaCl with or without food for 6 hr and placed on a chemotaxis assay plate on which NaCl …
Figure 1—figure supplement 1
Isolation and characterization of salt chemotaxis mutants JN572 and JN577.
(a) A schematic diagram of salt chemotaxis assay plate and calculation of the chemotaxis index. See Materials and methods for details. (b) Procedure for forward genetic screening to obtain …
Figure 1—figure supplement 2
Missense mutations in clh-1 responsible for salt chemotaxis defect.
(a) Gene structure of clh-1 and the positions of clh-1 mutations. The positions of pe572 (T to C) and pe577 (G to A) are noted by arrows. ok658 removes exons 3– 5, tm1243 removes exons 6 and 7, qa901…
Figure 1—figure supplement 3
Characterization of clh-1 mutants.
(a) Chemotaxis of clh-1 deletion mutants. Dots represent individual trials. Bars and the error bars represent mean +/- s.e.m., n ≧ 6 assays, compared with wild type, Dunnett’s test, **p<0.01. (b) pe5…
Figure 2 with 1 supplement
ClC genes redundantly function in salt chemotaxis.
(a) Chemotaxis of clh multiple mutants that carry clh-1(pe572) mutation with a deletion in other clh genes. Dots represent individual trials. Bars and the error bars represent mean +/- s.e.m., n ≧ 6 …
Figure 2—figure supplement 1
Effect of loss of ClC genes on salt chemotaxis.
(a) Comparison of C. elegans (C.e) CLHs, human (H.s), and murine (M.m) ClC-2. Amino acids altered in clh-1(pe) alleles (deep and light blue) and their flanking regions are shown. The ‘Proton …
Figure 3 with 1 supplement
clh-1 acts in the salt-sensing neuron ASER.
(a) Expression pattern of clh-1p::nls4::mTFP (green, bottom left) in an adult animal. At least three pairs of sensory neurons, AWA, AWC and ASE expressed the marker. eat-4p::nls4::tagRFP and DiI, …
Figure 3—figure supplement 1
clh-1 acts in ASER in salt chemotaxis, and mutations in clh-1 do not affect morphology of ASER.
(a) Rescue of clh-1(pe577) mutants by ASER-specific expression of clh-1(wt) cDNA. Dots represent individual trials. Bars and the error bars represent mean +/- s.e.m., n = 5 assays, Tukey’s test. …
Figure 4 with 2 supplements
Mutations in clh-1 affect chloride dynamics of ASER in response to salt down-step stimulus.
(a–e) Representative current traces from Xenopus oocytes that expressed cRNA for mock (a), clh-1(wt) (b), clh-1(pe572) (c), and clh-1(pe577) (d) clamped at voltages ranging from −160 mV to 80 mV and …
Figure 4—figure supplement 1
Extracellular pH-sensitivity and bicarbonate permeability of CLH-1.
(a–i) Representative current traces from Xenopus oocytes that expressed cRNA for clh-1(wt) (a–c), clh-1(pe572) (d–f), or clh-1(pe577) (g–i). Oocytes were perfused at pH 7 with Cl- as the dominant …
Figure 4—figure supplement 2
Mutant CLH-1 channels retained pH sensitivity and bicarbonate permeability.
(a–c) Comparison of current amplitudes at −130 mV between pH 7 and pH 5. clh-1(wt) (a, n = 20), clh-1(pe572) (b, n = 18), and clh-1(pe577) (c, n = 8). Two-tailed paired t-test, ***p<0.001, **p<0.01.
Figure 5 with 2 supplements
Calcium dynamics of ASER in response to repeated salt stimuli is altered in clh-1 mutants.
(a and c) Calcium responses of ASER stimulated by NaCl concentration changes between 50 mM and 25 mM after cultivation at 0 mM NaCl (a) or 100 mM NaCl (c) in the presence of food for 6 hr. A to I …
Figure 5—figure supplement 1
clh-1 missense mutations affect calcium dynamics of ASER.
Figure 5—figure supplement 2
Effect of clh-1(pe) mutations on calcium dynamics of ASER in starved animals.
(a and e) Calcium responses of ASER stimulated by NaCl concentration changes between 50 mM and 25 mM after cultivation at 0 mM NaCl (a) or 100 mM NaCl (e) for 6 hr in the absence of food. The shaded …
Figure 6 with 2 supplements
Missense mutations in clh-1 attenuate both klinotaxis and klinokinesis, as well as AIB response and reversal in response to salt increase.
(a and b) clh-1(pe) mutants show defects in migration bias in salt chemotaxis. Bias of klinotaxis (a) and klinokinesis (b), represented by weathervane index and pirouette index, respectively. In …
Figure 6—figure supplement 1
Quantification of the navigation behaviors.
(a and b) Chemotaxis after cultivation at 0 mM NaCl in the presence of food. Klinotaxis bias represented by curving rate (a) and klinokinesis bias represented by probability of pirouette (b). dC/dn …
Figure 6—figure supplement 2
Quantification of reversal behaviors in microfluidics arena.
Locomotion of wild type, clh-1(pe572), and clh-1(pe572) that express clh-1(wt) cDNA in ASER (pe572 + rescue). Locomotion of animals was categorized as either forward (white) or reversal (red). …
Figure 7
A model for the mechanism of impaired low salt chemotaxis of clh-1(pe) mutants.
Animals experience salt concentration fluctuation along with migration on salt gradient, which is depicted by blue waves (top). Salt information is sensed by the ASER neuron and translated into …
Author response image 1
Whole cell currents of the Xenopus oocytes that express CLH-1 under the presence of extracellular gluconate.
Tables
Author response table 1
The reversal potential of wild-type and mutant CLH-1 channels for Cl- and HCO3- at pH7.
Number of samples in parentheses, unit: mV.
| Cl- | HCO3- | |
|---|---|---|
| CLH-1(wt) | -20.89 ± 5.03 (20) | -26.07 ± 14.08 (19) |
| CLH-1(pe572) | -19.30 ± 4.44 (18) | -29.50 ± 16.36 (18) |
| CLH-1(pe577) | -14.20 ± 11.46 (8) | -19.69 ± 9.92 (8) |
Additional files
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Supplementary file 1
Strain list.
- https://cdn.elifesciences.org/articles/55701/elife-55701-supp1-v1.docx
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Supplementary file 2
Genotyping primer.
- https://cdn.elifesciences.org/articles/55701/elife-55701-supp2-v1.docx
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Supplementary file 3
Plasmid list and use.
- https://cdn.elifesciences.org/articles/55701/elife-55701-supp3-v1.docx
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Supplementary file 4
Plasmid sequences.
- https://cdn.elifesciences.org/articles/55701/elife-55701-supp4-v1.zip
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Supplementary file 5
Statistics.
- https://cdn.elifesciences.org/articles/55701/elife-55701-supp5-v1.zip
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55701/elife-55701-transrepform-v1.pdf
