1. Volume 84, Issue 5, Pages 983-996 (December 2014)
The Balance between Cytoplasmic and Nuclear CaM Kinase-1 Signaling Controls the Operating Range of Noxious Heat Avoidance 
Lisa C. Schild, Laurie Zbinden, Harold W. Bell, Yanxun V. Yu, Piali Sengupta, Miriam B. Goodman, Dominique A. Glauser 
Neuron 
Volume 84, Issue 5, Pages (December 2014)
DOI: /j.neuron
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Figure 1
Acclimation at High Temperature and cmk-1(pg58) Mutation Reduce Thermal Avoidance
(A) Impact of thermal acclimation on noxious heat avoidance in wild-type (N2). Worms were placed on the cool side of a thermogradient and attracted to the hot side with a spot of attractive odorant. The distribution of animals was scored after 15 min and plotted as a probability density function. Worms were conditioned for 2 hr at 20°C or 28°C prior to testing. Data were pooled from at least five independent assays, each scoring more than 100 animals.
(B) Heat avoidance index as a function of acclimation time at 28°C. Points are the mean ± SEM of n ≥ 5 assays. A one-way ANOVA showed a significant effect of acclimation time (p < 0.01). ∗p < by Dunnett’s post hoc tests versus baseline (at t = 0). Smooth line is a fit of the data to a single exponential decay.
(C) Distribution of wild-type (N2) animals and cmk-1(pg58) mutants in noxious heat thermogradients (29°C–37°C), tested as in (A). Data were pooled from at least 26 independent assays/genotype, each scoring more than 100 animals.
(D) Heat avoidance index of wild-type animals, cmk-1(pg58) gain-of-function mutants, and cmk-1(ok287) null mutants maintained at 20°C or acclimated 1 hr at 28°C. Bars are the mean ± SEM of at least five independent assays/genotype. A one-way ANOVA showed a significant genotype × acclimation interaction effect and was followed by Bonferroni post hoc tests: ∗p < 0.01; ns, not significant. See also Figure S1.
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3. Figure 2
cmk-1(pg58) Encodes a Truncated Protein that Accumulates in the Nucleus
(A) Structure of the cmk-1 genomic locus, position of deletion/mutations and corresponding CMK-1 open reading frames, and domain organization of the predicted CMK-1 protein isoforms. NES, nuclear export sequence; NLS, nuclear localization signal.
(B) Total mRNA in young adults analyzed with RT-qPCR targeting cmk-1 and the reference gene cdc-42. Results are mean ± SEM of three independent biological samples, each assayed in duplicate. A one-way ANOVA showed a significant genotype effect (p < 0.01) and was followed by Bonferroni post hoc tests: ∗p < 0.01 versus N2; ns, not significant.
(C) Fluorescence images of animals expressing either CMK-1(1–348)::GFP (full-length CMK-1, left) or CMK-1(1–304)::GFP (corresponding to the pg58 truncation, right). The constructs were expressed under the control of the cmk-1 promoter. Scale bars, 100 μm.
(D and E) Subcellular localization of full-length CMK-1(1–348) and truncated CMK-1(1–304) proteins expressed under the control of the cmk-1 promoter. Representative deconvolved images taken in the head (D) and the tail (E). Scale bars, 3 μm.
(F) Distribution of the subcellular localization of CMK-1 isoforms shown in (D) and (E). The numbers of neurons assessed are indicated to the right of each bar. ∗p < 0.01 by chi-square test. These data were obtained in the cmk-1(wt) genetic background. We observed similar results in the cmk-1(ok287) null background (Figure S2).
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4. Figure 3
pg58 Acts in the FLP Thermonociceptor Neurons to Regulate Noxious Heat Avoidance
(A) Heat avoidance index in wild-type, cmk-1(+/pg58) heterozygous, and cmk-1(pg58) homozygous animals carrying cell-specific rescue transgenes. cmk-1 cDNA expression was driven by the indicated promoters. The constructs were polycistronic and separately expressed fluorescent proteins through trans-splicing (SL2::mCherry or SL2::GFP) in order to label the cells expressing cmk-1. Based on the number and location of fluorescent cell bodies, expression patterns were consistent with those reported in the literature for each promoter tested. At least three independent stable lines per construct were examined, and the data were pooled across lines. Bars are the mean ± SEM. A one-way ANOVA showed significant differences across genotypes (p < 0.001) and was followed by Bonferroni post hoc tests. ∗p < 0.01 versus cmk-1(pg58) control in the absence of rescue.
(B) Noxious heat avoidance level is correlated with expression of CMK-1 in FLP, but not in PLM. Each point represents a single stable transgenic line carrying a [mec-3p::cmk-1] rescue construct in the cmk-1(pg58) background. As revealed by GFP fluorescence, expression was variable in FLP but consistently higher in the touch receptor neuron PLM. The heat avoidance index was plotted as a function of the rescue transgene expression penetrance in FLP (left panel) and in PLM (right panel). A significant correlation was found for FLP (Spearman’s rho = 1; p < 0.01), but not for PLM (Spearman’s rho = 0.36; p = 0.55).
(C) Transgenic animals expressing GFP in at least one FLP neuron (FLP+) were compared to those lacking any detectable GFP in FLP (FLP−) and to nontransgenic cmk-1(pg58) control animals. Data are the mean ± SEM (≥5 independent assays).∗p < 0.01; ns, not significant by Student’s t tests versus cmk-1(pg58) with no transgene.
(D) Heat avoidance index in animals where the FLP (and PLM) neurons were ablated with suicide transgenes. Bars are the mean ± SEM. A two-way ANOVA showed a significant ablation × cmk-1 allele interaction effect (p < 0.001). ∗p < 0.05, ∗∗p < by Bonferroni post hoc tests.
(E) Subcellular localization of CMK-1 in FLP and in PLM neurons of [mec-3p::CMK-1(wt)::GFP] transgenic animals. Worms were either maintained at 20°C or exposed to 28°C for 2 hr, a treatment known to reduce thermal avoidance behavior (Figures 1A and 1B). ∗∗p < 0.01; ns, not significant by chi-square test.
(F) Time course of CMK-1 nuclear translocation during acclimation at 28°C of [mec-3p::CMK-1(wt)::GFP] transgenic animals. ∗p < 0.01 versus t = 0 by Fisher’s exact tests with Bonferroni corrections for multiple testing.
See also Figure S3.
Neuron  , DOI: ( /j.neuron )
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5. Figure 4
Nuclear CMK-1 Reduces Avoidance, while Cytoplasmic CMK-1 Promotes Avoidance
(A) Schematic of the constructs used to manipulate CMK-1 protein subcellular localization. The ectopic NES sequence was a canonical sequence, while the NLS was from the C. elegans EGL-13 protein (See Experimental Procedures).
(B) Subcellular localization of the CMK-1 isoforms presented in (A). Transgene expression was driven by the endogenous cmk-1 promoter, and localization was scored as for Figures 2D–2F. ∗p < 0.01 as compared to untargeted control by chi-square tests. The numbers of neurons assessed are indicated.
(C) Truncated CMK-1(1-304) proteins were expressed in cmk-1(ok287) null mutants under the control of cmk-1 or mec-3 promoters. Mean values (±SEM) for the heat avoidance index were derived from at least two independent transgenic lines, each producing similar scores. A one-way ANOVA showed a significant genotype effect (p < 0.001) and was followed by Bonferroni post hoc tests: #p < 0.01 as compared to the cmk-1(ok287) control without transgene.
(D) The indicated constructs (schematized in A) were expressed in cmk-1(ok287) null mutants under the control of cmk-1 or mec-3 promoters. Mean values (±SEM) for the heat avoidance index were derived from at least two independent lines, each producing similar scores. A one-way ANOVA showed a significant genotype effect (p < 0.001) and was followed by Bonferroni post hoc tests: #p < 0.01 as compared to the cmk-1(ok287) control without transgene.
(E) The indicated constructs (schematized in A) were expressed in cmk-1(pg58) mutants under the control of cmk-1 or mec-3 promoters. Mean values (±SEM) for the heat avoidance index were derived from at least two independent lines, each producing similar scores. A one-way ANOVA showed a significant genotype effect (p < 0.001) and was followed by Bonferroni post hoc tests: ∗ ∗ p < as compared to the cmk-1(pg58) control without rescue; ∗p < as compared to wild-type (N2).
(F) The effect of 1 hr acclimation at 28°C on wild-type and transgenic animals overexpressing the cytoplasmic isoform CMK-1(1–348)+NES under the control of cmk-1 or mec-3 promoters. A two-way ANOVA showed a significant genotype × acclimation interaction effect (p < 0.01). ∗p < 0.01; ns, not significant by a priori contrasts.
(G and H) Noxious heat avoidance in transgenic animals expressing CMK-1(1–304) (G) or CMK-1(1–348) (H) with a mutated catalytic site residue (K52A). Bars are the mean ± SEM of n ≥ 10 independent assays, each scoring at least 100 animals. Separate one-way ANOVAs showed significant genotype effects (p < 0.01). ∗∗p < 0.01; ns, not significant versus the cmk-1(ok287) (G) or cmk-1(pg58) (H) control by a priori contrasts.
See also Figure S4.
Neuron  , DOI: ( /j.neuron )
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6. Figure 5
FLP Neurons in cmk-1(pg58) Have Intact Thermal Sensation but Fail to Increase Neuropeptide Secretion in Response to Heat
(A) Heat-evoked Ca2+ traces in FLP of wild-type and cmk-1(pg58) animals. Lines are the average (±SD) of ≥8 recordings per genotype.
(B) Peak values from data in (A). Bars are the mean ± SEM of ≥8 recordings per genotype.
(C and D) Neuropeptide secretion from wild-type and cmk-1(pg58) FLP neurons. A FLP-21::mCherry neuropeptide reporter was selectively expressed into FLP, and red fluorescence was measured in coelomocytes. Representative coelomocyte pictures are presented. Points are the average fluorescence intensity (±SEM) of at least 133 coelomocytes. Animals grown at 20°C were incubated for 1 hr at the indicated temperature (C) or shifted at 28°C at t = 0 and incubated for up to 2 hr (D). Two-way ANOVAs indicated significant temperature × genotype (C) and time × genotype (D) interaction effects (both p < 0.001) and were followed by Bonferroni post hoc tests: ∗∗p < 0.01 versus wild-type baseline (t = 0 or T = 15°C); #p < 0.05 and ##p < 0.01 versus corresponding wild-type values.
See also Figure S5.
Neuron  , DOI: ( /j.neuron )
Copyright © 2014 Elsevier Inc. Terms and Conditions

7. Figure 6 CKK-1 Acts Upstream of CMK-1 to Control Thermal Avoidance
(A) ckk-1 genetically interacts with cmk-1. Heat avoidance index as a function of genotype; bars are mean ± SEM (n ≥ 15 independent assays, each scoring at least 100 animals). A one-way ANOVA showed a significant genotype effect (p < 0.001) and was followed by Bonferroni post hoc tests: ∗p < 0.01 versus wild-type, #p < 0.01 versus cmk-1(pg58).
(B) Heat avoidance index as a function of genotype; bars are mean ± SEM (n ≥ 15 independent assays, each scoring at least 100 animals). A one-way ANOVA showed a significant genotype effect (p < 0.001) and was followed by Bonferroni post hoc tests: ∗p < 0.01 versus wild-type.
(C) Adaptation of heat avoidance after 1 hr acclimation at 28°C versus 20°C in wild-type animals and ckk-1(ok1033) null mutants. Bars are mean heat avoidance index ± SEM (n ≥ 10 independent assays, each scoring at least 100 animals). A two-way ANOVA showed a significant genotype × acclimation temperature interaction effect (p < 0.001). ∗p < 0.01; ns, not significant by Bonferroni post hoc tests.
(D) Impact of ckk-1 knockout and of the T197A mutation on the ability of truncated CMK-1(1–304) isoforms to alter heat avoidance. Transgenic proteins expressed under the control of the endogenous cmk-1 promoter. Bars are the mean (±SEM) heat avoidance indices derived from n ≥ 10 independent assays, each scoring at least 100 animals. Separate Student’s t tests were performed, and results were corrected for multiple testing with a Bonferroni correction. ∗p < 0.01; ns, not significant.
(E) Impact of the T179A mutation on the ability of full-length CMK-1(1–348) proteins to rescue heat avoidance in the cmk-1(pg58) mutant. Full-length isoforms expressed under the control of the cmk-1 and mec-3 promoters, as indicated. Bars are mean ± SEM of n ≥ 10 independent assays, each scoring at least 100 animals. A one-way ANOVA showed a significant genotype effect (p < 0.001). ∗p < 0.01 versus no transgene control; ns, not significant by Bonferroni post hoc tests.
(F) Nuclear translocation time course of CMK-1(1–348)::GFP in the FLP neurons during acclimation in wild-type and ckk-1(ok1033) animals. Scored as in Figure 3F. ∗p < 0.01 versus corresponding wild-type data by Fisher’s exact tests with Bonferroni corrections for multiple testing.
(G) Impact of knocking out ckk-1 and of mutating the CKK-1 target residue T179 of CMK-1 on the subcellular localization of CMK-1(1-348) in FLP neurons after acclimation at 28°C. ∗p < versus CMK-1(1–348) by chi-square tests.
(H) Impact of knocking out ckk-1 and of mutating the CKK-1 target residue T179 of CMK-1 on the subcellular localization of CMK-1(1-304) in FLP neurons. ns, not significantly different from CMK-1(1–304) by chi-square tests.
(I) Impact of the T179A mutation on the ability of nuclear isoforms of CMK-1 to reduce thermal avoidance in cmk-1(ok287) mutants. Bars are mean heat avoidance indices ± SEM (n ≥ 10 independent assays, each scoring at least 100 animals). Two one-way ANOVAs showed significant genotype effects (each p < 0.001). ∗∗p < 0.01; ∗p < 0.05; ns, not significant versus the no transgene control by Bonferroni post hoc tests.
(J) Model for the control of thermal avoidance by CKK-1/CMK-1 signaling in FLP thermal nociceptors. In wild-type animals that have not experienced noxious temperatures, CMK-1 is found primarily in the cytoplasm and promotes thermal avoidance in a CKK-1-dependent manner from this subcellular locus. Upon prolonged exposure to heat, CMK-1 translocates to the nucleus to produce apparent analgesia and decrease thermal avoidance. Both temperature-dependent translocation of CMK-1 and its activity in the nucleus require an intact CKK-1 kinase.
Neuron  , DOI: ( /j.neuron )
Copyright © 2014 Elsevier Inc. Terms and Conditions