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. 2021 Nov 12;10:e71443. doi: 10.7554/eLife.71443

Figure 5. Engineered mutations affecting CMK-1 NLS71-78 and NES288-294 impact the adaptation of FLP-dependent behavior.

(A) Schematic of CMK-1 protein highlighting the position of the deletion in ok287 mutant and of the single-point mutations in syb1435 and syb1375 CRISPR/Cas9 engineered alleles affecting NLS71-78 and NES288-294, respectively. (B) Avoidance response to 4 s heat pulses (at 0.3–0.6 W /m2, see Materials and methods) in adult animals of the indicated genotypes (average ± SEM, n ≥ 21 plates, each scoring at least 50 worms). #p<0.001 versus wild-type by Bonferroni contrasts. (C) Noxious heat thermogradient assays with animals of the indicated genotype maintained at 20°C (blue) or adapted for 1 hr at 28°C (red). Dispersal of animals reported as the temperature corresponding to the third quartile of the worm distribution in the spatial thermogradient. Vertical axis minimum was set to the lowest temperature in the thermogradient (29.5°C). Data as average (± SEM) of n ≥ 12 assays, each scoring more than 100 animals. ##p<0.001 by Bonferroni contrasts. ns, not significant. (D) FLP optogenetic analysis: light dose–response curves in young adult [FLP::CoChR] animals in response to 0.5 s light stimuli. Data as fraction of trials producing a response (n = 90 trials per genotype) showing a reduced response in cmk-1(syb1375) with constitutive nuclear CMK-1 expression. *p<0.001 versus cmk-1(wt) by Fisher’s exact tests. (E) FLP optogenetic analysis: light avoidance rescue experiment in cmk-1(syb1375) mutant. Data acquired as in (D), using 100 W/m2 light stimuli, and expressed as fraction of trials producing reversal or not. n ≥ 100 trials per genotype, each aggregating data from three independent rescue lines. *p<0.01 versus wild-type by Fisher’s exact tests. Transgenes containing cmk-1p and mec-3p promoter both produced a rescue effect. (F) FLP optogenetic analysis: impact of heat adaptation. Data as in (E), in animals maintained at 20°C (naïve) or incubated for 90 min at 28°C (adapted), showing impaired adaptation in cmk-1(syb1475) mutants. n ≥ 120 light trials per condition. **p<0.01 versus naïve control by Fisher’s exact test. ns, not significant.

Figure 5—source data 1. Summary statistics and raw data for Figure 5.

Figure 5.

Figure 5—figure supplement 1. Optogenetic experiment controls.

Figure 5—figure supplement 1.

(A, B) FLP optogenetic analysis: light dose–response curves in young adult [FLP::CoChR] animals in response to 0.5 s light stimuli. Data as fraction of trials producing a response (n = 120 trials per condition). No reversal response above the spontaneous reversal rate is seen for red light stimuli or for blue light stimuli in the absence of ATR.