Figure 3. In vivo tests for nuclear export sequence (NES) masking and nuclear localization signal (NLS) unmasking models.

(A) Schematic of the rejected model in which NES^288-294 would be masked by Ca²⁺/CaM binding. (B) Subcellular localization of CMK-1::mNG reporters carrying the indicated mutations in the FLP neurons of animals exposed for 90 min at 28°C. Data as nuclear/cytoplasmic signal ratio average (± SEM), showing that the CaM-binding-disrupting mutation W305S prevents nuclear localization independently of the NES^288-294 element. (C) Schematic of the retained model in which NLS^71-78 would be unmasked by Ca²⁺/CaM binding. (D) Subcellular localization of CMK-1(wt)::mNG and the NLS^71-78-disrupting mutant CMK-1(K71A/R74Q/R77S)::mNG at 20 and 28°C in both wild-type and unc-68(dom13) backgrounds. Data as nuclear/cytoplasmic signal ratio average (± SEM), showing that the NLS^71-78-disrupting mutations counteract the impact of the unc-68 gain-of-function mutation. *p<0.001 versus wild-type; ^#p<0.001 versus CMK-1(wt) for the corresponding condition by Bonferroni post-hoc tests. Experiments were run in parallel to those reported in Figure 2B, and CMK-1(wt) data are common across the two figures. (E) Subcellular localization of CMK-1::mNG reporters carrying the indicated mutations disrupting either NES^288-294, NLS^71-78, or both of them. Data as nuclear/cytoplasmic signal ratio average (± SEM), showing no effect of NLS^71-78 disruption at 20°C, even when the NES^288-294 is impaired. ns, not significant.

Figure 3—figure supplement 1. CMK-1 localization data distributions and statistical test results.

Violin plots with superimposed datapoints depicting the distributions of nucleus/cytoplasmic ratios of the indicated CMK-1::mNG reporters. Red bars: average. n: number of observations. p-Values obtained with Bonferroni post-hoc tests. (A) Data corresponding to Figure 3B. (B) Data corresponding to Figure 3D. (C) Data corresponding to Figure 3E.