KIN-29(S517A) mutant does not translocate to the nucleus during sleep and does not rescue the short-sleeping phenotype of kin-29.

(A) Confocal images of KIN-29::GFP in odr-4(+) neurons (green) of WT animals. Nuclei are identified by blue DAPI staining. One hour prior to L1 lethargus and 1 hr after L1 lethargus (n = 3–5 animals for each condition), KIN-29::GFP is mostly cytoplasmic. During mid and late L1 lethargus (n = 5–7 animals for each condition), KIN-29, but not the KIN-29(S517A) mutant, localizes to the nucleus of a subset of odr-4(+) neurons. Scale is 10 μm. (B) Percentage of KIN-29::GFP in the subcellular compartment of odr-4(+) neurons before, during, and after lethargus/DTS of the L1 stage. Shown is the percentage of animals that show fully nuclear, intermediate (nuclear > cytoplasmic, and cytoplasmic > nuclear), and fully cytoplasmic location of GFP (S1 Data, Sheet 7B). (C) The KIN-29(S517A) mutant does not rescue the reduced L4 lethargus/DTS of kin-29 null mutants. Left graph: The fraction of quiescence in a 10-min moving window is shown with n = 3 animals for the WT trace, n = 4 animals for the kin-29 trace, n = 8 animals for the Podr-4::kin-29(S517A) trace, and n = 6 animals for the Podr-4::kin-29(WT) trace. The x-axis represents hours from the start of recording in the late L4 stage. The data from individual worms were aligned such that the start of lethargus quiescence occurred simultaneously. Shading indicates SEM. Right graph: Total body movement quiescence during L4 lethargus/DTS determined from the time-course data. Data are represented as the mean ± SEM. ***p < 0.001 by an ANOVA with Tukey multiple-comparisons test (S1 Data, Sheet 7C). DAPI, 4,6-diamidino-2-phenylindole; DTS, developmentally timed sleep; GFP, green fluorescent protein; L1 stage, first larval stage; L4 stage; fourth larval stage; ns, not significant; WT, wild type.