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oai:figshare.com:article/34049524 2026-10-01T17:47:00Z category_4 category_12 category_13 category_14 category_15 category_16 category_64 category_128 portal_5 item_type_1 month_year_10_2026

A forward genetic screen revealed that egl-2(G574E) mutants are defective in sleep. Xinyu Huang (679541) Sudharsan Kannan (25158064) Gail A. Robertson (15007385) Han Wang (254423) Biochemistry Cell Biology Genetics Molecular Biology Neuroscience Physiology Cancer Hematology regulate neuronal excitability promoting neuron ala influence channel gating hallmark cyclic nucleotide genetic analysis demonstrated forward genetics reveals electrophysiological analysis showed dependent channel activation calcium imaging showed binding homology domains adjacent channel subunits accelerates activation kinetics subfamily kcnh channels conserved gating mechanism important sleep regulator findings identify egl div >< p caenorhabditis elegans </ similarly suppresses sleep 2 functions cell elegans </ suppresses sleep kcnh voltage vivo </ conserved cnbhd central sleep shifts voltage second substitution negative potentials intrinsic ligand g574r ), function effects corresponding residue (A) Schematic of the forward genetic screen to isolate egl-2(sy985). The parental strain (P0) contains the HS::EGF transgene, which drives worms into EGF-induced sleep after a brief heat shock. Phsp-16.41 is a heat-shock-inducible promoter. F2 progeny of EMS-mutagenized parental animals were screened for sleep-defective mutants that exhibited feeding and locomotion after heat shock. The worm illustration was created in BioRender. Wang, H. (2026) <a href="https://BioRender.com/4ukdkrg" target="_blank">https://BioRender.com/4ukdkrg</a>. (B) Representative chromatograms from Sanger sequencing of the genomic DNA from egl-2(+) and egl-2(sy985) animals. A G-to-A point mutation (highlighted in the red box) was identified in the egl-2 coding sequence of the sy985 allele, resulting in a glycine-to-glutamic acid substitution at position 574 (G574E). (C-D) Quantification of sleep behavior as the fraction of quiescent worms for the indicated genotypes and conditions. All strains contain the transgene (HS::EGF) for conditional overexpression of EGF via 15-min heat shock (HS) at 33 °C to induce sleep. Pre and post refer to before and 2 h after heat shock, respectively. Each dot represents one independent assay containing 15–25 animals on a single plate. Mean ± SEM are shown. ns, not significant; ****, p < 0.0001. One-way ANOVA with Dunnett’s multiple-comparisons test for post-heat shock samples, comparing each genotype with wild type. (C) gDNA rescue 1 and 2 are two independent extrachromosomal arrays carrying the same fosmid containing the wild-type egl-2 genomic sequence. (D) egl-2(tan200) is a CRISPR-generated allele carrying the same G574E substitution as egl-2(sy985). (E) Time course of endogenous stress-induced sleep in animals of the indicated genotypes, shown as the fraction of quiescent worms at each time point. Pre refers to before heat shock. N2 and egl-2(tan200) animals were used as wild type and egl-2(G574E), respectively. Data were collected from three independent replicates, with 15–25 animals of each strain assayed on separate plates in each replicate. Mean ± SEM are shown. ns, not significant; *, p < 0.05; **, p < 0.001. Two-tailed unpaired Student’s t-test for comparing wild type with egl-2(tan200) mutants at each time point. 2026-10-01T17:54:06Z Image Figure 10.1371/journal.pgen.1012255.g001 https://figshare.com/articles/figure/_p_A_forward_genetic_screen_revealed_that_i_egl-2_G574E_i_mutants_are_defective_in_sleep_p_/34049524 CC BY 4.0