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. 2019 Sep 16;9:13315. doi: 10.1038/s41598-019-49633-9

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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CED-4 EF-hand mutations do not disrupt CED-4 apoptosis functions. (a) Amino acid substitutions in the predicted CED-4 Ca²⁺-binding domains, EF-hand 1 (EFH1) and EF-hand 2 (EFH2) were introduced via CRISPR genome editing. Amino acids labeled green are potential Ca²⁺-binding amino acids; in each domain, we replaced two of these residues with alanine (red label). ced-4(∆) is a likely null allele that could potentially only express a partial CARD domain followed by a random short sequence (labeled purple). Actual allele names are: EFH1 ced-4(bz401); EFH2 ced-4(bz410); EFH(1 + 2) ced-4(bz406); ∆ced-4(bz404); CARD: caspase recruitment domain; HD: Helical domain; WHD: Winged-helix domain. (b) Representative pictures of the tail touch receptor neurons labeled by P_mec-4GFP in the background of various ced-4 alleles. In WT, two PLM tail neurons survive; in ced-4 null mutants, as many as two additional neurons survive and differentiate, so 3–4 cells may be visualized. (c) The PLM tail touch receptor neuron count for ced-4 mutants, n = 40 animals for each group in one trial. ****p < 0.0001 as compared to the wild type, Chi-square test.