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. 2023 Nov 3;14:7066. doi: 10.1038/s41467-023-42735-z

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© The Author(s) 2023

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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Fig. 6 — Steady-state ATPase activity of Hsp70 alone (white), and incubated with DNAJB6 WT (dark-gray) or LGMDD1-associated mutants (colored). DNAJB6^QPN mutant (³¹HPD³³ > ³¹QPN³³), deficient in Hsp70 binding, was used as a negative control (cyan). Data are means ± SEM (n = 3). b Titration of Hsp70^AF488 with full-length DNAJB6 WT (dark-gray) or LGMDD1-associated disease mutants (colored), monitored by steady-state fluorescence anisotropy shown in arbitrary units. Data are presented as mean values ± SEM, (n = 3 independent experiments). c Schematic representation of the different C. elegans DNAJB6 variants used. DNAJB6 WT corresponds to DNJ-24 protein with intact JD-GF inhibition and Hsp70 binding sites. DNAJB6^Open variant corresponds to DNAJ-24 in which 4 residues in the GF helix V were mutated to release the JD-GF inhibition (see Supplementary Fig. 9 for details). DNAJB6^Open-QPN is a double DNAJ-24 mutant that contains the mutations artificially lifting the JD-GF inhibition and mutations that abolish the ability of this variant to bind to Hsp70 (DNJ-24^QPN). d C. elegans muscle-fiber morphology visualized by Phalloidin-FITC staining, labeling F-actin for DNAJB6 WT, DNAJB6^Open mutant, and DNAJB6^Open-QPN double mutant. Animals expressing WT DNAJB6 display healthy muscle morphology, while DNAJB6^Open alters muscle morphology to disorganized myofibrils with an undulated appearance. The introduction of a second mutation to the Hsp70 binding region, DNAJB6^Open-QPN, rescues the abnormal phenotype, restoring healthy muscle morphology. Scale bars represent 10 µm. e Quantification of animals with the undulated phenotype from (d). Number of animals: Wild-type, n = 15; DNAJB6^Open, n = 18, DNAJB6^Open-QPN, n = 16. Statistics is Fisher’s exact test followed by Dunn’s multiple comparison test. f C. elegans muscle-fiber morphology visualized by Phalloidin-FITC staining, labeling F-actin for DNAJB6^Open (dnj-24(ety13)) and DNAJB6^Open with muscle-specific overexpression of Hsp70 (myo-3::hsp-1). Overexpression of Hsp70 restores the healthy phenotype of the muscles. g Quantification of animals with the undulated phenotype from (f). Statistics is Fisher’s exact test, two-tailed. Number of animals: Wild-type, n = 23; DNAJB6^Open, n = 18, DNAJB6^Open-QPN, myo-3::hsp-1 n = 24.