Figure 1. A muscle-specific ubiquitin fusion degradation (UFD) model substrate for monitoring myosin-associated proteostasis:(A) The UFD model substrate UbV-mCherry monitors ubiquitin-dependent proteasomal degradation specifically in the C. elegans body wall muscle (BMW). (B, C) The muscle-specific UFD model substrate is stabilized by proteasome inhibition using bortezomib (B) and RNAi depletion of indicated regulators (C). Representative Western blots of duplicate (n = 2) worm lysates with the indicated treatments for detection of UbV-mCherry (RFP) and tubulin. (D) The myosin chaperone UNC-45 folds myosin and assembles it into myofilaments. Loss-of-function (lof) mutations in UNC-45 (*) lead to misfolding and disorganization of myosin. (E) The muscle-specific UFD model substrate is stabilized by a temperature-sensitive (ts)
unc-45(
m94) allele that leads to myosin misfolding and increased proteasomal degradation at elevated cultivation temperatures. WT: wild-type. Scale bar: 200 μm. (F) At the permissive temperature, the muscle-specific UFD model substrate is only slightly stabilized by the
unc-45(
m94) ts allele, but more strongly stabilized by a
skn-1a(
mg570) lof allele. Representative Western blot of worm lysates with the indicated genotypes for detection of UbV-mCherry (RFP) and tubulin. (G) Quantification of UbV-mCherry bands in Western blots from n = 7 independent experiments. Data show means ± SEM of n = 7 independent experiments; ***p < 0.001; ratio paired T-test. (H) A transcriptional reporter under control of the SKN-1A-responsive proteasomal subunit gene promoter
rpt-3 is upregulated by the
unc-45(
m94) allele at all temperatures. Representative Western blot of worm lysates with the indicated genotypes and treatments detecting GFP and tubulin. (I) Transgenic expression of the human pathogenic variant CeUNC-45(I422P) corresponding to HsUNC-45B(S403P) shows a dominant negative effect on motility of adult worms. Data show mean values ± SEM obtained from n = 3 independent experiments; **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA with Dunn's post-hoc test. (J, K) Transgenic expression of the human pathogenic variant CeUNC-45(I422P) stabilizes the muscle-specific UFD model substrate already in the WT genetic background. (J) Representative Western blot of worm lysates with indicated genotypes detecting the muscle-specific UFD substrate (RFP), tubulin, total UNC-45 and transgenic UNC-45-FLAG. (K) Quantification of UbV-mCherry signals in Western blots of n = 3 biological replicates. Data show mean values ± SEM obtained from n = 3 independent experiments; *p < 0.05; ratio paired T-test.