Figure 1. Functional analysis of pathogenic VPS45 variants in C elegans:(A) Structures of human VPS45 and C. elegans VPS-45 proteins based on I-TASSER structural modeling. The 3D models with the highest confidence score are shown. The VPS45 proteins contain three domains, with domain 3 split into D3a and D3b. The SCN5-associated missense mutations are clustered in the putative hinge region. (B) Alignment of VPS45 protein sequences from Homo sapiens (NP_009190), Mus musculus (NP_038869.1), Gallus gallus (XP_040508302.1), Danio rerio (NP_001243585.1), Drosophila melanogaster (AAF54403.1), Caenorhabditis elegans (NP_741714.1), Saccharomyces cerevisiae (CAA96801.1). The SCN5-associated residues are boxed, and the amino acid residues conserved among the VPS45 homologs are highlighted in gray. (C) Rescue experiments of SCN5-associated hVPS45 mutants for ts larval lethality. Bar graphs and dots represent mean and individual body lengths of animals cultured at the restrictive temperature (25
x2103;), respectively. Error bars indicate SEM. A total of 40-60 animals from two independent lines were measured for each hVPS45 construct (WT or variant). ****p<0.0001, ns:not significant. (D) Proportions of developmental stages of animals measured in (C). (E) Fluorescence micrographs of adult hermaphrodites of wild-type,
vps-45 (
tm246), and hVPS45 strains (WT and variant) carrying the arIs37 [
myo-3p::ssGFP]. The left column shows worms at low magnification and the right column shows individual coelomocytes at high magnification. In the
vps-45 (
tm246) mutant, the secreted GFP is not endocytosed in the coelomocytes and accumulates in the body cavity. The endocytosis defects were rescued by expression of hVPS45 (wild-type or variant). Arrows indicate coelomocytes endocytosing GFP. Individual worms and coelomocytes are indicated by white lines. Scale bars, 100 µm (left panels) and 10 µm (right panels).