Mitochondrial translation and morphology in response to RP haploinsufficiency in C. elegans. (A) Log₂ fold change estimates in RNA and translation efficiency (TE) levels were plotted along the y-axis for mitochondrial-encoded (mito) or nuclear-encoded (nuclear) electron transport chain (ETC) genes, as well as for a randomly selected gene set, for each RP haploinsufficient mutant. Log₂ fold changes for RNA and TE were plotted on the left and right sections, respectively, and are labeled at the top. Each row represents a different RP haploinsufficient mutant, with the genotype labeled on the right-most side. On the x-axis, “mito” and “nuclear” represent mitochondrially and nuclear-encoded ETC component genes, respectively. The “random” set represents a group of random genes, equal in size to the nuclear-encoded ETC component gene set. For statistical comparison of RNA and TE expression differences in mitochondrial or nuclear-encoded ETC genes. ROAST multivariate gene expression analysis was conducted (Wu et al., 2010). The P values derived from the ROAST analysis are indicated on each box plot. According to this analysis, mitochondrially encoded transcripts are translated significantly less in rpl-5(0)/+ and rpl-33(0)/+ mutants (P < 0.005). While the overall distribution is lower in rps-10 (0)/+ and rps-23(0)/+, the changes are more modest and not statistically significant (P > 0.05). Superscript numbers denote the specific wild type balancer chromosomes and are used to compare an RP mutant and the wild-type counterpart. Balancer chromosomes are denoted as follows: +¹ = tmC20, +² = tmC5, +³ = mIn1. (B) Representative mitochondrial morphology images using mitochondrial and nuclear-localized GFP in body wall muscle cells in RP mutants as well as stage-matched wild-type controls. Day 3 adult animals, which were transferred from 16°C to 23°C on the last 2 days were imaged. RP mutants in this analysis do not have a balancer chromosome. Images were taken with Stellaris Confocal with a 63× objective, and the scale bar represents 10 µm. (C) Distribution of mitochondrial morphological measurements of mutant and control animals. GFP signal localized to mitochondria was used to identify mitochondrial objects. Each mitochondrion was measured using convexity (degree to which shape differs from its convex hull), defect area (area outside of convex hull), and skeleton branch length. Mean measurements are represented as points, and the black dashed line corresponds to the wild-type mean. A two-sided Welch’s two-sample t test was used to compare differences in the mean measurement across each mutant relative to the wild type. Experiments in B and C were conducted with three biological replicates, analyzing multiple body wall muscle cells from at least nine animals per group (with >10³ mitochondria measurements per strain). Two mitochondria images per mutant and wild type were shown in B to represent variability of mitochondrial morphology per body wall cell.