Homeostatic Responses Regulate Selfish Mitochondrial Genome Dynamics in C. elegans

Abstract

Mutant mitochondrial genomes (mtDNA) can be viewed as selfish genetic elements that persist in a state of heteroplasmy despite having potentially deleterious metabolic consequences. We sought to study regulation of selfish mtDNA dynamics. We establish that the large 3.1-kb deletion-bearing mtDNA variant uaDf5 is a selfish genome in Caenorhabditis elegans. Next, we show that uaDf5 mutant mtDNA replicates in addition to, not at the expense of, wild-type mtDNA. These data suggest the existence of a homeostatic copy-number control that is exploited by uaDf5 to "hitchhike" to high frequency. We also observe activation of the mitochondrial unfolded protein response (UPR(mt)) in uaDf5 animals. Loss of UPR(mt) causes a decrease in uaDf5 frequency, whereas its constitutive activation increases uaDf5 levels. UPR(mt) activation protects uaDf5 from mitophagy. Taken together, we propose that mtDNA copy-number control and UPR(mt) represent two homeostatic response mechanisms that play important roles in regulating selfish mitochondrial genome dynamics.

Keywords: C. elegans; droplet digital PCR; evolution; heteroplasmy; mitochondrial unfolded protein response; mitophagy; mtDNA; mtDNA copy number control; selfish genetic element.

Figure 1. Mutant mtDNA uaDf5 can be forced out from a stably persisting heteroplasmy in C. elegans

(A) Schematic of C. elegans mtDNA showing the uaDf5 and mptDf1 deletions (long and short red bars, respectively). Grey arrows show protein and rRNA-encoding genes and their orientation. White boxes show genes encoding tRNAs. (B) Schematic illustrating the selection strategy to force loss of uaDf5 mtDNA from a heteroplasmic C. elegans line. Each generation, the progeny of individuals with the lowest uaDf5 levels were selected for subsequent propagation. (C) Single worm PCR of wildtype and uaDf5 mtDNA. Successive propagation of individual worms with low uaDf5 levels (red boxes) results in complete loss of uaDf5 mtDNA from the population over multiple generations. (D) ddPCR data from single worms confirming complete loss of uaDf5. Positive droplets containing uaDf5-specific PCR product exhibit increased fluorescence intensity (blue) compared to negative droplets that contain no uaDf5 mtDNA (gray). For each droplet, the droplet reader detects droplet size, shape, and fluorescence intensity, and automatically distinguishes positive from negative droplets on the basis of these criteria. Sample 1, control containing uaDf5.

Figure 2. Quantification of mtDNA copy number dynamics reveals mtDNA copy number control

(A) Histogram showing uaDf5 frequency (%) distribution in individuals from a population stably maintaining uaDf5 mtDNA. Heteroplasmy frequency was determined using ddPCR to quantify wildtype and uaDf5 mtDNA copy number in single individuals. (B) mtDNA levels in individual day 4 adult worms, normalized to actin and rank-ordered by uaDf5 mtDNA copy number. (C) Wider variation in uaDf5 relative to wildtype copy number (p<0.05) suggests that wildtype mtDNA, but not uaDf5 mtDNA, is subject to homeostatic copy number control. Grey data points show mtDNA copy number from single individuals. Box and whisker plot shows the median, lower and upper quartile (boxes), and minimum and maximum (error bars) mtDNA copy number. (D) mptDf1 frequency distribution obtained from single individuals from a population stably maintaining mptDf1 heteroplasmy. (E) mtDNA levels in individual L4 worms, normalized to actin and rank-ordered by mptDf1 copy number. (F) Similar to uaDf5, wider variation in mptDf1 relative to wildtype copy number (p<0.05) suggests that wildtype mtDNA, but not mptDf1 mtDNA, is subject to homeostatic copy number control. AU, arbitrary units.

Figure 3. Mitochondrial function is perturbed in uaDf5 animals

(A) Schematic showing expected expression of mtDNA-encoded transcripts. The presence of uaDf5 mtDNA is expected to result in stoichiometric imbalance of gene expression, as the expression of uaDf5 and wildtype mtDNA copies (red and blue lines, respectively) combine to generate total expression (black line) at elevated levels for genes located outside the deletion but at wildtype levels for genes missing from the uaDf5 mtDNA. (B) Animals heteroplasmic for uaDf5 exhibit expression levels similar to that of wildtype animals for mtDNA-encoded genes affected by the deletion (CYTB and ND1), as well as a nuclear-encoded mitochondrial gene (NUO2) and actin. However, uaDf5 heteroplasmy results in overexpression for mtDNA-encoded genes located outside the uaDf5 deletion (COXI, COXII, COXIII, ND4, and ND5). All transcript levels are normalized to wildtype. Error bars represent standard deviation. (C) Mitochondrially targeted GFP (GFP^mt), but not cytosolic GFP (cGFP^cyt), is significantly reduced in uaDf5 heteroplasmic individuals. (D) Western blot analysis of wildtype and uaDf5 heteroplasmic animals expressing GFP^mt reveals reduced levels in uaDf5 heteroplasmic individuals relative to actin. Data are shown from two biological replicates each for wildtype and uaDf5 strain. (E) Fluorescence increase in uaDf5 animals stained with mitochondrial membrane potential independent dye MitoTracker Green FM and (F) membrane potential dependent dye TMRE. Error bars represent standard deviation. AU, arbitrary units.

Figure 4. UPR^mt is activated in heteroplasmic animals carrying uaDf5 mtDNA

(A) Transcription of two UPR^mt-activated molecular chaperones (hsp-60 and hsp-6) is increased in individuals with uaDf5 compared to wildtype individuals. (B) Quantification of fluorescence between wildtype homoplasmic and uaDf5 heteroplasmic animals shows increased activation of the UPR^mt marker hsp-60∷GFP in the presence of uaDf5 mtDNA. Each data point is from a single individual picked randomly from a population. (C) Visual comparison of GFP fluorescence between uaDf5 and wildtype animals, each expressing hsp-60∷GFP. Wildtype animals were picked at random from a population but only uaDf5 animals with apparent fluorescence were picked to show UPR^mt activation. (D) Positive relationship between uaDf5 frequency and hsp-60∷GFP fluorescence (trendline) indicates that UPR^mt activation increases at higher uaDf5 frequency. Each data point corresponds to a single individual. Error bars represent standard deviation. AU, arbitrary units.

Figure 5. Loss of UPR^mt activation results in decreased uaDf5 levels but does not affect mtDNA copy number control

(A) Growth under RNAi-mediated knockdown of atfs-1, required for UPR^mt activation, results in a shift to lower uaDf5 frequency relative to growth under control conditions (p<0.05). (B) uaDf5 frequency decreases in heteroplasmic animals homozygous for the atfs-1(tm4525) loss-of-function allele compared to heteroplasmic animals that express wildtype atfs-1, in which high uaDf5 levels are stably maintained. uaDf5 frequency decreases further in the atfs-1 null animals after multiple generations but is not lost completely. (C) Quantification of mtDNA copy number in individual day 4 adult animals homozygous for the atfs-1 loss-of-function allele, normalized to actin and rank-ordered by uaDf5 mtDNA copy number. (D) Wider variation in uaDf5 relative to wildtype copy number (p<0.05) in atfs-1 null animals suggests that mtDNA copy number control persists in absence of UPR^mt. (E) PCR of single heteroplasmic individuals against the atfs-1 wildtype or atfs-1 null nuclear background shows that uaDf5 is retained in both lines but is at lower levels in the null animals after about 30 generations. Note that because mutant and wildtype templates compete for amplification, the wildtype band appears fainter when uaDf5 levels are high but does not actually reflect reduced wildtype mtDNA levels (see Fig. 2B). Error bars represent standard deviation. AU, arbitrary units.

Figure 6. Persistence of uaDf5 at high frequency depends in part on UPR^mt activation

(A) Growth under RNAi-mediated knockdown of atfs-1 across seven generations reduces average uaDf5 frequency. However, restoration of atfs-1 expression by returning atfs-1 knockdown animals to control conditions results in recovery of elevated uaDf5 frequency in a single generation. (B) When starting uaDf5 frequency is high (75-80%), constitutive UPR^mt activation in individuals heterozygous for an atfs-1 gain-of-function allele causes no further rise in average uaDf5 frequency; (C) however, uaDf5 frequency rises when the atfs-1 gain-of-function allele is crossed into a strain harboring lower uaDf5 levels (∼30%). Error bars represent standard deviation.

Figure 7. UPR^mt protects uaDf5 from mitophagy

(A) Heteroplasmic individuals exhibit delayed growth: as 100% of progeny from wildtype parents reach adulthood in three days, approximately 10% of uaDf5 progeny remain in the larval stage. Knockdown of atfs-1 showed no effect on development in homoplasmic wildtype animals and did not further enhance developmental delay in uaDf5 heteroplasmic animals. (B) No significant difference was observed between uaDf5 and wildtype animals, or between atfs-1 knockdown and control conditions, on the percentage of embryos that remain unhatched after one day or (C) on the percentage of lethality among day 4 adults. (D) Quantification of Pink-1∷GFP fluorescence shows increased mitophagy in uaDf5 animals upon pdr-1;atfs-1 double knockdown compared to knockdown of pdr-1 alone. AU, arbitrary units. (E) Crossing scheme employed to isolate uaDf5 animals in wildtype, atfs-1 null, pdr-1 null, and atfs-1;pdr-1 double mutant backgrounds. (F) Quantification of uaDf5 levels shows recovery of uaDf5 levels in atfs-1;pdr-1 double mutants compared to atfs-1 single mutant animals. uaDf5 recovers to the highest levels in pdr-1 single mutants. Error bars represent standard deviation. AU, arbitrary units.