Disruption of mitochondrial dynamics affects behaviour and lifespan in Caenorhabditis elegans

Abstract

Mitochondria are essential components of eukaryotic cells, carrying out critical physiological processes that include energy production and calcium buffering. Consequently, mitochondrial dysfunction is associated with a range of human diseases. Fundamental to their function is the ability to transition through fission and fusion states, which is regulated by several GTPases. Here, we have developed new methods for the non-subjective quantification of mitochondrial morphology in muscle and neuronal cells of Caenorhabditis elegans. Using these techniques, we uncover surprising tissue-specific differences in mitochondrial morphology when fusion or fission proteins are absent. From ultrastructural analysis, we reveal a novel role for the fusion protein FZO-1/mitofusin 2 in regulating the structure of the inner mitochondrial membrane. Moreover, we have determined the influence of the individual mitochondrial fission (DRP-1/DRP1) and fusion (FZO-1/mitofusin 1,2; EAT-3/OPA1) proteins on animal behaviour and lifespan. We show that loss of these mitochondrial fusion or fission regulators induced age-dependent and progressive deficits in animal movement, as well as in muscle and neuronal function. Our results reveal that disruption of fusion induces more profound defects than lack of fission on animal behaviour and tissue function, and imply that while fusion is required throughout life, fission is more important later in life likely to combat ageing-associated stressors. Furthermore, our data demonstrate that mitochondrial function is not strictly dependent on morphology, with no correlation found between morphological changes and behavioural defects. Surprisingly, we find that disruption of either mitochondrial fission or fusion significantly reduces median lifespan, but maximal lifespan is unchanged, demonstrating that mitochondrial dynamics play an important role in limiting variance in longevity across isogenic populations. Overall, our study provides important new insights into the central role of mitochondrial dynamics in maintaining organismal health.

Keywords: Caenorhabditis elegans; DRP-1; DRP1; EAT-3; FZO-1; Mitochondria; Mitochondrial dynamics; Mitofusin 1; Mitofusin 2; OPA1; Transmission electron microscopy.

Fig. 1

Mitochondrial morphology in body wall muscles. a Schematic of the body wall muscle cells of C. elegans, which are arranged in four longitudinal bundles comprised 95 cells. Inset shows the mitochondrial network within a single muscle cell. b Representative images of body wall muscle with fluorescently labelled mitochondria (Pmyo-3::MLS::GFP). Arrows designate nuclei, which are also labelled with GFP in this transgenic strain. drp-1(tm1108) animals show elongated mitochondria similar to wild type as seen more closely in insets i and ii. eat-3(ad426) and fzo-1(cjn020) mutants exhibit more fragmented mitochondrial networks compared to wild type (iii, iv compared with i). Images are representative of  ≥ 10 muscle cells from nine worms analysed per genotype, visualised via confocal microscopy. Scale bars 20 μm. Insets are enlargements (× 3) of the indicated circular areas. c Mean size (µm²) of mitochondria in the body wall muscles as determined using object segmentation (SQUASSH). d Mean circularity of mitochondria in the body wall muscles, calculated by fitting each object to a perfect circle and measuring deviation using the following formula (4 × π) × (Area/Perimeter²). A value of 1 represents a perfect circle, and 0 a straight line. Histogram and Gaussian distribution of mitochondria circularity scores for each genotype. All three variances were statistically different (P < 0.0001) when compared to wild type (F test for variances). All imaging was performed on L4 animals. Data are represented as mean ± SEM. *P < 0.05, ****P < 0.0001 from one-way ANOVA with Dunnett’s post hoc tests for multiple comparisons; n ≥ 2525 mitochondria for quantitative analysis

Fig. 2

Ultrastructural analysis of mitochondria. Transverse sections captured with electron microscopy of mitochondria from the body wall muscles of L4 stage; a wild type, bdrp-1(tm1108), ceat-3(ad426), and dfzo-1(cjn020) mutant animals. Images representative of a total of 39 mitochondria imaged for wild type, 31 for drp-1(tm1108), 34 for eat-3(ad426), and 51 for fzo-1(cjn020). Further examples for each genotype are displayed in Supplementary Fig. 2. Scale bars 200 nm

Fig. 3

Disruption of mitochondrial dynamics affects animal movement. a Quantified movement across populations of L4-stage animals using a WMicrotracker instrument. The WMicrotracker records movement in multi-well plates using infrared beams. Data represent the mean ± SEM across 15 individual wells; n ≥ 900 worms per genotype. ***P < 0.001 compared to WT. b Quantification of the movement of populations of 3DOA animals using a WMicrotracker instrument. Data represent the mean ± SEM across 12 individual wells; n ≥ 360 worms per genotype. ***P < 0.001 compared to WT. c Number of thrashes per minute in liquid. At L4 stage eat-3(ad426) and drp-1(tm1108) show reduced thrashes per minute (29% and 13.6%, respectively). At 3DOA stage, the fusion mutants, fzo-1(cjn020) and eat-3(ad426), show ≥ 60% reduction in thrashes per minute, and drp-1(tm1108) shows no significant reduction. Analysis of 7DOA and 11DOA animals reveals defects in drp-1(tm1108) animals (46% and 52%, respectively). Symbols represent individual animals over three replicate experiments; n ≥ 30 worms. d Quantification of the number of body bends for drp-1(tm1108), eat-3(ad426), and fzo-1(cjn020) compared to WT. All three mutants show reduced sinusoidal bends per minute (on solid, not liquid media) across all ages tested. Symbols show individual animals from three replicates; n ≥ 30 worms. Data are represented as the mean, ± SEM. **P < 0.01, ****P < 0.0001 from one-way ANOVA with Dunnett’s post hoc tests for multiple comparisons

Fig. 4

Muscle strength is disrupted by loss of mitochondrial fission and fusion. a Schematic representation of the burrowing assay used to quantify muscle strength in C. elegans. An agar-filled serological pipette is injected with worms at one end, and an attractant (diacetyl) at the other. Worms burrow through the agar towards the attractant and the distance (in cm) moved is measured for each animal after 4 h. b Burrowing assay for 1DOA drp-1(tm1108) worms show no significant difference in distance moved compared to wild type. ceat-3(ad426) and dfzo-1(cjn020) show significantly reduced distance burrowed at 1DOA compared to wild type. edrp-1(tm1108) show significantly reduced distance burrowed at 5DOA. Data is represented as mean ± SEM cumulative distance covered across three replicate experiments. n ≥ 36 worms. ^nsP > 0.05, *P < 0.05, ***P < 0.001 from multiple t tests per row using the Holm–Sidak method

Fig. 5

Mitochondrial morphology in PLM neurons. a Schematic of the C. elegans mechanosensory neurons, with the bilateral pair of PLM neurons in the tail of the animal. The red rectangle indicates the approximate location of the sections highlighted in b, which show representative images of mitochondria fluorescently labelled with a GFP reporter (Pmec-4::MLS::GFP) in the PLM axons. Green puncta indicate mitochondria. drp-1(tm1108)ii have more elongated mitochondria compared to wild type, iiieat-3(ad426) are unchanged, and ivfzo-1(cjn020) show smaller, rounder mitochondria. Images are representative sections of n = 12 PLMs from 12 worms analysed per genotype; scale bars 10 μm. c Mean size (µm²) of mitochondria in the PLM axon as determined using object segmentation (SQUASSH) at L4 stage. d Mean circularity of mitochondria in the PLM axon at L4 stage. e Mean size (µm²) of mitochondria in the PLM axon in 3DOAs. f Mean circularity of mitochondria in the PLM axon in 3DOAs. g Mitochondrial load: the total area (µm²) of mitochondria per PLM, normalised to PLM length. h Histogram and Gaussian distribution showing the variance of mitochondrial size in 3DOAs. All three genotypes show a significantly difference in variance (F test, P < 0.01) compared to WT. i Histogram and Gaussian distribution showing the variance of mitochondrial circularity in 3DOA. F test was performed on variance differences for both mitochondrial size and circularity. In all graphs; red = WT, yellow = drp-1(tm1108), green = eat-3(ad426), and blue = fzo-1(cjn020). Data is represented as mean ± SEM. ^nsP > 0.05, ****P < 0.0001 from one-way ANOVA with Dunnett’s post hoc tests for multiple comparisons; n ≥ 330 mitochondria for quantitative analysis, n ≥ 6 worms

Fig. 6

Mitochondrial dynamics proteins are crucial for maintaining neuronal function. a Quantification of anterior mechanosensory neuron function. Light touch assays performed by gently stroking worms across the head with an eyebrow hair and their response recorded. At 3DOA stage, drp-1(tm1108) mutants show no significant reduction in response to head touch. eat-3(ad426) and fzo-1(cjn020) both show significant reductions in response. Symbols represent individual animal responses from three replicate experiments; n ≥ 30 worms. b Quantification of posterior mechanosensory neuron function using the light touch assay. In 3DOAs, drp-1(tm1108) mutants show no significant reduction; eat-3(ad426) and fzo-1(cjn020) both show reductions in response; n ≥ 30 worms. cdrp-1(tm1108) shows a reduction in head and tail touch at 7DOA stage; n ≥ 30 worms. Bars represent mean, ± SEM; ^nsP > 0.05, ****P < 0.0001 from one-way ANOVA with Dunnett’s post hoc tests for multiple comparisons

Fig. 7

Simultaneous disruption of fusion and fission proteins rescues mitochondrial and behavioural phenotypes. Images of a body wall muscle with fluorescently labelled mitochondria (Pmyo-3::MLS::GFP) in a an eat-3(ad426); drp-1(tm1108) double mutant animal and b a fzo-1(cjn020) animal with RNAi knockdown of drp-1. Arrows designate nuclei, which are also labelled with GFP in this transgenic strain. Insets (i) are enlargements (× 3) of the indicated circular areas. Scale bars 20 μm. c, d Histogram and Gaussian distribution of mitochondria circularity scores for eat-3(ad426); drp-1(tm1108) double mutants (c) and fzo-1(cjn020) animals with RNAi knockdown of drp-1 (d). All imaging was performed on L4 stage animals; P values calculated using F tests for variances; n ≥ 1832 mitochondria for quantitative analysis. e, f Quantified movement across populations of L4-stage animals using a WMicrotracker instrument. Data represents the mean ± SEM across ten individual wells; n ≥ 600 worms per genotype. *P < 0.05, **P < 0.01, ****P < 0.0001 compared to WT from one-way ANOVA with Dunnett’s post hoc tests. g Quantification of anterior mechanosensory neuron function using light touch assays. Symbols represent individual animal responses from three replicate experiments; n ≥ 30 worms. h Quantification of posterior mechanosensory neuron function using the light touch assay; n ≥ 30 worms. Bars represent mean, ± SEM; ^nsP > 0.05, **P < 0.01, ****P < 0.0001 from one-way ANOVA with Dunnett’s post hoc tests for multiple comparisons

Fig. 8

Reduced lifespan in the mitochondrial fusion/fission mutants. a Kaplan–Meier survival plot of mitochondrial fission/fusion mutants. All mutants (drp-1(tm1108), eat-3(ad426) and fzo-1(cjn020)) show reduced survival (P < 0.0001) compared to the wild type {strain QH3135 [zdIs5(Pmec-4::GFP)]}. b Median survival day of each mutant. eat-3(ad426) and fzo-1(cjn020) have a median survival of 12 and 13 days, respectively; drp-1(tm1108) has a median survival of 15.6 days; all of which are significantly less than the median survival of the wild type (20 days). Data are represented as the average of the median of each replicate, ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 from log-rank (Mantel–Cox) tests to compare the curves; three replicates with n ≥ 207 worms per genotype (individual replicates are shown in Supplementary Fig. 4)