A lysosomal switch triggers proteostasis renewal in the immortal C. elegans germ lineage

Abstract

Although individuals age and die with time, an animal species can continue indefinitely, because of its immortal germ-cell lineage. How the germline avoids transmitting damage from one generation to the next remains a fundamental question in biology. Here we identify a lysosomal switch that enhances germline proteostasis before fertilization. We find that Caenorhabditis elegans oocytes whose maturation is arrested by the absence of sperm exhibit hallmarks of proteostasis collapse, including protein aggregation. Remarkably, sperm-secreted hormones re-establish oocyte proteostasis once fertilization becomes imminent. Key to this restoration is activation of the vacuolar H⁺-ATPase (V-ATPase), a proton pump that acidifies lysosomes. Sperm stimulate V-ATPase activity in oocytes by signalling the degradation of GLD-1, a translational repressor that blocks V-ATPase synthesis. Activated lysosomes, in turn, promote a metabolic shift that mobilizes protein aggregates for degradation, and reset proteostasis by enveloping and clearing the aggregates. Lysosome acidification also occurs during Xenopus oocyte maturation; thus, a lysosomal switch that enhances oocyte proteostasis in anticipation of fertilization may be conserved in other species.

Extended Data Figure 1. Pattern of germline protein aggregates

a–c, Full gonad images of three aggregation-prone proteins in young hermaphrodites and females. Enlarged regions of different parts of the gonad are also shown. Bars, 10 µm.

Extended Data Figure 2. Signals from sperm reduce protein aggregation in oocytes

a–f, Time-lapse images of aggregation-prone proteins in oocytes of females, or females after mating. g–i, Localized fluorescence intensities in oocytes of mated females and non-mated controls. Mean ± s.d. for n = 10 aggregate sites. NS, not significant. ****P < 0.0001. j, k, GFP::RHO-1-aggregation in females and sperm-defective hermaphrodite mutants that still produce MSPs. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. Bars, 10 µm.

Extended Data Figure 3. V-ATPase suppression of oocyte protein aggregation

a, Lifespan, mean lifespans in parentheses. b–d, GFP::RHO-1-expressing hermaphrodites with catalytic (V₁) or membrane-anchoring (V₀) V-ATPase subunits knocked down. Animals with oocyte protein aggregation counted. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. e, Mating after vha-13 knockdown. f, GFP::RHO-1-expressing gsa-1(ce94gf) hermaphrodites following control or vha-13 RNAi. g, Percentage of gsa-1(ce94gf) animals with oocyte protein aggregation. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. h, DMSO- or bafilomycin A1-injected germlines. i, Control or NH₄Cl-treated germlines. Bars, 10 µm.

Extended Data Figure 4. Regulation of lysosomal acidity in the germline

a, Hermaphrodite and female worms stained with LysoSensor Blue DND-99. Gonads are outlined. b, Percentage of puncta that are positive for LysoTracker and/or GFP::VHA-13. Mean ± s.d. from n = 10 proximal oocytes. c, Co-localization (arrows) of GFP::VHA-13 and LysoTracker puncta in an oocyte (enlarged region). d, Proximal oocyte before and after mating in a GFP::VHA-13-expressing female. Bars, 5 µm.

Extended Data Figure 5. Aggregates are not cleared by macroautophagy

a, GFP::LGG-1-expressing germlines. b, Number of autophagosomes (mean ± s.d.) in the most proximal oocyte. c, Schematic of macroautophagy. d, GFP::LGG-1 after control or lgg-1 RNAi. e,f, Quantification of macroautophagy gene expression by RT-PCR. Normalized expression (mean ± s.d.) was scored for three biological replicates. ****P < 0.0001. The gel source data are shown in Supplementary Figure 1. g, h, GFP::RHO-1-expressing hermaphrodites treated with macroautophagy-gene RNAi. Percentage of animals with oocyte protein aggregation. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. i, Matings after macroautophagy-gene RNAi. j, Time-lapse images of aggregate clearance. Bars, 5 µm.

Extended Data Figure 6. Proteasome involvement in germline proteostasis

a, LysoTracker-stained dissected germlines. b, GFP::PBS-1 localization. c, d, Schematic and imaging of proteasome sensor Ub^G76V::GFP. Active proteasomes degrade Ub^G76V::GFP, unless inhibited by MG132. e–g, GFP::RHO-1-aggregation following control or proteasomal pbs-1 RNAi. The gld-1(q485) mutation precluded aggregation following pbs-1 RNAi. This finding fits the model that the proteasome degrades GLD-1, but not the aggregates, consistent with aggregate engulfment by lysosomes. However, we note that proximal gld-1 germ cells, which form tumors, could potentially be non-permissive for aggregation. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. Bars, 10 µm.

Extended Data Figure 7. Sperm-induced changes in mitochondrial morphology and ROS levels require V-ATPase function

a, MitoLS::GFP in germ cells of hermaphrodites. b, Different z-planes for MitoLS::GFP in the same distal germline. c, Proximal:distal MitoLS::GFP fluorescence ratios (mean ± s.d.) for n = 10 germlines. d, e, Proximal oocytes from MitoLS::GFP-expressing or MitoTracker CM-H₂TMRos-stained females. f, g, Mitochondria from proximal oocytes in MitoLS::GFP-expressing females before and after mating. Mitochondrial lengths (mean ± s.d.). ****P < 0.0001. h, i, Proximal oocytes from MitoLS::GFP-expressing or MitoTracker CM-H₂TMRos-stained hermaphrodites after vha-13 RNAi. Bars, 10 µm.

Extended Data Figure 8. Regulation of mitochondrial membrane potential in the germline

a, DiOC6(3)-stained germlines from control (ethanol solvent)- or antimycin-treated hermaphrodites. b, Percentage of DiOC6(3)-stained germlines. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. c, Real-colour and heatmap images of DiOC6(3)-stained germlines. d, JC-1-stained mitochondria in the distal and proximal germline of a wild-type hermaphrodite. e, JC-1-stained proximal germline mitochondria following control or vha-13 RNAi. Bars, 5 µm.

Extended Data Figure 9. ATP-synthase inhibition prevents the reduction in mitochondrial membrane potential in proximal oocytes and blocks aggregate clearance

a, Real-colour and heatmap images of DiOC6(3)-stained germlines after RNAi of genes encoding ATP synthase subunits. b, c, Aggregation-prone proteins in control (ethanol solvent)- or oligomycin-treated hermaphrodites. Percentage of animals with oocyte protein aggregation. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. d, LysoTracker reveals lysosome acidification in GFP::RHO-1-expressing hermaphrodites following asb-1 knockdown. Dotted line, intestine. Bars, 10 µm.

Extended Data Figure 10. Activation of germ cell metabolism

a, Immature germ cells arrest with a high ATP:ADP ratio and a high energy charge, which are reversed in response to sperm signals as ADP levels rise and unlock the ATP synthase. These changes reflect a shift from a resting to an active metabolic state,. b, 488 nm-excited fluorescence of PercevalHR and cpmVenus. c, Heatmap of the PercevalHR λ_high/λ_low ratio after vha-13 knockdown. d, Peredox fluorescence in a hermaphrodite germline, with line profiles. e, f, GFP::GLD-1 and mitochondrial morphology in ADP- or vehicle-injected female oocytes. Bars, 10 µm.

Figure 1. Sperm signalling enhances oocyte proteostasis

a, C. elegans germline. b, Aggregation-prone proteins in oocytes. Aged hermaphrodites deplete sperm. c, Percentage of animals with oocyte protein aggregation; herm., hermaphrodites; fem., females. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. d–f, Photobleaching of aggregation-prone proteins. Fluorescence recovery (mean ± s.d.) of each protein was measured for three hermaphrodites or three females. g, Proteostat-labelled oocytes. h, Proteostat-positive germlines. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. i, Aggregation-prone proteins after mating. j, GFP::RHO-1-expressing females following control or goa-1 RNAi. Bars, 5 µm.

Figure 2. Sperm-activated lysosomes clear protein aggregates

a, Overview of the screen. b, c, Aggregation-prone proteins following control or vha-13RNAi. Percentage of animals with oocyte protein aggregation. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. d, Oocytes stained with LysoTracker. Dotted line, intestine. e, Percentage of animals with lysosomal acidity in oocytes. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. f, LysoTracker-stained, GFP::VIT-2-expressing hermaphrodites. Dotted line, intestine. g, h, Expression of LMP-1::GFP and GFP::VHA-13 in the germline. i, Line profiles of GFP::VHA-13 fluorescence. j, Co-localization of GFP::VHA-13 with LMP-1::mCherry in oocytes of hermaphrodites. k, l, Aggregate degradation in lysosomes after mating. Bars, 5 µm.

Figure 3. Sperm trigger proteasome-dependent GLD-1 loss, releasing the block on synthesis of the lysosomal V-ATPase

a, b, Schematic of V-ATPase localization in the germline (a, as shown in Fig. 2h), and reciprocal GFP::GLD-1 localization (b). c, Percentage of animals with proximal GFP::GLD-1 expression. Mean ± s.d. from five biological replicates, each of n = 50 animals. ****P <0.0001. d, Ratios (mean ± s.d.) of distal:proximal GFP::VHA-13 fluorescence intensities. n = 10 germlines per genotype. ****P < 0.0001. e–g, Expanded GFP::VHA-13 expression in germlines lacking GLD-1 repression. h, Proximal germline of a GFP::GLD-1-expressing female following mating. i, GFP::GLD-1 expression in hermaphrodites treated with DMSO or the proteasome inhibitor MG132. Bars, 10 µm.

Figure 4. Mitochondria aid proteostasis enhancement

a, Mitochondria labelled with MitoLS::GFP. b, Lengths of mitochondria (mean ± s.d.) in distal and proximal germlines. c, Germlines stained with MitoTracker CM-H₂TMRos. Sp, sperm. d, ROS-positive germlines. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. e, DiOC6(3) heat maps. f, Proximal:distal DiOC6(3) fluorescence ratios (mean ± s.d.). ****P < 0.0001. g, h, GFP::RHO-1-aggregation in hermaphrodites after knockdown of ATP synthase subunits. Mean ± s.d. from three biological replicates, each of n = 50 animals. ****P < 0.0001. i, GFP::ATP-2 ATP synthase localization. j, PercevalHR λ_high/λ_low heat map. k, l, DiOC6(3) staining of oocytes, showing mitochondrial membrane-potential fluorescence following ADP injection. n = 5 oocytes per condition. m, Aggregate movement. Bars, 10 µm.

Figure 5. Conservation and model of a germline lysosomal switch

a, LysoTracker-stained Xenopus oocytes. Bar, 20 µm. b, In C. elegans, arrested oocytes exhibit a relaxation in proteostasis, which is reversed just before fertilization when sperm signalling relieves GLD-1-mediated repression of V-ATPase synthesis. Activated lysosomes enhance oocyte proteostasis by engulfing and clearing protein aggregates, and by promoting a metabolic shift from a primed, quiescent state accompanied by elevated ROS to an active metabolic state that supports aggregate mobilization for removal. This mechanism may also underlie the sperm-dependent clearance of carbonylated proteins.