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

An Addendum to this article was published on 20 March 2020

Abstract

Although individuals age and die with time, an animal species can continue indefinitely, because of its immortal germ-cell lineage1. 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 sperm2 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 lysosomes3. Sperm stimulate V-ATPase activity in oocytes by signalling the degradation of GLD-1, a translational repressor4 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.

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Figure 1: Sperm signalling enhances oocyte proteostasis.
Figure 2: Sperm-activated lysosomes clear protein aggregates.
Figure 3: Sperm trigger proteasome-dependent GLD-1 loss, releasing the block on synthesis of the lysosomal V-ATPase.
Figure 4: Mitochondria aid proteostasis enhancement.
Figure 5: Conservation and model of a germline lysosomal switch.

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Acknowledgements

We thank members of the Kenyon laboratory and our colleagues at Calico for discussions and comments on the manuscript, and the Calico microscopy core, especially M. Ingaramo, for help with microscopy and sensors. We thank the E. Blackburn and P. O’Farrell laboratories for sharing equipment at UCSF. K. Sato provided the Ppie-1::gfp::lgg-1 strain. Other strains were provided by the CGC, funded by the NIH Office of Research Infrastructure Programs (P40 OD010440). Research was performed at UCSF and then Calico, and was supported at UCSF by the George and Judy Marcus Family Foundation, the Life Extension and Chuan Lyu Foundations, and by NIH grant R37/R01 AG11816 to C.K. C.K. is now Vice President of Aging Research at Calico Life Sciences, which supported the research done at Calico. K.A.B. is an Honorary Fellow of the Jane Coffin Childs Memorial Fund.

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K.A.B. and C.K. designed experiments, interpreted data, and wrote the manuscript. K.A.B. performed all experiments.

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Correspondence to Cynthia Kenyon.

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Extended data figures and tables

Extended Data Figure 1 Pattern of germline protein aggregates.

ac, 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.

af, Time-lapse images of aggregation-prone proteins in oocytes of females, or females after mating. gi, 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. Source data

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

a, Lifespan, mean lifespans in parentheses. bd, 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. Source data

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. Source data

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. Source data

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 UbG76V::GFP. Active proteasomes degrade UbG76V::GFP, unless inhibited by MG132. eg, 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 tumors20, 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. Source data

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. Source data

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. Source data

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. Source data

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 state22,23. b, 488 nm-excited fluorescence of PercevalHR and cpmVenus. c, Heatmap of the PercevalHR λhigh/λlow 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. Source data

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Supplementary Figure 1

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Bohnert, K., Kenyon, C. A lysosomal switch triggers proteostasis renewal in the immortal C. elegans germ lineage. Nature 551, 629–633 (2017). https://doi.org/10.1038/nature24620

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