Hernando-Rodriguez, B. | Search bioRxiv

bioRxiv2019

Prohibitin depletion suppresses mitochondrial, lipogenic and autophagic defects of SGK-1 mutant extending lifespan; implication of autophagy and the UPRmt

Mitochondrial prohibitins (PHB) are highly conserved proteins with a peculiar effect on lifespan. While PHB depletion shortens lifespan of wild type animals, it enhances longevity of a plethora of metabolically compromised mutants, including target of rapamycin complex 2 (TORC2) mutants sgk-1 and rict-1. Here, we show that sgk-1 mutants have impaired mitochondrial homeostasis, lipogenesis, yolk formation and autophagy flux due to alterations in membrane lipid and sterol homeostasis. Remarkably, all these features are suppressed by PHB depletion. Lifespan analysis shows that autophagy and the mitochondrial unfolded protein response (UPRmt), but not mitophagy, are required for the enhanced longevity caused by PHB depletion in sgk-1 mutants. We hypothesize that UPRmt induction upon PHB depletion extends lifespan of sgk-1 mutants through autophagy. Our results strongly suggest that PHB depletion suppresses the autophagy defects of sgk-1 mutants by altering membrane lipid composition at ER-mitochondria contact sites, where TORC2 localizes.

cell biology↗

bioRxiv2017

Combined flow cytometry and high throughput image analysis for the study of essential genes in Caenorhabditis elegans

BackgroundThe advancement in automated image based microscopy platforms coupled with high throughput liquid workflows has facilitated the design of large scale screens utilizing multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high throughput approaches.\n\nResultsIn C. elegans, non-conditional lethal mutations can be maintained in heterozygosis using chromosome balancers, commonly labelled with GFP in the pharynx. Moreover gene-expression is typically monitored by the use of fluorescent reporters marked with the same fluorophore. Therefore, the separation of the different populations of animals at early larval stages represents a challenge. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at early larval stages. Because sorting is not completely error-free, we develop an automated high-throughput image-analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image-analysis in a functional genomic RNAi screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while, homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both, known and new PHB genetic interactors affecting the UPRmt and growth.\n\nConclusionsA systematic way to analyse genetic interactions of essential genes in multicellular organisms is lacking. The method presented here allows the study of balanced lethal mutations in a high-throughput manner and can be easily adapted depending on the users requirements. Therefore, it will serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.

genetics↗