Olek, K. | Search bioRxiv

bioRxiv2025

Mitochondrial redox homeostasis links organellar stress surveillance to germline and somatic integrity in Caenorhabditis elegans

Mitochondrial redox homeostasis is essential for cellular metabolism and organismal development. To investigate the consequences of disrupting redox homeostasis in this organelle in a metazoan organism, we generated a double mutant lacking mitochondrial glutathione reductase (gsr-1a) and thioredoxin reductase (trxr-2) genes in Caenorhabditis elegans. While gsr-1a or trxr-2 single mutants are phenotypically normal, double gsr-1a trxr-2 mutants displayed small body size, gonadal migration defects, reduced brood size, and prolonged egg-laying period, without developmental delay or lethality. Transcriptomic analysis revealed strong induction of ATFS-1-dependent stress and detoxification genes. Consistent with this, gsr-1a trxr-2 worms exhibited constitutive ATFS-1 nuclear localization and robust Phsp-6::gfp expression. Triple gsr-1a trxr-2; atfs-1 mutants were nonviable, demonstrating that unfolded protein response (UPRmt) activation is essential under mitochondrial redox stress. Despite the induction of a stress response at the transcriptional level, gsr-1a trxr-2 double mutants were not more resistant to oxidative or pathogen stressors. Moreover, these mutants maintained normal respiration, ATP and ROS production while displaying altered mitochondrial morphology in a tissue-specific manner, independent of mitophagy genes but dependent on mitochondrial fission or fusion machinery. Functionally, gsr-1a trxr-2 mutants showed impaired motility, reduced calcium uptake upon carbachol stimulation, enhanced hypodermal wound repair, and decreased fertilization efficiency associated with lower muscle exopher production. Overall, our data show that simultaneous loss of mitochondrial GSR-1a and TRXR-2 compromises growth, fertility and muscle performance and triggers a constitutive ATFS-1-dependent UPRmt that sustains viability revealing mitochondrial redox control as a core determinant of organismal proteostasis. HighlightsO_LIgsr-1a or trxr-2 single mutants have no overt phenotypes. C_LIO_LIgsr-1a trxr-2 double mutants are viable but show small size, gonad migration defects and reduced progeny. C_LIO_LILoss of both reductases in mitochondria triggers a constitutive ATFS-1-dependent UPRmt. C_LIO_LIATFS-1 is essential for gsr-1a trxr-2 worms survival. C_LIO_LIgsr-1a trxr-2 animals remodel mitochondrial morphology in a tissue-specific manner. C_LIO_LIgsr-1a trxr-2 double mutants exhibit impaired muscle and sperm function but enhanced wound healing. C_LI Graphical abstract (to be incorporated)

genetics↗

bioRxiv2025

p70S6 kinase-dependent phosphorylation of the μ2 subunit of the AP2 adaptor complex is needed for clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) internalizes cell-surface receptors via clathrin-coated invaginations of the plasma membrane. Both clathrin and endocytic cargo are recruited to these sites by the adaptor protein complex AP2. AP2 cycles between a closed cytoplasmic conformation and an open membrane-bound state, and efficient CME requires both conformations and their dynamic interconversion. The mechanisms regulating these conformational changes, which include post-translational modifications of the AP2, remain incompletely understood. Here, we report that p70S6 kinase phosphorylates the {micro}2 subunit of the AP2 and that the phosphorylation of serine 45 (S45) depends on p70S6K activity. Loss of S45-{micro}2 phosphorylation results in decreased internalization of canonical CME cargo such as transferrin and PDGF receptors. In Caenorhabditis elegans, lack of S45-{micro}2 phosphorylation produces directionally similar but markedly weaker phenotypes than AP2 loss of function. Live imaging and in silico dynamic modelling suggest that S45-2 phosphorylation has impact on the conformational changes of the AP2 complex. These findings identify a p70S6K-dependent mechanism that modulates AP2 function and further strengthen the importance of post-translational regulation in controlling CME. Summary statementClathrin-mediated endocytosis is essential for receptor internalization and cell signaling. This study identifies a novel regulatory mechanism in which p70S6K-mediated phosphorylation of the AP2 subunit 2 at Ser45 modulates AP2 conformational dynamics and facilitates receptor endocytosis.

cell biology↗