The Rheb-TORC1 signaling axis functions as a developmental checkpoint

Abstract

In many eukaryotes, the small GTPase Rheb functions as a switch to toggle activity of TOR complex 1 (TORC1) between anabolism and catabolism, thus controlling lifespan, development and autophagy. Our CRISPR-generated, fluorescently tagged endogenous Caenorhabditis elegans RHEB-1 and DAF-15/Raptor are expressed ubiquitously and localize to lysosomes. LET-363/TOR and DAF-15/Raptor are required for development beyond the third larval stage (L3). We observed that deletion of RHEB-1 similarly conferred L3 arrest. Unexpectedly, robust RNAi-mediated depletion of TORC1 components caused arrest at stages prior to L3. Accordingly, conditional depletion of endogenous DAF-15/Raptor in the soma revealed that TORC1 is required at each stage of the life cycle to progress to the next stage. Reversal of DAF-15 depletion permits arrested animals to recover to continue development. Our results are consistent with TORC1 functioning as a developmental checkpoint that governs the decision of the animal to progress through development.

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans

“…Strains grown on IAA arrested at L2, whereas strains grown on K-NAA arrested at L3 ( Table 3 and Supplementary Table S6 ). Based on previous work ( Duong et al 2020 ), these results suggests that K-NAA is producing slower or less complete DAF-15::mNG::AID* depletion than IAA. For DAF-15::mNG::AID*, IAA appeared to perform better.…”

“…It is unclear what is behind the discrepancy between these results; we will need to determine in which cells NHR-25 is acting to promote larval development and how IAA and K-NAA affect its depletion in this cell type. Additionally, daf-15::mNG::AID* animals grown on IAA arrested at an earlier larval stage than animals treated with K-NAA ( Table 3 ) consistent with faster and/or more robust depletion ( Duong et al 2020 ). Our results do not indicate that one form of auxin is consistently superior, but rather that one needs to empirically test each auxin, depending on the AID-tagged gene, the TIR1 transgene, and the tissue in which depletion is occurring.…”

“…Finally, we wished to test an additional cytoplasmic AID*-tagged protein for depletion phenotypes. We chose DAF-15/Raptor, a lysosome-localized factor which causes larval arrest when depleted ( Duong et al 2020 ). Both eft-3p::TIR1::mRuby2 and eft-3p::TIR1::F2A::BFP::AID*::NLS caused a completely penetrant larval arrest in animals carrying the daf-15::mNeonGreen::AID* ( Table 3 and Supplementary Table S6 ).…”

“…Another approach may be to fuse the Skp1 subunit of the SCF ubiquitin ligase to TIR1, which has resulted in enhanced degradation efficiency of AID-tagged proteins ( Kanke et al 2011 ). Determining which components of the system are limiting and boosting TIR1 activity has the potential to address the reported issue of incomplete degradation of some AID-tagged proteins and a failure to obtain null phenotypes ( Patel and Hobert 2017 ; Serrano-Saiz et al 2018 ; Duong et al 2020 )…”

Parallel Rap1>RalGEF>Ral and Ras signals sculpt theC. elegansnervous system

“…Ral also activates TORC1 in parallel to Rheb to control lifespan in C. elegans and invasion in mammalian cells (Duong et al, 2020;Martin et al, 2014).…”