C. elegans Practical course

While daf-15-RNAi downregulates the mTORC1 scaffolding protein Raptor, rict-1-RNAi downregulates the mTORC2 scaffolding protein Rictor 4

DAF-16 = FOXO (daf-16 mutant = dauer defective)

DAF-2 = Insulin receptor

DAF-18 = PTEN (kinase inhibitor)

DAF-15 = Raptor

rict-1 = Rictor

assay for recessive mutations

Complementation means restoring the function of a recessive mutant by a second functional allele -> non-complementation happens if a mutant is crossed to another mutant, that together cannot restore the normal function

abnormal dauer formation -> 3 unknown mutations (daf-2, daf-7 or daf-16) crossed with daf-2 males

successful crossing -> 50 % males in offspring!

one mutant no dauer = dauer defective -> must be daf-16 [can be seen in control]

Incubation at 25 °C -> at this temp the temperature sensitive daf-2 mutants will go into dauer!

to distinguish between daf-2 and daf-7 you need to look at the crossing -> daf-2 / daf-2 should go into dauer because daf-2 = dauer constitutive -> non-complementation

the crossing with daf-7 leads to complementation because there the offspring inherits one functional allel which is enough so the worm should not go into dauer (some might be in dauer because some offspring could be from selffertilization)

the strain was grown on rict-1 RNAi, daf-15 RNAi (=raptor) and sgk-1 RNAi -> 3 L4 individual let them have offspring and kill the old ones

-> synchronysation and then let them develope

-> after 3 days analyze the developement:

rict-1 RNAi shows normal developement (like WT) -> same

daf-15 more L3 and L4 individuals -> very delayed

sgk-1 a little L3 L4 but more adults -> dalayed

DAF-15 is part of the TOR (Target of Rapamycin) signaling pathway, specifically interacting with the mTORC1 complex (mammalian Target of Rapamycin Complex 1). This pathway is crucial for regulating cell growth, proliferation, and development in response to nutrient availability and other environmental cues. -> without RAPTOR no TOR1 signaling that means autophagy not inhibited and the translation (S6K) and translation initiation (by inhibition of 4EBP) is reduced! that is bad for developement

sgk-1 target of mTORC2 which would have let to conclusion that it should be same effect as the RICTOR RNAi BUT sgk-1 also target downstream in the insulin pathway where it is part of the kinase cascade (with AKT etc.) that inhibits FOXO

Only dauers survive because they have no pharings movement so they dont take up the toxin

GFP fused strains

Starvation leads to daf-16 nuclear localization

Heat stress even more!

Daf-2 mutants lack the insulin receptor so the kinase cascade is never active and cannot inhibit DAF-16 (FOXO) nuclear localization so it is always in nucleas BUT other pathways still act on it and the stress conditions do not change the localization (thats why not stronger after HS)

survival of daf-2, daf-16 and daf-16;daf-2 mutants will be compared to the wildtype

daf-2 mutants are known to have enhanced stress resistance, including heat stress, due to reduced IIS pathway signaling, which increases DAF-16 activity (because the kinase cascade inhibiting DAF-16 is not active without the DAF-2 receptor signal!)

• DAF-16 promotes the expression of heat shock proteins and other stress-responsive genes, enhancing survival under stressful conditions

Loss of DAF-16 function impairs the activation of stress response genes -> worse survival

daf-2:daf-16 -> the increased survival seen in daf-2 mutants under stress is expected to be abolished when DAF-16 is not present. Therefore, the double mutants are likely to show a survival rate similar to daf-16 single mutants, indicating the necessity of DAF-16 for the stress resistance conferred by daf-2 mutations

To observe the activity of the DAF-16 target gene sod-3, the coding sequence of GFP was used under the control of the sod-3-Promoter.

DAF-2 receptor represses the DAF-16 transcription factor. daf-2-mutations are temperature-sensitive, meaning that at permissive temperatures (15°C) DAF-2 is still mainly active while it is inactive at restrictive temperatures (25°C) 11 .The DAF-16 main transcription factor of insulin/IGF signaling promotes and represses a lot of target genes. The gene for the superoxide dismutase sod-3 is such a target gene of DAF-16. sod-3 signal tested for WT, daf-2 and daf-2:daf-16

daf-2 mutant = temp sensitive -> at 25 °C the receptor is inactive leading to no more inactivation of FOXO (DAF-16) so it is active and as the strong fluoresense shows it expresses the superoxide dismutase!

in WT it just expresses it maybe because of the light heat stress at 25 °C

in the double mutant DAF-16 is inactive -> no sod-3 transcription!

A further question would be, if this is a direct effect of DAF-16 or its antagonistic PQM-1 transcription factor -> under stress conditions PQM-1 is relocalized out of the nucleus (antagonist to FOXO)

PVD neurons in C-elegans are activated by CS, with the PVD:mCherry mutant the neuron is visualised -> for some groups a systemic reporter was used and for other groups a Promotor only active in the neurons was used

-> systemic reporter had way to much background fluoresence

CS causes neuronal degeneration which can be observed as beading phenotype -> pre CS starvation can reduce the beading

Control without starvation

-> grown on RNAi plates then starved 24 h then CS overnight then analysis under fluorecent microscope: