Mobilization of cholesterol induces the transition from quiescence to growth in Caenorhabditis elegans through steroid hormone and mTOR signaling

Recovery from the quiescent developmental stage called dauer is an essential process in C. elegans and provides an excellent model to understand how metabolic transitions contribute to developmental plasticity. Here we show that cholesterol bound to the small secreted proteins SCL-12 or SCL-13 is sequestered in the gut lumen during the dauer state. Upon recovery from dauer, bound cholesterol undergoes endocytosis into lysosomes of intestinal cells, where SCL-12 and SCL-13 are degraded and cholesterol is released. Free cholesterol activates mTORC1 and is used for the production of dafachronic acids. This leads to promotion of protein synthesis and growth, and a metabolic switch at the transcriptional level. Thus, mobilization of sequestered cholesterol stores is the key event for transition from quiescence to growth, and cholesterol is the major signaling molecule in this process.

exiting dauers on RNAi against rab-7 compared to EV (dotted lines), treated with 1 mM cholesterol (turquoise) or 13 µM cholesterol (black/orange) and with (green) or without 400 µM auxin (light green).Average ± SD of one experiment with a minimum of 25 individual worms per condition, two-way ANOVA with post hoc Tukey's test comparing rab-7 with rab-7 high chol.showed p < 0.0001, rab-7 HC with rab-7 high chol.+ auxin p < 0.0001, EV with EV + auxin p < 0.0001, EV with EV high chol.+ auxin p < 0.0001, EV high chol.with EV high chol.+ auxin p < 0.0001.Individual values can be found in the raw data file 6e_S4h.

Figure S1 :
Figure S1: Characterization of SCL-12 expression during dauer entry and exit a Genomic engineering of scl-12&13 and scl-12 knock-out mutants and SCL-12 reporter animals.b SCL-12::mScarlet;daf-2 reporter animals expressing SCL-12::mScarlet as indicated by appearance of fluorescence after synchronization in % per population.c Representative fluorescent micrographs of SCL-12::mScarlet;daf-2 reporter animals while entering dauer at 44 h, 50 h and 56 h after synchronization of eggs.44 h and 50 h insets are brightness adjusted images for increased visibility.Scale bar: 15 µm.d SCL-12::mScarlet reporter starvation dauers (wt) while exiting dauer at 1.5 h, 3 h and 6 h after introduction of food.3 h and 6 h insets are brightness adjusted images.Scale bar: 25 µm.e Mean fluorescence intensity of SCL-12::mScarlet reporter starvation dauers (0 h) and while exiting dauer via introduction of food.Black: Fluorescence in the gut lumen.Red: Fluorescence outside of the lumen.Means and individual values of at least 10 individual worms per time point.

Figure S2 :
Figure S2: Characterization of scl-12&13 mutants in dauer state a Lenght in mm of daf-2 (black) and scl-12&13;daf-2 (gray) dauer larvae.Average and individual values of 5 independent experiments with at least 20 worms per strain, unpaired two-tailed t-test showed p < 0.0001.b Oxygen consumption rate (OCR) in pM (picomolar)/min/μg protein of daf-2 and scl-12&13;daf-2 dauers as worms exit the state.Average and individual values of 3 measurements of 6 technical replicates in one representative experiment.Two-way RM ANOVA with Geisser-Greenhouse correction comparing the strains showed p = 0.8572.c Dauer survival.Average and individual values of one experiment with 3 -4 biological replicates.d Time to reach fertility as indicated by egg laying after dauer exit induced by temperature switch to 15°C.Average ± SD and individual values of 10 worms per strain, paired two-tailed t-test showed p = 0.0034.e Growth/length in mm of daf-2, single mutants scl-12;daf-2, and double mutants scl-12&13;daf-2 after induction of dauer exit by temperature switch to 15°C.Average and individual values of 2 independent experiments with a minimum of 10 worms per condition, two-way RM ANOVA with Geisser-Greenhouse correction comparing daf-2 versus scl-12;daf-2 showed p = 0.0364, and daf-2 versus scl-12&13;daf-2 p = 0.0053.

Figure S3 :
Figure S3: Dauer exit depends on intact lysosomes a Percentage of daf-2 populations that develop into L4 larvae 48 h after dauer recovery was induced by temperature switch to 15°C on RNAis against cav-2 (green), rme-1 (blue), glo-

Figure S5 :
Figure S5: Orthologues of SLC38A9 and NPC1 do not affect dauer exit Growth/length in mm in daf-2 exiting dauers induced by temperature switch to 15°C on RNAis against an OD-adjusted mix of ncr-1 and ncr-2 (teal) and F13H10.3(purple), compared to EV (black).Means and individual values of 1 experiment with a minimum of 15 worms per condition, two-way ANOVA test comparing EV versus ncr-1;ncr-2 showed p = 0.1146 and EV versus F13H10.3p = 0.0653.