A novel sphingolipid-TORC1 pathway critically promotes postembryonic development in Caenorhabditis elegans

  1. Huanhu Zhu
  2. Huali Shen
  3. Aileen K Sewell
  4. Marina Kniazeva
  5. Min Han  Is a corresponding author
  1. Howard Hughes Medical Institute, University of Colorado, Boulder, United States
  2. University of Colorado, Boulder, United States
  3. Fudan University, China
4 figures, 1 table and 1 additional file

Figures

Iso-branched d17iso-sphinganine rescues elo-5(-) L1 arrest.

(A) Sphingolipid biogenesis pathway in C. elegans, including the catalytic enzymes (blue) and corresponding genes (red) used in this study. Molecular structures in green and in light blue indicate …

https://doi.org/10.7554/eLife.00429.003
Figure 2 with 2 supplements
ku540 mutant suppresses L1 arrest of mmBCFA and sphingolipid biosynthetic mutants without restoring the levels of mmBCFAs.

(A) Screening strategy to isolate elo-5(−) suppressors. Green-colored C. elegans carry the extrachromosomal array containing copies of the elo-5(+), sur-5-gfp, and rol-6(dn) genes. (B) Percentages …

https://doi.org/10.7554/eLife.00429.004
Figure 2—figure supplement 1
Quantification of GC and mass spectrometry analyses of elo-5(−) ku540 animals.

(A) Tables show relative intensities of the five strongest signals by mass spectrometry precursor scan (m/z = −241.2) in wild-type animals (second right column) and their normalized relative …

https://doi.org/10.7554/eLife.00429.005
Figure 2—figure supplement 2
Suppression of fath-1(−) by nprl-3(ku540).

Microscopic images showing that nprl-3(ku540) suppresses the L1 arrest phenotype of fath-1 (injection RNAi) with or without C17ISO supplementation.

https://doi.org/10.7554/eLife.00429.006
Figure 3 with 1 supplement
ku540 is a loss-of-function missense mutation of nprl-3.

(A) Predicted structure and position of the ku540 mutation in the nprl-3 gene (F35H10.7). (B) Abbreviated alignment of C. elegans NPRL-3 with its orthologs in other organisms. (C) C. elegans images …

https://doi.org/10.7554/eLife.00429.007
Figure 3—figure supplement 1
Mapping and expression of nprl-3.

(A) A simplified diagram of the mapping process. ku540 is genetically linked to the E03H12 SNP marker on chromosome IV (upper section). Further three-point mapping narrowed the ku540 locus to near un…

https://doi.org/10.7554/eLife.00429.008
Figure 4 with 5 supplements
TORC1 activation is sufficient for mmBCFAs-mediated growth regulation.

(A) Representative florescent images showing that elo-5(−) animals with each of four RFP-marked transgenes, which constitutively activated TORC1, bypass L1 arrest to reach beyond L3 stage …

https://doi.org/10.7554/eLife.00429.010
Figure 4—figure supplement 1
Microscopic images of C. elegans with raga-1(RNAi) treatment.

Animals without C17ISO supplement were developmentally arrested at L1 stage (arrows). Animals with C17ISO supplement reached adulthood (arrowhead), suggesting that the arrest depends on mmBCFA …

https://doi.org/10.7554/eLife.00429.011
Figure 4—figure supplement 2
Leucine could not promote postembryonic development independent of the mmBCFA/d17isoGlcCer/TORC1 pathway.

Normalized percentages of elo-5(−) animals that reached adulthood on various supplements. While 1 mM C17ISO could suppress the L1 arrest of elo-5(−), 10 mM leucine could not. Error bar: SD.

https://doi.org/10.7554/eLife.00429.012
Figure 4—figure supplement 3
mmBCFA/GlcCer/TORC1 pathway is independent of the DAF-7/TGF-β pathway.

(A) Cartoon illustration of a simplified DAF-7/TGF-β pathway in C. elegans. Mutations in daf-3, daf-5, or bra-1 have been shown to cause constitutive activity of the TGF-β pathway and suppress dauer …

https://doi.org/10.7554/eLife.00429.013
Figure 4—figure supplement 4
Lysosome integrity is not disrupted in mmBCFA-deficient animals.

(A)–(H) DIC and GFP images illustrating LMP-1::GFP (AD) and GLO-1::GFP (EH) expression patterns are similar throughout the intestine of young wild-type or elo-5(RNAi) larvae. Both LMP-1::GFP (AD) …

https://doi.org/10.7554/eLife.00429.014
Figure 4—figure supplement 5
Neutral red staining of let-363 -and elo-5-deficient animals.

(A)–(H) DIC and Rhodamine channel fluorescence images of larvae stained with Neutral red. (AD) let-363 homozygous L3 animals have increased size and intensity of Neutral red stained lysosomes (D) …

https://doi.org/10.7554/eLife.00429.015

Tables

Table 1

Intact TORC1 function is necessary for mmBCFA-mediated growth regulation

https://doi.org/10.7554/eLife.00429.009
GenotypeRNAiDietary C17ISONormalized % of F1 reached adulthoodNp
elo-5(−)Vector0208
elo-5(−)Vector+86.92050
elo-5(−) nprl-3(−)Vector71.569
elo-5(−) nprl-3(−)Vector+111.51030.24
elo-5(−) nprl-3(−)raga-1 (a)35.556
elo-5(−) nprl-3(−)raga-1 (a)+102.5830.031
elo-5(−) nprl-3(−)raga-1 (b)25.5214
elo-5(−) nprl-3(−)raga-1 (b)+88.02790.00001
elo-5(−) nprl-3(−)rheb-1 (a)14.2351
elo-5(−) nprl-3(−)rheb-1 (a)+64.65260.0000001
elo-5(−) nprl-3(−)rheb-1 (b)32.5201
elo-5(−) nprl-3(−)rheb-1 (b)+86.01160.0037
elo-5(−) nprl-3(−)rsks-1 (a)15.068
elo-5(−) nprl-3(−)rsks-1 (a)+72.5830.022
elo-5(−) nprl-3(−)rsks-1 (b)19.0183
elo-5(−) nprl-3(−)rsks-1 (b)+61.01800.0034
elo-5(−) nprl-3(−)ife-2 (a)10.5144
elo-5(−) nprl-3(−)ife-2 (a)+71.5490.003
elo-5(−) nprl-3(−)ife-2 (b)0103
elo-5(−) nprl-3(−)ife-2 (b)+103.51500
elo-5(−) nprl-3(−)let-3630>100
elo-5(−) nprl-3(−)let-363+0>100NA
  1. Percentages of elo-5(−);nprl-3(ku540) homozygotes with the indicated RNAi treatments that reached adulthood, where (a) and (b) indicate two different RNAi constructs targeting different parts of the same gene. The presented percentage of elo-5(−) nprl-3(ku540) animals that reached adulthood was calculated by normalizing against the percentage of elo-5(−/+) nprl-3(ku540)/+ heterozygotes (see ‘Materials and methods’ for detail). Without C17ISO supplementation, RNAi knockdown of multiple TORC1 components reverted elo-5(−);nprl-3(ku540) animals to larval arrest.

Additional files

Supplementary file 1

Detailed description of phenotypes listed in Figure 1B.

https://doi.org/10.7554/eLife.00429.016

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