Fig 1.
The paqr-2 mutant is sensitive to several glycolysis-related metabolites.
(A) Outline of the metabolic pathway connecting the substrates tested, which are indicated with red text. (B-F) Length of wild-type N2 and paqr-2 mutant worms cultivated for 72 hours on various concentrations of metabolite, with representative images shown in (G). Note that the initial length of the L1s in these experiments is approximately 0.23 mm (indicated by dashed line in B-F): any length greater than this therefore represents growth.
Fig 2.
De novo lipogenesis precursors cause reduced membrane fluidity in paqr-2 mutants as measured by FRAP.
(A) Confocal image of the prenylated GFP marker enriched on the plasma membrane of intestinal cells. Clear membrane stretches are indicated by arrowheads, and the circle indicate the size of a region bleached during FRAP analysis. (B-F) FRAP results from worms grown under different conditions. Thalf values refer to the time by which half of the maximal fluorescence recovery is achieved (shown in B). Note the reduced membrane fluidity of the paqr-2 mutant grown on glycerol or pyruvate, as evidenced by the lengthened Thalf.
Fig 3.
E. coli is responsible for the conversion of precursors into saturated fatty acids.
(A) Glucose is not toxic to paqr-2 mutants fed a glucose uptake-deficient OP50 E. coli ΔPTS mutant. (B) Several mutations that affect the metabolism of fatty acid of precursors abrogate their toxicity. Mutations tested are indicated with orange text. (C) Representative images of E. coli mutants that support growth of the C. elegans paqr-2 mutant in the presence of glucose or glycerol. Note the glossy and discolored appearance of the pfkA mutant. (D) Growth of the paqr-2 mutant on 0.5% glycerol plates seeded with the parental BW25113 and five E. coli mutants that effectively prevent glycerol toxicity. The dashed line in (A) and (D) represents the approximate length of the L1s at the start of the experiments.
Fig 4.
PAQR-2 is required to prevent the toxicity of a diet with a high SFA/MUFA ratio.
(A) Even chain SFA/MUFA and 16:0/18.1 ratios in the PEs of E. coli samples from different strains or culture conditions. Conditions where the bars reached higher than the dashed red line (i.e. ratio of ~2:1 or higher) caused growth arrest and lethality of the paqr-2 mutant. (B) SFA fraction within the PEs of worms grown on control plates (NGM) or plates containing the indicated additives. Note the excessive enrichment of SFAs in paqr-2 mutants cultivated on glucose, glycerol or pyruvate.
Fig 5.
Palmitic acid causes membrane rigidity in the paqr-2 mutant.
(A) Outline of the experimental design. (B) PA content and PA/OA ratio among the PEs of E. coli grown with or without PA; vehicle is ethanol. (C) E. coli grown pre-loaded with PA inhibits the growth of the paqr-2 mutant but not of wild-type N2 worms. (D) PA content among the PEs and PCs of worms fed control or PA-loaded E. coli. Note that the paqr-2 mutant accumulates more PA than wild-type N2 worms (E-F) FRAP of N2 and paqr-2 mutants on normal plates or plates seeded with PA-loaded E. coli, respectively. Note the loss of membrane fluidity in paqr-2 worms fed PA-loaded E. coli. (G) The growth of paqr-2 is greatly ameliorated by pre-loading E. coli with both PA and OA rather than PA alone, with photographed worms shown in (H). (I) FRAP showing that the rigidifying effects of PA pre-loaded dietary E. coli on the paqr-2 mutant are abrogated by pre-loading the E coli with both PA and OA. The dashed line in (C) and (G) represents the approximate length of the L1s at the start of the experiments.
Fig 6.
Mammalian AdipoR2 prevents rigidification by palmitic acid.
(A) FRAP analysis showing that 400 μM PA causes reduced membrane fluidity in HEK293 cells and that 400 μM OA causes increased membrane fluidity even when PA is included. (B) Thalf values from FRAP analyses performed with different concentrations of PA and OA. (C) Quantitative PCR results showing the degree of siRNA inhibition of the indicated genes. (D-G) The morphology of HEK293 cells is altered by PA when AdipoR2 is knocked down. Note the presence of numerous circular structures in the BODIPY-labeled cells treated with AdipoR2 siRNA (arrows). Nuclei are indicated by the letter "N", and the circle in (D) indicates the size of the area bleached in FRAP experiments with HEK293 cells. (H-K) FRAP analyses showing that AdipoR2 knockdown greatly increases the rigidifying effect of PA. (L-O) Lipidomics analysis of HEK 293 cells cultivated 24 hours in serum free media or serum-free media containing PA, OA or PA + OA and treated with non-target siRNA (NT) or AdipoR1 or AdipoR2 siRNA, as indicated. Note that PA alone causes a dramatic increase in SFAs among PCs, PEs and TAGs, and that this effect is increased by AdipoR2 knockdown. (P-Q) FRAP analyses showing that AdipoR2 knockdown greatly increases the rigidifying effect of 200 μM PA. (R-S) Lipidomics analysis showing that AdipoR2 siRNA also causes an excess of SFAs among PCs and PEs when the cells are incubated with 200 μM PA. Statistical analysis in L-O and R-S were done by comparing siRNA-treated cells with the non-target siRNA cultivated under the same conditions.
Table 1.
Thalf values from FRAP experiments in HEK293 cells.
Fig 7.
Model of membrane fluidity regulation by PAQR-2/AdipoR2.
This revised model proposes that PAQR-2 (AdipoR2 in mammals) and IGLR-2 (homolog unknown in human) act as sensors that are activated by low membrane fluidity caused by cold (in C. elegans) or exogenous SFAs (in C. elegans and human cells), and act on downstream effectors to restore fluidity by promoting changes in fatty acid metabolism, including promoting the activity of fatty acid desaturases. In this visualization, the red head groups indicate phospholipids with UFAs resulting from PAQR-2/AdipoR2 activity.