(A) Positions and effects of mutant fld-1 alleles; transmembrane domains predicted by Phobius are indicated as blue boxes, and the Pfam PF03798 TLC domain is underlined in green. (B–D) Introduction …
The alignment produces 86 conserved positions, including, 34 identical position, out of 350 aa. The alignment was generated using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/). The boxed regions …
(A) The fld-1(gk653147) carries a premature STOP codon and suppresses the glucose intolerance and tail tip defect of the paqr-2 mutant. (B–C) Western blot detection of the FLD-1 protein in the …
(A) Confocal image of a live C. elegans L1 larva expressing the Pfld-1::GFP translational reporter. Note the ubiquitous and clear plasma membrane localization which outlines the shape of each cell. …
(A) Structure of FLD-1::GFP expression constructs driven by different promoters whereby the fld-1 promoter is ubiquitously expressed, the elt-3 promoter is hypodermis-specific and the ges-1 promoter …
The single mutants fld-1(et48) and fld-1(et49) had no obvious phenotype though they often suppressed the paqr-2 mutant defects for the following traits: (A) brood size, (B) pharyngeal pumping rate, …
(A) The five fld-1 alleles tested improve the growth of the paqr-2 mutant on nematode growth media (NGM) and suppressed the paqr-2 growth arrest phenotype on NGM media containing 20 mM glucose. (B) …
(A–B) In vivo FRAP measurements show that fld-1(et48) suppresses the membrane fluidity defect in paqr-2 mutant worms grown on 20 mM glucose or fed PA-loaded E. coli, respectively; note the …
Lipidomics data for panel F.
Lipidomics data for panel G.
(A) MUFA levels are reduced in all mutants grown on 20 mM glucose. (B) The paqr-2 mutant shows an excess of SFAs among the PEs when fed E. coli cultivated on 2% ethanol (vehicle for PA) or E. coli …
Lipidomics data for panel A.
Lipidomics data for panel B.
Lipidomics data for panel C.
Lipidomics data for panel D.
All FRAP experiments were performed on synchronized L1 worms and each panel shows the results from separate experimental sessions on control NGM plates (CONTROL row), worms grown overnight on 20 mM …
The fat-2 has a growth defect on control plates (A) and on plates containing 20 mM glucose (B) that is corrected by the fld-1(et48) mutation; the fld-1(et48) allele also corrects the growth of paqr-2…
Lipidomics data for panel D.
(A–B) FRAP analysis showing that siRNA against TLCD1 or TLCD2 prevent membrane rigidification by 400 μM PA in HEK293 human cells; note the significantly lower Thalf value in the TLCD1 and TLCD2 …
Lipidomics data for panel C.
Lipidomics data used in panels D-E.
Lipidomics data for panels H-I.
Note the difference in scale between TLCD1 (A) and TLCD2 (B), which shows a much wider range of expression levels for TLCD2.
(A) QPCR quantification of TLCD1 and TLCD2 48 or 72 hr after siRNA treatment and normalized to cells treated with NT siRNA. (B) Image of BODIPY 500/510 C1, C12-labelled HEK293 cells. Note the …
Lipidomics data for panels K-L.
siRNA knockdown does not cause large changes in the FA composition of PCs (A), PEs (B), PC/PE ratio (C), free cholesterol-to-PC ratio (D), or ceramide levels, which are strongly affected by …
Lipidomics data for panels A-B.
Lipidomics data for panels C-D.
Lipidomics data for panels E-H.
Lipidomics data for panels I-J.
(A–C) FRAP analysis showing that siRNA against TLCD2 or inclusion of 5 μM EPA protects against the rigidifying effects of 200 μM PA in cells where AdipoR2 is also knocked down. Note the …
Lipidomics data used in panel D.
Lipidomics data for panel E.
(A) qPCR quantification of the siRNA knockdown efficiency against AdipoR2 and TLCD2. (B–E) Effect of AdipoR2, TLCD1, TLCD2 knockdown and 5 μM EPA on ceramide composition. Note that exogenous TLCD1 …
Lipidomics data used in panels B-E
Loss of membrane fluidity, which may result from lowered temperature or SFA-rich diets, is sensed by the PAQR-2/IGLR-2 complex that then signals to promote FA desaturation, hence restoring fluidity; …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (C. elegans) | N2 | C. elegans Genetics Center (CGC) | ||
Strain, strain background (C. elegans) | CL2166 (dvIs19 [(pAF15)gst- 369 4 p::GFP::NLS] | C. elegans Genetics Center (CGC); PMID: 12078522 | ||
Strain, strain background (C. elegans) | HA1842 [rtIs30(pfat-7::GFP)] | PMID: 22035958 | Gift from Amy Walker | |
Genetic reagent (C. elegans) | fld-1(et45); fld-1(et46); fld-1(et47); fld-1(et48); fld-1(et49); fld-1(et50); fld-1(et51); fld-1(et53) | This paper | et45, et48 and et49 will be deposited at CGC | |
Genetic reagent (C. elegans) | SJ4005 [zcIs4 (hsp-4::GFP)] | C. elegans Genetics Center (CGC) | ||
Genetic reagent (C. elegans) | fld-1(gk653147) | C. elegans Genetics Center (CGC) | From strain VC40470 | |
Genetic reagent (C. elegans) | paqr-2(tm3410) | C. elegans Genetics Center (CGC); doi:10.1371/ journal.pone.0021343 | ||
Genetic reagent (C. elegans) | iglr-2(et34) | C. elegans Genetics Center (CGC); PMID 27082444 | ||
Genetic reagent (C. elegans) | mdt-15(et14) | C. elegans Genetics Center (CGC); DOI 10.1371/journal. pgen.1003801.s013 | ||
Genetic reagent (C. elegans) | cept-1(et10) | C. elegans Genetics Center (CGC); DOI 10.1371/journal. pgen.1003801.s013 | ||
Genetic reagent (C. elegans) | hacd-1(et12) | C. elegans Genetics Center (CGC); DOI 10.1371/journal.pgen.1003801.s013 | ||
Genetic reagent (C. elegans) | Y48G8AL.13 (ok3097) | C. elegans Genetics Center (CGC) | ||
Genetic reagent (C. elegans) | F41H10.5 (gk530235) | C. elegans Genetics Center (CGC) | From strain VC40240 | |
Cell line (Homo sapiens) | HEK293 | |||
Antibody | Rabbitt anti-FLD-1 | This paper | Raised by GeneScript against this peptide: TQVGDVESGPLRTQ. Used at 1:500 dilution for Western blot. | |
Recombinant DNA reagent | Pfld-1::FLD-1 | This paper | ||
Recombinant DNA reagent | Pfld-1::FLD-1::GFP | This paper | ||
Recombinant DNA reagent | Pelt-3::FLD-1::GFP | This paper | ||
Recombinant DNA reagent | Pges-1::FLD-1::GFP | This paper | ||
Sequence- based reagent | TLCD1 siRNA | Dharmacon | J-015483–10 | |
Sequence- based reagent | TLCD2 siRNA | Dharmacon | J-180826–09; J-180826–16; J- 180826–17 | |
Sequence- based reagent | AdipoR2 siRNA | Dharmacon | J-007801 | |
Sequence- based reagent | NT siRNA | Dharmacon | D-001810–10 | Non-target control |
Sequence- based reagent | FADS2 siRNA | Dharmacon | J-008211–09 | |
Sequence- based reagent | PPIB siRNA | Dharmacon | D-001820–10 | |
Commercial assay or kit | FITC Annexin V Apoptosis Detection Kit I | BD Bioscience | Cat No 556547 | |
Commercial assay or kit | RevertAid H Minus First Strand cDNA Synthesis Kit | ThermoFisher | K1631 | |
Commercial assay or kit | High Capacity cDNA Reverse Transcription Kit | Applied Biosystem | 10400745 | |
Commercial assay or kit | Hot FIREPol EvaGreen qPCR Supermix | Solis Biodyne | 08-36-00001 | |
Commercial assay or kit | Viromer Blue | Lipocalyx | VB-01LB-01 | |
Chemical compound, drug | Palmitic acid | Sigma-Aldrich | P0500 | |
Chemical compound, drug | Linoleic acid | Sigma-Aldrich | L1376 | |
Chemical compound, drug | Eicosapentaenoic acid | Sigma-Aldrich | E2011 | |
Chemical compound, drug | [9,10-3H(N)]-Palmitic Acid | Perkin Elmer | NET043001MC | |
Chemical compound, drug | Linoleic acid-[13C18] | IsoSciences | S14495-1.0 | IsoSciences provided a stock dissolved in DMSO |
Chemical compound, drug | BODIPY 500/510 C1,C12 | Invitrogen | D3823 |