Membrane fluidity is regulated by the C. elegans transmembrane protein FLD-1 and its human homologs TLCD1/2

7 figures, 1 table and 1 additional file

Figures

Figure 1 with 6 supplements
Mutant alleles, homology comparisons and expression profile of C.elegans fld-1.

(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 …

https://doi.org/10.7554/eLife.40686.003
Figure 1—figure supplement 1
Description of the novel fld-1 mutant alleles.
https://doi.org/10.7554/eLife.40686.004
Figure 1—figure supplement 2
Alignment of Y63D3A.8 (i.e. FLD-1), its closest C. elegans homolog (K12H6.6) and two closest human homologs (TLCD1 and TLCD2).

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 …

https://doi.org/10.7554/eLife.40686.005
Figure 1—figure supplement 3
Effect of independently isolated mutant alleles of fld-1, Y48G8AL.13 and F41H10.5.

(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 …

https://doi.org/10.7554/eLife.40686.006
Figure 1—figure supplement 4
Expression profile of the in vivo reporter Pfld-1::GFP.

(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. …

https://doi.org/10.7554/eLife.40686.007
Figure 1—figure supplement 5
Tissue-specific expression of FLD-1::GFP.

(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 …

https://doi.org/10.7554/eLife.40686.008
Figure 1—figure supplement 6
fld-1 single mutant worms have no obvious phenotype.

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, …

https://doi.org/10.7554/eLife.40686.009
Comparison and genetic interaction of the novel fld-1 alleles with other paqr-2 and iglr-2 suppressors.

(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) …

https://doi.org/10.7554/eLife.40686.010
Figure 3 with 3 supplements
fld-1(et48) corrects membrane phospholipid composition and several membrane defects of the paqr-2 mutant.

(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 …

https://doi.org/10.7554/eLife.40686.011
Figure 3—source data 1

Lipidomics data for panel F.

https://doi.org/10.7554/eLife.40686.020
Figure 3—source data 2

Lipidomics data for panel G.

https://doi.org/10.7554/eLife.40686.021
Figure 3—figure supplement 1
The fld-1(et48) mutation ameliorates the paqr-2 mutant lipid profiles and enhances the protective effects of mdt-15(et14).

(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

https://doi.org/10.7554/eLife.40686.012
Figure 3—figure supplement 1—source data 1

Lipidomics data for panel A.

https://doi.org/10.7554/eLife.40686.013
Figure 3—figure supplement 1—source data 2

Lipidomics data for panel B.

https://doi.org/10.7554/eLife.40686.014
Figure 3—figure supplement 1—source data 3

Lipidomics data for panel C.

https://doi.org/10.7554/eLife.40686.015
Figure 3—figure supplement 1—source data 4

Lipidomics data for panel D.

https://doi.org/10.7554/eLife.40686.016
Figure 3—figure supplement 2
The fld-1(et48) mutation protects paqr-2 mutants against membrane rigidification.

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 …

https://doi.org/10.7554/eLife.40686.017
Figure 3—figure supplement 3
The fld-1(et48) mutation suppresses the growth defect of the fat-2(wa17) mutant and promotes accumulation of the LCPUFA eicosapentaenoic acid (EPA).

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…

https://doi.org/10.7554/eLife.40686.018
Figure 3—figure supplement 3—source data 1

Lipidomics data for panel D.

https://doi.org/10.7554/eLife.40686.019
Figure 4 with 6 supplements
The mammalian TLCD1 and TLCD2 proteins regulate membrane composition and fluidity.

(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 …

https://doi.org/10.7554/eLife.40686.022
Figure 4—source data 1

Lipidomics data for panel C.

https://doi.org/10.7554/eLife.40686.031
Figure 4—source data 2

Lipidomics data used in panels D-E.

https://doi.org/10.7554/eLife.40686.032
Figure 4—source data 3

Lipidomics data for panels H-I.

https://doi.org/10.7554/eLife.40686.033
Figure 4—figure supplement 1
Relative expression levels of TLCD1 and TLCD2 among various mouse tissues.

Note the difference in scale between TLCD1 (A) and TLCD2 (B), which shows a much wider range of expression levels for TLCD2.

https://doi.org/10.7554/eLife.40686.023
Figure 4—figure supplement 2
Specificity of the TLCD1/2 siRNA effects.

(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 …

https://doi.org/10.7554/eLife.40686.024
Figure 4—figure supplement 2—source data 1

Lipidomics data for panels K-L.

https://doi.org/10.7554/eLife.40686.025
Figure 4—figure supplement 3
Effect of TLCD1/2 knockdown on FA composition and membrane fluidity.

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 …

https://doi.org/10.7554/eLife.40686.026
Figure 4—figure supplement 3—source data 1

Lipidomics data for panels A-B.

https://doi.org/10.7554/eLife.40686.027
Figure 4—figure supplement 3—source data 2

Lipidomics data for panels C-D.

https://doi.org/10.7554/eLife.40686.028
Figure 4—figure supplement 3—source data 3

Lipidomics data for panels E-H.

https://doi.org/10.7554/eLife.40686.029
Figure 4—figure supplement 3—source data 4

Lipidomics data for panels I-J.

https://doi.org/10.7554/eLife.40686.030
Figure 4–video 1
Movie of a FRAP experiment on NT siRNA-treated HEK293 cells.
https://doi.org/10.7554/eLife.40686.034
Figure 4–video 2
Movie of a FRAP experiment on NT siRNA-treated HEK293 cells cultivated in the presence of 400 μM PA.
https://doi.org/10.7554/eLife.40686.035
Figure 4–video 3
Movie of a FRAP experiment on TLCD2 siRNA-treated HEK293 cells cultivated in the presence of 400 μM PA.
https://doi.org/10.7554/eLife.40686.036
Figure 5 with 1 supplement
TLCD2 knockdown protects against AdipoR2 knockdown.

(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 …

https://doi.org/10.7554/eLife.40686.037
Figure 5—source data 1

Lipidomics data used in panel D.

https://doi.org/10.7554/eLife.40686.040
Figure 5—source data 2

Lipidomics data for panel E.

https://doi.org/10.7554/eLife.40686.041
Figure 5—figure supplement 1
Protective effects of TLCD1/2 knockdown against AdipoR2 knockdown.

(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 …

https://doi.org/10.7554/eLife.40686.038
Figure 5—figure supplement 1—source data 1

Lipidomics data used in panels B-E

https://doi.org/10.7554/eLife.40686.039
Updated model of membrane fluidity regulation in C. elegans.

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; …

https://doi.org/10.7554/eLife.40686.042

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain
background
(C. elegans)
N2C. 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: 22035958Gift 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 paperet45, 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
AntibodyRabbitt anti-FLD-1This paperRaised by GeneScript
against this
peptide:
TQVGDVESGPLRTQ.
Used
at 1:500 dilution for Western blot.
Recombinant
DNA reagent
Pfld-1::FLD-1This paper
Recombinant
DNA reagent
Pfld-1::FLD-1::GFPThis paper
Recombinant
DNA reagent
Pelt-3::FLD-1::GFPThis paper
Recombinant
DNA reagent
Pges-1::FLD-1::GFPThis paper
Sequence-
based reagent
TLCD1 siRNADharmaconJ-015483–10
Sequence-
based reagent
TLCD2 siRNADharmacon
J-180826–09; J-180826–16; J-
180826–17
Sequence-
based reagent
AdipoR2 siRNADharmaconJ-007801
Sequence-
based reagent
NT siRNADharmaconD-001810–10Non-target control
Sequence-
based reagent
FADS2 siRNADharmaconJ-008211–09
Sequence-
based reagent
PPIB siRNADharmaconD-001820–10
Commercial
assay or kit
FITC Annexin V Apoptosis
Detection
Kit I
BD BioscienceCat No 556547
Commercial
assay or kit
RevertAid H Minus First Strand
cDNA Synthesis Kit
ThermoFisherK1631
Commercial
assay or kit
High Capacity cDNA Reverse
Transcription Kit
Applied Biosystem10400745
Commercial
assay or kit
Hot FIREPol
EvaGreen qPCR
Supermix
Solis Biodyne08-36-00001
Commercial
assay or kit
Viromer BlueLipocalyxVB-01LB-01
Chemical
compound, drug
Palmitic acidSigma-AldrichP0500
Chemical
compound, drug
Linoleic acidSigma-AldrichL1376
Chemical
compound, drug
Eicosapentaenoic
acid
Sigma-AldrichE2011
Chemical
compound, drug
[9,10-3H(N)]-Palmitic AcidPerkin ElmerNET043001MC
Chemical
compound, drug
Linoleic acid-[13C18]IsoSciencesS14495-1.0IsoSciences
provided a stock
dissolved in DMSO
Chemical
compound, drug
BODIPY 500/510 C1,C12InvitrogenD3823

Additional files

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