1. Cell Biology
  2. Structural Biology and Molecular Biophysics

Pre-existing bilayer stresses modulate triglyceride accumulation in the ER versus lipid droplets

  1. Valeria Zoni
  2. Rasha Khaddaj
  3. Pablo Campomanes
  4. Abdou Rachid Thiam
  5. Roger Schneiter
  6. Stefano Vanni  Is a corresponding author
  1. University of Fribourg, Department of Biology, Switzerland
  2. Laboratoire de Physique de l’École Normale Supérieure, ENS, Université PSL, CNRS, Sorbonne Université, Université de Paris, France
https://doi.org/10.7554/eLife.62886
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  1. Valeria Zoni
  2. Rasha Khaddaj
  3. Pablo Campomanes
  4. Abdou Rachid Thiam
  5. Roger Schneiter
  6. Stefano Vanni
(2021)
Pre-existing bilayer stresses modulate triglyceride accumulation in the ER versus lipid droplets
eLife 10:e62886.
https://doi.org/10.7554/eLife.62886
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6 figures, 1 video, 2 tables and 2 additional files

Figures

Figure 1 with 1 supplement
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Diacylglycerol (DAG) promotes nucleation of triglyceride (TG) blisters.

(A) Fluorescence microscopy images of WT, dga1Δlro1Δare1Δare2Δ (4Δ) and pah1Δare1Δare2Δ yeast cells. Left panels: neutral lipids (NLs) are stained by BODIPY, right panels: ER-DAG sensor staining. Microscope intensity settings in the three panels are identical to allow for quantitative comparison. (B) Setup used to investigate TG nucleation in molecular dynamics (MD) simulations. TG at different concentrations are randomly distributed in a bilayer and simulations are run until formation of blisters or for a total length of 1.5 µs. TG concentrations are reported as ratio between TG and phospholipids (PLs). (C) Rate of formation of TG blisters at different TG concentrations, obtained from MD simulations. (D) Rate of formation of TG blisters at different TG concentrations in the presence of DAG (10 mol%, 20 mol%), from MD simulations. TG concentrations are reported as ratio between TG and PLs. (E) Top view of TG nucleation from simulations of lipid bilayers with two coexisting DAG concentrations (6 mol% and 13 mol%). The two bilayer regions with different DAG concentrations have identical surface areas.

Figure 1—figure supplement 1
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Diacylglycerol (DAG) promotes nucleation of triglyceride (TG) blisters.

Concentration of DAG in different regions over time in one replica. DAG molecules in the region with the highest concentration are colored in cyan, and DAG molecules in the region with the lowest concentration are colored in magenta.

Figure 2 with 1 supplement
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Triglyceride (TG) blister formation in lipid bilayers is consistent with a phase separation process.

(A) Setup used to measure the amount of diluted TG via spontaneous diffusion from a pre-formed blister and (B) quantification over time. The area in which TG is assumed as diluted is highlighted in light red. (C) Time evolution of the percentage of diluted TG inside the bilayer. The injection of additional TG molecules was performed after 3 μs of dynamics. (D) Comparison between the values of diluted TG before and after the insertion of TG in the bilayer. (E) Quantification of diluted TG in oil blisters of different sizes. (F) Time evolution of blister dissolution when the total TG concentration in the system is below the threshold observed in (B and C). (G) Percentage of diluted TG in lipid bilayers enriched in diacylglycerol (DAG) lipids. In all the panels, TG concentrations are reported as ratio between TG and phospholipid (PL).

Figure 2—figure supplement 1
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Choice of the radius for the calculation of diluted triglycerides (TG).

We chose the smallest radius (black) above which we got always the same value of diluted TG. 

Figure 3
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Endoplasmic reticulum (ER) membrane lipids promote triglyceride (TG) blister formation.

(A, D, G, J, M) Lipid compositions tested and chemical structures of the various lipids involved in the mixtures. (B, E, H, K, N) Corresponding rates of blister formation, and (C, F, I, L, O) percentage of diluted TG. In all the panels, TG concentrations are reported as ratio between TG and phospholipid (PL).

Figure 4
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Pre-existing bilayer stresses modulate in-bilayer diluted triglyceride (TG) concentration.

(A–C) Depletion-enrichment maps for (A) DOPC + 20 mol% DOPE, (B) DOPC + 20 mol% diacylglycerol (DAG), and (C) DOPC + 60 mol% DPPC bilayers in the presence of a TG blister (dashed line). (D) Correlation between the relative nucleation energy (Enucl) for different bilayer compositions (with respect to Enucl of DOPC bilayers) and excess TG. (E) Lateral pressure profile of bilayers containing different lipid compositions. (F and G) Correlation between chain pressure πCH (F) or monolayer curvature stress (G) and TG equilibrium concentration for bilayer mixtures containing DOPC, DOPE, DAG, DPPC, DLPC, DLPE, and cholesterol. All the percentage in the graph referring to bilayer compositions are to be intended as mol%, while TG concentrations are reported as ratio between TG and phospholipid (PL).

Figure 5
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Lipid saturation promotes triglyceride (TG) accumulation in the endoplasmic reticulum (ER).

(A) Chemical structure of unsaturated TG used in molecular dynamics (MD) simulations. Black ovals indicate the presence of double bonds in the acyl chain. (B) Equilibrium 'diluted' concentration of different TG in DOPC bilayers. TG concentrations are reported as ratio between TG and phospholipids (PLs). (C and D) Fluorescence microscopy images of wild-type and temperature-sensitive ole1ts at 24°C (C) and 37°C (D). Strains were cultivated in SC medium containing palmitic acid (C16:0) or palmitoleic acid (C16:1) and cells were stained with the neutral lipid (NL) marker BODIPY. The blue arrowheads highlight lipid droplets (LDs), and the pink arrows point to the cortical and perinuclear ER membrane. (E and F) Quantification of fluorescent intensities of LDs and membranes (n = 50) in the different conditions shown in (C and D): Values represent fluorescent intensity relative to total cellular fluorescence. Asterisks denote statistical significance (Student’s t-test, *p < 0.05, ***p < 0.001), n.s., non-significant.

Figure 6
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Short-chain lipids promote triglyceride (TG) accumulation in the endoplasmic reticulum (ER).

(A and B) Fluorescence microscopy images of wild-type and elo1∆ mutant cells cultivated in SC medium containing lauric acid (C12:0, 2 mM) or palmitic acid (C16:0, 2 mM) for the indicated period of time. Cells were stained with BODIPY and visualized by fluorescence microscopy. Lipid droplets (LDs) are indicated by blue arrowheads and ER stained with BODIPY is marked by purple arrows. Scale bar, 5 µM. (C and D) Quantification of BODIPY fluorescence of LD and cellular membranes (n = 50). Values represent fluorescent intensity relative to total cellular fluorescence. Asterisks denote statistical significance (Student’s t-test, *p < 0.05, ***p < 0.001), n.s., non-significant.

Videos

Video 1
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Triglyceride (TG) lens dissolution in a DOPC bilayer.

The concentration of TG is below (<1%) the calculated ‘free TG’ threshold in a DOPC bilayer (1.1 ± 0.1%).

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (S. cerevisiae)BY4741EuroscarfRRID: SCR_003093Mata, his3∆1, leu2∆0, met15∆0, ura3∆0.
Chemical compound, drugPalmitic acidSigma-AldrichP0500
RRID:SCR_008988		2 mM
Chemical compound, drugPalmitoleic acidSigma-AldrichP9417
RRID:SCR_008988		2 mM
Chemical compound, drugLauric acidSigma-Aldrich8053330100
RRID:SCR_008988		2 mM
Chemical compound, drugBrij58Sigma-AldrichP5884
RRID:SCR_008988		1%
Software, algorithmVisiViewVisitron systems GMBH4.2.0
Software, algorithmImageJhttps://imagej.nih.gov/ij/RRID:SCR_003070Schneider et al., 2012
Software, algorithmPhotoshopAdobe, Mountain ViewRRID:SCR_014199
Software, algorithmLAMMPShttps://lammps.sandia.govRRID:SCR_015240MD simulations software
Software, algorithmGROMACShttp://www.gromacs.orgRRID:SCR_014565MD simulations software
Software, algorithmVMDhttp://www.ks.uiuc.edu/Research/vmd/RRID:SCR_001820
OtherBODIPY493/503 stainInvitrogenD3922
RRID:SCR_008988		1 µg/mL
Table 1
List of all the molecular dynamics (MD) setups, with bilayer composition, number of triglyceride (TG) molecules, number of replicas, and length of simulations.
TG lenses formation
SystemBilayer composition
(no. of molecules)		No. of TGNo. of replicasLength (μs)
100% DOPC3200 DOPC6431.5
3200 DOPC12831.5
3200 DOPC1923<1.5
3200 DOPC2563<1.5
3200 DOPC3203<1.5
+60 mol% POPC1280 DOPC + 1920 POPC6431.5
1280 DOPC + 1920 POPC12831.5
1280 DOPC + 1920 POPC1923<1.5
1280 DOPC + 1920 POPC2563<1.5
1280 DOPC + 1920 POPC3203<1.5
+ 60 mol% DPPC1280 DOPC + 1920 DPPC6431.5
1280 DOPC + 1920 DPPC12831.5
1280 DOPC + 1920 DPPC1923<1.5
1280 DOPC + 1920 DPPC2563<1.5
1280 DOPC + 1920 DPPC3203<1.5
+ 60 mol% DLPC1280 DOPC + 1920 DLPC6431.5
1280 DOPC + 1920 DLPC12831.5
1280 DOPC + 1920 DLPC19231.5
1280 DOPC + 1920 DLPC2563<1.5
1280 DOPC + 1920 DLPC3203<1.5
+ 60 mol% DOPE1280 DOPC + 1920 DOPE6431.5
1280 DOPC + 1920 DOPE1283<1.5
1280 DOPC + 1920 DOPE1923<1.5
1280 DOPC + 1920 DOPE2563<1.5
1280 DOPC + 1920 DOPE3203<1.5
+30 mol% DOPE2240 DOPC + 960 DOPE6431.5
2240 DOPC + 960 DOPE12831.5
2240 DOPC + 960 DOPE1923<1.5
2240 DOPC + 960 DOPE2563<1.5
2240 DOPC + 960 DOPE3203<1.5
+ 60 mol% DLPE1280 DOPC + 1920 DLPE6431.5
1280 DOPC + 1920 DLPE1283<1.5
1280 DOPC + 1920 DLPE1923<1.5
1280 DOPC + 1920 DLPE2563<1.5
1280 DOPC + 1920 DLPE3203<1.5
+10 mol% Diacylglycerol (DAG)2880 DOPC + 320 DAG6431.5
2880 DOPC + 320 DAG1283<1.5
2880 DOPC + 320 DAG1923<1.5
2880 DOPC + 320 DAG2563<1.5
2880 DOPC + 320 DAG3203<1.5
+20 mol% DAG2560 DOPC + 640 DAG6431.5
2560 DOPC + 640 DAG1283<1.5
2560 DOPC + 640 DAG1923<1.5
2560 DOPC + 640 DAG2563<1.5
2560 DOPC + 640 DAG3203<1.5
+10 mol% CHOL2880 DOPC + 320 CHOL6431.5
2880 DOPC + 320 CHOL1283<1.5
2880 DOPC + 320 CHOL1923<1.5
2880 DOPC + 320 CHOL2563<1.5
2880 DOPC + 320 CHOL3203<1.5
+20 mol% CHOL2560 DOPC + 640 CHOL6431.5
2560 DOPC + 640 CHOL1283<1.5
2560 DOPC + 640 CHOL1923<1.5
2560 DOPC + 640 CHOL2563<1.5
2560 DOPC + 640 CHOL3203<1.5
Endoplasmic reticulum (ER) like1856 DOPC + 960 DOPE + 192 CHOL + 192 DAG6431.5
1856 DOPC + 960 DOPE + 192 CHOL + 192 DAG1283<1.5
1856 DOPC + 960 DOPE + 192 CHOL + 192 DAG1923<1.5
1856 DOPC + 960 DOPE + 192 CHOL + 192 DAG2563<1.5
1856 DOPC + 960 DOPE + 192 CHOL + 192 DAG3203<1.5
Calculation of ‘free TG’
100% DOPC6050 DOPC183623
+ 60 mol% POPC2420 DOPC + 3630 POPC183623
+ 60 mol% DPPC2420 DOPC + 3630 DPPC183623
+ 60 mol% DLPC2420 DOPC + 3630 DLPC183623
+ 60 mol% DOPE2420 DOPC + 3630 DOPE183623
+ 30 mol% DOPE4235 DOPC + 1815 DOPE183623
+ 60 mol% DLPE2420 DOPC + 3630 DLPE183623
+10 mol% DAG5445 DOPC + 605 DAG183623
ER like3509 DOPC + 1815 DOPE + 363 DAG + 363 CHOL183623
+20 mol% DAG4840 DOPC + 1210 DAG183623
+10 mol% CHOL5445 DOPC + 605 CHOL183623
+20 mol% CHOL4840 DOPC + 1210 CHOL183623
Insertion of TG6050 DOPC193423
Different sizes11,250 DOPC183623
11,250 DOPC550823
11,250 DOPC918023
16,200 DOPC13,66523
Dissolution
Dissolution6050 DOPC5010.8
Effect of saturation in TG chains
DOPC+TOOP6050 DOPC1836 TOOP23
DOPC+TOPP6050 DOPC1836 TOPP23
Lateral pressure profile
DOPC3200030.2
+10 mol% CHOL2880 DOPC + 320 CHOL030.2
+20 mol% DAG2560 DOPC + 640 DAG030.2
+10 mol% DAG2880 DOPC + 320 DAG030.2
+30 mol% DOPE2240 DOPC + 960 DOPE030.2
+20 mol% DOPE2560 DOPC + 640 DOPE030.2
+60 mol% DLPC1280 DOPC + 1920 DLPC030.2
+60 mol% DLPE1280 DOPC + 1920 DLPE030.2
+60 mol% DPPC1280 DOPC + 1920 DPPC030.2

Additional files

Supplementary file 1

Additional information.

Supplementary table 1. Non-bonded parameters derived for this study. Supplementary table 2. Calculated values of πCH for each bilayer composition and the relative percentage change with respect to the composition ‘100% DOPC’. Supplementary table 3. Calculated values of κb for each bilayer composition with two different methods: from the real-space analysis of the instantaneous surface deformations (κb ReSIS) and from the Fourier-space analysis of the membrane fluctuations (κb Fluct). Supplementary table 4: S. cerevisiae strains used in this study.

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  1. Valeria Zoni
  2. Rasha Khaddaj
  3. Pablo Campomanes
  4. Abdou Rachid Thiam
  5. Roger Schneiter
  6. Stefano Vanni
(2021)
Pre-existing bilayer stresses modulate triglyceride accumulation in the ER versus lipid droplets
eLife 10:e62886.
https://doi.org/10.7554/eLife.62886