Exceptional stability of a perilipin on lipid droplets depends on its polar residues, suggesting multimeric assembly
- Institut Jacques Monod, CNRS, Université de Paris, France
- Université Paris-Saclay, France
- Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Slovenia
- CRBM, University of Montpellier and CNRS, France
- Université Côte d’Azur, CNRS, IPMC, France
- Chair of Microprocess Engineering and Technology – COMPETE, University of Ljubljana, Slovenia
Figures
Figure 1 with 1 supplement
Plin4 AH forms very stable oil particles.
(A) Helical wheel representations of Plin4 and Plin4 4T > S AHs. (B) Summary of LD localization of Plin4 AH and mutants (Čopič et al., 2018). (C) A 10 µl drop of triolein was added to 190 μl of HK …
Figure 1—figure supplement 1
Summary of FRAP experiments on oil droplets.
(A) Examples of FRAP experiments on oil droplets formed with Plin4 4T > S mutant. Emulsions of triolein were prepared with a solution of 4T > S (0.5 mg/ml) and Alexa488-4T > S (0.025 mg/ml). FRAP …
Figure 2 with 2 supplements
Real-time monitoring of protein–oil interaction in a microfluidic system shows irreversible adsorption of Plin4 12mer-A488 on triolein.
(A) Scheme of the microfluidics experimental set-up. (B) Top row: confocal images of the triolein-buffer interface as formed in the microfluidic system after adsorption of Alexa488-labeled Plin4 …
Figure 2—figure supplement 1
Microfluidic experiments demonstrate that Plin4 12mer lowers the surface tension of oil.
Figure 2—figure supplement 2
Plin4 mutant 4T > S-A488 also adsorbs to the oil surface, but is outcompeted by Plin4 12mer-A488.
(A) Confocal images of the oil-buffer interface in the microfluidic system after adsorption of Plin4 4mer mutant 4T > S-A488 (0.1 mg/ml) on the oil surface and after rinsing with buffer. The …
Figure 3 with 1 supplement
Comparison of the LD-binding properties of perilipin AHs in yeast.
(A) Helical wheel representation of the AHs of Plin1 (aa 110–189 aa), Plin2 (aa 101–191 aa), Plin3 (aa 114–204), and Plin4 (aa 246–377, corresponding to the Plin4 4mer construct). In the case of …
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Figure 3—source data 1
Comparison of the LD-binding properties of perilipin AHs in yeast and their effect on LD size.
- https://cdn.elifesciences.org/articles/61401/elife-61401-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Plin AH fusion proteins co-localize with LDs in yeast and are expressed at similar levels.
(A) Co-localization of Plin1 AH, Plin2 AH, Plin3 AH, and Plin4 12mer mCherry fusions and LDs, stained with Bodipy in pet10Δ cells grown in oleic acid. Scale bar: 5 µm. (B) Western blot analysis of …
Figure 4 with 1 supplement
Dynamics of perilipin AH-GFP fusions on LDs or at the plasma membrane in yeast.
(A) Dynamics of AH-GFP fusions on LDs in pet10Δ cells grown for 48 hr in oleic acid. Images show two representative FRAP time-courses in cells expressing Plin3 AH-GFP (top panels) or Plin4 12mer-GFP …
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Figure 4—source data 1
Dynamics of perilipin AH-GFP fusions on LDs or at the plasma membrane in yeast assessed by FRAP.
- https://cdn.elifesciences.org/articles/61401/elife-61401-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Cell-to-cell variability in the recovery of Plin4 12mer-GFP after photobleaching of LDs in Drosophila S2 cells.
FRAP was performed on oleic acid-induced LDs in S2 cells stably transfected with Plin4 12mer-GFP. (A) Fluorescence recovery curves from individual cells in the same experiment performed in control …
Figure 5 with 1 supplement
Plin3 AH interacts much less strongly with oil than Plin4 AH.
(A) Turbidity assays with 0.5 mg/ml protein solutions of Plin4 12 mer, Plin4 4mer, Plin4 4mer mutant [4T > S], or Plin3 AH after vigorous vortexing with triolein (15 µl for 285 µl of protein …
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Figure 5—source data 1
Analysis of protein content and protein exchange in oil emulsions.
- https://cdn.elifesciences.org/articles/61401/elife-61401-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Plin3 AH purification and CD analysis.
(A) Tricine SDS-PAGE showing the purification of Plin3 AH after expression in E. coli. The lanes show the proteins present in the total bacteria extract (total) and after a four-step protocol, which …
Figure 6 with 2 supplements
The polar face of Plin4 AH is key for specific and stable coating of LDs in cells.
(A) Weblogo plot of the AH region of human Plin4 as determined by aligning its 29 33-mer repeats. The vertical arrows indicate the mutated aa: the NQ pair (gray), which was mutated into NN, QQ or QN;…
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Figure 6—source data 1
The polar face of Plin4 AH is key for specific and stable coating of LDs in cells.
- https://cdn.elifesciences.org/articles/61401/elife-61401-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Plin4 4mer mutants are not affected in their helical folding.
(A) CD spectra showing mean residue ellipticity (MRE) of purified Plin4 4mer (19 μM), 4T > S (19 μM) and NN (6 μM) mutants in Tris buffer (blue lines) or in buffer with 50% TFE (green lines). (B) …
Figure 6—figure supplement 2
Additional data for Plin4 charge-swap mutant.
(A) SDS-PAGE analysis of purified Plin4 12mer and csw 12mer. Gel stained with Coomassie Blue. (B) Western-blot analysis of Plin4 12mer-GFP and csw 12mer-GFP expression in WT yeasts, showing similar …
Figure 7
The distribution of charged residues in the polar face of Plin4 AH is key for stable coating of triolein in vitro.
(A) Fifteen µl triolein was added to 285 µl of HK buffer containing Plin4 12mer wild-type or the csw mutant (0.5 mg/ml each). After vigorous vortexing, the samples were photographed. (B) DLS …
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Figure 7—source data 1
Redistribution of charged residues of Plin4 AH affects particle size in oil emulsions and dynamics of protein-oil interaction.
- https://cdn.elifesciences.org/articles/61401/elife-61401-fig7-data1-v2.xlsx
Figure 8
Plin4 adopts a helical conformation at the surface of oil and forms a dense monolayer.
(A) CD analysis of three independent samples of Plin4 12mer-oil emulsion purified by sucrose step gradients (left panel), compared with CD spectra Plin4 12mer in a similar concentration range in …
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Figure 8—source data 1
Estimate of Plin4 12mer density on oil surface.
- https://cdn.elifesciences.org/articles/61401/elife-61401-fig8-data1-v2.xlsx
Author response image 1
Image showing a HeLa cell expressing Plin4 AH before FRAP.
The area that was bleached is marked.
Videos
Video 1
Adsorption of Plin4 onto the triolein interface.
A confocal time-lapse recording of diffusion of Alexa-488-labeled Plin4 12mer into the side microfluidic channel and its adsorption onto the triolein interface. The relative fluorescence intensity …
Video 2
Plin4 remains bound onto the triolein interface after rinsing.
A confocal time-lapse recording of rinsing the side microfluidic channel with buffer after Alexa-488-labeled Plin4 12mer adsorbed to the triolein interface (see Video 1). The fluorescence intensity …
Video 3
Alexa-488 dye does not adsorb onto the triolein interface.
A confocal time-lapse recording of Alexa-488 dye diffusion into the side microfluidic channel toward the triolein meniscus. The relative fluorescence intensity profile along the channel center is …
Video 4
Rinsing completely removes Alexa-488.
A confocal time-lapse recording of rinsing Alexa-488 dye out of the side microfluidic channel. The fluorescence intensity profile along the channel center is shown at the bottom. Scale bar: 100 µm.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | PLIN1 | Jacquier et al., 2013 | PLIN1_HUMAN AAH31084.1 | Supplementary file 1 |
| Gene (Homo sapiens) | PLIN2 | Jacquier et al., 2013 | PLIN2_HUMAN AAH05127.1 | Supplementary file 1 |
| Gene (Homo sapiens) | PLIN3 | Jacquier et al., 2013 | PLIN3_HUMAN AAC39751.1 | Supplementary file 1 |
| Strain, strain background (Escherichia coli) | BL21(DE3) | ThermoFisher | C600003 | Chemically competent cells |
| Strain, strain background (Saccharomyces cerevisiae) | BY4742 | Euroscarf | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 | |
| Strain, strain background (S. cerevisiae) | pet10Δ | Euroscarf | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 pet10Δ::KANMX4 | |
| Strain, strain background (S. cerevisiae) | Pet10-GFP | Huh et al., 2003 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 PET10-GFP::HisMX | |
| Cell line (D. melanogaster) | S2 | ThermoFisher | R69007 | |
| Cell line (Homo sapiens) | HeLa | ATCC | CCL-2 | |
| Antibody | Anti-GFP (rabbit polyclonal) | Thermo Fisher Scientific | A11122 | (1:5000) |
| Antibody | Anti-rabbit (goat polyclonal, HRP conjugate) | Sigma-Aldrich | A6154 | (1:5000) |
| Antibody | Anti-Vps10 (mouse monoclonal) | Molecular probes | A-21274 | (1:100) |
| Antibody | Anti-mouse (donkey, HRP conjugate) | GE Healthcare | NA934V | (1:10000) |
| Peptide, recombinant protein | Plin4 12mer | Čopič et al., 2018 | Human Plin4 (aa510-905) | Supplementary file 2 |
| Peptide, recombinant protein | Plin4 4mer | Čopič et al., 2018 | Human Plin4 (aa246-377) | Supplementary file 2 |
| Peptide, recombinant protein | Plin4(4T > S) (4mer) | This study | Supplementary file 2 | |
| Peptide, recombinant protein | Plin4(NN) (4mer) | This study | Supplementary file 2 | |
| Peptide, recombinant protein | Plin4 csw (12mer) | This study | Supplementary file 2 | |
| Peptide, recombinant protein | Plin3 AH | This study | Human Plin3 (aa113 – 205) | Supplementary file 2 |
| Chemical compound, drug | SyproOrange | ThermoFisher | S6651 | |
| Chemical compound, drug | Alexa488 C5 maleimide | ThermoFisher | A10254 | |
| Chemical compound, drug | Alexa568 C5 maleimide | ThermoFisher | A20341 | |
| Chemical compound, drug | Bodipy 493/503 | ThermoFisher | 11540326 | |
| Chemical compound, drug | Diphytanoyl-phosphatidylserine | Avanti Lipids | AVA-850408C-25Mg | |
| Chemical compound, drug | Diphytanoyl-phosphatidylcholine | Avanti Lipids | AVA-850356C-200Mg | |
| Other | Glass microfluidic chip with a T-junction | Dolomite | part # 3000086 | |
| Other | Glass microfluidic chip with a T-junction | Dolomite | part # 3000024 | |
| Other | Silica Microspheres,5.00 μm, SS05N | Bang laboratories | SS05003-0.5 |
Table 1
Plasmids used in this study.
| Name | Insert | Region (aa) * | Vector | Host † | Source |
|---|---|---|---|---|---|
| pCLG03 | Plin4 4mer | hPlin4(246-377) | pET21b | E. coli | Čopič et al., 2018 |
| pKE23 | Plin4 12mer | hPlin4(510-905) | pET21b | E. coli | Čopič et al., 2018 |
| pSB49 | 4T > S (4mer) | 4x[246–278 M5t] ‡ | pmCherry-N1 | Mamm | Čopič et al., 2018 |
| pMGA9 | 4T > S (4mer) | 4x[246–278 M5t] ‡ | pET21b | E. coli | This study |
| pGFP-Plin1 | Human Plin1 | Full cDNA | pGREG576 (ADH1pr, GFP) | Yeast | Jacquier et al., 2013 |
| pGFP-Plin2 | Human Plin2 | Full cDNA | pGREG576 (ADH1pr, GFP) | Yeast | Jacquier et al., 2013 |
| pGFP-Plin3 | Human Plin3 | Full cDNA | pGREG576 (ADH1pr, GFP) | Yeast | Jacquier et al., 2013 |
| pRHT140 | ADHpr-mcs-GFP | pRS416 (CEN-URA3) | Yeast | S. Leon | |
| pMGA4 | ADHpr-mcs-mCherry (swap of GFP in pRHT140) | pRS416 (CEN-URA3) | Yeast | This study | |
| pMGA11 | Plin1 AH-GFP | hPlin1(aa108-194) | pRHT140 | Yeast | This study |
| pMGA10 | Plin1 AH-mChe | hPlin1(aa108-194) | pMGA4 | Yeast | This study |
| pMGA6 | Plin2 AH-GFP | hPlin2(aa100-192) | pRHT140 | Yeast | This study |
| pMGA5 | Plin2 AH-mCherry | hPlin2(aa100-192) | pMGA4 | Yeast | This study |
| pMGA7 | Plin3 AH-GFP | hPlin3(aa113-205) | pRHT140 | Yeast | This study |
| pMGA29 | Plin3(87-205)-GFP | hPlin3(aa87-205) | pRHT140 | Yeast | This study |
| pMGA28 | Plin3(87-205)-mCherry | hPlin3(aa87-205) | pMGA4 | Yeast | This study |
| pMGA19 | Plin3 AH | hPlin3(aa113 – 205) | pET21b | E. coli | This study |
| pKE31 | Plin4 4mer-GFP | hPlin4(aa246-377) | pRHT140 | Yeast | Čopič et al., 2018 |
| pKE33 | Plin4 12mer-GFP | hPlin4(aa510-905) | pRHT140 | Yeast | Čopič et al., 2018 |
| pMGA16 | Plin4 12mer-mCherry | hPlin4(aa510-905) | pMGA4 | Yeast | This study |
| pCLG26 | Plin4 8mer | hPlin4(aa246-509) | pmCherry-N1 | Mamm | Čopič et al., 2018 |
| pMGA31 | Plin4 6mer-GFP | hPlin4(aa246-433) | pRHT140 | Yeast | This study |
| pMGA23 | Plin4 8mer-GFP | hPlin4(aa246-509) | pRHT140 | Yeast | This study |
| pACJ22 | Plin4 4mer | hPlin4(aa246-377) | pmCherry-N1 | Mamm | Čopič et al., 2018 |
| pSB58 | 2D > E (4mer) | 4x[246–278 M17e] ‡ | pmCherry-N1 | Mamm | This study |
| pSB60 | NN (4mer) | 4x[246–278 M18q] ‡ | pmCherry-N1 | Mamm | This study |
| pMGA32 | NN (4mer) | 4x[246–278 M18q] ‡ | pET21b | E. coli | This study |
| pSB83 | QN (4mer) | 4x[246–278 M19qn] ‡ | pmCherry-N1 | Mamm | This study |
| pSB65 | QQ (4mer) | 4x[246–278 M20q2] ‡ | pmCherry-N1 | Mamm | This study |
| pSB86 | 3K > R | 4x[246–278 M21r] ‡ | pmCherry-N1 | Mamm | This study |
| pCLG62 | Plin4 12mer | hPlin4(aa510-905) | pmCherry-N1 | Mamm | Čopič et al., 2018 |
| pSB06 | csw 12mer | Charge swap of hPlin4(aa510-905) | pmCherry-N1 | Mamm | This study |
| pCLG36 | Plin4 2T > V 4mer | 4x[246–278 M10t2] ‡ | pmCherry-N1 | Mamm | Čopič et al., 2018 |
| pACJ41 | Plin4 csw 2T > V 4mer | Charge swap and 2T > V of 4x[246–278 M7kt] ‡ | pmCherry-N1 | Mamm | Čopič et al., 2018 |
| pSB41 | Plin4 12mer-GFP | hPlin4(aa510-905) | pMTWG | Dros. | Čopič et al., 2018 |
| pMGA3 | csw 12mer | Charge swap of hPlin4(aa510-905)-GFP | pRHT140 | Yeast | This study |
| pMGA17 | csw 12mer | Charge swap of hPlin4(aa510-905)-mCherry | pMGA4 | Yeast | This study |
| pMGA1 | csw 12mer | Charge swap of hPlin4(aa510-905) | pET21b | E. coli | This study |
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* Position of amino acids (aa) in human perilipin sequences.
† Mamm: mammalian cells, Dros.: Drosophila cells.
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‡ All mutants are four repeats of the same amino acid sequence.
Additional files
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Supplementary file 1
Summary of synthetic gene sequences and protein sequences used in this study.
- https://cdn.elifesciences.org/articles/61401/elife-61401-supp1-v2.xlsx
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Supplementary file 2
Calculation of Plin4 12mer density on oil, related to Figure 8B–D.
Table summarizes input values and calculations for five experimental conditions (A-E) used to standardize the fluorescence of Alexa488 labeled Plin4 12mer on lipid surface, using bead-supported diphytanoyl bilayers as standards.
- https://cdn.elifesciences.org/articles/61401/elife-61401-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/61401/elife-61401-transrepform-v2.docx
