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. 2021 Apr 15;10:e61401. doi: 10.7554/eLife.61401

Figure 6. 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; the three K (blue), which were mutated into R, and the two D (red), which were mutated into E. (B) Co-localization of GFP-Plin4 4mer wild-type and 3K > R (in white) with LDs (purple) in HeLa cells. (C) Quantification of the percentage of LDs stained with the indicated protein per cell. These ‘SuperPlots’ (Lord et al., 2020) show all data fom three to four independent experiments, each with a different color; each light dot represents one cell, whereas each triangle shows the mean from one experiment. The black bars show the mean ± SE of three to four independent experiments. (D) Helical wheels of Plin4 WT and csw mutant. (E) Localization of Plin4 12mer wild-type or csw mutant in HeLa cells. The insets show extended views with the protein in purple and LDs in green (stained with Bodipy). The super plots show the mean ± SE of the % of LDs positive for the indicated protein per HeLa cell as determined from three independent experiments. (F) CD spectra of Plin4 12mer (5 μM) and csw 12mer (7.5 μM) in solution (blue) or in an equal volume of buffer and TFE (green). (G) Light microscopy images of mCherry fusions of Plin4 12mer wild-type or csw mutant in yeast. Top: mCherry fluorescence (mCh); bottom co-localization of mCherry (purple) with LDs stained with bodipy (green). The relative fluorescence signal of mCherry fusions of Plin4 12mer wild-type or csw mutant on LDs and at the PM in PET10-GFP yeast strain was used to build the SuperPlots shown on the right. Data are from three independent experiments, with n ≥ 25 for each condition in each assay. (H) Fluorescence recovery curves of mCherry fusions of Plin4 4mer 2T > V (green points) and 2T > V csw mutants (blue points) on LDs in HeLa cells. Each curve represents FRAP of a single LD in one cell.

Figure 6—source data 1. The polar face of Plin4 AH is key for specific and stable coating of LDs in cells.

Figure 6.

Figure 6—figure supplement 1. Plin4 4mer mutants are not affected in their helical folding.

Figure 6—figure supplement 1.

(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) Tricine SDS-PAGE analysis of the relative concentrations of the purified proteins. Gel was stained with Coomassie Blue.
Figure 6—figure supplement 2. Additional data for Plin4 charge-swap mutant.

Figure 6—figure supplement 2.

(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 expression levels. Vps10 was used as loading control. (C) Influence of redistribution of charge in Plin4 12mer on LD size in yeast. Plot shows distribution of LD size in pet10Δ cells incubated with OA for 24 hr and expressing GFP fusions of Plin4 12mer or csw 12mer mutant. Graph shows a representative of three independent experiments, with 270 LDs measured for each construct. Pixel size: 0.091 μm x 0.091 μm.