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

Figure 4. 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 (bottom panels); time (in seconds) is indicated on top. Cells are outlined in white and the bleached areas containing one LD are outlined in green. Graph shows recovery curves for Plin1 AH-GFP (n = 29), Plin2 AH-GFP (n = 14), Plin3 AH-GFP (n = 16), or Plin4 12mer-GFP (n = 24). (B) Recovery curves for Plin1 AH-GFP (n = 11) or Plin4 12mer-GFP (n = 5) on LDs in pet10Δ cells in late exponential phase (small LDs). (C) FRAP of Plin1 AH (n = 14) and Plin4 12mer (n = 15) at the PM in exponentially growing pet10Δ cells. Images show two representative time-courses for Plin1 AH (top) and Plin4 12mer (bottom). Bleached areas are outlined in green. (D) FRAP of Plin4 6mer-GFP (n = 12), Plin4 8mer-GFP (n = 7) and Plin4 12mer-GFP (n = 7) at the PM in exponentially growing wild-type cells. All graphs show the mean ± SD of the fluorescence recovery curves from n FRAP measurements on different LDs or different regions of the PM, as shown in the images. Scale bar: 5 µm.

Figure 4—source data 1. Dynamics of perilipin AH-GFP fusions on LDs or at the plasma membrane in yeast assessed by FRAP.

Figure 4.

Figure 4—figure supplement 1. Cell-to-cell variability in the recovery of Plin4 12mer-GFP after photobleaching of LDs in Drosophila S2 cells.

Figure 4—figure supplement 1.

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 cells (RNAi against luciferase). Each curve represents mean ± SD from FRAP on three LDs in the same cell, as exemplified in the two images below the graph, showing two cells before bleaching. Areas that were bleached are marked with green circles. Fast fluorescence recovery was observed in the cell on the left, slow in the cell on the right. The fluorescence signal in the two images was acquired under the same settings, showing that the total Plin4-GFP expression level in the two cells was similar. However, note the difference in the amount of cytosolic signal. Scale bar: 5 μm. (B) Same as A, except that FRAP was performed on cells in which CCT1 has been depleted by RNAi. (C) Correlation between the ratio of LD/cytosolic Plin4 12mer-GFP signal and the half-time of recovery, as determined from the curves shown in A and B. Each dot represents one cell, data is from two independent experiments. Asterisks denote cells which had very slow recovery kinetics (half-time >100 s; in this case the value was set to 100 s).