Arf1/COPI machinery acts directly on lipid droplets and enables their connection to the ER for protein targeting
- Yale University School of Medicine, United States
- Ecole Normale Supérieure de Paris, Université Pierre et Marie Curie, Université Paris Diderot, Centre National de la Recherche Scientifique, France
- University of Heidelberg, Germany
- Gladstone Institute of Cardiovascular Disease, United States
- University of California, San Francisco, United States
Figures
Figure 1 with 1 supplement
The COPI machinery is required for LD targeting of specific proteins.
(A) The bimodal size distribution of control cells (black line) with few large LDs and many small LDs shifts a monodisperse size in Arf1/COPI-depleted cells (green and red line). The figure shows …
Figure 1—figure supplement 1
The COPI machinery is required for LD targeting of specific proteins.
(A) Targeting of GPAT4 from the ER to LDs depends on Arf1/COPI. GFP-GPAT4 localizes to LDs stained with lipidtox in control-treated cells, but not in the absence of COPI machinery subunits, Arf79F …
Figure 2 with 1 supplement
Arf1/COPI mediate LD protein targeting by establishing connections between the ER and LDs.
(A) Schematic representation of cell–cell fusion experiments. (B) Fusion of βCOP depleted cells expressing GFP-GPAT4 and induced LDs with WT cells rapidly rescues GFP-GPAT4 targeting to LDs. Soluble …
Figure 2—figure supplement 1
Mathematical model for GPAT4 targeting to LDs through bridges with the ER.
(A) Measurement of GPAT4-GFP diffusion in the ER. D = 0.035μm2/sec denotes for the apparent diffusion coefficient of GPAT4 in the ER, determined by FRAP recovery experiments. Normalized fluorescence …
Figure 3 with 1 supplement
The COPI machinery localizes to the LD surface.
(A) The endogenous COPI machinery stained with αCOP or garz antibodies (red) localizes to LDs in S2 cells. Frequencies of colocalization of αCOP and garz spots with LDs from experiments are higher …
Figure 3—figure supplement 1
COPI machinery localizes to the surface of LDs.
(A) The probability P of having n colocalization events from nA type A dots (A = βCOP, αCOP, clathrin, KDELR) follows a binomial distribution. ns is the fraction with the highest probability of …
Figure 4 with 1 supplement
Arf1/COPI bud nano-LDs from purified, cellular LDs.
(A) Purified LDs from S2 cells were incubated with components of the Arf1/COPI machinery in the presence or absence of GTPγS. Representative electron micrographs reveal abundant nano-LDs formed in …
Figure 4—figure supplement 1
Purified LDs were incubated with Arf1-Y35A, coatomer, ARNO and GTPγS, upon budding conditions shown in Figure 4A.
The left panel shows an electron micrograph reaction products with budded nano-LDs as shown in the inlay. Dimerization deficient Arf1-Y35A is able to induce nano-LDs formation similarly to Arf1. …
Figure 5 with 1 supplement
Lack of Arf1/COPI increases phospholipids on LDs, abolishing GPAT4 LD targeting.
(A) PC and PE, but not TG levels are increased in LDs from βCOP depleted cells compared with WT cells. (B) LD (green) targeting of endogenous CCT1 (red) is delayed in cells depleted of βCOP. Time = …
Figure 5—figure supplement 1
Depletion of COPI machinery components is efficient.
Expression of indicated subunits were measured by quantitative real-time CR. Primers used are listed in Table 1. Means ± SD of three experiments are shown.
Figure 6 with 1 supplement
LD surface properties modulate GPAT4 LD targeting.
(A) Addition of exogenous PC to S2 cells inhibited GPAT4 LD targeting in βCOP or control RNAi-treated cells. Cholesterol (chol) addition to cells restored GPAT4 LD targeting in βCOP-depleted cells. …
Figure 6—figure supplement 1
Cholesterol leads to an increase of surface tension at a TG/buffer interface.
(A) Cholesterol increases the surface tension of PC/PE monolayer at a TG/buffer interface. Surface tension was measured by a drop weight method for the indicated phospholipid/cholesterol ratios. …
Videos
Video 1
Time lapse analysis of GFP-GPAT4 targeting to LDs in cell–cell fusion experiments.
https://doi.org/10.7554/eLife.01607.007
Video 2
Time lapse video of Arf1/COPI coated nano-LDs formed from cellular LDs.
Nano-LDs are visualized by fluorescence microscopy detecting Arf1 (red), COPI (green) and LDs (MDH labeled, blue). Shifts between channels are due to short time delays between channel acquisitions, …
Tables
Table 1
Sequences of primers used for RNAi experiments
| Gene | Gene ID | Forward | Reverse |
|---|---|---|---|
| garz | CG8487 | TTGCACAAACTTTGATTCCTG | CATATCGGCGCACTATAATC |
| Arf79F | CG8385 | TAGCGATTAGCGTTCTTCA | CTGCCAAATGCAATGAACG |
| αCOP | CG7961 | AGGAAGCTAAGCTTGTCAAA | GGACGAGTCTGGAGTGTTT |
| βCOP | CG6223 | CCAGTCAGTTGGGTGACCTT | CCTAGCAAGCCCATAACCAA |
| β’COP | CG6699 | ATCTTGCTTCCCACAACGTC | CCGAAGGACAACAACACCTT |
| γCOP | CG1528 | ATTACGTTCACAGCACGCAG | CAGAGGAGGGCTATGACGAC |
| ζCOP | CG3948 | CCGTCGCAGATCTCGTC | GCATCCTGGCCAAGTACTA |
| εCOP | CG9543 | AGGTGCCAGATGTTGGTCTC | CCAACTCGGTGCTATTCGAT |
| δCOP | CG14813 | AAGCTGTCTGCGCCATAAAT | TCCAGTGGCACATTCCAATA |
| CCT1 | CG1049 | ACATCTATGCTCCT1CTCAAGGC | CTCTGCAGACTCTGGTAACTGC |
| pBluescript | AATTCGATATCAAGCTTATCGAT | TAAATTGTAAGCGTTAATATTTTG |
Table 2
Sequences of primers used for RT-PCR
| Gene | Gene ID | Forward | Reverse |
|---|---|---|---|
| GAPDH1 | CG12055 | TTGTGGATCTTACCGTCCG | ACCTTAGCCTTGATTTCGTC |
| Arf79F | CG8385 | TTACAGTGTGGGATGTGGG | GAAGATAAGACCTTGTGTATTCTGG |
| βCOP | CG6223 | GACTTCTGCAATATCAAGGCC | GGTTTCGTAAACAATATTGCCG |
| CCT1 | CG1049 | GATACGGAGTGCGTCA | AATTCATCGGACAGAGTCCA |
