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First published online 24 May 2005
doi: 10.1242/jcs.02401

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Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane

Shintaro Ozeki^1,*, Jinglei Cheng^1,*, Kumi Tauchi-Sato¹, Naoya Hatano², Hisaaki Taniguchi^2,3 and Toyoshi Fujimoto^1,

¹ Department of Anatomy and Molecular Cell Biology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa, Nagoya 466-8550, Japan
² Harima Institute at SPring-8, RIKEN, Mikazuki, Sayo, Hyogo 679-5148, Japan
³ Institute for Enzyme Research, The University of Tokushima, Tokushima 770-8503, Japan

View larger version (58K): [in a new window]   Fig. 1. (A) Double labeling of Rab proteins (red) and LDs (green) in HepG2 cells. Cells were transfected with cDNA of tagged Rab proteins. LDs were stained with BODIPY 493/503. Rab1, Rab5, Rab7, Rab10 and Rab18 were tagged with FLAG, and Rab2 was tagged with Myc. Rab18 was consistently concentrated around LDs, whereas other Rabs were observed in the vicinity of LDs only infrequently. Bars, 10 µm. (B) The proportion of Rab-positive LDs among all the LDs stained with BODIPY 493/503. Only cells showing positive Rab labeling were selected and counted. More than 500 LDs in 10 random areas were counted in two independent experiments. (C) The proportion of cells in which the Rab-positive LDs were more than 10% of the total detected LDs. Only cells showing positive Rab labeling were selected and counted. More than 30 cells in 10 random areas were counted in two independent experiments. (D) Double labeling of EGFP-tagged Rab18 (green) and LDs (red) in HepG2 cells. The distributions of wild-type Rab18(WT) and GTPase-deficient Rab18(Q67L) were confined to LDs stained with Sudan III, while constitutively GDP-bound Rab18(S22N) was observed diffusely in the cytoplasm. Scale bars: 10 µm.

View larger version (61K): [in a new window]   Fig. 2. (A) Western blotting of sucrose density-gradient fractions from HepG2. An equal volume from each fraction was loaded. Both ADRP and Rab18 were strongly present in the top floating LD fraction (#1). There was also some Rab18 in the bottom fractions containing soluble and membrane proteins (#7, #8). EEA1, Lamp1, syntaxin 6, and calnexin were detected only in the bottom fractions (#6-8). (B) Double labeling of endogenous Rab18 (red) and LDs (green) in HepG2 cells. LDs were stained with BODIPY 493/503. Antibody labeling for Rab18 was concentrated around LDs. Note that there were LDs that were not associated with anti-Rab18 (arrowheads). Scale bar: 10 µm. (C) Double labeling of endogenous Rab18 (green) and endosomal markers (red). EEA1 and transferrin receptor (Tf-R) (upper panel), and lysobisphosphatidic acid (LBPA) and Lamp1 (lower panel) were used as markers for early and late endosomes, respectively. Rab18 did not show co-distribution with any of the endosomal markers. Scale bars: 10 µm.

View larger version (92K): [in a new window]   Fig. 3. Time course of LD formation (stained with BODIPY 493/503) in relation to ADRP and Rab18 in 3T3 cells loaded with oleic acid. Cells were cultured in medium containing 2% LPDS for 2 days, and then transferred to medium with 10% FBS with 200 µM oleic acid-BSA complex (OA/FBS). LDs, ADRP and Rab18 were not detected in the cells kept in LPDS. As early as 6 hours after transfer to OA/FBS medium, LDs and ADRP were observed as bright dots that increased in number and brightness thereafter. In contrast, Rab18 was barely visible as dots at 6 or 12 hours in OA/FBS medium, and was seen clearly only after 24 hours in OA/FBS medium.

View larger version (61K): [in a new window]   Fig. 4. (A). HepG2 cells were triple labeled for endogenous Rab18 (red), ADRP (blue), and LDs (green). BODIPY 493/503-stained LDs were labeled for Rab18 and ADRP, but the labeling intensity for Rab18 and ADRP appeared to be reciprocal in most cases. LDs showing strong Rab18 and weak ADRP staining are indicated by arrowheads, and those with the reverse pattern are indicated by arrows. Scale bar: 10 µm. (B) HepG2 cells transfected with EGFP-Rab18(WT) cDNA were labeled with anti-ADRP. Both EGFP-Rab18(WT) (green) and ADRP (red) are visible as small rings of fluorescence, but there is very little overlap. Some Rab18-positive, ADRP-negative ring-shaped fluorescent signals are marked by arrowheads. Scale bar: 10 µm. (C) Western blotting of ADRP. HepG2 cells were transfected with empty vector or Rab18 cDNA. Equal amounts (20 µg) of the total cell lysates were electrophoresed and probed with anti-ADRP antibody. Expression of Rab18 caused a reduction of ADRP. (D) 3T3 cells, expressing EGFP, EGFP-Rab18(WT), EGFP-Rab18(Q67L), or EGFP-Rab18(S22N) (green), were further labeled with anti-ADRP (blue) and Sudan III (red). Merged images of the three colors are shown. EGFP and EGFP-Rab18(S22N) were distributed diffusely in the cytoplasm, and all the Sudan III-positive LDs were labeled with anti-ADRP. In contrast, in cells expressing EGFP-Rab18(WT) or EGFP-Rab18(Q67L), LDs were labeled for either EGFP-Rab18 or ADRP, and those showing both labels were scarce. Scale bar: 10 µm. (E) LDs isolated from HepG2-expressing EGFP-Rab18(WT) (green) were labeled with anti-ADRP (red). LDs with strong EGFP-Rab18(WT) fluorescence generally showed weak ADRP labeling. EGFP-Rab18(WT)-positive, ADRP-negative LDs are indicated by arrowheads. Scale bar: 10 µm.

View larger version (138K): [in a new window]   Fig. 5. (A) Immunogold electron microscopy of HepG2 cells expressing EGFP-Rab18(WT). Ultrathin cryosections were labeled with anti-GFP antibody. Most LDs appeared as vacant round spaces because lipid esters were not retained well in the sections. Gold labeling was observed along the rim of LDs. Notably, thin membrane cisternae were often seen adjacent to the labeled LD (arrows). Scale bar: 100 nm. (B) Conventional electron microscopy of 3T3 cells expressing EGFP-Rab18(WT). LDs were frequently apposed to thin membrane cisternae (arrows). The direct continuity of the membrane cisterna and the rough ER (double arrows in the upper left, upper right and middle right figures), and the ribosomes in the membrane cisterna (double arrows in the lower right figure) were observed in many cases. Scale bars: 100 nm. (C) Conventional electron microscopy of control 3T3 cells. Small ER cisternae were occasionally found adjacent to LDs (arrowheads), but contacts between them were seldom extensive. Scale bar: 100 nm. (D) 3T3 cells treated with siRNA for knockdown of the expression of ADRP. (D-1) Western blotting of ADRP. Cells were treated with control random siRNA or ADRP siRNA, and equal amounts of the total cell lysates (20 µg) were electrophoresed. The expression of ADRP was reduced by more than 50% by this procedure. (D-2) In cells treated with ADRP siRNA, LDs in apposition with thin membrane cisternae were frequently observed (arrows). Scale bar: 100 nm. (E) Conventional electron microscopy of 3T3 cells treated with brefeldin A for 5 hours. Most LDs were surrounded by thin membrane cisternae (arrows). Scale bar: 100 nm. (F) Frequency of 3T3 cells with LDs in close membrane apposition. More than 30 cells were chosen randomly, and only LDs with membrane cisternae apposed to more than half its circumference were counted as positive. The transfection efficiency was no less than 60% as determined by fluorescence microscopy.