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ARFGAP1 is dynamically associated with lipid droplets in hepatocytes.

Gannon J, Fernandez-Rodriguez J, Alamri H, Feng SB, Kalantari F, Negi S, Wong AH, Mazur A, Asp L, Fazel A, Salman A, Lazaris A, Metrakos P, Bergeron JJ, Nilsson T - PLoS ONE (2014)

Bottom Line: In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells.Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation.Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: The Research Institute of the McGill University Health Centre, McGill, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada.

ABSTRACT
The ARF GTPase Activating Protein 1 (ARFGAP1) associates mainly with the cytosolic side of Golgi cisternal membranes where it participates in the formation of both COPI and clathrin-coated vesicles. In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells. Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation. Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes.

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Quantification of ARFGAP1 associated with lipid droplets. Endogenous ARFGAP1 as revealed by indirect immunofluorescence was quantified before (-OA) and 4 hours after addition of oleate (+OA) in HepG2 and McA-RH7777 cells expressed as % cells with ARFGAP1 staining (as revealed through indirect immunofluorescence) or % Bodipy-stained droplets associated with ARFGAP1 per cell. On the right, total area of Bodipy-stained lipid droplets per µm2 per cell.
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pone-0111309-g007: Quantification of ARFGAP1 associated with lipid droplets. Endogenous ARFGAP1 as revealed by indirect immunofluorescence was quantified before (-OA) and 4 hours after addition of oleate (+OA) in HepG2 and McA-RH7777 cells expressed as % cells with ARFGAP1 staining (as revealed through indirect immunofluorescence) or % Bodipy-stained droplets associated with ARFGAP1 per cell. On the right, total area of Bodipy-stained lipid droplets per µm2 per cell.

Mentions: To investigate when ARFGAP1 redistributes to lipid droplet structures, McA-RH7777 cells were incubated with oleate for various lengths of time (Fig. 6). This revealed that though most of ARFGAP1 localized to lipid droplets 4 hours after addition of oleate, much less was observed at later time points suggesting a transient association. At 10 hours after oleate addition, over half of ARFGAP1 still remained associated with lipid droplet structures whereas after 24 hours, ARFGAP1 was exclusively located at the juxta nuclear Golgi area. The shift in distribution of endogenous ARFGAP1 from the Golgi apparatus to lipid droplets was quantified 4 hours after addition of oleate in 350 cells per experiment in 5 independent experiments using ImageJ 1.45 s software (see Materials and Methods). In HepG2 and McA-RH7777 cells, the percentage of cells that had ARFGAP1 associated with lipid droplets increased from 0% to 80% and 0% to 75% at 4 hours after addition of oleate, respectively (Fig. 7-left). About 65% and 50% of Bodipy-stained structures were associated with ARFGAP1 per cell at 4 hours after oleate addition in HepG2 and McA-RH7777 cells, respectively (Fig. 7, middle). Addition of oleate caused a marked increase in the area of Bodipy-positive lipid droplets corresponding to a 10 and 5 fold increase in HepG2 and McA-RH7777 cells, respectively (Fig. 7, right). To confirm that ARFGAP1 indeed localizes to lipid droplets upon addition of oleate, HepG2 cells were fixed and processed for immuno-gold transmission electron microscopy (see Materials and Methods). Figure 8 shows single antigen gold-labeled thin frozen sections after 4 hours incubation with oleate incubated with a control antibody (MOCK), a goat polyclonal antibody to ADRP (PLIN2), a lipid droplet marker, or a rabbit polyclonal antibody to ARFGAP1 [30]. Arrowheads in black and white point to gold particles detected revealing staining at the periphery of lipid droplets of both ADRP and ARFGAP1. On occasion, gold particles for ADRP were detected also within the lipid droplet structure.


ARFGAP1 is dynamically associated with lipid droplets in hepatocytes.

Gannon J, Fernandez-Rodriguez J, Alamri H, Feng SB, Kalantari F, Negi S, Wong AH, Mazur A, Asp L, Fazel A, Salman A, Lazaris A, Metrakos P, Bergeron JJ, Nilsson T - PLoS ONE (2014)

Quantification of ARFGAP1 associated with lipid droplets. Endogenous ARFGAP1 as revealed by indirect immunofluorescence was quantified before (-OA) and 4 hours after addition of oleate (+OA) in HepG2 and McA-RH7777 cells expressed as % cells with ARFGAP1 staining (as revealed through indirect immunofluorescence) or % Bodipy-stained droplets associated with ARFGAP1 per cell. On the right, total area of Bodipy-stained lipid droplets per µm2 per cell.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232254&req=5

pone-0111309-g007: Quantification of ARFGAP1 associated with lipid droplets. Endogenous ARFGAP1 as revealed by indirect immunofluorescence was quantified before (-OA) and 4 hours after addition of oleate (+OA) in HepG2 and McA-RH7777 cells expressed as % cells with ARFGAP1 staining (as revealed through indirect immunofluorescence) or % Bodipy-stained droplets associated with ARFGAP1 per cell. On the right, total area of Bodipy-stained lipid droplets per µm2 per cell.
Mentions: To investigate when ARFGAP1 redistributes to lipid droplet structures, McA-RH7777 cells were incubated with oleate for various lengths of time (Fig. 6). This revealed that though most of ARFGAP1 localized to lipid droplets 4 hours after addition of oleate, much less was observed at later time points suggesting a transient association. At 10 hours after oleate addition, over half of ARFGAP1 still remained associated with lipid droplet structures whereas after 24 hours, ARFGAP1 was exclusively located at the juxta nuclear Golgi area. The shift in distribution of endogenous ARFGAP1 from the Golgi apparatus to lipid droplets was quantified 4 hours after addition of oleate in 350 cells per experiment in 5 independent experiments using ImageJ 1.45 s software (see Materials and Methods). In HepG2 and McA-RH7777 cells, the percentage of cells that had ARFGAP1 associated with lipid droplets increased from 0% to 80% and 0% to 75% at 4 hours after addition of oleate, respectively (Fig. 7-left). About 65% and 50% of Bodipy-stained structures were associated with ARFGAP1 per cell at 4 hours after oleate addition in HepG2 and McA-RH7777 cells, respectively (Fig. 7, middle). Addition of oleate caused a marked increase in the area of Bodipy-positive lipid droplets corresponding to a 10 and 5 fold increase in HepG2 and McA-RH7777 cells, respectively (Fig. 7, right). To confirm that ARFGAP1 indeed localizes to lipid droplets upon addition of oleate, HepG2 cells were fixed and processed for immuno-gold transmission electron microscopy (see Materials and Methods). Figure 8 shows single antigen gold-labeled thin frozen sections after 4 hours incubation with oleate incubated with a control antibody (MOCK), a goat polyclonal antibody to ADRP (PLIN2), a lipid droplet marker, or a rabbit polyclonal antibody to ARFGAP1 [30]. Arrowheads in black and white point to gold particles detected revealing staining at the periphery of lipid droplets of both ADRP and ARFGAP1. On occasion, gold particles for ADRP were detected also within the lipid droplet structure.

Bottom Line: In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells.Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation.Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: The Research Institute of the McGill University Health Centre, McGill, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, Quebec, Canada.

ABSTRACT
The ARF GTPase Activating Protein 1 (ARFGAP1) associates mainly with the cytosolic side of Golgi cisternal membranes where it participates in the formation of both COPI and clathrin-coated vesicles. In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells. Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation. Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes.

Show MeSH
Related in: MedlinePlus