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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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ARFGAP1 co-localizes with PLIN3 on lipid droplets.Endogenous ARFGAP1 staining (red) was compared with antibody staining of endogenous PLIN3 (green). An enlarged region of interest (ROI) is shown in the bottom right panel of the merged image (ARFGAP1/PLIN3). Bar  = 5µm.
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pone-0111309-g004: ARFGAP1 co-localizes with PLIN3 on lipid droplets.Endogenous ARFGAP1 staining (red) was compared with antibody staining of endogenous PLIN3 (green). An enlarged region of interest (ROI) is shown in the bottom right panel of the merged image (ARFGAP1/PLIN3). Bar  = 5µm.

Mentions: To investigate the localization of ARFGAP1 upon oleate addition more precisely; we fixed and processed HepG2 cells for indirect immunofluorescence both before and 4 hours after addition of 0.5 mM oleate (final concentration). As shown in Figure 3 (upper panels), there was no apparent co-localization between endogenous ARFGAP1 and structures staining positive for Bodipy before addition of oleate. In contrast, after 4 hours of incubation with oleate, most of the ARFGAP1 staining was seen surrounding Bodipy-positive lipid droplets (Fig. 3 lower panels). The staining for ARFGAP1 was compared with a known lipid droplet marker, PLIN3 (Perilipin 3/TIP47) which has also been implicated in Golgi/endosomal trafficking [32]. As shown in Figure 4 and in the enlarged region of interest (ROI), there was considerable co-localization (yellow) between ARFGAP1 (red) and PLIN3 (green). This shows that ARFGAP1 localizes to lipid droplet structures 4 hours after oleate addition partly overlapping with PLIN3. As ARFGAP1 was first identified as an activator protein for the small GTPase ARF1 in the context of coat regulation of COPI coatomer responsible for the generation of COPI transport vesicles and retrograde transport in the Golgi apparatus (for review, see [32]), it was of further interest to test whether these and other Golgi markers also localized to lipid droplet structures upon addition of oleate. Figure 5 row of upper panels show the distribution of COPB1 (βCOP), a component of the 7 subunit coatomer complex as revealed by the mouse monoclonal antibody CM1A10 specific for one of its components [33] before (-OA) and 4 hours after addition of oleate (4 hr OA). Most staining forCOPB1 appeared as juxta nuclear Golgi staining with additional punctate staining throughout the cytoplasm, a staining pattern typical of coatomer. No apparent co-localization with lipid droplets was observed under these conditions. ARF1 was probed for in the hepatoma cell line, McA-RH7777 using a rabbit polyclonal (Fig. 5, αARF1) [30] resulting in a predominant juxta nuclear Golgi staining with occasional small punctate structures throughout the cytoplasm of cells before addition of oleate. At 4 hours after addition of oleate, staining remained predominantly juxta nuclear with an increase in staining of cytoplasmic structures. A minor portion of such cytoplasmic structures appeared close to lipid droplets as revealed by Bodipy stain. It is likely that the antibody used here recognizes additional ARFs due to their close homology. From above experiments, it appears that ARFGAP1 distributes to lipid droplets upon addition of oleate whereas coatomer and most of ARF1 do not. At 4 hours after addition of oleate, the Golgi apparatus appears intact. This was confirmed in HepG2 cells using an antibody to TMED7 (gp27) (Fig. 5, αTMED7), a member of the gp25L/emp24 family of small transmembrane proteins of the early secretory pathway [34]. Exclusive juxta nuclear Golgi staining was observed before and 4 hours after addition of oleate. An antibody to the cytoplasmic domain of CANX (calnexin) was used to test for changes of the ER in HepG2 cells 4 hours after addition of oleate. No discernable difference was observed (Fig 5, αCANX). This shows that the gross architecture of the Golgi apparatus or the ER is not affected at 4 hours after addition of oleate despite loss of ARFGAP1 to lipid droplets.


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)

ARFGAP1 co-localizes with PLIN3 on lipid droplets.Endogenous ARFGAP1 staining (red) was compared with antibody staining of endogenous PLIN3 (green). An enlarged region of interest (ROI) is shown in the bottom right panel of the merged image (ARFGAP1/PLIN3). Bar  = 5µm.
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Related In: Results  -  Collection

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

pone-0111309-g004: ARFGAP1 co-localizes with PLIN3 on lipid droplets.Endogenous ARFGAP1 staining (red) was compared with antibody staining of endogenous PLIN3 (green). An enlarged region of interest (ROI) is shown in the bottom right panel of the merged image (ARFGAP1/PLIN3). Bar  = 5µm.
Mentions: To investigate the localization of ARFGAP1 upon oleate addition more precisely; we fixed and processed HepG2 cells for indirect immunofluorescence both before and 4 hours after addition of 0.5 mM oleate (final concentration). As shown in Figure 3 (upper panels), there was no apparent co-localization between endogenous ARFGAP1 and structures staining positive for Bodipy before addition of oleate. In contrast, after 4 hours of incubation with oleate, most of the ARFGAP1 staining was seen surrounding Bodipy-positive lipid droplets (Fig. 3 lower panels). The staining for ARFGAP1 was compared with a known lipid droplet marker, PLIN3 (Perilipin 3/TIP47) which has also been implicated in Golgi/endosomal trafficking [32]. As shown in Figure 4 and in the enlarged region of interest (ROI), there was considerable co-localization (yellow) between ARFGAP1 (red) and PLIN3 (green). This shows that ARFGAP1 localizes to lipid droplet structures 4 hours after oleate addition partly overlapping with PLIN3. As ARFGAP1 was first identified as an activator protein for the small GTPase ARF1 in the context of coat regulation of COPI coatomer responsible for the generation of COPI transport vesicles and retrograde transport in the Golgi apparatus (for review, see [32]), it was of further interest to test whether these and other Golgi markers also localized to lipid droplet structures upon addition of oleate. Figure 5 row of upper panels show the distribution of COPB1 (βCOP), a component of the 7 subunit coatomer complex as revealed by the mouse monoclonal antibody CM1A10 specific for one of its components [33] before (-OA) and 4 hours after addition of oleate (4 hr OA). Most staining forCOPB1 appeared as juxta nuclear Golgi staining with additional punctate staining throughout the cytoplasm, a staining pattern typical of coatomer. No apparent co-localization with lipid droplets was observed under these conditions. ARF1 was probed for in the hepatoma cell line, McA-RH7777 using a rabbit polyclonal (Fig. 5, αARF1) [30] resulting in a predominant juxta nuclear Golgi staining with occasional small punctate structures throughout the cytoplasm of cells before addition of oleate. At 4 hours after addition of oleate, staining remained predominantly juxta nuclear with an increase in staining of cytoplasmic structures. A minor portion of such cytoplasmic structures appeared close to lipid droplets as revealed by Bodipy stain. It is likely that the antibody used here recognizes additional ARFs due to their close homology. From above experiments, it appears that ARFGAP1 distributes to lipid droplets upon addition of oleate whereas coatomer and most of ARF1 do not. At 4 hours after addition of oleate, the Golgi apparatus appears intact. This was confirmed in HepG2 cells using an antibody to TMED7 (gp27) (Fig. 5, αTMED7), a member of the gp25L/emp24 family of small transmembrane proteins of the early secretory pathway [34]. Exclusive juxta nuclear Golgi staining was observed before and 4 hours after addition of oleate. An antibody to the cytoplasmic domain of CANX (calnexin) was used to test for changes of the ER in HepG2 cells 4 hours after addition of oleate. No discernable difference was observed (Fig 5, αCANX). This shows that the gross architecture of the Golgi apparatus or the ER is not affected at 4 hours after addition of oleate despite loss of ARFGAP1 to lipid droplets.

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