Direct interaction of Plin2 with lipids on the surface of lipid droplets: a live cell FRET analysis

Abstract

<jats:p>Despite increasing awareness of the health risks associated with excess lipid storage in cells and tissues, knowledge of events governing lipid exchange at the surface of lipid droplets remains unclear. To address this issue, fluorescence resonance energy transfer (FRET) was performed to examine live cell interactions of Plin2 with lipids involved in maintaining lipid droplet structure and function. FRET efficiencies ( E) between CFP-labeled Plin2 and fluorescently labeled phosphatidylcholine, sphingomyelin, stearic acid, and cholesterol were quantitated on a pixel-by-pixel basis to generate FRET image maps that specified areas with high E (&gt;60%) in lipid droplets. The mean E and the distance R between the probes indicated a high yield of energy transfer and demonstrated molecular distances on the order of 44–57 Å, in keeping with direct molecular contact. In contrast, FRET between CFP-Plin2 and Nile red was not detected, indicating that the CFP-Plin2/Nile red interaction was beyond FRET proximity (&gt;100 Å). An examination of the effect of Plin2 on cellular metabolism revealed that triacylglycerol, fatty acid, and cholesteryl ester content increased while diacylglycerol remained constant in CFP-Plin2-overexpressing cells. Total phospholipids also increased, reflecting increased phosphatidylcholine and sphingomyelin. Consistent with these results, expression levels of enzymes involved in triacylglycerol, cholesteryl ester, and phospholipid synthesis were significantly upregulated in CFP-Plin2-expressing cells while those associated with lipolysis either decreased or were unaffected. Taken together, these data show for the first time that Plin2 interacts directly with lipids on the surface of lipid droplets and influences levels of key enzymes and lipids involved in maintaining lipid droplet structure and function.</jats:p>

FOXO3 Growth Inhibition of Colonic Cells Is Dependent on Intraepithelial Lipid Droplet Density

“…As in yeast cell cycle kinases, Cdk1/Cdc28 and Cdk1 regulate the flow of lipid precursors into LDs (48), and as our study reveals that LDs control the cell cycle in human cells, LDs and the cell cycle most likely have feedback regulation among different species. However, the role of the LD coat protein in modulation of the cell cycle is not understood, as PLIN2 was found to control the levels of lipids involved in maintaining LDs (23,49). The increased number of LDs, together with the elevated expression of the primary enzyme catalyzing the synthesis of fatty acids in some tumors (22,50), and the effective treatment of some tumors with inhibitors of fatty acid synthesis (51) underscore the importance of understanding the interplay between LDs and the cell cycle.…”

“…19 and 20). PLINs are key regulators of lipid metabolism, and their expression correlates with LD status (21)(22)(23). Moreover, LD surfaces are associated with vimentin filaments forming a vimentin cage, which most likely stabilize LDs, suggesting that changes in LDs status involves cytoskeletal reorganization in the cell (24).…”

Plin2 Inhibits Cellular Glucose Uptake through Interactions with SNAP23, a SNARE Complex Protein

“…Cells were maintained in high glucose DMEM containing 10% FBS and antibiotics (100 units/ml penicillin and 100 units/ml streptomycin) under 5% CO 2 at 37 o C. Plin2 overexpressing L cells were generated by stable transfection using a plasmid containing the full coding region of mouse Plin2 cDNA N-terminally fused to CFP as described elsewhere [13]. It should be noted that the N-terminal CFP tag had no effect on Plin2 targeting to lipid droplets as assessed by laser scanning confocal microscopy, consistent with earlier reports [34].…”

“…Plin1 is the most studied lipid droplet protein with known function involving TG hydrolysis [3–5], but the full physiological significance of the rest of the Plin family remains less defined. With regard to Plin2, several reports describe increased TAG accumulation and lipid droplet formation when Plin2 is overexpressed in cells [10–13]. Conversely, knockdown of Plin2 in macrophages was shown to decrease cellular lipids and lipid droplet size and number [10].…”

Epidermal growth factor receptor mediated proliferation depends on increased lipid droplet density regulated via a negative regulatory loop with FOXO3/Sirtuin6

“…They contain a mixture of lipids enclosed by a family of regulatory proteins known as PERILIPINS (PLIN1-5) [10], which are involved in the regulation of lipid metabolism and correlate with LD status [11]. Elevated LDs are formed at the endoplasmic reticulum following increases in lipid synthesis and maturation steps [12].…”