Direct interaction of Plin2 with lipids on the surface of lipid droplets: a live cell FRET analysis
Direct interaction of Plin2 with lipids on the surface of lipid droplets: a live cell FRET analysis
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DOI：
10.1152/ajpcell.00448.2011
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发表时间：
2012-10-01
影响因子：
5.5
通讯作者：
Atshaves, Barbara P.
中科院分区：
文献类型：
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作者：
McIntosh, Avery L.;Senthivinayagam, Subramanian;Atshaves, Barbara P.
McIntosh AL，Senthivinayagam S，Moon KC，Gupta S，Lwande JS，Murphy CC，Storey SM，Atshaves BP. Plin 2与脂滴表面脂质的直接相互作用：活细胞FRET分析。Am J Physiol Cell Physiol 303：C728-C742，2012.首次发表于2012年6月27日; doi：10.1152/ajpcell.00448.2011。尽管人们越来越意识到与细胞和组织中过量脂质储存相关的健康风险，但对脂滴表面脂质交换事件的了解仍不清楚。为了解决这个问题，进行荧光共振能量转移（FRET）以检查Plin 2与参与维持脂滴结构和功能的脂质的活细胞相互作用。在逐像素的基础上定量CFP标记的Plin 2和荧光标记的磷脂酰胆碱、鞘磷脂、硬脂酸和胆固醇之间的FRET效率（E），以生成FRET图像图，其指定脂滴中具有高E（>60%）的区域。探针之间的平均E和距离R表明能量转移的高产量，并证明分子距离在44-57埃的量级，与直接分子接触一致。相比之下，未检测到CFP-Plin 2和尼罗红之间的FRET，表明CFP-Plin 2/尼罗红相互作用超出FRET接近度（>100埃）。Plin 2对细胞代谢的影响的检查显示，在CFP-Plin 2过表达细胞中，三酰甘油、脂肪酸和胆固醇酯含量增加，而二酰甘油保持恒定。总磷脂也增加，反映了磷脂酰胆碱和鞘磷脂增加。与这些结果一致，参与三酰甘油，胆固醇酯和磷脂合成的酶的表达水平在CFP-Plin 2表达细胞中显著上调，而与脂解相关的酶的表达水平降低或不受影响。总之，这些数据首次表明Plin 2直接与脂滴表面的脂质相互作用，并影响参与维持脂滴结构和功能的关键酶和脂质的水平。
McIntosh AL, Senthivinayagam S, Moon KC, Gupta S, Lwande JS, Murphy CC, Storey SM, Atshaves BP. Direct interaction of Plin2 with lipids on the surface of lipid droplets: a live cell FRET analysis. Am J Physiol Cell Physiol 303: C728-C742, 2012. First published June 27, 2012; doi:10.1152/ajpcell.00448.2011.-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 (>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 angstrom, 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 (>100 angstrom). 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.