Ferretti A, Knijn A, Iorio E, Pulciani S, Giambenedetti M, Molinari A, Meschini S, Stringaro A, Calcabrini A, Freitas I, Strom R, Arancia G, Podo F
Laboratory of Cell Biology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.
Biochim Biophys Acta. 1999 Jun 10;1438(3):329-48. doi: 10.1016/s1388-1981(99)00071-2.
Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.
通过核磁共振（NMR）、尼罗红荧光和电子显微镜技术，研究了在NIH - 3T3成纤维细胞及其转染了大量癌基因拷贝的H - ras转化细胞（3T3ras）中，1H - NMR可检测到的流动性脂质结构域（ML）的性质和亚细胞定位。在未转化的成纤维细胞中，较高的ML水平（(CH2)n/CH3峰面积比R = 1.56±0.33）与以下两者相关：（a）直径约60 nm、与小窝不同的膜内无定形脂质囊泡；（b）被自身膜包围、具有膜内颗粒的细胞质嗜锇脂质体。二维NMR图谱表明，ML包含单不饱和脂肪酸链和多不饱和脂肪酸链。在3T3ras细胞（R = 0.76±0.37）中，在各种细胞生长条件下检测到较低的ML信号。在R<0.4的3T3ras细胞的细胞质或膜区室中，很少（如果有的话）检测到脂质体和囊泡，而只有膜内脂质囊泡与中等R值相关。通过选择性抑制内源性磷脂酶C（PC - plc）或暴露于细菌PC - plc，证明了磷脂酰胆碱水解参与ML的生成。这项研究表明：（1）细胞质脂质体和膜囊泡（可能处于相互动态交换中）都可能（尽管程度不同）对ML信号有贡献；（2）高水平的ras转染要么抑制ML的形成，要么促进它们从细胞中排出。
