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首页> 外文会议>Optical Investigations of Cells In Vitro and In Vivo >Fluorescence recovery after photobleaching measured by confocal microscopy as a tool for the analysis of vesicular lipid transport and plasma membrane mobility
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Fluorescence recovery after photobleaching measured by confocal microscopy as a tool for the analysis of vesicular lipid transport and plasma membrane mobility

机译：共聚焦显微镜测量光漂白后的荧光恢复，作为分析囊泡脂质转运和质膜迁移率的工具

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Abstract: The vesicular transport of lipids from the endoplasmic reticulum via the Golgi apparatus affects the composition of the plasma membrane. The purpose of our study was to develop an in vitro test system for characterization of vesicular lipid transport kinetics by using confocal microscopy and fluorescence recovery after photobleaching (FRAP). Fibroblasts from two patients homozygous for the hypercatabolic HDL deficiency syndrome Tangier disease and 4 control subjects were pulsed with the C$-6$/-NBD-ceramide for 30 minutes. Chase incubation at room temperature resulted in the metabolic accumulation of fluorescent C$-6$/-NBD-sphingolyelin and C$- 6$/-NBD-glycosylceramides in the medial- and trans-Golgi region. Cells were analyzed with an inverted Leica TCS microscope. Calibration was performed through the analysis of diffusion of 50 nm microparticles embedded in media of different viscosity. An acousto optical tunable filter (AOTF) was used for the selective bleaching of the medial- and trans- Golgi region followed by analysis of the fluorescence recovery for 4 minutes. Post-bleach fluorescence recovery through the trans-Golgi-oriented transport of NBD-sphingomyelin was calculated from 2-dimensional scans. Tangier fibroblasts displayed a retarded recovery of fluorescence in the trans- Golgi region. This suggests that the vesicular transport of sphingomyelin and cholesterol is disturbed in Tangier disease confirming data from our laboratory generated with radiometabolites on whole cells. Our data suggest that FRAP analysis allows a sensitive kinetic and spatially resolved analysis of disturbances of vesicular lipid transport. !26
机译：摘要：脂质通过高尔基体从内质网囊泡运输影响质膜的组成。我们研究的目的是通过使用共聚焦显微镜和光漂白后的荧光恢复（FRAP）来开发表征囊泡脂质转运动力学的体外测试系统。将来自两名高催化HDL缺乏综合征丹吉尔病患者纯合子的成纤维细胞和4名对照受试者的C $ -6 $ /-NBD-神经酰胺脉冲30分钟。在室温下追逐孵育导致荧光C $ -6 $ /-NBD-鞘氨醇和C $ -6 $ /-NBD-糖基神经酰胺在中高尔基区和反高尔基区的代谢积累。用倒置的徕卡TCS显微镜分析细胞。通过分析嵌入不同粘度介质中的50 nm微粒的扩散来进行校准。使用声光可调滤光片（AOTF）对内侧和反高尔基体区域进行选择性漂白，然后分析4分钟的荧光回收率。通过二维扫描计算通过NBD-鞘磷脂的反高尔基体定向转运的漂白后荧光恢复。丹吉尔成纤维细胞在反高尔基体区域显示出荧光的恢复缓慢。这表明丹吉尔病中鞘磷脂和胆固醇的囊泡运输受到干扰，这证实了我们实验室用全细胞放射性代谢物产生的数据。我们的数据表明，FRAP分析可以对囊泡脂质运输的干扰进行灵敏的动力学和空间分辨分析。 ！26

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