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首页> 外文会议>Conference on 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：通过共聚焦显微镜测量的光漂白后的荧光回收作为分析囊泡脂质传输和质膜迁移率的工具

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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.
机译：通过GOLGI装置从内质网的脂质的抗体传输影响质膜的组成。我们研究的目的是通过使用聚焦显微镜和光漂白后的荧光回收来发展囊泡脂质输送动力学的体外测试系统。来自两种纯合的纯合的高毒性HDL缺乏综合征andier疾病和4个对照受试者的成纤维细胞用C $ -6 $ / - NBD-Ciramide脉冲30分钟。在室温下追加孵育导致荧光-6 $ / - NBD-Spopolyelin和C $ - 6 $ / - NBD-糖基在内侧和Trans-Golgi地区中的代谢累积产生的。用倒的LeICA TCS显微镜分析细胞。通过分析嵌入不同粘度的培养基中的50nm微粒的扩散来进行校准。使用声光可调过滤器（AOTF）用于中介和转毒醇和转胶区域的选择性漂白，然后分析荧光回收率4分钟。通过二维扫描计算通过反式高尔基鞘蛋白的反式高尔基型转运的漂白剂荧光恢复。突变成纤维细胞在Trans-Golgi区域中显示出荧光的延迟回收。这表明肌肉素和胆固醇的凹凸传输在痴呆症中被干扰，确认我们的实验室与全细胞上的辐射素质产生的实验室产生的数据。我们的数据表明，FRAP分析允许敏感动力学和空间解决的血管脂质运输扰动分析。

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