A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor.
A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor.
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DOI：
10.1083/jcb.113.6.1267
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发表时间：
1991-06
期刊：
影响因子：
--
通讯作者：
Haugland RP
中科院分区：
文献类型：
--
作者：
Pagano RE;Martin OC;Kang HC;Haugland RP
合成了一系列含氟基团二吡咯甲基二氟化硼（BODIPY）的神经酰胺类似物，作为高尔基体的重要染色剂，并作为研究高尔基体与活细胞质膜之间脂质运输的工具进行了评价。对脂质囊泡中几种BODIPY标记的神经酰胺的光谱特性的研究表明，荧光发射最大值强烈依赖于膜中探针的摩尔密度。这一点在N-[5-(5,7-二甲基BODIPY)-1-戊烷基]- d -红-鞘氨醇（C5-DMB-Cer）中表现得尤为明显，随着浓度的增加，其最大发射波长从绿色（515 nm积分）向红色（620 nm积分）转变。当C5-DMB-Cer用于标记活细胞时，这一特性使我们能够将含有高浓度荧光脂质及其代谢物（鞘磷脂和葡萄糖神经酰胺的相应类似物）的膜与存在少量探针的细胞其他区域区分开来。使用这种方法，在活的人皮肤成纤维细胞以及其他细胞类型中，可以看到高尔基体、高尔基体相关的管状泡过程和假定的高尔基体运输囊泡的显著红色荧光标记。基于荧光比成像显微镜，我们估计C5-DMB-Cer及其代谢物存在于高尔基体膜中，浓度高达5- 10%。此外，C5-DMB-Cer的浓度依赖光谱特性被用来监测c5 - dmb -脂质在37℃下向细胞表面的运输。
A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY- labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N-[5-(5,7- dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), which exhibited a shift in its emission maximum from green (integral of 515 nm) to red (integral of 620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller amounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that C5-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration- dependent spectral properties of C5-DMB-Cer were used to monitor the transport of C5-DMB-lipids to the cell surface at 37 degrees C.
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DOI：
10.1083/jcb.108.6.2169
发表时间：
1989-06
期刊：
The Journal of cell biology
影响因子：
--
作者：
Koval M;Pagano RE
通讯作者：
Pagano RE
影响因子：
7.8
作者：
Koval, M;Pagano, R E
通讯作者：
Pagano, R E
影响因子：
64.5
作者：
COOPER, MS;CORNELLBELL, AH;SMITH, SJ
通讯作者：
SMITH, SJ
影响因子：
2.9
作者：
KLAUSNER, RD;WOLF, DE
通讯作者：
WOLF, DE
影响因子：
2.9
作者：
DRAGSTEN, PR;WEBB, WW
通讯作者：
WEBB, WW