A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.
A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.
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荧光脂质类似物可用于监测分泌活性和分离哺乳动物分泌突变体。
DOI：
10.1091/mbc.6.2.135
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发表时间：
1995
影响因子：
3.3
通讯作者：
Roth,MG
中科院分区：
文献类型：
--
作者：
Ktistakis,NT;Kao,CY;Wang,RH;Roth,MG
使用报告蛋白来研究分泌调节常常因翻译后加工事件而变得复杂，这些事件影响某些蛋白质的分泌，但不是特异性调节分泌的细胞机制的一部分。这对于哺乳动物分泌突变体的分离来说是一个特殊的限制，该分离通常是一个缓慢的过程。为了提供独立于蛋白质加工事件的分泌活性报告，细胞用荧光脂质类似物 C5-DMB-神经酰胺（与荧光团二吡咯亚甲基二氟化物偶联的神经酰胺）标记，并通过荧光显微镜和荧光激活细胞分选来跟踪其分泌。 Brefeldin A 严重抑制中国仓鼠卵巢细胞的分泌，阻断 C5-DMB-神经酰胺的分泌。在高温下，HRP-1 细胞中 C5-DMB-神经酰胺的输出受到抑制，HRP-1 细胞存在条件性分泌缺陷。使用 C5-DMB-神经酰胺作为分泌活性的报告基因，设计了几种不同的脉冲追踪方案，选择对布雷菲德菌素 A 药物具有抗药性的突变中国仓鼠卵巢细胞以及其他在糖蛋白转运至细胞表面方面存在缺陷的细胞。在诱变群体中以 10(-6) 的频率鉴定出任一类型的突变细胞。因此，荧光脂质C5-DMB-神经酰胺可用作分泌活性的特异性标记物，为分离分泌途径缺陷的哺乳动物细胞提供有效、通用的方法。
The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.