Sleight R G, Abanto M N
Department of Molecular Genetics, University of Cincinnati College of Medicine, Ohio 45267-0524.
J Cell Sci. 1989 Jun;93 ( Pt 2):363-74. doi: 10.1242/jcs.93.2.363.
The transport and metabolism of a fluorescent phosphatidylcholine analog, 1-palmitoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)- aminocaproyl-phosphatidylcholine [palmitoyl, C6-NBD)-PC), in BHK, CHO-K1, CHO-15B, MDCK, VA-2, Vero, V79 and WI-38 cells has been investigated. When liposomes containing (palmitoyl, C6-NBD)-PC were incubated with cells at 2 degrees C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells' plasma membranes occurred. Most of the lipid transferred to the cells could be removed by incubating the cells in the presence of nonfluorescent liposomes or media containing 10% serum, suggesting that the fluorescent probe resided exclusively in the outer leaflet of the plasma membrane at 2 degrees C. After insertion into the plasma membrane, internalization of (palmitoyl, C6-NBD)-PC occurred when the cells were warmed to 37 degrees C. This resulted in four different labeling patterns: (1) little or no internalization of (palmitoyl, C6-NBD)-PC into punctate vesicles was observed in Vero cells. (2) Transport of (palmitoyl, C6-NBD)-PC to the region of the Golgi apparatus and to a small number of intracellular vesicles was observed in both V79 and CHO-K1 cell lines. (3) A large number of fluorescently labeled intracellular vesicles with little or no labeling in the region of the Golgi apparatus appeared after the internalization of (palmitoyl, C6-NBD)-PC in BHK, CHO-15B, MDCK and WI-38 cell lines. (4) Accumulation of (palmitoyl, C6-NBD)-PC in small vesicles, mitochondria and the nuclear envelope was observed in VA-2 cells. In addition, cells having a defect in glycoprotein processing and those transformed with simian virus 40 (SV40) internalized the fluorescent lipid probe differently compared with parental lines. Neither differences in rates of endocytosis nor rates of (palmitoyl, C6-NBD)-PC degradation between cell types appears to cause the observed dissimilarities in intracellular lipid transport. We suggest that these different cell types may have dissimilar pathways of intracellular lipid trafficking or differential regulation of a common transport pathway, and that the predominant pathway of lipid translocation can be altered in cells by changing the composition of their glycoproteins or by viral transformation.
研究了一种荧光磷脂酰胆碱类似物1-棕榈酰-2-（N-4-硝基苯并-2-恶唑-1,3-二氮杂环戊二烯）-氨基己酰基-磷脂酰胆碱[棕榈酰，C6-NBD)-PC]在BHK、CHO-K1、CHO-15B、MDCK、VA-2、Vero、V79和WI-38细胞中的运输和代谢。当含有（棕榈酰，C6-NBD)-PC的脂质体在2℃下与细胞孵育时，荧光脂质从脂质体自发转移到细胞质膜上。转移到细胞中的大部分脂质可以通过在非荧光脂质体或含有10%血清的培养基存在下孵育细胞来去除，这表明荧光探针在2℃时仅存在于质膜的外小叶中。插入质膜后，当细胞升温至37℃时，（棕榈酰，C6-NBD)-PC发生内化。这导致了四种不同的标记模式：（1）在Vero细胞中未观察到（棕榈酰，C6-NBD)-PC很少或没有内化到点状小泡中。（2）在V79和CHO-K1细胞系中均观察到（棕榈酰，C6-NBD)-PC运输到高尔基体区域和少量细胞内小泡中。（3）在BHK、CHO-15B、MDCK和WI-38细胞系中，（棕榈酰，C6-NBD)-PC内化后出现大量荧光标记的细胞内小泡，高尔基体区域几乎没有或没有标记。（4）在VA-2细胞中观察到（棕榈酰，C6-NBD)-PC在小泡、线粒体和核膜中积累。此外，糖蛋白加工有缺陷的细胞和用猴病毒40（SV40）转化的细胞与亲本系相比，荧光脂质探针的内化方式不同。细胞类型之间内吞速率和（棕榈酰，C6-NBD)-PC降解速率的差异似乎都不会导致观察到的细胞内脂质运输的差异。我们认为，这些不同的细胞类型可能具有不同的细胞内脂质运输途径或对共同运输途径的不同调节，并且通过改变其糖蛋白组成或病毒转化可以改变细胞中脂质转运的主要途径。
