Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms
Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms
复制标题
葡萄糖神经酰胺荧光类似物对人皮肤成纤维细胞高尔基体的内化和分类：利用内吞和非内吞运输机制
作者：
O. Martin;R. Pagano
我们研究了荧光葡萄糖神经酰胺类似物N-[5-（5，7-二甲基BODIPYTM）-1-戊酰基]-葡萄糖鞘氨醇（C5-DMB-GlcCer）在人皮肤成纤维细胞中的摄取和细胞内转运，并将其行为与鞘磷脂、半乳糖神经酰胺和乳糖神经酰胺的相应荧光类似物进行了比较。在4 ℃孵育期间，所有四种荧光类似物都容易从脱脂BSA转移到质膜上。当用C5-DMB-GlcCer处理的细胞被洗涤，升温至37 ℃，随后与脱脂BSA孵育以除去细胞表面的荧光脂质时，在高尔基体处观察到强荧光，以及在核膜和其他细胞内膜处较弱的标记。用C5-DMB-半乳糖基神经酰胺获得类似的结果，除了高尔基体的标记弱于用C5-DMB-GlcCer。C5-DMB-GlcCer的内化不受各种处理的抑制，包括ATP耗竭或升温至19 ℃，并且生化分析表明脂质在其内化期间不代谢。然而，当用葡萄糖神经酰胺的非荧光类似物处理细胞时，高尔基体处C5-DMB-GlcCer的积累减少，表明高尔基体处C5-DMB-GlcCer的积累是可饱和的过程。相比之下，用鞘磷脂或乳糖神经酰胺的C5-DMB类似物处理的细胞在37摄氏度的升温过程中将荧光脂质内化成点状荧光图案，并且该过程是温度和能量依赖性的。用C5-DMB-鞘磷脂和C5-DMB-乳糖神经酰胺得到的这些结果类似于用鞘磷脂的另一种荧光类似物得到的结果，其中记录了内吞囊泡的标记和质膜脂质再循环（Koval，M.，和R. E.帕加诺1990. 111：429-442）。穿孔细胞与C5-DMB-鞘磷脂的孵育导致核膜和其他细胞内膜的显著标记，类似于在完整细胞中用C5-DMB-GlcCer观察到的模式。这些观察结果是一致的transbilayer运动的荧光类似物的葡萄糖神经酰胺和半乳糖神经酰胺在质膜和早期的内体的人皮肤成纤维细胞，并表明，内吞和nonendocytic途径被用于这些脂质从质膜的内化。
We examined the uptake and intracellular transport of the fluorescent glucosylceramide analogue N-[5-(5,7-dimethyl BODIPYTM)-1-pentanoyl]- glucosyl sphingosine (C5-DMB-GlcCer) in human skin fibroblasts, and we compared its behavior to that of the corresponding fluorescent analogues of sphingomyelin, galactosylceramide, and lactosylceramide. All four fluorescent analogues were readily transferred from defatted BSA to the plasma membrane during incubation at 4 degrees C. When cells treated with C5-DMB-GlcCer were washed, warmed to 37 degrees C, and subsequently incubated with defatted BSA to remove fluorescent lipid at the cell surface, strong fluorescence was observed at the Golgi apparatus, as well as weaker labeling at the nuclear envelope and other intracellular membranes. Similar results were obtained with C5-DMB- galactosylceramide, except that labeling of the Golgi apparatus was weaker than with C5-DMB-GlcCer. Internalization of C5-DMB-GlcCer was not inhibited by various treatments, including ATP depletion or warming to 19 degrees C, and biochemical analysis demonstrated that the lipid was not metabolized during its internalization. However, accumulation of C5-DMB-GlcCer at the Golgi apparatus was reduced when cells were treated with a nonfluorescent analogue of glucosylceramide, suggesting that accumulation of C5-DMB-GlcCer at the Golgi apparatus was a saturable process. In contrast, cells treated with C5-DMB-analogues of sphingomyelin or lactosylceramide internalized the fluorescent lipid into a punctate pattern of fluorescence during warming at 37 degrees C, and this process was temperature and energy dependent. These results with C5-DMB-sphingomyelin and C5-DMB-lactosylceramide were analogous to those obtained with another fluorescent analogue of sphingomyelin in which labeling of endocytic vesicles and plasma membrane lipid recycling were documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429-442). Incubation of perforated cells with C5-DMB- sphingomyelin resulted in prominent labeling of the nuclear envelope and other intracellular membranes, similar to the pattern observed with C5-DMB-GlcCer in intact cells. These observations are consistent with the transbilayer movement of fluorescent analogues of glucosylceramide and galactosylceramide at the plasma membrane and early endosomes of human skin fibroblasts, and suggest that both endocytic and nonendocytic pathways are used in the internalization of these lipids from the plasma membrane.
磷脂酰丝氨酸和磷脂酰乙醇胺的荧光类似物在培养细胞质膜上的跨双层运动。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Martin,OC;Pagano,RE
通讯作者：
Pagano,RE
神经酰胺的荧光衍生物：物理性质及其在研究动物细胞高尔基体中的用途。
影响因子：
--
作者：
Pagano,RE
通讯作者：
Pagano,RE
影响因子：
56.9
作者：
LIPSKY, NG;PAGANO, RE
通讯作者：
PAGANO, RE