Selective caveolin-1-dependent endocytosis of glycosphingolipids
Selective caveolin-1-dependent endocytosis of glycosphingolipids
复制标题
DOI：
10.1091/mbc.e02-12-0809
复制
发表时间：
2003-08-01
影响因子：
3.3
通讯作者：
Pagano, RE
中科院分区：
文献类型：
--
作者：
Singh, RD;Puri, V;Pagano, RE
我们研究了荧光鞘糖脂（GSL）类似物在各种细胞类型的内吞作用，使用通路特异性抑制剂和共定位研究与内吞标记物和DsRed小窝蛋白-1（cav-1）。基于抑制剂研究，所有测试的GSL主要（>80%）通过网格蛋白非依赖性、小窝相关机制内化，而与细胞类型无关。此外，荧光乳糖神经酰胺（LacCer）与DsRed-cav-1共定位在大鼠成纤维细胞内吞后的囊泡结构中。GSL的内化机制不受不同的碳水化合物头基或鞘氨醇主链长度的影响;然而，荧光磷脂酰胆碱类似物没有通过小窝内化，这表明GSL神经酰胺核心可能对小窝摄取很重要。荧光LacCer的内化在具有低cav-1的细胞类型中减少80-90%，但被cav-1过表达显著刺激。然而，即使在具有低水平cav-1的细胞中，残留的LacCer内化也是不依赖于网格蛋白的。相反，霍乱毒素B亚基（Ctx B），结合内源性GM（1），在高cav-1表达的细胞中通过网格蛋白非依赖性内吞作用内化，而在低cav-1表达的细胞中发生显著的网格蛋白依赖性摄取。荧光GM 1，通常由在HeLa细胞中具有低cav-1的网格蛋白非依赖性内吞作用内化，在CtxB的存在下通过网格蛋白途径诱导部分内化。这些结果表明，GSL类似物在大多数细胞类型中通过小窝相关机制选择性内化，而CtxB在cav-1水平低时可能发生“通路转换”。
We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (>80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80-90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM(1), was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM1, normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo "pathway switching" when cav-1 levels are low.