Induction of endocytic vesicles by exogenous C6-ceramide
Induction of endocytic vesicles by exogenous C6-ceramide
复制标题
DOI：
10.1074/jbc.274.30.21121
复制
发表时间：
1999-07-23
影响因子：
4.8
通讯作者：
Ladisch, S
中科院分区：
文献类型：
--
作者：
Li, RX;Blanchette-Mackie, EJ;Ladisch, S
神经酰胺是一种新发现的第二信使，已被证明可导致细胞生长阻滞和凋亡。本研究表明，外源性添加的c -6-神经酰胺可诱导晚期内体和溶酶体的扩大，10 μ M的c -6-神经酰胺可在成纤维细胞（3T3-L1和3T3-F442A）中形成许多大小不等（2-10 μ M）的囊泡，且无毒性作用。c -6神经酰胺诱导的囊泡形成具有时间和剂量依赖性，快速且可逆。10 μ M c -6神经酰胺处理后8 h内出现大量小泡，随时间增大，核周可见大泡，细胞外周可见小泡。在治疗24小时内，大约30%的细胞表现出这些囊泡。从培养基中去除神经酰胺导致囊泡消失，再加入神经酰胺后囊泡重新出现，使用抗溶酶体相关膜蛋白抗体的共聚焦免疫荧光显微镜分析鉴定增大的囊泡为晚期内体/溶酶体。荧光c -6- nbd神经酰胺，一种对高尔基体至关重要的染色剂，没有染色这些囊泡。对囊泡形成的影响受神经酰胺结构的影响；d - red -c -6神经酰胺是活性最高的神经酰胺类似物。外源性添加短链神经酰胺代谢物，如鞘氨醇、鞘氨醇- 1-磷酸、n -己醇-鞘氨酰基磷胆碱、n -乙酰精神碱和c -2-神经酰胺G（M3）、(G（M3）、n -乙酰神经酰胺- α（2,3)-半乳糖- β(1,4)-葡萄糖神经酰胺）对囊泡形成无活性。这些研究表明，外源性c -6-神经酰胺诱导小鼠成纤维细胞内吞囊泡形成，导致晚期内溶体和溶酶体增大。
Ceramide is a newly discovered second messenger that has been shown to cause cell growth arrest and apoptosis, Here, we present evidence that exogenously added C-6-ceramide induces enlargement of late endosomes and lysosomes, 10 mu M C-6-ceramide caused the formation of numerous vesicles of varying sizes (2-10 mu m) in fibroblasts (3T3-L1 and 3T3-F442A), without toxic effects. Vesicle formation induced by C-6-ceramide was time- and dose-dependent, rapid, and reversible. Numerous small vesicles appeared within 8 h of treatment with 10 mu M C-6-ceramide, They enlarged with time, with large vesicles found in the perinuclear region and small ones observed at the cell periphery. Within 24 h of treatment, similar to 30% of the cells exhibited these vesicles. Removal of ceramide from the culture medium caused disappearance of the vesicles, which reappeared upon readdition of ceramide, Confocal immunofluorescence microscopic analysis using an anti-lysosome-associated membrane protein antibody identified the enlarged vesicles as late endosomes/lysosomes. The fluorescent C-6-NBD-ceramide, a vital stain for the Golgi apparatus, did not stain these vesicles. The effect on vesicle formation was influenced by ceramide structure; D-erythro-C-6-ceramide was the most active ceramide analogue tested. Short chain ceramide metabolites, such as sphingosine, sphingosine 1-phosphate, N-hexanoyl-sphingosylphosphorylcholine, N-acetylpsychosine, and C-2-ceramide G(M3), (G(M3), N-acetylneuraminosyl-alpha(2,3)-galactosyl-beta (1,4)-glucosylceramide), were inactive in causing vesicle formation when added exogenously, Together, these studies demonstrate that exogenous C-6-ceramide induces endocytic vesicle formation and causes enlarged late endosomes and lysosomes in mouse fibroblasts.