Non-autophagic GFP-LC3 puncta induced by saponin and other detergents
Non-autophagic GFP-LC3 puncta induced by saponin and other detergents
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DOI：
10.4161/auto.4843
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发表时间：
2007-11-01
期刊：
影响因子：
13.3
通讯作者：
Tolkovsky, Aviva M.
中科院分区：
文献类型：
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作者：
Cieehomska, Iwona A.;Tolkovsky, Aviva M.
GFP-LC 3的表达现在被广泛用于可视化培养细胞中的自噬。最近，Kuma等人（Autophagy 2007; 3：323-8）强调了使用GFP-LC 3的一些并发症，证明含有GFP-LC 3的点状点并不总是代表自噬结构。我们在这里报告，GFP-LC 3也可以迅速聚集成自噬体外观相似的结构时，细胞与皂苷透化细胞固定前。用皂苷处理减少了弥散的胞质和核GFP-LC 3，但引起每个细胞的荧光点的数量和强度增加，而不管细胞是否被诱导进行自噬。皂苷还在用GFP-LC 3转染的Atg 5（-/-）MEF中诱导GFP-LC 3斑点，其中不产生LC 3-II，表明斑点是自噬体非依赖性的。当将蛋白质样品标准化为细胞数时，GFP-LC 3斑点的增加与通过免疫印迹检测到的内源性LC 3-II或GFP-LC 3-II的增加不匹配。当细胞用常用于膜透化的其他去污剂（如CHAPS、Triton X-100或毛地黄皂苷）处理时，观察到定性类似的效果。我们还注意到，微管蛋白不能用于在施用皂苷后对印迹上的蛋白质加载进行标准化，因为它选择性地从未处理的细胞中提取，而不是从用长春碱处理的细胞中提取。当使用温和的洗涤剂去除背景荧光时，我们建议使用膜相关蛋白，如ATP合成酶p进行归一化。因此，在固定之前使用的洗涤剂可能使GFP-LC 3聚集成看起来是自噬体的结构，因此应谨慎使用。
Expression of GFP-LC3 is now in widespread use to visualize autophagy in cultured cells. Recently, Kuma et al. (Autophagy 2007; 3:323-8) highlighted some complications using GFP-LC3, demonstrating that punctate dots containing GFP-LC3 do not always represent autophagic structures. We report here that GFP-LC3 can also rapidly aggregate into autophagosome look-alike structures when cells are permeabilized with saponin before cell fixation. Treatment with saponin reduced diffuse cytosolic and nuclear GFP-LC3 but caused an increase in the number and intensity of fluorescent puncta per cell regardless of whether the cells were induced to undergo autophagy. Saponin also induced GFP-LC3 puncta in Atg5(-/-) MEF transfected with GFP-LC3, where no LC3-II is produced, demonstrating that the puncta are autophagosome-independent. The increase in GFP-LC3 puncta was not matched by an increase in endogenous LC3-II or GFP-LC3-II detected by immunoblotting when protein samples were normalized to cell number. A qualitatively similar effect was observed when cells were treated with other detergents commonly used for membrane permeabilization, such as CHAPS, Triton X-100 or digitonin. We also noted that tubulin could not be used to normalize for protein loading on blots after applying saponin as it was selectively extracted from untreated cells but not from cells treated with vinblastine. When using mild detergents to remove background fluorescence, we recommend using a membrane-associated protein such as ATP synthase p for normalization. Thus, detergents used prior to fixation may precipitate GFP-LC3 aggregation into structures that appear autophagosomal and so should be used with caution.