LC3-positive structures are prominent in autophagy-deficient cells
LC3-positive structures are prominent in autophagy-deficient cells
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DOI：
10.1038/s41598-019-46657-z
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发表时间：
2019-07-12
影响因子：
4.6
通讯作者：
Rubinsztein, David C.
中科院分区：
文献类型：
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作者：
Runwal, Gautam;Stamatakou, Eleanna;Rubinsztein, David C.
自噬是真核生物中一个进化保守的过程，它通过传递到溶酶体来降解货物，如易聚集蛋白质，病原体，受损的细胞器和大分子。这个过程涉及形成双膜自噬体，吞噬注定要降解的货物，有时在自噬受体如p62的帮助下，它们本身就是自噬底物。LC 3-II是自噬体的标准标记物，是通过胞质LC 3-I与新生自噬体表面的磷脂酰乙醇胺（PE）偶联产生的。由于LC 3-II相对特异性地与自噬体和自溶酶体相关（在不存在刺激LC 3相关吞噬作用的条件下），因此LC 3阳性斑点的定量被认为是评估细胞中自噬体数量的金标准测定。在这里，我们发现，内源性LC 3阳性斑点在自噬体形成被废除的细胞中变得更大，并且即使在LC 3-II未形成时也是突出的。这甚至在自噬体生物发生的瞬时和不完全抑制下也会发生。这种现象是由于LC 3-I螯合到p62聚集体，当自噬受损时，p62聚集体积累。这一观察结果质疑了LC 3-免疫荧光测定在具有受损自噬的细胞中的可靠性。
Autophagy is an evolutionarily conserved process across eukaryotes that degrades cargoes like aggregate-prone proteins, pathogens, damaged organelles and macromolecules via delivery to lysosomes. The process involves the formation of double-membraned autophagosomes that engulf the cargoes destined for degradation, sometimes with the help of autophagy receptors like p62, which are themselves autophagy substrates. LC3-II, a standard marker for autophagosomes, is generated by the conjugation of cytosolic LC3-I to phosphatidylethanolamine (PE) on the surface of nascent autophagosomes. As LC3-II is relatively specifically associated with autophagosomes and autolysosomes (in the absence of conditions stimulating LC3-associated phagocytosis), quantification of LC3-positive puncta is considered as a gold-standard assay for assessing the numbers of autophagosomes in cells. Here we find that the endogenous LC3-positive puncta become larger in cells where autophagosome formation is abrogated, and are prominent even when LC3-II is not formed. This occurs even with transient and incomplete inhibition of autophagosome biogenesis. This phenomenon is due to LC3-I sequestration to p62 aggregates, which accumulate when autophagy is impaired. This observation questions the reliability of LC3-immunofluorescence assays in cells with compromised autophagy.