Induction of macroautophagy by exogenously introduced calcium
Induction of macroautophagy by exogenously introduced calcium
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DOI：
10.4161/auto.6360
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发表时间：
2008-08-16
期刊：
影响因子：
13.3
通讯作者：
Yin, Xiao-Ming
中科院分区：
文献类型：
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作者：
Gao, Wentao;Ding, Wen-Xing;Yin, Xiao-Ming
大自噬可以通过广泛的试剂和细胞操作来激活。在使用磷酸钙方法进行细胞转染时，我们注意到磷酸钙沉淀物（CPP）可以诱导LC 3点样。由于这种转染方法在哺乳动物细胞中的辅助使用以及钙在自噬诱导中的潜在意义，我们研究了CPP是否可以特异性地诱导大自噬。我们发现，CPP诱导的LC 3点是依赖于钙离子，可以被细胞外或细胞内的钙离子螯合剂中和。点状不是由于LC 3的非特异性聚集，因为它依赖于氨基酸残基甘氨酸120，这是LC 3与磷脂酰乙醇胺（PE）结合所特别需要的。因此，PE缀合形式的LC 3也显著增加。电子显微镜证实CPP处理后典型的自噬体的积累。流量分析表明：CPP诱导但不抑制自噬降解。最后，CPP诱导的自噬依赖于经典的自噬机制，包括Beclin 1，III类磷酸肌醇-3激酶和Atg 5。因此，我们的研究表明，外源性钙离子以CPP的形式导入细胞后，可以特异性地诱导巨自噬，这对于利用CPP将基因导入细胞，以及作为模型系统研究自噬机制具有重要的实际意义。
Macroautophagy can be activated by a broad range of agents and cellular manipulations. In performing cellular transfection using the calcium phosphate method, we noticed that the calcium phosphate precipitates (CPP) could induce LC3 punctation. Because of the Aide use of this transfection method in mammalian cells and the potential significance of calcium in autophagy induction, we investigated whether CPP could specifically induce macroautophagy. We found that CPP-induced LC3 punctation was dependent on calcium and could be neutralized by an extra-celluar or intracellular calcium chelator. The punctation was not due to nonspecific aggregation of LC3 since it depended on the amino acid residue Glycine120, which is specifically required for LC3 to conjugate to phosphatidylethanolamine (PE). Consistently, there was also a significant increase of the PE-conjugated form of LC3. Electron microscopy confirmed the accumulation of typical autophagosomes following CPP treatment. Flux analysis indicated that: CPP induced but did not inhibit autophagic degradation. Finally CPP-induced autophagy depended on the classical macroautophagy machinery including Beclin 1, the class III phosphoinositide-3 kinase and Atg5. Our studies thus indicate that exogenously introduced calcium in the form of CPP could specifically induce macroautophagy, which may have the practical significance in the use of this agent for introducing genes into cells, and for studying the mechanism of autophagy as a model system.