Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells
Monitoring autophagic flux by an improved tandem fluorescent-tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs autophagic flux in cancer cells
复制标题
通过改进的串联荧光标记 LC3 (mTagRFP-mWasabi-LC3) 监测自噬通量表明，高剂量雷帕霉素会损害癌细胞的自噬通量
DOI：
10.4161/auto.20284
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发表时间：
2012-08-01
期刊：
影响因子：
13.3
通讯作者：
Lin, Jian
中科院分区：
文献类型：
--
作者：
Zhou, Cuihong;Zhong, Wu;Lin, Jian
监测自噬通量对于分析自噬具有重要意义。串联荧光标记LC3（mRFP-EGFP-LC3）是基于EGFP和mRFP荧光蛋白的不同pH稳定性用于监测自噬通量的方便测定。然而，已有报道，在酸性环境（pH在4和5之间）或酸性溶酶体中，EGFP仍有微弱的荧光。因此，自噬溶酶体可能被黄色信号（GFP + RFP+斑点）标记，这导致误解自噬通量结果。因此，在自噬通量的测定中，希望选择比EGFP更酸敏感的单体绿色荧光蛋白。在这里，我们报告了mTagRFP-mWasabi-LC 3报告基因，其中mWasabi比EGFP对酸更敏感，并且在酸性溶酶体中没有荧光。同时，构建mTagRFP-mWasabi-LC3 Δ G作为本试验的阴性对照。与mRFP-EGFP-LC3相比，该报告基因在检测自噬体和自溶体聚集方面更灵敏、更准确。使用这个报告基因，我们发现高剂量的雷帕霉素（30 μ M）将损害自噬通量，在HeLa细胞中诱导比自溶酶体更多的自噬体，而低剂量的雷帕霉素（500 nM）具有相反的效果。此外，其他化学自噬诱导剂（顺铂、星形孢菌素和Z18）也在高剂量下比在低剂量下引起更多的自噬体。结果提示，自噬诱导剂的剂量对细胞自噬通量有明显的影响。
Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP+RFP+ puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 μM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.