An Autophagic Flux Probe that Releases an Internal Control
An Autophagic Flux Probe that Releases an Internal Control
复制标题
DOI：
10.1016/j.molcel.2016.09.037
复制
发表时间：
2016-11-17
期刊：
影响因子：
16
通讯作者：
Mizushima, Noboru
中科院分区：
文献类型：
--
作者：
Kaizuka, Takeshi;Morishita, Hideaki;Mizushima, Noboru
大自噬是一种利用自噬体将细胞质组分递送至溶酶体的细胞内降解系统。测量自噬活性至关重要，但仍然复杂且具有挑战性。在这里，我们开发了GFP-LC3-RFP-LC3 Delta G，一种用于评估自噬通量的荧光探针。该探针被内源性ATG 4蛋白酶切割成等摩尔量的GFP-LC 3和RFP-LC 3 Delta G。GFP-LC3通过自噬降解，而RFP-LC3 Delta G保留在胞质溶胶中，作为内部对照。因此，可以通过计算GFP/RFP信号比来估计自噬通量。使用这种探针，我们重新评估了先前报道的自噬调节化合物，对批准的药物库进行了高通量筛选，并鉴定了自噬调节剂。此外，我们成功地测量了斑马鱼和小鼠胚胎和组织中的诱导和基础自噬通量。GFP-LC3-RFP-LC3 Delta G探针是一种简单且定量的方法，用于评估培养细胞和整个生物体中的自噬通量。
Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3 Delta G, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3 Delta G. GFP-LC3 is degraded by autophagy, while RFP-LC3 Delta G remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3 Delta G probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.