A Sensitive and Quantitative Technique for Detecting Autophagic Events Based on Lysosomal Delivery
A Sensitive and Quantitative Technique for Detecting Autophagic Events Based on Lysosomal Delivery
复制标题
DOI：
10.1016/j.chembiol.2011.05.013
复制
发表时间：
2011-08-26
影响因子：
--
通讯作者：
Miyawaki, Atsushi
中科院分区：
文献类型：
--
作者：
Katayama, Hiroyuki;Kogure, Takako;Miyawaki, Atsushi
我们试图开发一种灵敏的定量技术，能够监测涉及溶酶体膜融合的自噬的整个通量。我们观察到Keima的溶酶体隔室内的积累，Keima是一种珊瑚衍生的酸稳定荧光蛋白，在酸性和中性pH值下发出不同颜色的信号。累积荧光读数可用于在单个时间点定量自噬。值得注意的是，该技术使我们能够表征Atg 5缺陷细胞中的自噬途径，其中传统的基于LC 3的自噬体探针无效。由于Keima的大斯托克斯位移，这种自噬探针可以与其他发绿光的荧光团一起可视化。我们研究了线粒体自噬作为一种选择性的自噬过程;靶向Keima和GFP-Parkin的时间推移成像使我们能够同时观察到膜去极化后线粒体的Parkin招募和自噬降解。
We sought to develop a sensitive and quantitative technique capable of monitoring the entire flux of autophagy involving fusion of lysosomal membranes. We observed the accumulation inside lysosomal compartments of Keima, a coral-derived acid-stable fluorescent protein that emits different-colored signals at acidic and neutral pHs. The cumulative fluorescent readout can be used to quantify autophagy at a single time point. Remarkably, the technique led us to characterize an autophagy pathway in Atg5-deficient cells, in which conventional LC3-based autophagosome probes are ineffective. Due to the large Stokes shift of Keima, this autophagy probe can be visualized in conjunction with other green-emitting fluorophores. We examined mitophagy as a selective autophagic process; time-lapse imaging of mitochondria-targeted Keima and GFP-Parkin allowed us to observe simultaneously Parkin recruitment to and autophagic degradation of mitochondria after membrane depolarization.