Visualization of reticulophagy in living cells using an endoplasmic reticulum-targeted p62 mutant

作     者：Liang Wang 

作者机构：Britton Chance Center for Biomedical Photonics Wuhan National Laboratory for Optoelectronics Huazhong University of Science and Technology Wuhan 430074 China MoE Key Laboratory for Biomedical Photonics Department of Biomedical Engineering Huazhong University of Science and Technology Wuhan 430074 China 

出 版 物：《Science China(Life Sciences)》 (中国科学（生命科学英文版）)

年 卷 期：2017年第60卷第4期

页      面：333-344页

核心收录：

学科分类：07[理学] 08[工学] 

基　　金：supported by the Major Research Plan of the National Natural Science Foundation of China (91442201) the National Science Fund for Distinguished Young Scholars (81625012) the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (61421064) 

主　　题：endoplasmic reticulum targeted mutant aggregate puncta homeostasis autophagy Figure lapse 

摘      要：Reticulophagy is a type of selective autophagy in which protein aggregate-containing and/or damaged endoplasmic reticulum(ER)fragments are engulfed for lysosomal degradation, which is important for ER homeostasis. Several chemical drugs and mutant proteins that promote protein aggregate formation within the ER lumen can efficiently induce reticulophagy in mammalian ***, the exact mechanism and cellular localization of reticulophagy remain unclear. In this report, we took advantage of the self-oligomerization property of p62/SQSTM1, an adaptor for selective autophagy, and developed a novel reticulophagy system based on an ER-targeted p62 mutant to investigate the process of reticulophagy in living cells. LC3 conversion analysis via western blot suggested that p62 mutant aggregate-induced ER stress triggered a cellular autophagic response. Confocal imaging showed that in cells with moderate aggregation conditions, the aggregates of ER-targeted p62 mutants were efficiently sequestered by autophagosomes, which was characterized by colocalization with the autophagosome precursor marker ATG16L1, the omegasome marker DFCP1, and the late autophagosomal marker LC3/GATE-16. Moreover, time-lapse imaging data demonstrated that the LC3-or DFCP1-positive protein aggregates are tightly associated with the reticular structures of the ER, thereby suggesting that reticulophagy occurs at the ER and that omegasomes may be involved in this process.