Department of Pediatrics and Adolescent Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
Department of Endocrinology and Metabolism, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan; Center for Therapeutic Innovations in Diabetes, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
Biochem Biophys Res Commun. 2019 Aug 27;516(3):686-692. doi: 10.1016/j.bbrc.2019.06.129. Epub 2019 Jun 26.
Autophagy is a mechanism of bulk protein degradation that plays an important role in regulating homeostasis in many organisms. Among several methods for evaluating its activity, a fluorescent reporter GFP-LC3-RFP-LC3ΔG, in which GFP-LC3 is cleaved by ATG4 following autophagic induction and degraded in lysosome, has been used for monitoring autophagic flux, which is the amount of lysosomal protein degradation. In this study, we modified this reporter by exchanging GFP for pHluorin, which is more sensitive to low pH, and RFP to mCherry, to construct pHluorin-LC3-mCherry reporter. Following starvation or mTOR inhibition, the increase of autophagic flux was detected by a decrease of the fluorescent ratio of pHluorin to mCherry; our reporter was also more sensitive to autophagy-inducing stimuli than the previous one. To establish monitoring cells for mouse genome-wide screening of regulators of autophagic flux based on CRISPR/Cas9 system, after evaluating knockout efficiency of clones of Cas9-expressing MEFs, we co-expressed our reporter and confirmed that autophagic flux was impaired in gRNA-mediated knockout of canonical autophagy genes. Finally, we performed genome-wide gRNA screening for genes inhibiting starvation-mediated autophagic flux and identified previously reported genes such as Atgs. Thus, we have successfully established a system for screening of genes regulating autophagic flux with our pHluorin-LC3-mCherry reporter in mice.
自噬是一种批量蛋白降解的机制，在许多生物中对调节内稳态起着重要作用。在几种评估其活性的方法中，荧光报告 GFP-LC3-RFP-LC3ΔG 已被用于监测自噬流，即溶酶体蛋白降解的量，该报告 GFP-LC3 在自噬诱导后被 ATG4 切割并在溶酶体中降解。在本研究中，我们通过将 GFP 替换为对低 pH 更敏感的 pHluorin，将 RFP 替换为 mCherry，对该报告进行了修饰，构建了 pHluorin-LC3-mCherry 报告。在饥饿或 mTOR 抑制后，通过 pHluorin 与 mCherry 的荧光比值降低来检测自噬流的增加；与之前的报告相比，我们的报告对自噬诱导刺激也更敏感。为了建立基于 CRISPR/Cas9 系统的监测细胞对自噬流调节因子的小鼠全基因组筛选，在评估 Cas9 表达 MEF 克隆的敲除效率后，我们共表达了我们的报告，并证实 gRNA 介导的经典自噬基因敲除会损害自噬流。最后，我们对抑制饥饿诱导自噬流的 gRNA 进行了全基因组筛选，并鉴定了以前报道的基因，如 Atgs。因此，我们已经成功地在小鼠中建立了使用我们的 pHluorin-LC3-mCherry 报告筛选调节自噬流的基因的系统。
