【摘要】 目的 探讨自噬接头蛋白(Sequestosome 1,SQSTM1/p62)与磷脂酰肌醇3-激酶(Phosphatidylinositide 3-kinase, PI3K)复合物的结合蛋白WD重复结构域磷酸肌醇互作蛋白2(WD repeat domain phosphoinositide-interacting protein 2,WIPI2)在空间上定位的调控关系。方法 使用CRISPR-Cas9技术敲除正常大鼠肾上皮细胞(Normal rat kidney, NRK)中的自噬相关基因2A(Autophagy-related gene,Atg2A)、自噬相关基因2B(Autophagy-related gene,Atg2B)和p62基因，建立Atg2A和Atg2B基因敲除的细胞系(Atg2AB double knockout,Atg2AB DKO)以及p62基因敲除细胞系(p62 knockout,p62 KO);透射电镜观察Atg2AB DKO细胞中p62体周围囊泡的定位；活细胞成像观察Atg2AB DKO细胞内p62体与自噬相关蛋白WIPI2的定位变化；光漂白实验观察相变蛋白p62与WIPI2的荧光漂白恢复；通过免疫荧光观察WT、p62 KO和Atg2AB DKO细胞中WIPI2与p62的定位关系；通过蛋白免疫印记检测WT和p62 KO细胞中WIPI2表达量的变化。结果 建立了Atg2AB DKO和p62 KO细胞系；透射电镜显示Atg2AB DKO细胞中p62附近聚集了大量囊泡；在Atg2AB DKO中，通过活细胞成像观察到tdTomato-p62与WIPI2-GFP高度共定位；荧光漂白实验观察到WIPI2具有流动性；通过免疫荧光观察到，与WT细胞相比，在Atg2AB DKO中WIPI2点的数量明显增多(P<0.000 1);与WT细胞相比，p62 KO细胞中WIPI2点的数量和表达量无明显变化(P>0.05)。结论 无膜细胞器p62能够与具有流动性的WIPI2阳性囊泡动态融合，促进自噬体的形成。更多还原

【Abstract】 Objective To investigate the regulation of the spatial localization of autophagy junction protein(SQSTM1/p62) to the binding protein of the phosphatidylinositol 3-kinase complex(PI3K),WD repeat structural domain phosphosphoinositide-interacting protein 2(WIPI2). Methods CRISPR-Cas9 was used to knockout the autophagy-related gene 2A(Atg2A), autophagy related gene 2B(Atg2B) and p62 gene in normal rat kidney epithelial cells(NRK), and established cell lines of Atg2AB DKO and p62 KO.The location of vesicles around p62 in Atg2AB DKO cells was observedby transmission electron microscopy. The location changes of p62 bodies and autophagyrelated protein WIPI2 in Atg2AB DKO cells were observed by live cell imaging.The restoration of phase change protein p62 bodiesand WIPI2 by fluorescence bleaching was observed by photobleaching experiment.The location relationship between WIPI2 and p62 in WT, p62 KO and Atg2AB DKO cellswas observed by immunofluorescence.The expression of WIPI2 expression in WT and p62 KO cellswas detected by immunoimprinting. Results Atg2AB DKO and p62 KO cell lines were established; transmission electron microscopy showed that a large number of vesicles were aggregated near p62 in Atg2AB DKO cells; tdTomato-p62 was observed to be highly co-localized with WIPI2-GFP by live-cell imaging in Atg2AB DKO; fluorescence bleaching assay observed that WIPI2 was mobile; a significant increase in the number of WIPI2 sites was observed by immunofluorescence in Atg2AB DKO compared to WT cells(P<0.000 1); there was no significant change in the number and expression of WIPI2 sites in p62 KO cells compared with WT cells(P>0.05). Conclusions The membrane-free organelle p62 is able to dynamically fuse with WIPI2-positive vesicles with mobility to promote autophagosome formation.更多还原