Study on mechanism of recruitment of autophagyassociated protein WD repeat domain phosphoinositide-interacting protein 2(WIPI2)by p62
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 lo-cation 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 bleach-ing 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 ag-gregated near p62 in Atg2AB DKO cells;tdTomato-p62 was observed to be highly co-localized with WI-PI2-GFP by live-cell imaging in Atg2AB DKO;fluorescence bleaching assay observed that WIPI2 was mo-bile;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 or-ganelle p62 is able to dynamically fuse with WIPI2-positive vesicles with mobility to promote autophago-some formation.
autophagosomesequestosome 1(p62/SQSTM1)WD repeat domain phosphoinositide-interac-ting protein 2