Autophagy Eliminates ER Membrane Reorganization Bcl-2Inhibitor Induced In Hela Cells

ObjectiveThe Endoplasmic Reticulum (ER) is a membranous network within cells that isimportant for several cellular functions including translation and folding of secretoryand membrane proteins, lipid biogenesis and sequestration of Ca2+. Disruption of ERstructure might affect the normal physiology of the cell. In yeast, expansion of the ERis observed under unfolded protein response (UPR) and subsequently inducesautophagy initiated from the ER. In this study, we demonstrated a drastic and specificER membrane reorganisation (EMR), characterized by the clustering of the ERmembrane into large and compact aggregates and occuring independent of UPR inHela cells treated with S1combined with ABT-737. Subsequently, treatment of S1andABT-737triggered autophagy. Herein, we reported a key step for removal of damagedand superfluous cellular constituents, whereby amechanistic link between ERaggregation and autophagic activation. Our study may help us to analyze autophagy indisease.MethodIn our study, we used of fluorescence microscopy to monitor ER membraneaggregation. A clustering of ER membrane proteins (Bap31/Calnexin), was observedin cells exposed to S1combined with ABT-737. We also found ER membranereorganisation in knockdown of Bcl-2and Mcl-1with small interfering RNA in Helacells. Furthermore, TUDCA an ER stress reducers, failed to abolish combined withABT-737induced ER membrane reorganization in Hela cells. ER membrane proteinsmarkedly reincreased under the effect of3-MA in Hela cells. Atg12-Atg5and Atg8 (LC3), the two ubiquitin-like conjugation systems, are required for the initiation andexpansion of autophagosomal membranes. When autophagy occurs, LC3proteinappears as dots, and the soluble form of LC3(LC3-I) changes into the lipidated andautophagosome-associated form (LC3-II). Hela cells stained with an antibody againstCalnexin or Bap31to label the ER and with antibodys against Beclin-1, Atg12orLC-3to label the autophagosomes. Analysis of cells by high-resolution imagingrevealed extensive colocalization between autophagosomes and membranereorganised ER by the S1and ABT-737combination in Hela cells.ResultWhen the two inhibitors were applied together, there was an increased ERmembrane reorganisation phenomenon at4h. A clustering of ER membrane proteins(Bap31/Calnexin), was observed in cells exposed to S1combined with ABT-737. Wealso found ER membrane reorganisation in knockdown of Bcl-2and Mcl-1with smallinterfering RNA in Hela cells.The expression of Bip was upregulated following treatment with S1combinedwith ABT-737in Hela cells. Our results showed that S1combined with ABT-737upregulated eIF2a.In this study we found S1combined with ABT-737markedlydecreased the detection of Calnexin and Bap31,the ER membrane proteins,as early as4h. As expected, after S1combined with ABT-737treatment for4h, ER membraneproteins markedly reincreased under the effect of3-MA in Hela cells, but MG-132had no effact on them.When autophagy occurs, LC3protein appears as dots, and the soluble form ofLC3(LC3-I) changes into the lipidated and autophagosome-associated form (LC3-II).Compared with the control group, S1and ABT-737increased the expression ofLC3-II in Hela cells. The S1and ABT-737combination also increased the expressionof Beclin-1after4h in Hela cells. In addition, visible LC3dots in the cytoplasm wereobserved in Hela cells treated with S1combined with ABT-737. In this study,phosphorylation of proteins involved in the mTOR pathway remained relativelyunchanged in Hela cells treated with S1combined with ABT-737. Analysis of cells by high-resolution imaging revealed extensive colocalizationbetween autophagosomes and membrane reorganized ER by the S1and ABT-737combination in Hela cells. A pseudo-line scan analysis confirmed thatautophagosomes colocalized with membrane reorganized ER by the S1and ABT-737combination in Hela cells. Rapamycin, an autophagy activator, relieved ER membranereorganization S1with ABT-737induced in HeLa cells.ConclusionS1combined with ABT-737induces ER membrane remodeling in Hela cells. S1combined with ABT-737induces ER membrane remodeling before UPR-relatedchanges in hela cells. S1combined with ABT-737downregulates ER membraneproteins. S1combined with ABT-737induces autophagy in Hela cells. Membranereorganized ER are removed by autophagy.