Deacidification of the Endolysosomal System by the Vesicular Proton Pump V-ATPase Inhibitor Bafilomycin A1 Affects EGF Receptor Endocytosis Differently in Endometrial MSC and HeLa Cells.

It is well-known that EGF binding to EGFR stimulates signal transduction and endocytosis, with the latter leading to lysosomal degradation of EGFR. However, the majority of data on the regulation of endocytosis have been obtained in tumor-derived cells. Here, we perform a comprehensive analysis of the role of endolysosome acidification in the regulation of endocytic pathway in tumor cells and in endometrial MSCs as a model of proliferating, undifferentiated, non-immortalized cells. Using QD-labeled EGF, the dynamics of co-localization of EGF-receptor complexes with endocytic markers in the control and upon inhibition of V-ATPase by Bafilomycin A1 (BafA1) were studied using confocal microscopy. Image analysis showed that in HeLa and A549 cells, BafA1 significantly slowed down EGFR entry into and exit from EEA1-positive early endosomes without disrupting passage through Rab7, CD63, and Lamp1 compartments, but rather shifting it to later times. In enMSCs, only a portion of EGF-containing endosomes entered the degradation pathway, and lysosomal delivery was significantly delayed. Unlike HeLa, in enMSC early endosomes, BafA1 increased the association of EGF-QDs with EEA1, suggesting a lower pH level, which is suboptimal for EEA1-dependent fusions. It is concluded that, unlike HeLa, enMSCs form a population of pH-independent endosomes containing activated EGFR for a long time.