Quantification of 3D EEA1 object volume (m3) in EV-control and Rab4A-shRNA cells for MCF10A (M) and MDAMB231 (O). increased EGF-induced activation of EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). Rab4A over-expression in MCF10A cells produced EEA1-positive enlarged endosomes that displayed prolonged and amplified EGF-induced EGFR-p1068 activation. Knock-down of Rab4A lead to increased endosomal size in MCF10A but not in MDAMB231 cells. Nevertheless, Rab4A knock-down resulted in enhanced EGF-induced activation of EGFR-p1068 in MDAMB231 as well as downstream signaling in MCF10A cells. Altogether, this extensive characterization of early endosomes in breast cancer cells has identified a Rab4-modulated enlarged early endosomal compartment as the site of prolonged and increased EGFR activation. (4). As the regulation of EGFR trafficking and signaling involves multiple Rab proteins, the role of Rab4 in EGFR regulation DW-1350 remains unclear (24). EEs have been characterized as tubulo-vesicular compartments with an average diameter between 100C500 nm (9,25). In this study, super-resolution (SR) microscopy, DW-1350 time-lapse live-cell imaging, and 3D whole-cell quantitative microscopy approaches, as well as immunostaining and immunoblotting signaling assays were used to characterize an enlarged endosomal population that is also the site of increased and prolonged EGF-induced EGFR activation upon phosphorylation at tyrosine 1068 (EGFR-p1068), in aggressive triple-negative MDAMB231 DW-1350 cells but not in non-transformed mammary epithelial MCF10A cells MCF10A cells. Furthermore, Rab4A over-expression lead to an increase in EEA1-positive endosomal size as well as an extended and elevated EGF-induced EGFR-p1068 activation in those enlarged endosomes in MCF10A cells. Unexpectedly, Rab4A knockdown lead to an increased endosomal size in MCF10A but not in MDAMB231 cells. Nevertheless, Rab4A-depleted MDAMB231 cells DW-1350 displayed an elevated EGF-induced activation of EGFR-p1068. These findings establish enlarged EEs in breast cancer cells as central players in the regulation of cargo trafficking and cell signaling, Rabbit Polyclonal to MOBKL2B Here, we suggest a complex role for Rab4 in the regulation of endosomal size as well as of intensity and duration of EGFR activation. MATERIAL AND METHODS Cell culture Cells were purchased from ATCC (Manassas, VA) and tested routinely for mycoplasma. MCF10A cells were cultured in DMEM/F12 with 5% horse serum, 20 ng/ml EGF, 0.5 mg/mL hydrocortisone, 100 ng/ml cholera toxin, 10 g/mL bovine insulin and penicillin/streptomycin. Human breast cancer cells were cultured in DMEM with 10% fetal bovine serum (FBS), 4 mM L-glutamine and 10 mM HEPES, pH 7.4. Cells were passaged less than 15 times. No further cell line authentication was performed. For imaging, cells were plated onto poly-D-lysine coated glass-bottom dishes with 1.5 glass (Mattek, MA). Clear imaging (CI) medium consisted of phenol-free DMEM with 0.5% bovine serum albumin (BSA), 4 mM L-glutamine and 20 mM HEPES, pH 7.4. Chemicals used in solutions and kits are described in Table S1. Plasmids, transfection and lentiviral infection Transfections were performed by electroporation using Neon transfection system (ThermoFisher, MA) and Neon reagents with pulse-width optimized per cell line. Lentiviral particles were packaged and collected from HEK239 cells (Thermo Fisher, MA). Concentration of viral particles was performed with Vivcell100, 30 kDa concentrators (Sartorius, Germany). Immunoblots were used to confirm depletion DW-1350 or over-expression of Rab4. Plasmids were sequenced through Genewiz for confirmation of mutation sequences or successful cloning. Source of plasmids is explained in Table S2. Immunofluorescence, immunoblotting and antibody validation Cells were fixed for 15 min with 4% paraformaldehyde (PFA), permeabilized with 0.1% TX-100 in PBS for 15 min and blocked for 90 min in 2% fish pores and skin gelatin (FSG), 1% BSA in PBS. Washing and antibody obstructing were performed using 0.5% FSG, 0.05% TX-100 in PBS. Antibody validation was performed via cell transfection with target-fluorescent protein constructs, followed by immunostaining and quantitative colocalization analysis of confocal images. Pearsons correlation coefficient of 0.9 indicated a highly specific antibody. Whole cell lysates were processed for immunoblotting using standard methods (26). Antibodies are outlined in Table S3. EGF binding assays are explained in Supplementary Methods. Tumor xenografts All animal procedures were carried out with the authorization of the Institutional Animal Care.