Autophagosome formation and endosomal expansion caused by constitutively active Rab5 and endocytosis are disrupted in Becn1 deficient MEFs.
A. Reduction of Atg12 and WIPI2  puncta formation in Becn1 deficient MEFs. DAPI, endogenous Atg12, WIPI2. White dashed boxes represent zoomed regions. Scalebars = 20 µm. ‘con’ is control MEFs, ‘def’ is beclin 1 deficient MEFs. Quantification of an average of 91 cells per condition from three separate experiments (con: 91, 110, 110; def: 64, 84, 87). Bars represent mean +/− s.e.m. (n = 3); Atg12 p = 0.0039; WIPI2 p = 0.0183 using a one-tailed t-test. B. Rab5-CA mutant is mislocalized in Becn1 deficient MEFs and rescued with re-introduction of beclin 1. MEF cells were transfected with Rab5wt, CA or DN-GFP and visualized. White dashed boxes represent zoomed regions. Scale bars = 20 µm. C. PI(3)P is recruited to expanding endosome sites in control MEFs but no endosome expansion associated with Rab5CA-GFP was found in Becn1 deficient MEFs. MEF cells were transfected with Rab5CA-GFP, fixed, stained with PI(3)P antibody and visualized. Scalebars = 10 µm. Quantification of percentage of cells with no rings or rings (at least 30 transfected cells from at least 3 separate experiments). Control MEFs (7 cells with no rings, 37 cells with rings) vs. Becn1 deficient MEFs (36,5) significantly different (p<0.0001) and Becn1 deficient MEFs (36,6) vs. Becn1 revertant MEFs (11,33) significantly different (p<0.0001) using Fisher's exact test (two-sided).