Fig. 8. Beclin1 and MAM localization in WT and Ulk1^−/− MEFs.

A) Representative images of WT MEFs overexpressing the split-GFP MAM reporter plus Beclin1, Beclin1 S15A, or Beclin2. Cells were treated with DMSO or 10 μM FCCP for 6h. The fluorescence intensity of co-localization (yellow) signal normalized to total MAMs (green) was quantified in cells (n=30 cells/group from three independent experiments). B) Representative images of WT and Ulk1^−/− MEFs overexpressing Beclin1 or Beclin1S15D plus the split-GFP MAM reporter. Cells were treated with 10 μM FCCP for 6h. C) Quantification of Beclin1-MAM colocalization events/total MAMs on a per cell basis (n=30 cells/group from three independent experiments). D) Proposed model illustrating role of Ulk1-mediated phosphorylation of Beclin1 at Ser¹⁵ during mitophagy. Phosphorylated Beclin1 re-localizes to a mitochondria-ER associated membrane site where it initiates formation of the autophagosome membrane adjacent to a dysfunctional mitochondrion. Data are presented as means ± SEM. **p<0.01 by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Scale bars = 10 μm.