Journal: Autophagy

Article Title: PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation

doi: 10.1080/15548627.2016.1277309

Figure Lengend Snippet: BECN1 is not required to trigger the early steps of mitophagy. (A and B) Confocal analysis of PINK1 accumulation and PARK2 recruitment at mitochondria upon CCCP treatment in shSCR and shBECN1 SH-SY5Y cells, overexpressing PINK1-GFP (A) or YFP-PARK2 (B). Cells were treated with DMSO alone or with 25 μM CCCP for 3 h and mitochondria were immunostained using the mitochondrial marker TOMM20 (red). Scale bar: 10 μm. (C and D) Statistical analysis of data from A-B (mean ± SD of n = 3, 30 cells per experiment). Histograms report the percentage of cells showing colocalization of PINK1 (C) or PARK2 (D) with mitochondria. **p < 0.001. (E) WB of PINK1 and early mitophagic markers in shSCR and shBECN1 SH-SY5Y cells overexpressing HA-PARK2. Degradation (shorter exposure) and ubiquitination (longer exposure, upper band) of the mitochondrial proteins MFN2, OPA1 and VDAC1 were assessed at the indicated early time points of treatment with 25 μM CCCP. Values were normalized against TUBA/tubulin. Separated blots indicate that we joined together distant parts from the same gel.

Article Snippet: The following antibodies were used for western blotting, immunoprecipitation and immunofluorescence assays: rabbit anti-PINK1 (Novus Biologicals, BC100–494), mouse anti-HA (Sigma-Aldrich, H3663), rat anti-HA (Roche Applied Science, 10744700), mouse anti-BECN1 (BD Biosciences, BD612113), rabbit anti-BECN1 (Novus Biologicals,NB500–249), goat anti-MYC (Novus Biologicals, NB600–335), mouse anti-MYC (Santa Cruz Biotechnology, sc-40), mouse anti-MFN2 (Abcam, ab56889), mouse anti-OPA1 (BD Biosciences, BD612607), rabbit anti-VDAC (Cell Signaling Technology, 4661), mouse anti-TIMM23/TIM23 (BD Biosciences, BD611222), mouse anti-TOMM20/TOM20 (BD Biosciences, BD612278), mouse anti-COX4I1 (Abcam, ab14744), rabbit anti-PARK2 (Abcam, ab15954),rabbit anti-LC3B (Sigma-Aldrich, L7543), rabbit anti-cleaved PARP (Cell Signaling Technology, 95416), rabbit anti-CANX/calnexin (Sigma-Aldrich, C4731), rabbit anti-ACSL4/FACL4 (Abcam, ab137525), rabbit anti-HSPA9/GRP75 (Cell Signaling Technology, 3593), rabbit anti-GFP (Cell Signaling Technology, 2555) and mouse anti-TUBA/α-tubulin (Sigma-Aldrich, T9026).

Techniques: Marker

Journal: Autophagy

Article Title: PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation

doi: 10.1080/15548627.2016.1277309

Figure Lengend Snippet: PINK1 and BECN1 increase in the MAM fraction upon CCCP treatment. (A and B) co-IP analysis of PINK1-BECN1 interaction in cytoplasmic and crude mitochondrial fractions from SH-SY5Y cells, both in basal conditions and upon 25 µM CCCP treatment of 6 h. Cross-contaminations of cytoplasmic and mitochondrial-enriched fractions were assessed by evaluating TIMM23 and TUBA/tubulin as specific markers. (A) Endogenous BECN1 was immunoprecipitated using mouse anti-BECN1 antibody, followed by immunoblotting to detect co-immunoprecipitated endogenous PINK1 with rabbit anti PINK1. shBECN1 SH-SY5Y cells were used as control (CTR). (B) Overexpressed PINK1-HA was immunoprecipitated using mouse anti-HA antibody covalently attached to crosslinked agarose bead particles, and co-immunoprecipitated endogenous BECN1 was detected using mouse anti-BECN1. Preimmune IgG were used as negative control. (C-F) Confocal analysis of BECN1 and PINK1 colocalization with ER and mitochondrial markers upon treatment with 25 µM CCCP for 6 h. SH-SY5Y cells expressing BECN1-mCherry (left panels) or PINK1-mCherry (right panels) were co-transfected with ER-GFP (C and D) or stained with the mitochondrial marker TOMM20 (green) (E and F). Scale bar: 10 µm. (G) Histogram reporting Mander's overlap coefficients relative to BECN1 and PINK1 colocalization with ER and mitochondria (mean ± SD of n = 3, 10 cells per experiments). *p<0.05; **p<0.001. (H) Western blot of PINK1 and BECN1 distribution in Percoll-purified subcellular fractions isolated from SH-SY5Y cells exposed to DMSO or 25 µM CCCP for 6 h. Fractions were separated by SDS-PAGE and immunoblotted with specific antibodies for each fraction. VDAC1 and TUBA/tubulin were used as markers for pure mitochondrial and cytosolic fractions, respectively; CANX (calnexin) was considered as a marker of both ER and MAM, whereas ACSL4 was adopted as a MAM-specific marker. PNS, post-nuclear supernatant; Micro, microsome fraction; MitoP, pure mitochondria; MAM, mitochondria-associated membranes. Separated lines indicate that we joined together distant parts from the same gel.

Article Snippet: The following antibodies were used for western blotting, immunoprecipitation and immunofluorescence assays: rabbit anti-PINK1 (Novus Biologicals, BC100–494), mouse anti-HA (Sigma-Aldrich, H3663), rat anti-HA (Roche Applied Science, 10744700), mouse anti-BECN1 (BD Biosciences, BD612113), rabbit anti-BECN1 (Novus Biologicals,NB500–249), goat anti-MYC (Novus Biologicals, NB600–335), mouse anti-MYC (Santa Cruz Biotechnology, sc-40), mouse anti-MFN2 (Abcam, ab56889), mouse anti-OPA1 (BD Biosciences, BD612607), rabbit anti-VDAC (Cell Signaling Technology, 4661), mouse anti-TIMM23/TIM23 (BD Biosciences, BD611222), mouse anti-TOMM20/TOM20 (BD Biosciences, BD612278), mouse anti-COX4I1 (Abcam, ab14744), rabbit anti-PARK2 (Abcam, ab15954),rabbit anti-LC3B (Sigma-Aldrich, L7543), rabbit anti-cleaved PARP (Cell Signaling Technology, 95416), rabbit anti-CANX/calnexin (Sigma-Aldrich, C4731), rabbit anti-ACSL4/FACL4 (Abcam, ab137525), rabbit anti-HSPA9/GRP75 (Cell Signaling Technology, 3593), rabbit anti-GFP (Cell Signaling Technology, 2555) and mouse anti-TUBA/α-tubulin (Sigma-Aldrich, T9026).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Negative Control, Expressing, Transfection, Staining, Marker, Purification, Isolation, SDS Page