Figure 2.
Figure 2. Parkin is recruited by PINK1 to focal sites on mitochondria that harbor matrix-localized misfolded protein aggregates. (A) Tet-ON: WT OTC or ΔOTC-expressing HeLa cells were treated with DOX for 48 h and then fixed and stained with antibodies against OTC (red) and Tom20 (green). Bars: (left) 10 µm; (right) 2 µm. (B) Tet-ON: WT OTC or ΔOTC-expressing HeLa cells expressing YFP-Parkin (green) were treated with DOX for 48 h and then fixed and stained for OTC (red) and TOM20 (blue). The blue arrows indicate YFP-Parkin foci that colocalized with ΔOTC. Bars: (top and middle) 10 µm; (bottom) 5 µm. (C) Tet-ON: WT OTC or ΔOTC-expressing cells expressing YFP-Parkin (green) were treated with DOX for 48 h, labeled with MitoTracker deep red (red), and imaged live. Blue arrows indicate YFP-Parkin foci that localized on polarized mitochondria. Bar, 5 µm. (D) Tet-ON: ΔOTC-expressing cells expressing YFP-Parkin (green) were treated with DOX for 48 h, labeled with MitoTracker deep red (red), and imaged live. Bar, 2 µm. (E) HeLa cells expressing YFP-Parkin (green) were treated with actinonin for 4 h, labeled with MitoTracker CMXRos (red), and imaged live. Bar, 5 µm. (F) Tet-ON: ΔOTC-expressing HeLa cells expressing YFP-Parkin with or without a PINK1 KO background were treated with DOX for 48 h, fixed, and imaged. Blue arrows indicate YFP-Parkin foci that localized on mitochondrial subdomains. Bar, 10 µm. (G) Quantification of the percentage of cells with Parkin recruitment in Tet-ON: ΔOTC-expressing HeLa cells with or without a PINK1 KO background after treatment with DOX for 48 h. n = 3; N ≥ 400. ***, P < 0.001. The error bar indicates SD. (H) HeLa cells stably expressing YFP-Parkin with or without a PINK1 KO background expressing mito-RFP (red) were treated with vehicle (DMSO) or 150 μM actinonin for 6 h, fixed, and imaged. Bar, 10 µm. (I) PINK1 KO Tet ON: ΔOTC-expressing HeLa cells expressing PINK1-V5-His (green) and mito-RFP (red) with and without DOX treatment for 72 h were processed for immunostaining. Uninfected PINK1 KO cells were stained as a negative control. Bar, 10 µm.

Parkin is recruited by PINK1 to focal sites on mitochondria that harbor matrix-localized misfolded protein aggregates. (A) Tet-ON: WT OTC or ΔOTC-expressing HeLa cells were treated with DOX for 48 h and then fixed and stained with antibodies against OTC (red) and Tom20 (green). Bars: (left) 10 µm; (right) 2 µm. (B) Tet-ON: WT OTC or ΔOTC-expressing HeLa cells expressing YFP-Parkin (green) were treated with DOX for 48 h and then fixed and stained for OTC (red) and TOM20 (blue). The blue arrows indicate YFP-Parkin foci that colocalized with ΔOTC. Bars: (top and middle) 10 µm; (bottom) 5 µm. (C) Tet-ON: WT OTC or ΔOTC-expressing cells expressing YFP-Parkin (green) were treated with DOX for 48 h, labeled with MitoTracker deep red (red), and imaged live. Blue arrows indicate YFP-Parkin foci that localized on polarized mitochondria. Bar, 5 µm. (D) Tet-ON: ΔOTC-expressing cells expressing YFP-Parkin (green) were treated with DOX for 48 h, labeled with MitoTracker deep red (red), and imaged live. Bar, 2 µm. (E) HeLa cells expressing YFP-Parkin (green) were treated with actinonin for 4 h, labeled with MitoTracker CMXRos (red), and imaged live. Bar, 5 µm. (F) Tet-ON: ΔOTC-expressing HeLa cells expressing YFP-Parkin with or without a PINK1 KO background were treated with DOX for 48 h, fixed, and imaged. Blue arrows indicate YFP-Parkin foci that localized on mitochondrial subdomains. Bar, 10 µm. (G) Quantification of the percentage of cells with Parkin recruitment in Tet-ON: ΔOTC-expressing HeLa cells with or without a PINK1 KO background after treatment with DOX for 48 h. n = 3; N ≥ 400. ***, P < 0.001. The error bar indicates SD. (H) HeLa cells stably expressing YFP-Parkin with or without a PINK1 KO background expressing mito-RFP (red) were treated with vehicle (DMSO) or 150 μM actinonin for 6 h, fixed, and imaged. Bar, 10 µm. (I) PINK1 KO Tet ON: ΔOTC-expressing HeLa cells expressing PINK1-V5-His (green) and mito-RFP (red) with and without DOX treatment for 72 h were processed for immunostaining. Uninfected PINK1 KO cells were stained as a negative control. Bar, 10 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal