Fig. 2: Decreased mitochondrial protein import induces mitophagy without requiring depolarization. | Nature Communications

Fig. 2: Decreased mitochondrial protein import induces mitophagy without requiring depolarization.

From: Protein import motor complex reacts to mitochondrial misfolding by reducing protein import and activating mitophagy

Fig. 2

a MTS-EGFP-inducible HeLa FlpIn cells (PARKIN-expressing) were treated with HSPA9 siRNA for 96 h. Doxycycline (dox) added 15 h before imaging. Mitochondrial localization of EGFP was assessed by Mitotracker Deep Red FM staining and microscopy. Scale bar 25 µm. b Co-localization image analysis of a. 100 EGFP-positive cells examined over n = 3 biological replicates. Minimum-maximum values and mean as central line shown. c HeLa FlpIn TRex mt-mKEIMA wild type (WT) or PINK1 KO cells (PARKIN-expressing) were treated with HSPA9 siRNA for 96 h. Cells showing increased 561 nm/405 nm mt-mKEIMA ratios were considered mitophagy-positive. Mean ±s.d. for n = 3 shown. d Mitochondrial membrane potential measurement of HeLa FlpIn TRex PARKIN cells with HSPA9 siRNA for 96 h, dox for last 15 h. Cells were stained with TMRE and categorized by gating in TMRE-positive, as untreated or TMRE-negative as depolarized, CCCP treated cells. >5000 fluorescent cells per sample analyzed. Mean ±s.d. for n = 3 shown. e Schematic overview depicting the effects of HSPA9 knock-down on mitochondrial import, membrane potential and mitophagy induction (from a–d). f Monitoring mitochondrial protein import by pulsed-SILAC proteomics of extracted mitochondria after treatment with 10 µM oligomycin (oligo) or 1 mM deferiprone (DFP) compared to DMSO. Cell line as d. SILAC pulse for last 2 h applied. SILAC labeled peptides included in MitoCarta 2.0 used for protein import quantification. Workflow depicted in Supplementary Fig. 2g. Minimum-maximum values, mean as central line for n = 3 shown. g, h Assessment of mitophagy flux by mt-mKEIMA (g) or membrane potential by TMRE (h) in cells treated with DMSO, oligomycin or DFP. As depolarization control CCCP treatment for 2 h included. Mean ±s.d. for n = 3 shown. i Schematic illustration of oligomycin and DFP effects on mitophagy, protein import and membrane potential. For all experiments, two-sided unpaired t test method was performed.

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