Extended Data Figure 10. OPTN and NDP52 rescue DFCP1 and ULK1 recruitment deficit in pentaKOs.
a, Representative images of pentaKOs expressing mCherry-Parkin (mCh-Parkin), GFP-DFCP1 and the indicated FLAG/HA-tagged autophagy receptors immunostained for HA (n=2). Right-hand panels display co-localization of FLAG/HA-tagged constructs and GFP-DFCP1 by fluorescence intensity line measurement. b, Representative images of pentaKOs expressing mCherry-Parkin and GFP-ULK1 were rescued with FLAG/HA-OPTN, FLAG/HA-NDP52, and FLAG/HA-p62, and immunostained for HA and GFP. Arrows indicate HA-tagged receptor puncta (n=2). Right panels display colocalization of HA and GFP by fluorescence intensity line measurement. c, d, Representative images of pentaKOs stably expressing FRB-Fis1 and transiently expressing PINK1Δ110-YFP-2xFKBP and vector or myc-tagged receptors, were (c) untreated or (d) treated with rapalog and imaged live (n=3, see Figure 4h, i for quantification of c, d). OA, Oligomycin and Antimycin A. Scale bars, 10 μm. e, Old and new models of PINK1/Parkin mitophagy. The old model is dominated by Parkin ubiquitination of mitochondrial proteins. Here PINK1 plays a small initiator role whose main function is to bring Parkin to the mitochondria. The new model depicts Parkin-dependent and independent pathways leading to robust and low-level mitophagy, respectively. Based on our data, PINK1 is central to mitophagy both before and after Parkin recruitment by phosphorylating UB to recruit both Parkin and autophagy receptors mitochondria, to induce clearance. In the absence of Parkin (right panel), this occurs at a low level due to the relatively low basal UB on mitochondria. When Parkin is present it serves to amplify the PINK1 generated UB-PO4 signal, allowing for robust and rapid mitophagy induction.