Fig. 5

Non-disassembled mitoaggregates harbored reduced autophagosome generation and displayed delayed turnover. a Diagram illustrating the principle underlying photoclustering experiments. After 635 nm illumination, CRY2-Ub become conjugated to damaged mitochondria through Parkin catalyzed ubiquitination. 488 nm illumination drives CRY2 oligomerization, which helps to maintain the clustering of damaged mitochondria and delays disassembly. For clarity, representative CRY2-Ub were drawn. b A MitoTracker DeepRed FM-stained HeLa cell co-expressing EBFP2-Parkin, CRY2-EGFP-Ub, and TPC2-TagRFP was illuminated with 635 nm to locally activate Parkin-mediated mitophagy. Selected frames of mitoaggregates of interest 40–340 min after mitophagy initiation are shown. During mitophagy, a portion of the mitoaggregates were 488-nm illuminated (as indicated by white dotted circles) at both 80 and 140 min. c The progression of mitophagy in b 340 min after mitochondrial damage. EGFP-quenched and TPC2-positive Parkin-labeled mitochondria represents mature autophagolysosomes (yellow arrows). EGFP-fluorescence retaining and TPC2-negative Parkin-labeled mitochondria represents ubiquitinated mitochondria outside of lysosomes (blue arrows). Photoclustered mitoaggregates are outlined with a blue-dotted line. d Diagram illustrating the experimental flow of photoclustering experiments in single cells. e A MitoTracker DeepRed FM-stained HeLa cell co-expressing EBFP2-Parkin, CRY2-TagRFP-Ub, and hrGFP-BATS was illuminated with 635 nm to activate Parkin-mediated mitophagy at two separate locations (white circles) within a single cell. f Selected frames from a time-lapse movie (Supplementary Movie 7) on cellular region containing mitoaggregates in e 0–366 min after 635 nm illumination. 488 nm mediated clustering (green dotted circles) were triggered at both 77 and 154 min. The red arrows denote BATS-positive structures on the surfaces of photoclustered mitoaggregates. g Total number of BATS puncta in non 488-nm illuminated and 488-nm illuminated regions as shown in f. Scale bar: 10 μm