Fig. 5: p62 bodies regulate autophagosome formation by a spatially concentrated PI3K kinase subunit.

a TIRF images showing that droplets formed by LLPS of mCherry-p62 (50 μM), His-Ub8 (15 μM), PI3KC3-C1 (GFP-Becllin1) and GFP-ULK1 (1 μg/μl) fused upon contact in vitro. Scale bar, 5 μm. b Line profiling of a representative section of the p62 droplets, indicated by the white lines in panels (1)–(3) from (a). c Schematic diagram of the sedimentation assay to separate the condensed liquid droplets and the supernatant. In vitro phase separation assay of mCherry-p62 with linear His-Ub8. ULK1, PI3KC3-C1 and ATP were added after the phase separation. mCherry-p62, 50 µM; His-Ubx8: 15 µM; ULK1, (1 μg/μl); PI3KC3-C1, (1 μg/μl); ATP, 10 µM. d The sedimentation and supernatant were separated by centrifugation and analyzed by western blot using antibodies against p-Beclin1, Beclin1, p-ATG14, ATG14 and ULK1. S: supernatant, P: pellet. e The ratio of p-Beclin1 or p-ATG14 to total Beclin1 or ATG14 were analyzed from (d), Data are presented as mean ± SD, n = 3 times measurement of band intensity, p values were calculated using the two-tailed, unpaired t-test, ****p < 0.0001. f In vitro phase separation assay of GFP-p62 with linear His-Ub8, and then PI3KC3-C1 and ATP were added up to the phase separation system. Liposome and mCherry-FYVE were finally added into the above phase separation system. GFP-p62, 50 µM; His-Ub8: 15 µM; PI3KC3-C1, (1 μg/μl); ATP, 10 µM; Liposome, 1 µM; mCherry-FYVE, 1 µM. g Liposomes (PE-NDB) and mCherry-FYVE(X2) around the phase separated p62 bodies were observed from (c). Scale bar, 1 µm. h The number of mCherry-FYVE puncta colocalized with p62 droplet was quantified. Data are presented as mean ± SD, n = 3 independent experiments. w/o means without. p values were calculated using the two-tailed, unpaired t-test. ****p < 0.0001.