ULK1-ATG13 and their mitotic phospho-regulation by CDK1 connect autophagy to cell cycle
Fig 6
ULK1-ATG13 phosphorylation in mitosis promotes mitotic autophagy.
(A) The localization of mutant ULK1-11A and ATG13-4A in mitosis. Cells released from thymidine block for 10 hours were fixed with 3.7% formaldehyde for 20 minutes at room temperature and then for blocking and FLAG antibody, Alexa-488 conjugated secondary antibody, DAPI staining. Scale bar, 10 μm. (B) The ULK1 kinase activity is not affected by the 11A mutation. ATG13-Ser355 is the substrate of ULK1, which corresponds to Ser318 of ATG13 isoform 2. ULK1-KO 293T cells reconstituted with ULK1-WT, ULK1-11A, or ULK1-K46I were plated and lysed with M-PER. The western blot for ATG13-Ser355 indicated that ULK1 activity was not affected by 11A mutation. n = 3. (C) Autophagy activity was examined in ULK1&ATG13 double WT or mutant (“Mut”) cells expressing GFP-LC3-RFP by flow cytometry. ULK1&ATG13 double WT or mutant cells, HeLa, or HeLa- ULK1&ATG13 DKO cells stably expressing GFP-LC3-RFP were established by infection of retrovirus packaged by pMRX-IP-GFP-LC3-RFP and helper plasmids Vsvg and pMLV. Mitotic cells were collected by shake-off from thymidine and nocodazole synchronized cells for flow cytometry detection. The GFP/RFP ratio inversely correlated with autophagy activity. n = 4, ***p < 0.001, ****p < 0.0001. (D) The autophagic flux of HeLa-DKO cells stably overexpressing double WT or mutant FLAG-tagged mULK1 and ATG13. Cells synchronized to mitosis with thymidine and nocodazole were shaken off and treated with or without autophagy inhibitor 25 μM CQ for 1 hour. The left panel is a representative western blot, and the right panel is the statistical result for LC3B-II and p62. n = 3, *p < 0.05, **p < 0.01. (E) LC3B puncta number was counted, and the micrographs were captured by Zeiss LSM710 confocal microscope. Scale bar, 10 μm. n = 43, **p < 0.01. Numerical data underlying the figure panels are available in S1 Data. 4A, T342/T332/S44/S224A; 11A, S622&T635&T653&S479&S543&S413&T401&S403&S405&T282&T502A; ATG, autophagy-related; CQ, chloroquine; Ctrl, control; DAPI, 4 DAPdiamidino-2-phenylindole; DKO, double knockout; GFP, green fluorescent protein; KO, knockout; M-PER, Mammalian Protein Extraction Reagent; mULK1, mouse ULK1; n.s., not significant; RFP, red fluorescent protein; ULK1, unc-51-like autophagy activating kinase 1; WT, wild type.