ATG5-knockout mutants of Physcomitrella provide a platform for analyzing the involvement of autophagy in senescence processes in plant cells

<p>Autophagy is a pathway in which a cell degrades part of its cytoplasm in vacuoles or lysosomes. To identify the physiological functions of autophagy in plants, we disrupted <i>ATG5</i>, an autophagy-related gene, in <i>Physcomitrella</i>, and confirmed that <i>atg5</i> mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, <i>atg5</i> colonies turned yellow earlier than the wild-type (WT) colonies, showing that <i>Physcomitrella atg5</i> mutants, like yeast and <i>Arabidopsis</i>, are sensitive to nutrient starvation. In the dark, even under nutrient-sufficient conditions, colonies turned yellow and the net degradation of chlorophyll and Rubisco protein occurred together with the upregulation of several senescence-associated genes. Yellowing reactions were inhibited by the protein synthesis inhibitor cycloheximide, suggesting that protonemal colonies undergo dark-induced senescence like the green leaves of higher plants. Such senescence responses in the dark occurred earlier in <i>atg5</i> colonies than WT colonies. The sugar content was almost the same between WT and <i>atg5</i> colonies, indicating that the early-senescence phenotype of <i>atg5</i> is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between <i>atg5</i> and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the <i>atg5</i> mutants, irrespective of the early degradation of Rubisco protein. On nutrient-sufficient medium supplemented with casamino acids, the early-senescence phenotype was slightly moderated. We propose that the early-senescence phenotype in <i>atg5</i> mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in <i>Physcomitrella</i>.</p

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H⁺-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

<p>Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.</p

Additional file 1 of ATM depletion induces proteasomal degradation of FANCD2 and sensitizes neuroblastoma cells to PARP inhibitors

Additional file 1: Supplementary Figure S1. Generation of CRISPR/Cas9-mediated ATM-depleted NB cells. Supplementary Figure S2. Phenotypic analysis of ATM heterozygous CHP-134 NB cells. Supplementary Figure S3. ATM is required for both ATM/Chk2/p53 and ATR/Chk1 pathway activation. Supplementary Figure S4. Combination treatment (ATMi KU-55933 + PARPi Olaparib), reversed resistance to PARPi in ATM haploinsufficient CHP-134 cells. Supplementary Figure S5. Loss of function in ATM suppresses tumorigenicity and sensitizes NB cells to PARPi. Supplementary Table S1. List of primer sequences used in this study. Supplementary Table S2. Targeting sequences of shRNAs against human ATM used in this study. Supplementary Table S3. List of antibodies used in this study