Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum
Abstract
:1. Introduction
2. Materials and Methods
2.1. Dictyostelium Strains, Growth and Development
2.2. Primary Antibodies
2.3. SDS-PAGE and Western Blotting
2.4. Macropinocytosis and Phagocytosis Assays
2.5. Random Cell Motility Analysis
2.6. Proteasomal Activity Analysis
2.7. Fluorescence Microscopy
2.8. Bioinformatics and RNAseq
2.9. Statistics and Reproducibility
3. Results
3.1. D. discoideum ATG5 Is highly Conserved
3.2. Cellular Processes Dependent on Canonical Autophagy Are Similarly Impaired in the Different atg5 Knock-Out Strains
3.3. Cell Growth, Macropinocytosis and Phagocytosis Are Severely Affected in Strains Lacking ATG5
3.4. Protein Homeostasis Is Severely Disturbed in Mutant Strains
4. Discussion
4.1. ATG5 Is Required for Conjugation of ATG8 to the Autophagosomal Membrane and for ATG12 Stability
4.2. The Dictyostelium ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ Mutants Have Complex and Distinct Phenotypes
4.3. Protein Homeostasis Is Severely Impaired in ATG5 Deficient Strains
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Strains | Summary | References |
---|---|---|
AX2 | Axenically growing derivate of wild isolate NC-4 | [46] |
ATG5¯ | ATG5 null mutant | This work |
ATG5¯/12¯ | ATG5/12 double null mutant | This work |
ATG5¯/12¯/16¯ | ATG5/12/16 triple null mutant | This work |
ATG12¯ | ATG12 null mutant | [39] |
ATG16¯ | ATG16 null mutant | [40] |
ATG12¯/16¯ | ATG12/16 double null mutant | [39] |
Type | Cellular Process | Strains | ||
---|---|---|---|---|
ATG5¯ | ATG5¯/12¯ | ATG5¯/12¯/16¯ | ||
(i) | Development | − | − | − |
Cell viability | − | − | − | |
Conjugation of ATG8 to PE | − | − | − | |
(ii) | Generation time | − | −− | −−− |
Macropinocytosis | − | − | −− | |
Phagocytosis of yeast | − | − | −− | |
(iii) | Growth on K. aerogenes | ++ | − | + |
Phagocytosis/membrane association of bacteria | ++ | − | + | |
(iv) | Proteasomal activity | −− | −− | − |
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Karow, M.; Fischer, S.; Meßling, S.; Konertz, R.; Riehl, J.; Xiong, Q.; Rijal, R.; Wagle, P.; S. Clemen, C.; Eichinger, L. Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum. Cells 2020, 9, 1179. https://doi.org/10.3390/cells9051179
Karow M, Fischer S, Meßling S, Konertz R, Riehl J, Xiong Q, Rijal R, Wagle P, S. Clemen C, Eichinger L. Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum. Cells. 2020; 9(5):1179. https://doi.org/10.3390/cells9051179
Chicago/Turabian StyleKarow, Malte, Sarah Fischer, Susanne Meßling, Roman Konertz, Jana Riehl, Qiuhong Xiong, Ramesh Rijal, Prerana Wagle, Christoph S. Clemen, and Ludwig Eichinger. 2020. "Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum" Cells 9, no. 5: 1179. https://doi.org/10.3390/cells9051179