Journal: Cells

Article Title: Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum

doi: 10.3390/cells9051179

Figure Lengend Snippet: Schematic depiction of the components and their interrelations of the two ubiquitin-like conjugation systems in autophagy. The ATG12 (left) and the ATG8 (LC3 in mammals) (right) conjugation systems are represented. Similar to protein ubiquitination, the ubiquitin-like protein ATG12 is activated by the E1 enzyme ATG7 and transferred to the E2 enzyme ATG10. Finally, ATG12 is covalently attached to its target protein ATG5 and two ATG12~5 conjugates in turn associate non-covalently with an ATG16 dimer and form a heterohexameric complex. Likewise, the ubiquitin-like protein ATG8 is also activated by ATG7, transferred to the E2 enzyme ATG3, and finally conjugated to PE via an amide bond. The ATG12~5/16 complex catalyses via its E3-like activity the conjugation of ATG8 to PE at the isolation membrane. The different components of the two conjugation systems are not drawn to scale. IM-PE, isolation membrane containing phosphatidylethanolamine. Modified from . See text for further details.

Article Snippet: To analyse the ATG5 domain structure, InterPro version 72.0 [ ] was used after excluding the identified asparagine-rich regions.

Techniques: Ubiquitin Proteomics, Conjugation Assay, Activity Assay, Isolation, Membrane, Modification

Journal: Cells

Article Title: Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum

doi: 10.3390/cells9051179

Figure Lengend Snippet: D. discoideum strains used in this study.

Article Snippet: To analyse the ATG5 domain structure, InterPro version 72.0 [ ] was used after excluding the identified asparagine-rich regions.

Techniques: Mutagenesis

Journal: Cells

Article Title: Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum

doi: 10.3390/cells9051179

Figure Lengend Snippet: ATG5 domain composition, 3D structure and multiple sequence alignment of the helix-rich domain (HRD). ( A ) Schematic representation of the predicted ATG5 domains. Two ubiquitin-like domains (UblDs) flank the HRD, which contains Lys187 (green line), that forms the covalent bond with the C-terminal glycine of ATG12. Both UblDs are interrupted by N-rich, low-complexity regions. The domains and regions were predicted by InterPro and SMART , respectively. ( B ) Predicted 3D structure of the D. discoideum ATG12~5/16N complex as ribbon diagram by homology modelling using the human complex as template (PDB ID: 4gdk) . ATG12 is shown in yellow, the ATG16N in rose, and for ATG5 the same colors as in ( A ) were used, to emphasise the domains and regions. The amino acid side chain of ATG5 Lys187 is shown in green. ( C ) Multiple sequence alignment of the conserved HRD of ATG5 orthologues from different organisms. The multiple sequence alignment was performed with Clustal Omega . Amino acid residues are numbered on the right and sequence similarity is indicated by shading. Dark grey represents identical amino acid residues, medium grey highlights amino acids with very similar properties (roughly equivalent to > 0.5 scoring in the Gonnet PAM 250 matrix) and light grey amino acids with slightly similar properties (roughly equivalent to scoring ≤ 0.5 and > 0 in the Gonnet PAM 250 matrix). Sequence conservation is indicated below the alignment by bar sizes . Alpha helices as predicted in the homology model for D. discoideum ATG5 in ( B ) are marked with α above the alignment. The complete sequence alignment of ATG5 orthologs is shown in .

Article Snippet: To analyse the ATG5 domain structure, InterPro version 72.0 [ ] was used after excluding the identified asparagine-rich regions.

Techniques: Sequencing, Ubiquitin Proteomics

Journal: Cells

Article Title: Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum

doi: 10.3390/cells9051179

Figure Lengend Snippet: Verification of the different mutant strains by immunoblotting and immunofluorescence analysis of ATG8a-positive autophagosomes. ( A ) Immunoblotting of total cell lysates of wild-type AX2, ATG5¯, ATG12¯, ATG5¯/12¯, ATG16¯, ATG12¯/16¯, and ATG5¯/12¯/16¯ cells. The ATG12~5 conjugate was detected at about 68 kDa in AX2 and ATG16¯ cell lysates but not in atg5 and atg12 knock-out strains. Unconjugated ATG5 of about 46 kDa was detected in ATG12¯ and ATG12¯/16¯ cells. No unconjugated ATG12 of about 14 kDa was detectable. Actin was used as a loading control. ATG5, ATG12 and actin were visualised on the same membrane. Top row, ATG5 pAb; middle row, ATG12 mAb; bottom row, actin mAb. ( B ) Immunofluorescence microscopy of AX2, ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells. Cells were fixed with cold methanol and stained with the ATG8a pAb. Puncta representing ATG8a-positive autophagosomes (arrows) were only detected in AX2 wild-type cells. Cell boundaries in the enlarged insets are indicated by dotted lines. Nuclei were visualised by DAPI staining. Scale bar, 5 µm.

Article Snippet: To analyse the ATG5 domain structure, InterPro version 72.0 [ ] was used after excluding the identified asparagine-rich regions.

Techniques: Mutagenesis, Western Blot, Immunofluorescence, Knock-Out, Control, Membrane, Microscopy, Staining

Journal: Cells

Article Title: Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum

doi: 10.3390/cells9051179

Figure Lengend Snippet: Development and cell survival upon nitrogen starvation of AX2 and mutant strains. ( A ) Development of AX2, ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells on phosphate agar plates. 5 × 10 7 cells of each strain were plated homogeneously. On the left, the tipped mound stage for AX2 after 15 h, and for mutant cells after 30 h (top view), and on the right an exemplary fruiting body of AX2 after 24 h, and of mutant cells after 48 h (side view), is shown. The white dashed line encircles tipped mounds. Scale bars, 100 µm. Note the different scale for mutant fruiting bodies. ( B ) Cell survival of AX2, ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells upon nitrogen starvation. AX2 and mutant strains were grown in SIH medium without amino acids, and cell survival was determined every 24 h for 5 days. Relative cell survival after 24 h was set to 1 for each strain. Cell survival of mutant cells was significantly reduced in comparison to AX2 after 96 and 120 h. Mean values and standard errors of the mean of three independent experiments are shown. For statistical analysis, one-way ANOVA and Tukey’s test as post hoc analysis were used. *, significant ( p -value < 0.05); **, very significant ( p -value < 0.01).

Article Snippet: To analyse the ATG5 domain structure, InterPro version 72.0 [ ] was used after excluding the identified asparagine-rich regions.

Techniques: Mutagenesis, Comparison

Journal: Cells

Article Title: Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum

doi: 10.3390/cells9051179

Figure Lengend Snippet: Analysis of cell growth and macropinocytosis in AX2 and mutant strains. ( A ) Generation time of AX2, ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells in shaking culture. Cell titres of three parallel shaking cultures were determined every 24 h. Mean values and SEM of three (ATG5¯/12¯/16¯), four (AX2 and ATG5¯/12¯) and seven (ATG5¯) independent experiments are shown. For statistical analysis, one-way ANOVA and Tukey’s test as post hoc analysis were used. ( B ) Macropinocytosis of TRITC-labelled dextran. AX2, ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells were adjusted to 6 × 10 6 cells/mL and co-incubated with TRITC-labelled dextran. Intracellular fluorescence was determined at t0 and after 15, 30, 60, 90, and 120 min. The final fluorescence of AX2 was set to 1. Color assignment of strains is as in panel A. Mean values and SEM of three independent experiments are shown. For statistical analysis, two-way ANOVA and Tukey’s test as post hoc analysis were used. *, significant ( p -value < 0.05); **, very significant ( p -value < 0.01); ***, highly significant ( p -value < 0.001).

Article Snippet: To analyse the ATG5 domain structure, InterPro version 72.0 [ ] was used after excluding the identified asparagine-rich regions.

Techniques: Mutagenesis, Incubation, Fluorescence

Journal: Cells

Article Title: Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum

doi: 10.3390/cells9051179

Figure Lengend Snippet: Phagocytosis of yeast and bacteria by AX2 and mutant strains. ( A ) Phagocytosis of TRITC-labelled yeast. AX2, ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells were adjusted to 6 × 10 6 cells/mL and co-incubated with a six-fold excess of TRITC-labelled yeast for 180 min. Color assignment of strains is as in panel C. Analysis was done as for macropinocytosis (see B). ( B ) Growth on K. aerogenes . Representative images of plaques formed by AX2, ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells after 96 h growth on a lawn of K. aerogenes. Scale bar is 1 mm. ( C ) Quantitation of plaque growth for AX2, ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells. Three days after plating the cells, plaque diameters were determined every 24 h for five days and the average growth per 24 h was calculated. Mean values and SEM of three (ATG5¯/12¯ and ATG5¯/12¯/16¯), four (AX2) and seven (ATG5¯) independent experiments are shown. For statistical analysis, one-way ANOVA and Tukey’s test as post hoc analysis were used. ( D ) Representative images of phagocytosis of E. coli BioParticles by AX2, ATG5¯, ATG5¯/12¯ and ATG5¯/12¯/16¯ cells. Cells were co-incubated with fluorescently labelled E. coli for 30 min. After fixation of cells, fluorescence and phase contrast microscopy was performed. Some of the membrane bound E. coli are marked by arrows. Nuclei were stained with DAPI and cell outlines are indicated by white dashed lines. Scale bar, 5 µm. ( E ) and ( F ) Quantitation of phagocytosed and membrane bound E. coli . The amount of E. coli BioParticles within the cells ( E ) and at the cell membrane ( F ) was determined for 100 cells of each strain in each of three independent experiments. Every data point represents a single cell. Additionally, the median and interquartile range are shown. Color assignment of strains is as in panel C. For statistical analysis, Kruskal–Wallis test and the Dunn–Bonferroni test were used as post hoc analysis. *, significant ( p -value < 0.05); **, very significant ( p -value < 0.01); ***, highly significant ( p -value < 0.001).

Article Snippet: To analyse the ATG5 domain structure, InterPro version 72.0 [ ] was used after excluding the identified asparagine-rich regions.

Techniques: Bacteria, Mutagenesis, Incubation, Quantitation Assay, Fluorescence, Microscopy, Membrane, Staining

Journal: Cells

Article Title: Functional Characterisation of the Autophagy ATG12~5/16 Complex in Dictyostelium discoideum

doi: 10.3390/cells9051179

Figure Lengend Snippet: Protein homeostasis is disturbed. ( A ) Quantification of global protein ubiquitination of AX2, ATG5¯, ATG5¯/12¯, and ATG5¯/12¯/16¯ cells. Western blots of total cell lysates were stained with mAb P4D1 and signal intensities of ubiquitinated proteins were quantitated and normalised with the actin signal. The signal intensity of AX2 was set to 1. Mean values and SEM of five independent experiments are shown. ( B ) Immunofluorescence microscopy of AX2, ATG5¯, ATG5¯/12¯, and ATG5¯/12¯/16¯ cells. Cells were fixed with cold methanol and stained with the mAb P4D1. Ubiquitin-positive protein aggregates in mutant strains are marked by arrows. Nuclei were visualised by DAPI staining. Scale bar, 5 µm. ( C ) Proteasomal activity of AX2, ATG5¯, ATG5¯/12¯, ATG5¯/12¯/16¯, and ATG16¯ cells. The assay was performed as described . The chymotrypsin-like activity of AX2 was set to 1. Color assignment of strains is as in panel D. Mean values and SEM of five independent experiments are shown. ( D ) Quantification of the amount of the proteasomal subunit A7 (psmA7) in AX2, ATG5¯, ATG5¯/12¯, ATG5¯/12¯/16¯, and ATG16¯ cells. The psmA7 signal intensities were quantitated after Western blotting and normalised with the actin signal. The signal intensity of AX2 was set to 1. Mean values and SEM of five independent experiments are shown. For all statistical analyses, two-way ANOVA and Tukey’s test were used as post hoc analysis. n.s., not significant; ***, highly significant ( p -value < 0.001).

Article Snippet: To analyse the ATG5 domain structure, InterPro version 72.0 [ ] was used after excluding the identified asparagine-rich regions.

Techniques: Ubiquitin Proteomics, Western Blot, Staining, Immunofluorescence, Microscopy, Mutagenesis, Activity Assay