Open Access Highly Accessed Research article

Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila

Péter Lőw, Ágnes Varga, Karolina Pircs, Péter Nagy, Zsuzsanna Szatmári, Miklós Sass and Gábor Juhász*

  • * Corresponding author: Gábor Juhász szmrt@elte.hu

  • † Equal contributors

Author Affiliations

Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány P. s. 1/C, Budapest, H-1117, Hungary

For all author emails, please log on.

BMC Cell Biology 2013, 14:29  doi:10.1186/1471-2121-14-29

Published: 25 June 2013

Additional files

Additional file 1: Figure S1:

Aggregates of GFP-CL1 accumulate in proteasome RNAi cells. A-D) Expression of transgenic RNAi constructs in mosaic animals for Prosα1 (A), Prosα5 (B), Prosβ2 (C), and Rpt1 (D) results in accumulation of GFP-CL1 aggregates in larval fat body cells. Scale bar in A equals 20 μm for A-D.

Format: PDF Size: 112KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 2: Figure S2:

Aggregates of p62 and ubiquitinated proteins accumulate in proteasome RNAi cells. A-H) Knockdown of Prosα1 (A, B), Prosα5 (C, D), Prosβ2 (E, F), and Rpt1 (G, H) leads to the formation of large aggregates containing ubiquitinated proteins (A, C, E, G) and p62 (B, D, F, H). Boxed areas in A-H are shown enlarged. Scale bar in A equals 20 μm for A-H.

Format: PDF Size: 366KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 3: Figure S3:

Proteasome RNAi upregulates p62 transcription. Systemic depletion of Prosβ2 or Rpt1 leads to increased transcription of p62 relative to controls in RT-PCR experiments.

Format: PDF Size: 45KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 4: Figure S4:

Cell size decreases upon proteasome inactivation. Regression analysis reveals that GFP-CL1 level inversely changes with cell size in proteasome RNAi cells. Spearman’s correlation coefficient = -0.728, p < 0.001, R2 Linear = 0.332.

Format: PDF Size: 69KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 5: Figure S5:

Proteasome RNAi enhances starvation-induced autophagy. A-D) Knockdown of Prosα1 (A), Prosα5 (B), Prosβ2 (C), and Rpt1 (D) leads to increased punctate LTR staining in fat body cell clones of starved larvae compared to control non-GFP cells. Boxed areas in A-D are shown enlarged. Scale bar in A equals 20 μm for A-D.

Format: PDF Size: 185KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 6: Figure S:

Lamp1-GFP partially colocalizes with LTR. Colocalization of the reporter Lamp1-GFP that labels primary lysosomes, late endosomes and digesting lysosomes is not complete with LTR, a dye that stains acidic structures only. Boxed area is shown enlarged. Scale bar equals 20 μm.

Format: PDF Size: 151KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 7: Figure S7:

Proteasome RNAi induces autophagy in well-fed cells. A-H) Knockdown of Prosα1 (A, B), Prosα5 (C, D), Prosβ2 (E, F), and Rpt1 (G, H) induces the formation of LTR-positive autolysosomes in fat body cell clones (marked by Lamp1-GFP expression) compared to surrounding non-GFP control cells in well fed larvae (A, C, E, G), and also leads to increased generation of Atg8a-positive autophagosomes (B, D, F, H) in fat body cell clones of well fed larvae. Boxed areas in A-H are shown enlarged. Scale bar in A equals 20 μm for A-H.

Format: PDF Size: 368KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 8: Figure S8:

Punctate LTR staining is increased by proteasome inactivation. Regression analysis reveals that punctate LTR staining inversely changes with cell size in proteasome RNAi cells. R2 = 0.101, P < 0.001. The linearized equation of the curve is the following: ln(y) = ln(a) + b*ln(x), where a = -0.012 ± 0.002; p < 0.001 and b = 1.015 ± 0.002.

Format: PDF Size: 67KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 9: Figure S9:

Overexpressed Atg8a reporters are captured into p62 aggregates in proteasome RNAi cells. A-D) Overexpressed GFP-Atg8a is incorporated into large p62-positive aggregates in Prosα1 (A), Prosα5 (B), Prosβ2 (C), and Rpt1 (D) RNAi cells. Boxed areas in A-D are shown enlarged. Scale bar in A equals 20 μm for A-D.

Format: PDF Size: 186KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 10: Figure S10:

Autophagic flux is enhanced upon genetic inactivation of the proteasome. A-H) Knockdown of Prosα1 (A, B), Prosα5 (C, D), Prosβ2 (E, F), and Rpt1 (G, H) cells expressing the tandemly tagged mCherry-GFP-Atg8a reporter induces the formation of mCherry-labeled autolysosomes in fat body cell clones in well fed larvae (A, C, E, G). The lysosome inhibitor chloroquine blocks autophagy-dependent quenching of GFP, as now most puncta are positive for both mCherry and GFP (B, D, F, H). Boxed areas in A-H are shown enlarged. Scale bar in A equals 20 μm for A-H.

Format: PDF Size: 366KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 11: Figure S11:

Protein aggregates and autophagic structures form in fat body cells undergoing Rpn2 RNAi. Depletion of Rpn2 in whole fat bodies (mediated by the cg-Gal4 driver) results in the formation of protein aggregates (agg), double-membrane autophagosomes (arrow) and digesting autolysosomes (arrowhead) in cells. Scale bars equal 1 μm.

Format: PDF Size: 2.9MB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 12: Figure S12:

Accumulation of p62 aggregates is increased by proteasome inactivation. Regression analysis reveals that accumulation of p62 aggregates inversely changes with cell size in proteasome RNAi cells. Spearman’s correlation coefficient = -0.806, P < 0.001, R2 Linear = 0.412.

Format: PDF Size: 53KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 13: Figure S13:

LTR-positive autolysosomes do not colocalize with p62 and Atg8a reporters. A, B) p62-GFP (A) and GFP-Atg8a (B) display practically no colocalization with the lysosome marker LTR. Boxed areas in A and B are shown enlarged. Scale bar in A equals 20 μm for A and B.

Format: PDF Size: 252KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 14: Figure S14:

Caspases are not activated in proteasome or Vhl RNAi or sima overexpressing cells. A-H) No active caspase 3 immunoreactivity is detected in Prosα1 (A), Prosα5 (B), Prosβ2 (C), Rpt1 (D) and Rpn2 (E) RNAi fat body cells. (F) Similarly, no active caspase 3 immunolabeling is detected in Rpt1 RNAi cells (marked by Lamp1-GFP) in brains. Note that several control cells positive for active caspase 3 (arrowheads) are seen in this panel. Similarly, overexpression of sima (G) or depletion of Vhl (H) does not lead to activation of caspase 3 in fat body cells either. Boxed areas in A-H are shown enlarged. Scale bar in A equals 20 μm for A-H.

Format: PDF Size: 355KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 15: Figure S15:

The hypoxia reporter LDH-GFP is activated in proteasome or Vhl RNAi animals. Systemic depletion of Prosβ2, Rpn2, Rpt1 or Vhl all lead to a similar upregulation of the transcriptional hypoxia reporter transgene LDH-GFP, compared to control larvae. Per cent values refer to GFP fluorescence.

Format: PDF Size: 84KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 16: Figure S16:

List of Drosophila stocks used in this study.

Format: PDF Size: 278KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 17: Figure S17:

Original images for gels and western blots. Black boxes highlight cropped regions, which are shown in image panels as indicated.

Format: PDF Size: 207KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data