Stress-induced Cdk5 activity enhances cytoprotective basal autophagy in Drosophila melanogaster by phosphorylating acinus at serine437
- UT Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
Acn protein is stabilized by phosphorylation at Serine 437.
(A) Cartoon shows regulatory elements of Acn. Genomic Acn transgenes are Myc-tagged and expressed from the endogenous acnP promoter in the background of acn1 and acn27 null alleles. Green boxes …
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Figure 1—source data 1
Quantification of Acn proteins in Western blots.
- https://doi.org/10.7554/eLife.30760.005
Figure 1—figure supplement 1
AcnS437A and AcnS437D support normal Sevenless-mediated Boss endocytosis.
Micrographs of AcnWT (A), AcnS437A (B), or AcnS437D (C) larval eye discs stained for Boss and Acn. In eye discs that completely lack acn function, the Boss ligand cannot be detected in R7 cells (Habe…
Figure 2 with 1 supplement
Stabilized AcnS437D enhances basal autophagy.
(A–C) Micrographs of fed AcnWT, AcnS437A or AcnS437D larval eye discs stained for Atg8a. Scale bar: 40 µm in A-O. (D) Quantification of Atg8a punctae in eye discs from five larvae. (E–G, I–K, M–O) …
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Figure 2—source data 1
Relates to Figure 2D.
Quantification of Atg8a punctae in eye discs.
- https://doi.org/10.7554/eLife.30760.008
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Figure 2—source data 2
Relates to Figure 2H and L.
Quantification of integrated intensity of Atg8a punctae in fat bodies from fed and starved larvae with or without Chloroquine treatment.
- https://doi.org/10.7554/eLife.30760.009
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Figure 2—source data 3
Relates to Figure 2P.
Quantification of lysotracker punctae in fat bodies from fed and starved larvae.
- https://doi.org/10.7554/eLife.30760.010
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Figure 2—source data 4
Quantification of autolysosome and lysosome diameters and relative area.
- https://doi.org/10.7554/eLife.30760.011
Figure 2—figure supplement 1
Quantification of Atg8a punctae in fat bodies.
Quantification of Atg8a punctae in fed (A) or starved (B) fat bodies averaged from six to eight cells from four to five larvae from one representative experiment out of three repeats.
Figure 3
Stabilized AcnS437D enhances life span.
(A) Starvation-induced mortality of flies expressing the indicated Acn proteins. In parenthesis shown are numbers of initial flies in a single representative experiment out of three. (B) Survival …
Figure 4 with 2 supplements
Acn genetically interacts with p38b MAP kinase and Cdk5/p35.
SEM images of eye expressing the indicated transgenes under GMR-Gal4 control at 28°C. (A) Expression of UAS-AcnWT causes a rough eye. This eye-roughness is suppressed by knockdown of p38b MAPK (B) …
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Figure 4—source data 1
Relates to Figure 4—figure supplement 1.
Quantification of efficiency of RNAi-mediated Kinase knockdowns.
- https://doi.org/10.7554/eLife.30760.016
Figure 4—figure supplement 1
Knockdown efficiency of p38b MAPK, Cdk5 and p35 RNAi UAS-transgenes.
Indicated UAS-RNAi transgenes were expressed using Da-Gal4 or Arm-Gal4 drivers in adult flies. Transcript levels for the indicated genes were determined using RT-qPCR relative to the ribosomal …
Figure 4—figure supplement 2
Expression of dominant-negative mutant Cdk5 and p38b MAP kinases.
FLAG-tagged dominant negative mutant Cdk5 or p38b kinases as used in Figure 4 were expressed in eye discs under control of the GMR-Gal4 driver and detected with anti-FLAG antibodies. Scale bar: 50 µm.
Figure 5
Cdk5/p35-mediates phosphorylation of Acinus.
Projections (A, D, G–L) or individual optical sections (B,C,D,E) of confocal micrographs of eye discs stained for pS437-Acn or DNA from wild-type controls (A–C), or from larvae with knockdown for …
Figure 6
Acn-S437 is the critical target site for Cdk5/p35-mediated phosphorylation.
(A–H) SEM images of eyes expressing the indicated transgenes under UAS/GMR-Gal4 control at 25°. Under these conditions, expression of AcnWT (A) or AcnS437A (B) causes a mildly rough eye, but not …
Figure 7
Cdk5/p35-mediated phosphorylation of Acn regulates basal autophagy and longevity.
Projections of confocal micrographs of eye discs stained for Atg8a (A–E) or p62 (F–J) from wild-type controls (A,F), p35 mutant larvae (B,G) or p35 mutant larvae expressing AcnWT (C,H), AcnS437A (D,I…
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Figure 7—source data 1
Quantification of Atg8a and p62 punctae in eye discs.
- https://doi.org/10.7554/eLife.30760.020
Figure 8
Protein-aggregate-induced phosphorylation of Acn S437 depends on Cdk5/p35.
Projections of confocal micrographs of eye discs stained for pS437-Acn (A–E) or polyQ proteins (G–K) and DNA. Compared to a GMR-Gal4 control (A), GMR-Gal4-driven UAS-Htt.Q93 (B) or GMR-Htt.Q120 (D) …
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Figure 8—source data 1
Quantification of pS437 and polyQ-levels in eye discs.
- https://doi.org/10.7554/eLife.30760.022
Figure 9
Increased Acn-S437 phosphorylation is specific for a subset of neurodegeneration models.
Projections of confocal micrographs of eye discs stained for pS437-Acn (A–F) or DNA (A’–F’). Compared to GMR-Gal4 controls (A), eye discs with GMR-Gal4-driven UAS-SCA3.Q78 (B) or UAS-ATX1.Q82 (C) or …
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Figure 9—source data 1
Quantification of pS437-Acn in neurodegenerative model eye discs.
- https://doi.org/10.7554/eLife.30760.024
Additional files
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Supplementary file 1
Suppression of Acn-induced eye roughness by kinase knock down.
The effect on eye morphology of GMR-Gal4-driven expression of UAS-RNAi transgenes with or without UAS-Acinus was scored and recorded as the number of flies with normal eyes (score = 1), eye with mild errors (score = 2), rough eyes (score = 3) or severely rough eyes (score = 4). The percentage suppression or enhancement was calculated from the average score for each RNAi transgene compared to UAS-Acn in the absence of RNAi transgenes. Positive or negative numbers indicate suppression and enhancement, respectively. Green or red colors highlight UAS-transgenes with more than 50% suppression or enhancement. Numbers in parenthesis indicated stock numbers of the Bloomington Drosophila stock center or, if starting with a ‘V..', from the Vienna Drosophila Resource Center. All flies were raised at 28°C.
- https://doi.org/10.7554/eLife.30760.025
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Supplementary file 2
Genetic interactions of Acn with p38b and Cdk5/p35 loss and gain of function.
The effect on eye morphology of GMR-Gal4-driven expression of UAS-transgenes with or without UAS-Acinus was scored and recorded as the number of flies with normal eyes (score = 1), eye with mild errors (score = 2), rough eyes (score = 3) or severely rough eyes (score = 4). The percentage suppression or enhancement was calculated from the average score for each RNAi transgene compared to UAS-Acn in the absence of RNAi transgenes. Positive or negative numbers indicate suppression and enhancement, respectively. Green or red colors highlight UAS-transgenes with more than 50% suppression or enhancement. Numbers in parenthesis indicated stock numbers of the Bloomington Drosophila stock center. All flies were raised at 28°C.
- https://doi.org/10.7554/eLife.30760.026
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Supplementary file 3
Genotypes of flies used for each figure.
Relevant genotypes are listed for all Figures.
- https://doi.org/10.7554/eLife.30760.027
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Supplementary file 4
Key resources table.
- https://doi.org/10.7554/eLife.30760.028
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Transparent reporting form
- https://doi.org/10.7554/eLife.30760.029
