Acinus integrates AKT1 and subapoptotic caspase activities to regulate basal autophagy

Abstract

How cellular stresses up-regulate autophagy is not fully understood. One potential regulator is the Drosophila melanogaster protein Acinus (Acn), which is necessary for autophagy induction and triggers excess autophagy when overexpressed. We show that cell type-specific regulation of Acn depends on proteolysis by the caspase Dcp-1. Basal Dcp-1 activity in developing photoreceptors is sufficient for this cleavage without a need for apoptosis to elevate caspase activity. On the other hand, Acn was stabilized by loss of Dcp-1 function or by the presence of a mutation in Acn that eliminates its conserved caspase cleavage site. Acn stability also was regulated by AKT1-mediated phosphorylation. Flies that expressed stabilized forms of Acn, either the phosphomimetic Acn(S641,731D) or the caspase-resistant Acn(D527A), exhibited enhanced basal autophagy. Physiologically, these flies showed improvements in processes known to be autophagy dependent, including increased starvation resistance, reduced Huntingtin-induced neurodegeneration, and prolonged life span. These data indicate that AKT1 and caspase-dependent regulation of Acn stability adjusts basal autophagy levels.

Figure 1.

Acn is regulated on the protein level. (A) Schematic of acn locus and genomic transgenes all inserted at 96F3 for this study. RSB is the conserved RNPS1 and SAP18 binding site (Martelli et al., 2012). (B–E″) Projections of confocal sections of eye discs showing early stages of photoreceptor development. Regions of eye discs shown are just posterior to the furrow as in the white frame of Fig. 2 D. Images show wild-type discs stained for endogenous Acn (B) and DNA (B′) or discs with indicated transgenes stained for GFP (C and D) and DNA (C′ and D′) or for Myc (E) and GFP (E′). Arrows in E–E″ point to cells expressing GFP-KDEL but lacking Myc-Acn. Merged images are to the right. Bars, 20 µm. Detailed genotypes are given in Table S2.

Figure 2.

Acn is cleaved and inactivated at aspartate D527. (A) Conserved aspartate D527 of Drosophila melanogaster (Dm) Acn is shown in alignment with Drosophila yakuba (Dy), Anopheles gambiae (Ag), Xenopus tropicalis (Xt), mouse (Mus musculus [Mm]), and human (Homo sapiens [Hs]) proteins. Color code for amino acid groups: brown for aromatic, white for hydrophobic, salmon for hydroxyls, red for acidic, blue for basic, cyan for amido, and yellow for sulfur. (B) Western blot of lysates from larvae probed for Acn and Actin. (C) Quantification of blots for Acn levels relative to Actin (n = 3). Error bars show means ± SD. *, P < 0.05; ***, P < 0.001. (D) Micrograph of eye disc stained for DNA (magenta) and Acn (green). The frame just posterior to the furrow (arrow) indicates the region shown in high magnification images (E and F). (D′) Micrograph of an eye disc expressing nuclear GFP. Dotted lines encircle R3/4 and R2/5/8 photoreceptors just posterior to the furrow. (E–F″) Dynamic changes in Acn expression in photoreceptors visualized in projections of confocal sections of eye discs from acnP-Myc-Acn^wt (E) and acnP-Myc-Acn^D527A (F) larvae double labeled for Myc and C-terminal Acn epitopes of Myc-Acn (merged images are shown in E″ and F″). Arrows point to R3/4 photoreceptor pairs just posterior to the furrow. Bars, 10 µm. (G and H) Quantification of Acn and Myc levels in early R3/4 and R2/5/8 cells measured from ≥180 ommatidia from four eye discs, each for Acn^wt and Acn^D527A. (G) Cumulative frequency histograms of normalized fluorescence intensities for Myc and Acn immunoreactivity. (H) Intensity blots of normalized integrated intensities of Acn and Myc immunoreactivity for individual groups of R3/4 and R2/5/8 cells in Myc-Acn^wt and Myc-Acn^D527A discs and calculated Pearson correlation coefficients r. fluor. int., fluorescence intensities. (I–N) SEM images of wild-type control (I) and eyes expressing under GMR-Gal4 control the indicated UAS transgenes: none (J), UAS-Acn^wt (K), UAS-Acn^D527A (L), UAS-Acn^1–527 (M), and UAS-Acn^528–739 (N). Bar, 50 µm. Detailed genotypes and roughness quantification are given in Tables S1 and S2. wt, wild type.

Figure 3.

Stabilized Acn^D527A enhances basal autophagy. (A and B) Micrographs of fed Acn^wt or Acn^D527A larval eye discs stained for Atg8a. (C) Quantification of Atg8a punctae in eye discs from five larvae. (D) Western blot of fat body lysates from 96-h fed larvae probed for Acn and Actin. (E) Quantification of blots for Acn levels relative to Actin (n = 3). (F–G′ and I–J′) Micrographs of Acn^wt or Acn^D527A fat bodies encompassing six to eight cells from 96-h fed or starved size-matched larvae. (F–G′) Fat bodies stained with antibodies against Atg8a. (H) Quantification of Atg8a punctae averaged from five larvae from one representative experiment out of three repeats. (I–J′) GFP-Atg8a fluorescence detected in live fat bodies. (K) Quantification of GFP-Atg8a punctae averaged from five larvae from one representative experiment out of three repeats. (L–M″) TEMs of fed Acn^wt or Acn^D527A fat bodies. Examples of dense lysosomes (arrowheads) and membrane-enriched autolysosomes (arrows) are shown at higher magnification. (N) Quantification of percentage of autolysosomal area averaged from 25 images per genotype. (O) Quantification of diameters of ≥100 lysosomes and autolysosomes per genotype. Detailed genotypes are given in Table S2. wt, wild type. Error bars show means ± SD. **, P < 0.01; ***, P < 0.001.

Figure 4.

Acn levels and activity is regulated by Dcp-1. (A–G) Micrographs of wild type (A) and eyes in which GMR-Gal4 drives expression of Acn^wt (B), Acn^D527A (C), Acn^wt + Dcp-1–RNAi (D), Dcp-1-RNAi (E), Acn^wt + Dredd-RNAi (F), Acn^wt + Dronc-RNAi (G). Bar, 50 µm. (H–K′) Micrographs of larval eye discs stained for DNA and Acn (green) from OreR (H and H′), dcp-1 (I and I′), GMR-Gal4, UAS–Dcp-1–RNAi (J and J′), and GMR-Gal4, UAS-Dredd-1–RNAi (K and K′). (L–M″′) Micrographs of eye (L–L″′) and antennal (M–M″′) discs with clones of cells expressing Dcp-1–RNAi marked by RFP and stained for Acn and DNA. Arrow indicates the morphogenetic furrow and the broken line indicates the clone boundary. (N) Western blots of wild type (+/+) or dcp-1 larvae (−/−) expressing Myc-Acn^wt or Myc-Acn^D527A as indicated. Blots were probed for Myc or Actin. (O) Quantification of blots (n = 3) as shown in N. Bars: (H–M) 10 µm. Detailed genotypes and roughness quantification are given in Tables S1 and S2. Error bars show means ± SD. **, P < 0.01; ***, P < 0.001.

Figure 5.

Dcp-1 regulates basal autophagy. (A–E′) Micrographs show fat bodies from 96-h fed and starved size-matched larvae stained for Atg8a. Genotypes are wild type (A), dcp-1 (B), Da-Gal4, UAS–Dcp-1–RNAi (C), Da-Gal4, UAS–Dcp-1–RNAi + UAS-Acn-RNAi (D), and Da-Gal4, UAS-Dredd-RNAi (E). Each image encompasses six to eight cells. (F) Quantification of Atg8a punctae averaged from five larvae from one representative experiment out of three repeats. wt, wild type. See also Fig. S1 H for efficiency of Acn knockdown. Detailed genotypes are given in Table S2. Error bars show means ± SD. ***, P < 0.001.

Figure 6.

AKT1 phosphorylates Acn. (A–I) Micrographs of eyes expressing the indicated UAS transgenes under control of GMR-Gal4. Bar, 50 µm. (J) Western blots of anti-Myc immunoprecipitates of Myc-Acn^wt and Myc-Acn^S641,731A expressed in S2 cells and detected with antibodies against Acn or Acn-pS641. S2 cell lysates were probed for Actin. IP, immunoprecipitation; wt, wild type. (K) Western blots of lysates from S2 cells expressing Myc-Acn^wt that were treated without (−) or with calf intestinal phosphatase (CIP) and probed for Acn, Acn-pS641, or Actin. (L–M″) Apical most regions of eye discs that include cone cell nuclei (marked CC) are depicted in z projections of confocal sections. Eye discs were from larvae expressing Myc-Acn^wt (L–L″) or Myc-Acn^S641,731A (M–M″) and were stained for Acn-pS641, Myc, and DNA. Arrows point to unspecific staining in mitotic cells. (N–N″) Projection of confocal sections of eye disc expressing AKT1-RNAi within RFP-marked clones (magenta) and stained for Acn-pS641 (N′) and DNA (N″). The broken line indicates the clone boundary. Detailed genotypes and roughness quantification are given in Tables S1 and S2.

Figure 7.

AKT1 phosphorylation regulates Acn levels. (A–F) SEMs of eyes expressing the indicated UAS-Acn transgenes with or without UAS-AKT1 under control of GMR-Gal4. Bar, 50 µm. (G) Number of flies with the indicated degree of roughness for genotypes as in A–F. (H) Western blot of lysates from fed 75-h larvae with the indicated Acn transgenes probed for Acn. Bands of full-length and cleaved Acn are indicated. FL, full length; wt, wild type. (I and J) Acn expression in developing photoreceptors visualized in projections of confocal sections of eye discs from acnP-Myc-Acn^S641,731D (I) or acnP-Myc-Acn^S641,731A (J) larvae stained for Myc and Acn. Bar, 10 µm. (K and L) Quantification of Acn and Myc levels in early R3/4 and R2/5/8 cells from ≥120 ommatidia from three eye discs. (K) Box and whisker graphs of integrated intensities of Acn and Myc immunoreactivity for individual groups of R3/4 and R2/5/8 cells in discs with the genomic transgenes (genTgs) Myc-Acn^S641,731A and Myc-Acn^S641,731D. Box and whisker graphs show boxes with medians, 25 and 75 percentiles, and whiskers extending to minimal and maximal values. arb units, arbitrary units. (L) Intensity blots of normalized integrated intensities of Acn and Myc immunoreactivity for individual groups of R3/4 and R2/5/8 cells in Myc-Acn^S641,731A and Acn^S641,731D discs and calculated Pearson correlation coefficients r. flour. int., fluorescence intensities. Detailed genotypes are given in Table S2.

Figure 8.

AKT1-mediated phosphorylation of Acn modulates basal autophagy. (A and B) Micrographs of fed Acn^S641,731A or Acn^S641,731D eye discs stained for Atg8a. (C) Quantification of Atg8a punctae in eye discs from five larvae. (D–E′ and G–H′) Micrographs of Acn^S641,731A or Acn^S641,731D fat bodies encompassing six to eight cells from 96-h fed or starved size-matched larvae. Bars, 40 µm. (D–E′) GFP-Atg8a fluorescence detected in live fat bodies. (F) Quantification of GFP-Atg8a punctae averaged from five larvae from one representative experiment out of three repeats. (G–H′) Fat bodies stained for Atg8a. (I) Quantification of Atg8a punctae averaged from five larvae from a single representative experiment out of three repeats. (J) TEM images of fed 96-h fat bodies expressing Acn^S641,731D. Examples of dense lysosomes (arrowheads) and membrane-enriched autolysosomes (arrows) are shown at higher magnification (inset). (K) Quantification of diameters of ≥100 lysosomes and autolysosomes diameters per genotype. (L) Quantification of percentage of autolysosomal area averaged from 25 images per genotype. Wild-types images are shown in Fig. 3 L. Detailed genotypes are given in Table S2. wt, wild type. Error bars show means ± SD. **, P < 0.01; ***, P < 0.001.

Figure 9.

Acn regulates quality control autophagy and longevity. (A and B) ERGs of 1- or 2-wk-old flies expressing the indicated UAS transgenes (Tgs) under control of GMR-Gal4. (B) Quantification of sustained negative photoreceptor response averaged from three replicate experiments including at least 64 traces from eight flies. (C and D) ERGs of 2-wk-old flies all expressing UAS-HttQ93 under control of GMR-Gal4 (GMR>HttQ93) and the indicated genomic Acn transgenes. (D) Quantification of sustained negative photoreceptor response from three replicate experiments including at least 64 traces from eight flies. Avg. sust., average sustained. (E) Quantification of dot blots measuring aggregated HttQ93 protein in fly heads expressing UAS-HttQ93 under control of GMR-Gal4 (GMR>HttQ93) and the indicated genomic Acn transgenes. Flies expressing neither indicate background level of dot blot measurements (n = 3). rel., relative; arb units, arbitrary units. (F) Starvation-induced mortality of flies expressing the indicated Acn proteins. In the parenthesis shown are the numbers of initial flies in a single representative experiment. (G) Survival curves of flies expressing the indicated Acn proteins. In the parenthesis shown are the numbers of initial flies in a single representative experiment. Detailed genotypes are given in Table S2. wt, wild type. Error bars show means ± SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.