Figures and data in A phosphoswitch at acinus-serine437 controls autophagic responses to cadmium exposure and neurodegenerative stress

Figures
Tables
Additional files

6 figures, 1 table and 5 additional files

Figures

Figure 1 with 1 supplement

Download asset Open asset

A genetic screen identifies Nil as the Acinus-Serine⁴³⁷ phosphatase.

(A–N) Micrographs of eyes in which GMR-Gal4 drives expression of AcnWT (A), AcnWT+ microtubule star (mts)–RNAi (B), AcnWT+ Pp1-13 C RNAi (C), AcnWT+ PpD6 RNAi (D), AcnWT+ flapwing (flw) RNAi (E), AcnWT+ CG15035 RNAi (F), AcnWT+ nil RNAi (G), mts–RNAi (I), Pp1-13C–RNAi (J), PpD6–RNAi (K), flw–RNAi (L), CG15035–RNAi (M), nil–RNAi (N), and H represents GMR-Gal4 control. (O–T) Projection of confocal micrographs of larval eye discs stained for pS437-Acn (green) and DNA from Da-Gal4 (**O, O’**), Da-Gal4, Pp1-13C RNAi (**P, P’**), Da-Gal4, PpD6 RNAi (**Q, Q’**), Da-Gal4, CG15035 RNAi (**R, R’**), Da-Gal4, flw RNAi (**S, S’**), Da-Gal4, nil RNAi (**T, T’**). Scale bar in A is 100 µm for A-N, scale bar in O is 40 µm for O-T. Genotypes are listed in Supplementary file 3.

Figure 1—source data 1 Phosphatase RNAi lines were crossed to Da-Gal4 in order to measure knockdown of Phosphatases which are hits in the screen.: https://cdn.elifesciences.org/articles/72169/elife-72169-fig1-data1-v2.xlsx
Download elife-72169-fig1-data1-v2.xlsx

Figure 1—figure supplement 1

Download asset Open asset

qPCR analysis of phosphatase genes interacting with Acinus.

The indicated phosphatases were knocked down in wandering third instar larvae using the Da-Gal4 driver. For each phosphatase, bar graphs show transcript levels relative to wild-type larvae as analyzed by quantitative RT-PCR. The data were normalized to levels of ribosomal protein 49 transcription. Genotypes are listed in Supplementary file 3.

Figure 2 with 2 supplements

Download asset Open asset

Nil loss and gain of function regulates Acinus-Serine⁴³⁷ phosphorylation.

(A) Diagram depicting the Nil^WT protein with amino acids highly conserved in the PPM family of phosphatases, the *nil*¹ allele with its 3xP3-DsRed insertion, and the multicistronic *nil*^Ty1-G4 allele with Ty1 tag and T2A-coexpressed Gal4. (B–E) Projection of confocal micrographs of larval antennal discs (**B, C**) and fat bodies (**D, E**) stained for pS437-Acn (green) and DNA from w¹¹¹⁸ and *nil*¹. (F–J) Projection of confocal micrographs of larval eye discs stained for pS437-Acn (green) and DNA from GMR-Gal4/+ (**F, F’**), *nil*¹ (**G, G’**), GMR-Gal4, UAS-Nil^WT; *nil*¹ (**H, H’**), GMR-Gal4, UAS-hPPM1B; *nil*¹ (**I, I’**), GMR-Gal4, UAS-Nil^D231N; *nil*¹ (**J, J’**). (K–O) Projection of confocal micrographs of larval fat bodies from *nil*^Ty1-G4 all stained for Ty1(green) and Acn (magenta) (**K- K’’**), YFP-Rab5 (magenta) (**L-L’’**), Rab7 (magenta) (**M-M’’**), Arl8 (magenta) (**N- N’’**), or Atg8a (magenta) (**O-O’’**). Arrowheads in M-M’’ indicate colocalization of Nil-Ty1 with Rab7 in cytosolic punctae. Notice that projections in L–O represent apical regions largely excluding nuclei. Scale bar in B and D is 40 µm for B-E and in F and K is 20 µm for F-O. Genotypes are listed in Supplementary file 3.

Figure 2—figure supplement 1

Download asset Open asset

Effects of Nil overexpression depend on its phosphatase activity.

(A–F) Micrographs of eyes in which GMR-Gal4 drives expression of Acn^WT (A), Acn^WT+ Nil^WT (B), Acn^WT + Nil^D231N (C), Nil^WT (E), Nil^D231N (F); GMR-Gal4 control is in (D). Scale bar in D is 100 µm for A–F. (G–I) Projection of confocal micrographs of larval eye discs stained for pS437-Acn (green), V5 (cyan), and DNA from GMR-Gal4 (**G-G’’**), GMR-Gal4, UAS- Nil^WT (**H-H’’**), GMR-Gal4, UAS- Nil^D231N (**I-I’’**). Nil transgenes are V5 tagged. Scale bar in G is 20 µm for G–I. Genotypes are listed in Supplementary file 3.

Figure 2—figure supplement 2

Download asset Open asset

Nil expression in larval and adult tissues.

(**A–E**) Projection of confocal micrographs of larval eye discs (B) and fat bodies (**D, E**) from *nil*^Ty1-T2A-Gal4 (*nil*^Ty1-G4) stained for Ty1 (green). No Ty1 (green) staining is observed in 20XUAS-6XmCherry-HA controls (**A,C**). (**F–N**) Projection of confocal micrographs of larval eye discs (G) and fat bodies (**I, J**), adult brain (L), and testes (N) from animals expressing UAS-mCD8-GFP under control of the *nil*^Ty1-T2A-Gal4 driver (*nil*^Ty1-G4) stained for GFP (green) and DNA. Only background levels of GFP (green) staining are observed in UAS-mCD8-GFP controls (**F,H, K,M**). (O) Western blot of lysates from adult male *nil*^Ty1-G4 flies or 20XUAS-6XmCherry-HA controls probed for Ty1 and Hook as control. Scale bars: in A, C, F, H are 20 µm for A–J; in K is 50 µm for K–L; in M is 100 µm for M–N. Genotypes are listed in Supplementary file 3.

Figure 2—figure supplement 2—source data 1 Raw Western blot data with molecular weight markers for Figure 2—figure supplement 2O from lysates of adult male flies of nil^Ty1-G4 and appropriate control probed for Ty1 and Hook. Western blot analysis using anti-Ty1 and anti-Hook antibodies in lysates from adult males of nil^Ty1G4 and appropriate control. The parts of the raw image used in Figure 2—figure supplement 2O were marked with box.: https://cdn.elifesciences.org/articles/72169/elife-72169-fig2-figsupp2-data1-v2.zip
Download elife-72169-fig2-figsupp2-data1-v2.zip

Figure 3 with 1 supplement

Download asset Open asset

Loss of Nil enhances autophagic flux.

(**A–B**) Western blot of lysates from adult heads of w¹¹¹⁸ and *nil*¹ probed for ATG8a (A). Quantification of ATG8a-II to ATG8a-I ratio from Western blots as in (A). Data are from six different lysates from three experimental repeats (B). Normality of data distributions was assessed using the Kolmogorov-Smirnov test and statistical significance using two-tailed paired t test (p=0.006). Bar graphs show mean ± SD. (**C–E**) Projection of confocal micrographs of fed w¹¹¹⁸ and *nil*¹ larval eye discs (**C, D**) stained for Atg8a. (E) Quantification of Atg8a punctae in eye discs (**C, D**) of fed w¹¹¹⁸ and *nil*¹. Data are from 15 larvae taken from three experimental repeats. Normality of data distributions was assessed using the Kolmogorov-Smirnov test and statistical significance using two-tailed unpaired t test (p<0.001). Bar graphs show mean ± SD. (**F–H**) Projection of confocal micrographs of fed w¹¹¹⁸ and *nil*¹ antennal discs (**F, G**) stained for Atg8a. (H) Quantification of Atg8a punctae in antennal discs (**F, G**) of fed w¹¹¹⁸ and *nil*¹. Data are from 15 larvae from three experimental repeats. Normality of data distributions was assessed using the Kolmogorov-Smirnov test and statistical significance using two-tailed unpaired t test (p<0.001). Bar graphs show mean ± SD. (**I–P**) Projection of confocal micrographs encompassing 6–8 cells of w¹¹¹⁸ and *nil*¹ larval fat bodies aged 96 hr after egg laying, either fed (**I–L**) or amino acid starved for 4 hr in 20% sucrose solution (**M–P**) stained for Atg8a. Larvae were matched for size. To assess autophagic flux, for panels K, L and O, P lysosomal degradation was inhibited with chloroquine (CQ). (**Q–R**) Quantification of Atg8a punctae in fed (Q) or amino acid starved (R) w¹¹¹⁸ and *nil*¹ larval fat bodies untreated or treated with CQ averaged from six to eight cells each per fat body. Data are from 10 larvae from three experimental repeats. Normality of data distributions was assessed using the Kolmogorov-Smirnov test and statistical significance using one-way analysis of variance with Tukey’s correction for multiple comparisons. Bar graphs show mean ± SD. ***p<0.001. Scale bar in C, F, I is 20 µm for C–D, F–G, I–P. Genotypes are listed in Supplementary file 3.

Figure 3—source data 1 Raw Western blot data with molecular weight markers for Figure 3A from lysates of adult heads of w¹¹¹⁸ and nil¹ probed for ATG8a and Actin. Western blot analysis using anti-ATG8a and anti-actin antibodies in lysates from adult heads of w¹¹¹⁸ and nil¹. Boxes mark the parts of the raw image used in Figure 3A.: https://cdn.elifesciences.org/articles/72169/elife-72169-fig3-data1-v2.zip
Download elife-72169-fig3-data1-v2.zip

Figure 3—figure supplement 1

Download asset Open asset

p62/ Ref(2)P is not accumulated in adult heads of *nil*¹ animal.

(**A–B**) Western blot of lysates from adult heads of w¹¹¹⁸ and *nil*¹ probed for p62/Ref(2)P and actin as loading control (A). Quantification of blots for Ref(2)P levels relative to actin. Data are from six different lysates from three experimental repeats. (B) Normality of data distributions was assessed using the Kolmogorov-Smirnov test and statistical significance using two-tailed t test (p=0.122). Bar graphs show mean ± SD. Genotypes are listed in Supplementary file 3.

Figure 3—figure supplement 1—source data 1 Raw Western blot data with molecular weight markers for Figure 3—figure supplement 1A from lysates of adult heads of w¹¹¹⁸ and nil¹ probed for Ref(2)P and Actin. Boxes mark the parts of the raw image used in Figure 3—figure supplement 1A.: https://cdn.elifesciences.org/articles/72169/elife-72169-fig3-figsupp1-data1-v2.zip
Download elife-72169-fig3-figsupp1-data1-v2.zip

Figure 4

Download asset Open asset

Nil dephosphorylates acinus (acn) counteracting Cdk5-p35-mediated phosphorylation.

(A–F) Projection of confocal micrographs of w¹¹¹⁸ and *nil*¹ larval eye discs stained for p-ERK (green) and DNA (**A- B’**), p-SAPK/JNK (green) and DNA (**C-D’**), p-p38b MAPK (green) and DNA (**E-F’**). (**G–J**) Projection of confocal micrographs of larval eye discs stained for pS437-Acn (green) and DNA from w¹¹¹⁸ (**G, G’**), *p35*^20C (**H, H’**), *nil*¹ (**I, I’**), *p35*^20C; *nil*¹ (**J, J’**). (**K–N**) Projection of confocal micrographs of eye discs from Acn^WT larvae stained for pS437-Acn (green) and DNA after in situ dephosphorylation without addition (**K, K’**) or with addition of wild-type Nil (**L,L'**), wild-type Nil +100 µM CdCl₂ (**M,M'**), or inactive Nil^D231N (**N,N'**). Scale bar in A is 40 µm for A–F. Scale bar in G is 20 µm for G–N. Genotypes are listed in Supplementary file 3.

Figure 5 with 1 supplement

Download asset Open asset

Acinus-Serine⁴³⁷ phosphorylation is required for Cd²⁺-induced autophagy.

(**A–B**) Projection of confocal micrographs of w¹¹¹⁸ larval eye discs stained for pS437-Acn (green) and DNA from either 100 µM CdCl₂ treated (B) or untreated (A) larvae. (**C–D**) Projection of confocal micrographs of fed w¹¹¹⁸ larval eye discs either 100 µM CdCl₂ treated (D) or untreated (C) stained for Atg8a. (**E–H**) Projection of confocal micrographs of fed Acn^WT and Acn^S437A larval eye discs either 100 µM CdCl₂ treated (**G, H**) or untreated (**E, F**) stained for Atg8a. (**I,J**) Quantification of Atg8a punctae in either 100 µM CdCl₂ treated or untreated w¹¹¹⁸ larval eye discs (I) or Acn^WT and Acn^S437A larval eye discs (J). Data are from 10 larvae from three experimental repeats. Normality of data distributions was assessed using the Kolmogorov-Smirnov test and statistical significance using one-way analysis of variance with Tukey’s correction for multiple comparisons. Bar graphs show mean ± SD. ns, not significant; **p<0.01; ***p<0.001. Scale bar in A, C, E is 20 µm for A–H. Genotypes are listed in Supplementary file 3.

Figure 5—figure supplement 1

Download asset Open asset

Nil transcription or protein level does not change under conditions influencing starvation-independent or dependent autophagy.

(**A–B**) Western blot of lysates from adult males of w¹¹¹⁸, *nil*^Ty1-G4 fed without or with 250 µM Cd²⁺, probed for Ty1 (A). Total protein for the same blot is imaged for normalization (A). Quantification of blots for Ty1-tagged Nil relative to total protein loaded. Data are from five different lysates from three experimental repeats. Normality of data distributions was assessed using the Kolmogorov-Smirnov test and statistical significance using two-tailed t test (p=0.89). Bar graphs show mean ± SD. (**C–D**) Analysis of nil transcript level by quantitative RT-PCR from heads of w¹¹¹⁸ adults fed without or with 125 µM Cd²⁺ or 250 µM Cd²⁺ (C) and w¹¹¹⁸ third instar larvae (D) fed without or with 100 µM Cd²⁺, or amino acid starved (Stv). Data shown are three technical repeats from two sperate experiments each normalized to levels of Act5C transcription. Genotypes are listed in Supplementary file 3.

Figure 5—figure supplement 1—source data 1 Raw Western blot data with molecular weight markers for Figure 5—figure supplement 1A from adult males of w¹¹¹⁸, fed nil^Ty1-G4, and Cd²⁺(250 µM) fed nil^Ty1-G4 probed for Ty1. Raw data of total Protein with molecular weight marker from the same blot was also provided. Boxes mark the parts of the raw image used in Figure 5—figure supplement 1A.: https://cdn.elifesciences.org/articles/72169/elife-72169-fig5-figsupp1-data1-v2.zip
Download elife-72169-fig5-figsupp1-data1-v2.zip

Figure 6 with 1 supplement

Download asset Open asset

Loss of Nil function provides partial protection against Cd²⁺ poisoning and proteostasis stress.

(**A–C**) Survival curves for w¹¹¹⁸, *nil*¹, *acn*^S437A, and *acn*^S437A; *nil*¹ adult male flies either untreated (A) or treated with 125 µM CdCl₂ (B), 250 µM CdCl₂ (C). N was at least 99 (A), 82 (B), and 79 (C). Significance was assessed using log-rank Mantel-Cox test.:

ns, not significant. (**D–I**) Micrographs of adult eyes in which GMR-Gal4 drives expression of UAS-HTTex1.Q93 (D), UAS-HTTex1.Q93+ UAS Nil RNAi (E), UAS-HTTex1.Q93+ UAS-hPPM1B (F), UAS-Nil RNAi (H), UAS-hPPM1B (I), and (G) represents GMR-Gal4 control. Scale bar in D is 100 µm for D–I. (**J–M**) Projection of confocal micrographs of larval eye discs stained for PolyQ (green) and DNA from GMR-Gal4 (**J,J’**), GMR-Gal4, UAS-HTTex1.Q93 (**K,K’**), GMR-Gal4, UAS-HTTex1.Q93+ UAS Nil RNAi (**L,L’**), GMR-Gal4, UAS-HTTex1.Q93+ UAS-hPPM1B (**M,M’**). Scale bar in J is 20 µm for J–M. (N) Quantification of PolyQ accumulation in eye discs of the indicated genotypes from a constant area starting two to three rows posterior to the furrow containing around 100 ommatidial clusters. The Region of interest(ROI) is represented using a box in the figure panels (**J–M**). Normality of data distribution was assessed using the Kolmogorov-Smirnov test and statistical significance using one-way analysis of variance with Tukey’s correction for multiple comparisons. Bar graphs show mean ± SD of integrated densities from 10 larvae taken out of three experimental repeats. **p<0.01; ****p<0.0001. Genotypes are listed in Supplementary file 3.

Figure 6—figure supplement 1

Download asset Open asset

Survival curves for adult male w¹¹¹⁸ and *nil*¹ flies at different Cd²⁺ concentrations.

(**A–C**) Survival curves for w1118, and nil1 adult male flies either untreated (A) or treated with 375 µM CdCl₂ (B), 500 µM CdCl₂ (C). N was at least 130 (A), 74 (B), and 90 (C). Significance was assessed using log-rank Mantel-Cox test.: ns, not significant; ****p<0.0001. Genotypes are listed in Supplementary file 3.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene (Drosophila melanogaster)	Acinus (Acn)	GenBank	FLYB:FBgn0263198
Gene (Drosophila melanogaster)	Cyclin-dependent kinase 5 (Cdk5)	GenBank	FLYB:FBgn0013762
Gene (Drosophila melanogaster)	Cdk5 activator-like protein (p35)	GenBank	FLYB:FBgn0027491
Gene (Drosophila melanogaster)	CG6036	GenBank	FLYB:FBgn0039421
Genetic reagent (Drosophila melanogaster)	w¹¹¹⁸	Bloomington Drosophila Stock Center	BDSC:3605; FLYB:FBst0003605; RRID:BDSC_3605
Genetic reagent (Drosophila melanogaster)	UAS-hPPM1B	Bloomington Drosophila Stock Center	BDSC:76916; FLYB: FBst0076916; RRID:BDSC_76916
Genetic reagent (Drosophila melanogaster)	p35-/-	PMID:17368005	Gift from Edward Giniger, NINDS, Bethesda, Maryland, USA
Genetic reagent (Drosophila melanogaster)	GMR-GAL4	Bloomington Drosophila Stock Center	BDSC:1104; FLYB:FBst0001104; RRID: BDSC_1104
Genetic reagent (Drosophila melanogaster)	vas-Cas9(X)	BestGene	BDSC:1104; FLYB:FBst0055821; RRID:BDSC_55821
Genetic reagent (Drosophila melanogaster)	Da-GAL4	Bloomington Drosophila Stock Center	BDSC: 55851; FLYB:FBst0055851; RRID: BDSC_55851
Genetic reagent (Drosophila melanogaster)	UAS-HTT-exon1-Q93 (human)	PMID:11607033	Gift from Robin Hiesinger, Free University Berlin, Berlin, Germany
Genetic reagent (Drosophila melanogaster)	For phosphatase RNAi lines screened the information is contained in Supplementary file 1
Genetic reagent (Drosophila melanogaster)	YFP-MYC-Rab5	PMID:25942626	Gift from Marcos Gonzalez-Gaitan Marcos Gonzalez-Gaitan, MPI Dresden, Germany
Genetic reagent (Drosophila melanogaster)	UAS-mCD8-GFP		FLYB:FBti0012686
Genetic reagent (Drosophila melanogaster)	20xUAS-6xmCherry-HA		BDSC:52267; FLYB:FBst0052267; RRID:BDSC_52267
Genetic reagent (Drosophila melanogaster)	Nil¹	This paper		CRISPR/ Cas9 derived, details in “Methods” and Supplementary file 4
Genetic reagent (Drosophila melanogaster)	Acn^S437A	This paper		CRISPR/ Cas9 derived, details in “Methods” and Supplementary file 4
Genetic reagent (Drosophila melanogaster)	Nil^Ty1-T2A-Gal4	This paper		CRISPR/ Cas9 derived, details in “Methods” and Supplementary file 4
Cell line (Drosophila melanogaster)	S2		FBtc0000006DGRC Cat# 6, RRID:CVCL_TZ72
Transfected construct (Drosophila melanogaster)	pMT-Nil [WT]–3xFLAG-TST	This paper
Transfected construct (Drosophila melanogaster)	pMT-Nil [D231N]–3xFLAG-TST	This paper
Antibody	Anti-pS437-Acn (rabbit polyclonal)	Genemed Synthesis, PMID:29227247		1:1,000 (IF); Ab made against the Acn peptide H I V R D P- S(p)-P A R N R A S
Antibody	Guinea pig anti-Acn (aa 423–599) (guinea pig polyclonal)	PMID:20504956		1:1,000 (IF)
Antibody	Anti-Ty1 clone BB2 (mouse monoclonal)	Invitrogen	Invitrogen:MA5-23513, RRID:AB_2610644	1:2000 (WB), 1:500 (IF)
Antibody	Anti-hook (rabbit polyclonal)	PMID:8682859		1:5,000 (WB)
Antibody	Anti-Actin JLA20 (mouse monoclonal)	Developmental Studies Hybridoma Bank	DSHB:JLA20; RRID:AB_528068	1:2000 (WB)
Antibody	Anti-p62 (rabbit polyclonal)	PMID:22952930	Gift from G. Juhàsz, Eötvös Loránd University, Budapest, Hungary	1:5,000 (WB)
Antibody	Anti-GABARAP [EPR4805] (rabbit monoclonal)	Abcam	Abcam:ab109364; RRID:AB_10861928	1:200 (IF), 1:1,000 (WB)
Antibody	Anti-Polyglutamine-Expansion Diseases Marker Antibody, clone 5TF1-1C2(mouse monoclonal)	EMD Millipore	EMD Millipore:MAB1574; RRID:AB_94263	1:1,000(IF)
Antibody	Anti-V5 clone E10/V4RR(mouse monoclonal)	Invitrogen	Invitrogen:MA5-1525, RRID:AB_10977225	1:500 (IF)
Antibody	Anti-GFP clone B2 (mouse monoclonal)	Santa Cruz Biotechnology	SCB:sc-9996, RRID:AB_627695	1:100 (IF)
Antibody	Antiphospho-p44/42 MAPK (Erk1/2) (D13.14.4E) (Thr202/Tyr20) (rabbit monoclonal)	Cell Signaling Technology	CST:4,370 S, RRID:AB_2315112	1:200 (IF)
Antibody	Antiphospho-SAPK/JNK (G9) (Thr183/Tyr18) (mouse monoclonal)	Cell Signaling Technology	CST:9,255 S, RRID:AB_2307321	1:200 (IF)
Antibody	Antiphospho-p38 MAPK (D3F9) (Thr180/Tyr18) (rabbit monoclonal)	Cell Signaling Technology	CST:4,511 S, RRID:AB_2139682	1:500 (IF)
Antibody	Anti-Rab7 (rabbit polyclonal)	PMID:18272590	Gift from Akira Nakamura, RIKEN Center for Devel. Biol., Kobe, Japan	1:3,000 (IF);
Antibody	Anti-Arl8 (rabbit polyclonal)	PMID:30590083	Gift from……	1:300 (IF)
Antibody	Alexa 488- or 568- or 647 secondaries	Molecular Probes		1:500 (IF)
Antibody	IRDye 800CW and 680RD Secondary Antibodies	LI-COR Biosciences		1:15,000 (WB)
Sequence-based reagent	DNA oligonucleotides used are listed in Supplementary file 4	Integrated DNA Technologies
Commercial assay or kit	Revert 700 Total Protein Stain Kits	LI-COR Biosciences	LI-COR Biosciences:926–11010
Commercial assay or kit	High-Capacity cDNA Reverse Transcription kit	Applied Biosystems (now: Thermo Fischer Scientific)	Thermo Fischer Scientific:4368813
Chemical compound, drug	Tissue-Tek O.C.T. Compound	Sakura	Sakura:4,583
Chemical compound, drug	Cadmium Chloride Anhydrous	Fisher Scientific	Fischer Scientific: C10-500
Chemical compound, drug	H-XStable Prestained Protein Ladder	UBP-Bio	UBP-Bio:L2021
Chemical compound, drug	Fast SYBR Green Master Mix	Applied Biosystems	AB:4385610
Chemical compound, drug	VECTASHIELD Antifade Mounting Medium with DAPI	Vector Laboratories	Vector Laboratories:H-1200
Chemical compound, drug	cOmplete ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail	Roche	Roche:5892970001
Chemical compound, drug	Chloroquine	Sigma-Aldrich	Sigma-Aldrich:C6628-25G
Software, algorithm	Imaris software	Bitplane	RRID:SCR_007370
Software, algorithm	Adobe Photoshop	Adobe	RRID:SCR_014199
Software, algorithm	ImageJ	NIH	RRID:SCR_003070
Software, algorithm	Image Studio ver 5.2	LI-COR	RRID:SCR_015795
Software, algorithm	Prism	Graphpad	RRID:SCR_002798
Software, algorithm	SteREO Discovery.V12	Carl Zeiss
Software, algorithm	CZFocus	Carl Zeiss
Software, algorithm	Helicon Focus	Helicon Soft

Additional files

Supplementary file 1 Effect of knockdown of different phosphatases on eye roughness in a Acn gain-of-function model. All flies were raised at 28°C. Scores to calculate average roughness: normal = 1; mild = 2; rough = 3; strongly rough = 4. Positive or negative numbers indicate suppression and enhancement, respectively. Green or red colors highlight UAS-transgenes with more than 45% suppression or enhancement. Numbers in parenthesis indicated stock numbers of the Bloomington Drosophila stock center.: https://cdn.elifesciences.org/articles/72169/elife-72169-supp1-v2.docx
Download elife-72169-supp1-v2.docx
Supplementary file 2 Effect of loss and gain of phosphatase activity on eye pigmentation in a Drosophila Huntington’s model. All flies were raised at 28°C. Scores to calculate depigmentation and roughness: Score 1: No depigmentation and no roughness. Score 2: Mild depigmentation and no roughness. Score 3: Moderate depigmentation and roughness. Score 4: Extreme depigmentation and roughness.: https://cdn.elifesciences.org/articles/72169/elife-72169-supp2-v2.docx
Download elife-72169-supp2-v2.docx
Supplementary file 3 Genotypes of flies used for each figure. The table list relevant genotype for flies used in each figure.: https://cdn.elifesciences.org/articles/72169/elife-72169-supp3-v2.docx
Download elife-72169-supp3-v2.docx
Supplementary file 4 DNA oligonucleotides used. The table contains sequences of DNA oligonucleotides used for this study.: https://cdn.elifesciences.org/articles/72169/elife-72169-supp4-v2.docx
Download elife-72169-supp4-v2.docx
Transparent reporting form: https://cdn.elifesciences.org/articles/72169/elife-72169-transrepform1-v2.pdf
Download elife-72169-transrepform1-v2.pdf

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Mendeley

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Nilay Nandi
Zuhair Zaidi
Charles Tracy
Helmut Krämer

(2022)

A phosphoswitch at acinus-serine⁴³⁷ controls autophagic responses to cadmium exposure and neurodegenerative stress

eLife 11:e72169.

https://doi.org/10.7554/eLife.72169

Figures

A genetic screen identifies Nil as the Acinus-Serine⁴³⁷ phosphatase.

Figure 1—source data 1

qPCR analysis of phosphatase genes interacting with Acinus.

Nil loss and gain of function regulates Acinus-Serine⁴³⁷ phosphorylation.

Effects of Nil overexpression depend on its phosphatase activity.

Nil expression in larval and adult tissues.

Figure 2—figure supplement 2—source data 1

Loss of Nil enhances autophagic flux.

Figure 3—source data 1

p62/ Ref(2)P is not accumulated in adult heads of nil¹ animal.

Figure 3—figure supplement 1—source data 1

Nil dephosphorylates acinus (acn) counteracting Cdk5-p35-mediated phosphorylation.

Acinus-Serine⁴³⁷ phosphorylation is required for Cd²⁺-induced autophagy.

Nil transcription or protein level does not change under conditions influencing starvation-independent or dependent autophagy.

Figure 5—figure supplement 1—source data 1

Loss of Nil function provides partial protection against Cd²⁺ poisoning and proteostasis stress.

Survival curves for adult male w¹¹¹⁸ and nil¹ flies at different Cd²⁺ concentrations.

Tables

Additional files

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Transparent reporting form

Download links

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Be the first to read new articles from eLife

Share this article

Cite this article

A genetic screen identifies Nil as the Acinus-Serine437 phosphatase.

Figure 1—source data 1

qPCR analysis of phosphatase genes interacting with Acinus.

Nil loss and gain of function regulates Acinus-Serine437 phosphorylation.

Effects of Nil overexpression depend on its phosphatase activity.

Nil expression in larval and adult tissues.

Figure 2—figure supplement 2—source data 1

Loss of Nil enhances autophagic flux.

Figure 3—source data 1

p62/ Ref(2)P is not accumulated in adult heads of nil1 animal.

Figure 3—figure supplement 1—source data 1

Nil dephosphorylates acinus (acn) counteracting Cdk5-p35-mediated phosphorylation.

Acinus-Serine437 phosphorylation is required for Cd2+-induced autophagy.

Nil transcription or protein level does not change under conditions influencing starvation-independent or dependent autophagy.

Figure 5—figure supplement 1—source data 1

Loss of Nil function provides partial protection against Cd2+ poisoning and proteostasis stress.

Survival curves for adult male w1118 and nil1 flies at different Cd2+ concentrations.

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Transparent reporting form

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

A genetic screen identifies Nil as the Acinus-Serine⁴³⁷ phosphatase.

Nil loss and gain of function regulates Acinus-Serine⁴³⁷ phosphorylation.

p62/ Ref(2)P is not accumulated in adult heads of nil¹ animal.

Acinus-Serine⁴³⁷ phosphorylation is required for Cd²⁺-induced autophagy.

Loss of Nil function provides partial protection against Cd²⁺ poisoning and proteostasis stress.

Survival curves for adult male w¹¹¹⁸ and nil¹ flies at different Cd²⁺ concentrations.