Muscle function and homeostasis require cytokine inhibition of AKT activity in Drosophila
- Imperial College London, United Kingdom
- University of Freiburg, Germany
- Institute of Science and Technology, Austria
Figures
Figure 1 with 1 supplement
Dome inhibition in adult muscle reduces lifespan, disrupts homeostasis, and causes AKT hyperactivation.
(A) STAT activity in different muscles in 10xSTAT92E-GFP reporter fly. One fly out of 5 shown. Upper panel: lateral view, Scale bar = 500 µm. Lower panels: dorsal thorax (left); dorsal abdomen …
Figure 1—figure supplement 1
Further characterisation of the requirement for dome in adult muscle.
(A) STAT activity (10xSTAT92E-GFP) and muscle (MHC-RFP) colocalize in adult flies. One fly of 6 shown. Scale bar = 500 µm. (B) dome expression by qRT-PCR in thorax samples from 14-day-old 24B-Gal80ts…
Figure 2 with 1 supplement
Hop is required, but not sufficient, for Dome to control AKT.
(A) Lifespan of w1118 and hopTum-L flies at 29°C. Log-Rank test: χ2 = 0.3223, ns p=0.5702; Wilcoxon test: χ2 = 0.4756, ns p=0.4906. (B) Phospho-AKT in leg samples from 14-day-old w1118 and hopTum-L …
Figure 2—figure supplement 1
Interactions of dome with AMPK, MAPK, and FOXO signalling in adult muscle.
(A) Phospho-AKT in leg samples from 14-day-old 24B-Gal80ts/+, 24B-Gal80ts > AMPKα-IR, and 24B-Gal80ts > AMPKβ-IR flies. One of three independent experiments is shown. P values from unpaired T-Test. …
Figure 3 with 1 supplement
AKT hyperactivation causes pathology in 24B-Gal80ts > dome∆ flies.
(A) Lifespan of 24B-Gal80ts/+ and 24B-Gal80ts > myr-AKT at 29°C. Log-Rank test: χ2 = 115.5, ***p<0.0001; Wilcoxon test: χ2 = 123.6, ***p<0.0001. (B) Negative geotaxis assay of 14-day-old 24B-Gal80ts/…
Figure 3—figure supplement 1
Mutual regulation by AKT, Foxo, and Dome.
(A) dome expression by qRT-PCR in whole fly samples from 14-day-old 24B-Gal80ts/+, 24B-Gal80ts > dome∆, 24B-Gal80ts > Akt-IR, and 24B-Gal80ts > Akt-IR;dome∆ flies. (B–F) Gene xpression analysis of do…
Figure 4 with 2 supplements
Plasmatocytes promote muscle Dome activity.
(A) Muscle (MHCYFP) and plasmatocytes (srpHemo-3xmCherry) in 7-day-old flies. Plasmatocytes are found in close proximity to adult muscles. One representative fly of 5 is shown. Scale bar = 500 µm. (B…
Figure 4—figure supplement 1
Further characterisation of plasmatocyte-depleted flies.
(A) Plasmatocyte depletion in crq-Gal80ts > rpr flies (right) and controls (left) at 24 hr, 48 hr and 72 hr at 29°C. One out of four flies is shown. Scale bar = 250 µm. (B) Quantification of …
Figure 4—figure supplement 2
Further characterisation of requirements for specific Upds.
(A) Glucose + trehalose and glycogen in 7-day-old crq-Gal80ts/+, crq-Gal80ts > rpr, upd2Δ upd3Δ;crq-Gal80ts/+, and upd2Δ upd3Δ; crq-Gal80ts > rpr flies. (B) TLC of triglyceride in 7-day-old crq-Gal80…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | w1118; tubulin-Gal80ts/SM6a;24B-Gal4/TM6c, Sb1 | This study | Inserted Elements: P[w[+mC]=tubP-GAL80[ts]]; P[GawB]how24B | |
| Genetic reagent (D. melanogaster) | w1118; tubulin-Gal80ts/SM6a;Mef2-Gal4/TM6c, Sb1 | This study | Inserted Elements: P[w[+mC]=tubP-GAL80[ts]]; P[GAL4-Mef2.R]3 | |
| Genetic reagent (D. melanogaster) | w1118;;UAS-dome∆/TM6c, Sb1 | Brown et al., 2001 | Gift of James Castelli-Gair Hombría | |
| Genetic reagent (D. melanogaster) | w1118;UAS-dome∆/CyO | Brown et al., 2001 | Gift of James Castelli-Gair Hombría | |
| Genetic reagent (D. melanogaster) | w1118;;UAS-myr-AKT/TM6c, Sb1 | Stocker et al., 2002 | Gift of Ernst Hafen | |
| Genetic reagent (D. melanogaster) | w;UAS-AMPKα-IR | Vienna Drosophila Research Center (VDRC) | RRID:FlyBase_FBst0478025; VDRC 106200 | |
| Genetic reagent (D. melanogaster) | w;UAS-AMPKβ-IR | VDRC | RRID:FlyBase_FBst0476347; VDRC 104489 | |
| Genetic reagent (D. melanogaster) | w;UAS-rl-IR | VDRC | RRID:FlyBase_FBst0480887; VDRC 109108 | |
| Genetic reagent (D. melanogaster) | w;UAS-Dsor1-IR | VDRC | RRID:FlyBase_FBst0479098; VDRC 107276 | |
| Genetic reagent (D. melanogaster) | w1118;foxoGFP | BDSC | RRID:BDSC_38644 | Inserted Element: PBac[foxo-GFP.FLAG]VK00037 |
| Genetic reagent (D. melanogaster) | w;UAS-AKT-IR | VDRC | RRID:FlyBase_FBst0475561; VDRC 103703 | |
| Genetic reagent (D. melanogaster) | w1118;10xSTAT92E-GFP | BDSC Bach et al., 2007 | RRID:BDSC_26197 | Inserted Element: P[10XStat92E-GFP]1 |
| Genetic reagent (D. melanogaster) | w1118;MHC-Gal4,MHC-RFP/SM6a | BDSC | RRID:BDSC_38464 | Inserted Element: P[Mhc-GAL4.F3-580]2; P[Mhc-RFP.F3-580]2 |
| Genetic reagent (D. melanogaster) | w upd2 Δ upd3Δ;;; | BDSC | RRID:BDSC_55729 | |
| Genetic reagent (D. melanogaster) | w1118;;crq-Gal4/TM6 c, Sb1 | Gift of Nathalie Franc | ||
| Genetic reagent (D. melanogaster) | w1118;tub-Gal80ts;TM2/TM6 c, Sb1 | BDSC | RRID:BDSC_7108 | |
| Genetic reagent (D. melanogaster) | w1118;;UAS-rpr/TM6 c, Sb1 | BDSC | RRID:BDSC_5824 | |
| Genetic reagent (D. melanogaster) | w1118;UAS-CD8-mCherry | BDSC | RRID:BDSC_27391 | |
| Genetic reagent (D. melanogaster) | w1118;;srpHemo-3xmCherry/TM6c, Sb1 | Gyoergy et al., 2018 | ||
| Genetic reagent (D. melanogaster) | w;UAS-hop-IR | VDRC | RRID:FlyBase_FBst0463355; VDRC 40037 | |
| Genetic reagent (D. melanogaster) | w;UAS-upd1-IR/SM6a | VDRC | RRID:FlyBase_FBst0459787; VDRC 3282 | |
| Genetic reagent (D. melanogaster) | w;UAS-upd3-IR | VDRC | RRID:FlyBase_FBst0456774; VDRC 27134 | |
| Genetic reagent (D. melanogaster) | w1118;;UAS-upd3/TM6 c, Sb1 | Gift of Bruce Edgar | ||
| Genetic reagent (D. melanogaster) | w1118;UAS-2xeGFP/SM6a | BDSC | RRID:BDSC_6874 | |
| Genetic reagent (D. melanogaster) | w1118 hopTum-L/FM7h | BDSC | RRID:BDSC_8492 | backcrossed onto w1118 background |
| Sequence-based reagent | Akt1_forward | This study | PCR primers | 5’-ctttgcgagtattaactggacaga-3’ |
| Sequence-based reagent | Akt1_reverse | This study | PCR primers | 5’-ggatgtcacctgaggcttg-3’ |
| Sequence-based reagent | Ilp2_forward | This study | PCR primers | 5’-atcccgtgattccaccacaag-3’ |
| Sequence-based reagent | Ilp2_reverse | This study | PCR primers | 5’-gcggttccgatatcgagtta-3’ |
| Sequence-based reagent | Ilp3_forward | This study | PCR primers | 5’-caacgcaatgaccaagagaa-3’ |
| Sequence-based reagent | Ilp3_reverse | This study | PCR primers | 5’-tgagcatctgaaccgaact-3’ |
| Sequence-based reagent | Ilp4_forward | This paper | PCR primers | 5’-gagcctgattagactgggactg-3’ |
| Sequence-based reagent | Ilp4_reverse | This paper | PCR primers | 5’-tggaccggctgcagtaac-3’ |
| Sequence-based reagent | Ilp5_forward | This paper | PCR primers | 5’-gccttgatggacatgctga-3’ |
| Sequence-based reagent | Ilp5_reverse | This paper | PCR primers | 5’-agctatccaaatccgcca-3’ |
| Sequence-based reagent | Ilp6_forward | This paper | PCR primers | 5’-cccttggcgatgtatttcc-3’ |
| Sequence-based reagent | Ilp6_reverse | This paper | PCR primers | 5’-cacaaatcggttacgttctgc-3’ |
| Sequence-based reagent | Ilp7_forward | This paper | PCR primers | 5’-cacaccgaggagggtctc-3’ |
| Sequence-based reagent | Ilp7_reverse | This paper | PCR primers | 5’-caatatagctggcggacca-3’ |
| Sequence-based reagent | dome_forward | This paper | PCR primers | 5’-cggactttcggtactccatc-3’ |
| Sequence-based reagent | dome_reverse | This paper | PCR primers | 5’-accttgatgaggccaggat-3’ |
| Sequence-based reagent | upd1_forward | This paper | PCR primers | 5’-gcacactgatttcgatacgg-3’ |
| Sequence-based reagent | upd1_reverse | This paper | PCR primers | 5’- ctgccgtggtgctgtttt −3’ |
| Sequence-based reagent | upd2_forward | This paper | PCR primers | 5’-cggaacatcacgatgagcgaat-3’ |
| Sequence-based reagent | upd2_reverse | This paper | PCR primers | 5’-tcggcaggaacttgtactcg-3’ |
| Sequence-based reagent | upd3_forward | This paper | PCR primers | 5’-actgggagaacacctgcaat-3’ |
| Sequence-based reagent | upd3_reverse | This paper | PCR primers | 5’-gcccgtttggttctgtagat-3’ |
| Sequence-based reagent | Pepck_forward | This paper | PCR primers | 5’-ggataaggtggacgtgaag-3’ |
| Sequence-based reagent | Pepck_reverse | This paper | PCR primers | 5’-acctcctgcgaccagaact-3’ |
| Sequence-based reagent | Thor_forward | This paper | PCR primers | 5’-caggaaggttgtcatctcgga-3’ |
| Sequence-based reagent | Thor_reverse | This paper | PCR primers | 5’-ggagtggtggagtagagggtt-3’ |
| Sequence-based reagent | InR_forward | This paper | PCR primers | 5'-gcaccattataaccggaacc-3' |
| Sequence-based reagent | InR_reverse | This paper | PCR primers | 5'-ttaattcatccatgacgtgagc-3' |
| Sequence-based reagent | Akh_forward | This paper | PCR primers | 5’- agccgtgctcttcatgct-3’ |
| Sequence-based reagent | Akh_reverse | This paper | PCR primers | 5’-aaaggttccaggaccagctc-3’ |
| Sequence-based reagent | Hsl_forward | This paper | PCR primers | 5’-cttggaaatacttgaggggttg-3’ |
| Sequence-based reagent | Hsl_reverse | This paper | PCR primers | 5’-agatttgatgcagttctttgagc-3’ |
| Sequence-based reagent | bmm_forward | This paper | PCR primers | 5’-gtctcctctgcgatttgccat-3’ |
| Sequence-based reagent | bmm_reverse | This paper | PCR primers | 5’-ctgaagggacccagggagta-3’ |
| Sequence-based reagent | plin1_forward | This paper | PCR primers | 5’-gcgttctatggtagccttcag-3’ |
| Sequence-based reagent | plin1_reverse | This paper | PCR primers | 5’-gcgtccggatagaaagctg-3’ |
| Sequence-based reagent | plin2_forward | This paper | PCR primers | 5’-gcagaatggcaagagttctga-3’ |
| Sequence-based reagent | plin2_reverse | This paper | PCR primers | 5’-actgtgtgtaggactggatcctc-3’ |
| Sequence-based reagent | Rpl1_forward | This paper | PCR primers | 5’-tccaccttgaagaagggcta-3’ |
| Sequence-based reagent | Rpl1_reverse | This paper | PCR primers | 5’-ttgcggatctcctcagactt-3’ |
| Peptide, recombinant protein | Trehalase | Sigma Aldrich | T8778 | |
| Peptide, recombinant protein | β-Amyloglucosidase | Sigma Aldrich | 10115 | |
| Antibody | anti-phospho(Ser505)-AKT | Cell Signal Technology (CST) | Cat# 4054; RRID:AB_331414 | WB (1:1000) |
| Antibody | anti-AKT | Cell Signal Technology (CST) | Cat# 4691; RRID:AB_915783 | WB (1:1000) |
| Antibody | anti-phospho(Thr172)-AMPKα | Cell Signal Technology (CST) | Cat# 2535; RRID:AB_331250 | WB (1:1000) |
| Antibody | anti-phospho(Thr389)-p70 S6 kinase | Cell Signal Technology (CST) | Cat# 9206; RRID:AB_2285392 | WB (1:1000) |
| Antibody | anti-GFP | Cell Signal Technology (CST) | Cat# 2956; RRID:AB_1196615 | WB (1:1000) |
| Antibody | anti-phospho-p44/42 MAPK (Erk1/2) | Cell Signal Technology (CST) | Cat# 4370; RRID:AB_2315112 | WB (1:1000) |
| Antibody | anti-α-tubulin | Developmental Studies Hybridoma Bank) | Clone 12G10; RRID:AB_1157911 | WB (1:3000) |
| Antibody | HRP anti-rabbit IgG | Cell Signal Technology (CST) | Cat# 7074; RRID:AB_2099233 | WB (1:5000) |
| Antibody | HRP anti-mouse IgG | Cell Signal Technology (CST) | Cat# 7076; RRID:AB_330924 | WB (1:5000) |
| Commercial assay or kit | First Strand cDNA Synthesis Kit | Thermo Scientific | K1622 | |
| Commercial assay or kit | Sensimix SYBR Green no-ROX | Bioline | QT650-05 | |
| Chemical compound, drug | Bromophenol blue | Sigma Aldrich | SML1656 | |
| Chemical compound, drug | Xylene cyanol | Carl Roth | A513.1 | |
| Chemical compound, drug | Brilliant Blue FCF | Sigma Aldrich | 80717 | |
| Other | HCS Lipid Tox Deep Red | Thermo Fisher | H34477 | IF (1:200) |
| Other | Alexa Fluor 488 Phalloidin | Thermo Fisher | A12379 | IF (1:20) |
| Other | Fluoromount-G | ebioscience | 00-4958-02 | |
| Other | TRIzol | Invitrogen | AM9738 | |
| Other | Fixable Viability Dye 780 | ebioscience | 65-0865-18 | FC (1:1000) |
| Other | Supersignal West Pico Chemiluminescent Substrate | Thermo Scientific | 34077 | |
| Other | Supersignal West Femto Chemiluminescent Substrate | Thermo Scientific | 34094 | |
| Other | Glucose Reagent | Sentinel Diagnostics | 17630H | |
| Software, algorithm | ImageJ | ImageJ | RRID:SCR_003070 | |
| Software, algorithm | GraphPad Prism | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | FlowJo | FlowJo | RRID:SCR_008520) |
