Supplementary Figures

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1 J. Cell Sci. 128: doi: /jcs : Supplementary Material Supplementary Figures Fig. S1 Fig. S1. Description and/or validation of reagents used. All panels show Drosophila tissues oriented with anterior to the left, except the wing dics in panel B, which have anterior to the bottom. Genotypes are as marked. (A-C) Expression pattern of Gal4 lines used to drive UAS-transgene expression in gut (A), wing disc (B) and salivary glands (C). Stainings are as marked in panels using anti-ß-gal or anti-gfp Ab to detect Gal4-driven expression, except for GFP in panel A, which originates from the 4Mbox-GFP Mitf target. Staining for Dlg highlights cell membranes. In panel A, Malpighian tubules (MT), hindgut (HG), pylorus region join HG to midgut are marked. (D-G) Mitf RNAi reagents validation. (D) Overexpression of an HA tagged Mitf protein is severely decreased by co-expression of RNAi-VDRC TZ and VDRC dsrna transgenes in the eye disc under control of the strong driver GMR-Gal4. However, the UAS-Mitf RNAi-TZ1-3 RNAi-TZ4-8 is considerably more effective than either UAS-Mitf or UAS-Mitf, which show residual protein expression even when present in 3 copies (2xTZ1-3 plus 1xTZ4-8). Stainings in panels is as indicated for HA-Mitf (green; anti-ha Ab), Mitf (blue; anti-mitf Ab) or Elav (red; anti-elav); the latter protein marks all neurons of the developing eye field. (E) Mitf sequence targeted RNAi-VDRC ; corresponding changes to void RNAi targeting in Mitf-rescueSI1-RES are shown in red. (F) (xpression of by Mitf 4Mbox-GFP (green; left panel) is lost in the hindgut of byn-gal4 UAS-MitfRNAi-VDRC larvae (green; middle panel), but rescued by SI1-RES (green; right panel); Staining for ß-Galactosidase UHG showv domain of *al4 expression. (G) Schematic one copy of Mitf-rescue SI1-RES of crosses to induce UAS-MitfRNAi-VDRC expression in the hindgut with or without one copy of the Mitf-rescue genomic RNAi-VDRC progeny (which do not inherit the Gal4-suppressor Gal80) die at the L1-L2 stages; construct. The byn-gal4 UAS-Mitf the lethality is rescued by one copy of the Mitf-rescueSI1-RES construct, producing live flies. (H) The mutant protein MitfDelR cannot induce target gene expression. In the salivary glands, exogenous Mitf (bottom panel), but not MitfDelR (middle panel), can induce strong expression of the Vha55-lacZET enhancer-trap line.

2 Fig. S2 Fig. S2. Regulation of v-atpase gene expression and v-atpase activity by Mitf. In all panels, genotypes are as marked above and to the left of panels. (A-C) Regulation of Vha genes by Mitf-gain (A) and Mitf-loss (B-C). (A) Gal4-driven Mitf increases Vha gene expression in the pattern of the Gal4 driver. Expression of ET or GT Vha lines was compared in Gal4-only or Gal4 plus UAS-Mitf genetic backgrounds. In each case expression of the ET or GT reporter was robustly upregulated by Mitf. ET or GT expression is shown in green (anti-ß-gal or anti-gfp Ab; middle panels); Mitf expression is shown in red (anti-mitf Ab) (right panels). Bottom panels shows upregulation of the gene VhaAC39-1 by in situ hybridization; whereas Mitf is shown in red (anti-mitf Ab) in rightmost panel. (B-C) Effect of loss-of-mitf function on expression of Vha ET and GT lines. (B) The lines Vha26-lacZ ET and Vha55-lacZ ET are highly expressed in MG, HG, and MT of larvae (red; anti-ß-gal Ab), but expression is greatly diminished in all three tissues from Mitf TZ2 /Df(4)TZ. (C) Gal4-driven Mitf-RNAi decreases Vha gene expression in the pattern of the Gal4 driver (i.e. in the HG but not in the MT). In byn-gal4 UAS-GFP UAS-Mitf RNAi-VDRC larvae, expression of Vha26-lacZ ET, Vha55-lacZ ET, or Vha68-2-lacZ ET (red; anti-ß-gal Ab) is significantly reduced specifically in the HG, where the UAS-GFP transgene is expressed (green; anti-gfp Ab). In byn-gal4 UAS-Mitf RNAi-TZ1-3 UAS-Mitf RNAi-TZ4-8 larvae, expression of Vha13-GFP GT, Vha16-1-GFP GT or VhaSFD-GFP GT (green; anti-gfp Ab) is reduced specifically in HG, and not in MT. (D-E) Dominant suppression of Mitf-induced wing defects by Vha mutant alleles and expression of Vha55-GFP mrna in Mitf expressing eye discs. (D) Mutant alleles of Vha genes dominantly (-/+) suppress wing defects caused by dpp-gal4 UAS-Mitf ompare to and Mitf-expressing wings in Fig. 3A. Loss of one copy of Vha13, Vha26, Vha55, Vha68-2, or VhaSFD resulted in significant suppression of the wing defect. Notice that the Vha16-1 allele used here could not suppress the wing phenotype, is similar to the dpp-gal4 UAS-Mitf panel shown in Fig. 3A. (E) UAS-Vha55-GFP is similarly expressed at the mrna level in eye discs with and without Mitf. Panels show in situ hybridizations using an antisense GFP probe.

3 Fig. S3 Fig. S3. TORC1 regulates Mitf subcellular localization in Drosophila. (A) dpp-gal4 UAS-Mitf SG cells stained for exogenous Mitf (red; anti-mitf Ab) and nuclear DAPI (green). Knockdown of the TORC1 positive regulator RagA-B, the TORC1 component Raptor, or the cytoplasmic anchors shifts Mitf protein localization from cytoplasm to nucleus. Quantification of nuclear localization is shown in (A ) as percentage of total cells scored (Y-axis); one-tailed Student s t-test: ***p< or **p<0.01 as compared to dpp- Gal4 UAS-Mitf control. (B) Upregulation of TORC1 activity, through loss of negative (Gig/TSC2 and TSC1) or gain of positive (RagC-D) regulators, leads to suppression of the Mitf-induced wing phenotype (compare to WT wing and Mitf-induced defects in Fig. 3A); notice that heterozygocity for gig already has a strong suppression effect. (C) Quantification of nuclear localization of exogenous Mitf in SG cells after starvation of early L3 larvae. Dots show result for individual salivary glands.

4 Fig. S4 Fig. S4. Analysis of HA-MITF and lysosomal genes in 501mel melanoma cells and Proposed model of an adaptive Mitf/v-ATPase/TORC1 regulatory loop for cellular homeostasis. (A) MITF and HA-MITF expression in 501Mel. (B-C) ChIP-Seq peaks from HA-MITF bound at selected lysosomal loci (B) and summary of results from ChIP-Seq analysis (C) as compared to distribution of the CLEAR site among lysosomal loci (Sardiello et al., 2009); see Table S6A for gene list. Seventy-five of the 96 lysosomal genes showed binding by MITF. Binding was not limited to loci with CLEAR elements, nor were all CLEAR-containing loci bound by MITF (C). Nine genes reported to contain CLEAR sites showed no evidence of MITF binding and 16 loci without CLEAR sites showed up to 4 MITF peaks (Table S6A). Among the 59 genes that had CLEAR sites and that were bound by MITF, nearly half showed ChIP peaks in regions containing one or more CLEAR sequences, suggesting that in many but not all cases MITF binding may occur at or near TFEB binding sites. We believe that a considerable number of these binding events translate into increased gene expression, because 36 of these loci (out of the 75 MITF-bound) were reported as upregulated by at least 2 folds in MITF-transfected 501mel cells (Hoek et al., 2008). Given the relatively high cut-off (2 fold) used in this study, we consider this gene number an underestimate of the lysosome-related transcriptome of MITF in this cell line. (D) Proposed model for an adaptive Mitf/v-ATPase/TORC1 regulatory loop for cellular homeostasis. The Mitf/v- ATPase/TORC1 regulatory loop offers a dynamic mechanism for continuously optimizing metabolic activity as external conditions fluctuate. On one hand, as nutrients become scarce, active TORC1 levels drop and Mitf locates to the nucleus leading to enrichment in functional v-atpases at the lysosome as well as other factors involved in degradative pathways. This leads to enhancement of catabolic pathways over anabolic ones. Under these conditions, the enrichment in v-atpase at the lysosome would sensitize the nutritional sensing mechanism to amino acids increases, thereby priming the system to reset at a new normal through v-atpase-dependent recruitment of TORC1 to the lysosomal membrane and subsequent TORC1 activation. Such mechanism would set a limit on upregulation of catabolic pathways under low nutrient conditions thereby avoiding extensive cellular damage. On the other hand, when nutrients are abundant, TORC1 activity would remove Mitf to the cytoplasm and consequently v-atpase levels at the lysosome would decrease. This would ultimately impose a limit on TORC1 activation even in the presence of amino acids. At the same time, the sensing mechanism would adapt to these new conditions by lowering its sensitivity to amino acid, through the reduction in v-atpase at the lysosomal membrane.

5 Table S1 Click here to Download Table S1 Figure # Table S2: genotypes of samples in figures Genotype Temperature 1C w; Mitf 2.2 -GFP 25 C 1E-F w; 4Mbox-GFP 25 C 1G w; 4Mbox-GFP/+; Mitf TZ2 /Df(4)TZ 25 C 1H w; 4Mbox-GFP/UAS-Mitf RNAi-TZ1-3, UAS-Mitf RNAi-TZ4-8 ; byn-gal4/+ 25 C 1I w; 4Mbox-GFP/UAS-Mitf RNAi-VDRC ; byn-gal4/+ 25 C 2A 2E 2F 2G w; nub-gal4/+; UAS-Mitf/+ 25 C w; nub-gal4/+ 25 C w; en-gal4/+; Vha26-lacZ ET /tub-gal80 ts w; en-gal4/+; Vha26-lacZ ET /UAS-Mitf, tub-gal80 ts w; Ser-Gal4/Vha16-1-GFP GT ; tub-gal80 ts /+ w; Ser-Gal4/Vha16-1-GFP GT ; UAS-Mitf, tub-gal80 ts /+ w; Vha68-2-lacZ ET /+ 25 C w; Vha68-2-lacZ ET /+; Mitf TZ2 /Df(4)TZ 25 C 2H w; Vha68-2-lacZ ET / UAS-Mitf RNAi-TZ1-3, UAS-Mitf RNAi-TZ4-8 ; byn-gal4, UAS-GFP/+ 25 C 3A w; dpp-gal4/+ 25 C w; UAS-Mitf/+; dpp-gal4/+ 25 C w; Vha14 - /UAS-Mitf; dpp-gal4/+ 25 C 3C w; dpp-gal4/uas-vha55-gfp 25 C 3D w; UAS-Mitf/+; dpp-gal4/uas-vha55-gfp 25 C 3E w; en-gal4/+; UAS-Mitf, tub-gal80 ts /+ 3F w; Rab2 - /UAS-Mitf; dpp-gal4/+ 25 C w; Cp1 - /UAS-Mitf; dpp-gal4/+ 25 C w; spin - /UAS-Mitf; dpp-gal4/+ 25 C w; UAS-Mitf/+; dpp-gal4/uas-lamp1 RNAi 25 C

6 3G 3H 3I 3J 3K 3L w; en-gal4/+; tub-rab7-yfp/uas-mitf, tub-gal80 ts w; dpp-gal4/uas-spin-gfp 25 C w; UAS-Mitf/+; dpp-gal4/uas-spin-gfp 25 C w; byn-gal4/tub-rab7-yfp 25 C w; UAS-Mitf RNAi-TZ1-3, UAS-Mitf RNAi-TZ4-8 /+; byn-gal4/tub-rab7-yfp 25 C w; UAS-spin-GFP/+; byn-gal4/+ 25 C w; UAS-Mitf RNAi-TZ1-3, UAS-Mitf RNAi-TZ4-8 /UAS-spin-GFP; byn-gal4/+ 25 C w; dpp-gal4/tub-lamp1-gfp 25 C w; UAS-Mitf/+; dpp-gal4/tub-gfp-lamp1 25 C w; tub-gfp-lamp1 /+; byn-gal4/+ 25 C w; UAS-Mitf RNAi-TZ1-3, UAS-Mitf RNAi-TZ4-8 /tub-lamp1-gfp; byn-gal4/+ 25 C 4C w; 4Mbox-GFP/+; dpp-gal4/uas-mitf.myc 25 C 4E 4H 5A 5B 5C 5D 5E w; Vha68-2-GFP 25 C w; Vha68-2 -GFP 25 C w; Vha13-GFP 25 C w; Vha13 -GFP 25 C w; ato5 FL-Gal4/+; UAS-Mitf.myc/UAS-lacZ.nls 25 C w; ato5 FL-Gal4/+; UAS-Mitf DelN1.myc/UAS-lacZ.nls 25 C w; ato5 FL-Gal4/+; UAS-HA.Mitf/UAS-lacZ.nls 25 C w; ato5 FL-Gal4/+; UAS-HA.Mitf DelC1 /UAS-lacZ.nls 25 C w; ey-gal4/+; UAS-Mitf/+ 25 C/L2 w; ey-gal4/+; UAS-Mitf/UAS-gig RNAi 25 C/L2 w; ey-gal4/+; UAS-Mitf/UAS-RagA-B RNAi 25 C/L2 w; UAS-Mitf/+; dpp-gal4/+ 25 C w; UAS-Mitf/+; dpp-gal4/uas-vhasfd RNAi 25 C w; dpp-gal4/uas-mitf delr 25 C w; UAS-Mitf/+; dpp-gal4/uas-mitf DelR 25 C w; dpp-gal4/+ 25 C w; UAS-Mitf/+; dpp-gal4/+ 25 C w; UAS-Mitf/+; dpp-gal4/uas-vhasfd RNAi 25 C S1A w; 4Mbox-GFP/+; byn-gal4, UAS-lacZ/+ 25 C w; nub-gal4/+; UAS-GFP/+ 25 C S1B w; en-gal4/+; UAS-GFP/+ 25 C w; Ser-Gal4/+; UAS-GFP/+ 25 C w; dpp-gal4, UAS-GFP/+ 25 C S1C w; ato5 FL-Gal4/+; UAS-lacZ.nls/+ 25 C w; ey-gal4/+; UAS-GFP/+ 25 C

7 S1D S1F S1H S2A S2B S2C w; GMR-Gal4/+; UAS-HA.Mitf/UAS-dicer2 25 C w; GMR-Gal4/ UAS-Mitf RNAi-TZ1-3, UAS-Mitf RNAi-TZ4-8 ; UAS-HA.Mitf/UAS-Mitf RNAi-TZ1-3, UAS-dicer2 w; GMR-Gal4/UAS-Mitf RNAi-VDRC ; UAS-HA.Mitf/UAS-dicer2 25 C w; 4Mbox-GFP/+; byn-gal4, UAS-lacZ/+ 18 or 25 C w; 4Mbox-GFP/UAS-Mitf RNAi-VDRC ; byn-gal4, UAS-lacZ/+ 18 or 25 C w; 4Mbox-GFP/UAS-Mitf RNAi-VDRC ; byn-gal4, UAS-lacZ/Mitf-rescue Si1-RES 18 or 25 C w; dpp-gal4, Vha55-lacZ ET /+ 25 C w; dpp-gal4, Vha55-lacZ ET /UAS-Mitf DelR 25 C w; dpp-gal4, Vha55-lacZ ET /UAS-Mitf 25 C w; en-gal4/+; Vha55-lacZ ET /tub-gal80 ts w; en-gal4/+; Vha55-lacZ ET /UAS-Mitf, tub-gal80 ts w; en-gal4/vha68-2-lacz ET ; tub-gal80 ts /+ w; en-gal4/vha68-2-lacz ET ; UAS-Mitf, tub-gal80 ts /+ w; Ser-Gal4/+; Vha13-GFP GT /tub-gal80 ts w; Ser-Gal4/+; Vha13-GFP GT /UAS-Mitf, tub-gal80 ts w; Ser-Gal4/VhaSFD-GFP GT ; tub-gal80 ts /+ w; Ser-Gal4/VhaSFD-GFP GT ; UAS-Mitf, tub-gal80 ts /+ 25 C w; dpp-gal4/+ 25 C w; UAS-Mitf/+; dpp-gal4/+ 25 C w; Vha26-lacZ ET /+ 25 C w; Vha26-lacZ ET /+; Mitf TZ2 /Df(4)TZ 25 C w; Vha55-lacZ ET /+ 25 C w; Vha55-lacZ ET /+; Mitf TZ2 /Df(4)TZ 25 C w; byn-gal4, UAS-GFP/Vha26-lacZ ET 18 C w; UAS-Mitf RNAi-VDRC /+; byn-gal4, UAS-GFP/Vha26-lacZ ET 18 C w; byn-gal4, UAS-GFP/Vha55-lacZ ET 18 C w; UAS-Mitf RNAi-VDRC /+; byn-gal4, UAS-GFP/Vha55-lacZ ET 18 C w; Vha68-2-lacZ ET /+; byn-gal4, UAS-GFP/+ 18 C w; Vha68-2-lacZ ET /UAS-Mitf RNAi-VDRC ; byn-gal4, UAS-GFP/+ 18 C w; byn-gal4/vha13-gfp GT 25 C w; UAS-Mitf RNAi-TZ1-3, UAS-Mitf RNAi-TZ4-8 /+; byn-gal4/vha13-gfp GT 25 C w; Vha16-1-GFP GT /+; byn-gal4/+ 25 C w; Vha16-1-GFP GT / UAS-Mitf RNAi-TZ1-3, UAS-Mitf RNAi-TZ4-8 ; byn-gal4/+ 25 C

8 w; VhaSFD-GFP GT /+; byn-gal4/+ 25 C w; VhaSFD-GFP GT / UAS-Mitf RNAi-TZ1-3, UAS-Mitf RNAi-TZ4-8 ; byn-gal4/+ 25 C w; UAS-Mitf/+; dpp-gal4/vha13-25 C w; UAS-Mitf/+; dpp-gal4/vha26-25 C S2D w; UAS-Mitf/+; dpp-gal4/vha55-25 C w; Vha /UAS-Mitf; dpp-gal4/+ 25 C w; VhaSFD - /UAS-Mitf; dpp-gal4/+ 25 C w; Vha /UAS-Mitf; dpp-gal4/+ 25 C S2E w; dpp-gal4/uas-vha55-gfp 25 C w; UAS-Mitf/+; dpp-gal4/uas-vha55-gfp 25 C w; UAS-Mitf/+; dpp-gal4/+ 25 C w; UAS-Mitf/+; dpp-gal4/uas-raga-b RNAi 25 C S3A w; UAS-Mitf/+; dpp-gal4/uas-raptor RNAi 25 C w; UAS-Mitf/+; dpp-gal4/uas ζ RNAi 25 C w; UAS-Mitf/+; dpp-gal4/uas RNAi 25 C w; UAS-Mitf/+; dpp-gal4/uas-gig RNAi 25 C S3B w; UAS-Mitf/+; dpp-gal4/uas-tsc1 RNAi 25 C w; UAS-Mitf/+; dpp-gal4/gig - 25 C w; UAS EP -RagC-D/UAS-Mitf; dpp-gal4/+ 25 C S3C w; UAS-Mitf/+; dpp-gal4/+ 25 C Table S3 Click here to Download Table S3 Table S4 Click here to Download Table S4

9 Table S5 Click here to Download Table S5 Table S6 Click here to Download Table S6 Table S7 Click here to Download Table S7

10 TABLE S8:DNA primers used in this work Name KO5 F2 KO5 R1 KO3 F1 KO3 R1 5 KO1 White-R KO3TR1 KO3CF2 Mbox1forw Mbox1rev MitfPF1 MitfPR TZ1for TZ3rev TZ4for TZ8rev MitfCDS5 MitfCDS3 KO5 F2 In1R In2F Mitf3R2 Vha13Chip-F Vha13Chip-R Vha14-1Chip-F Vha14-1Chip-R Vha26Chip-F Vha26Chip-R Vha36-1Chip-F Vha36-1Chip-R Vha68-2Chip-1F Vha68-2Chip-1R 4MboxChIP-F 4MboxChIP-F atochip-f atochip-r Sequence 5 -CGCGGATCCGGTATTATGTACATAGAAGATTAGGC-3 5 -CGCGGATCCGCCTCAGCAGTTAAAGATTCGG-3 5 -CGCGCGGCCGCGTCGATCACTTTTTGACCAACC-3 5 -CGCGCGGCCGCAATATAATCATCGAATGTGTAC-3 5 -CAAGATATGTAAGACGATGATCCTCG-3 5 -TTAGCTTGGCTGCAGGTCGA-3 5 -CCCAAGCAGTTAGTTGAGTG-3 5 -CCGTCCATAATAGATTAACAT-3 5 -GCTCTAGACGGGAGGTACAGT-3 5 -CGCGGATCCAGATGC AGATCTCGAG-3 5 -CACTCCGCGGAAATACCTTATCGATAAA TTCAGATATG-3 5 -CGCGGATCCATTTAGGATTAAATTTTTTATACTTAG G-3 5 -TGACGGAATCTGGAATCGATTTG-3 5 -TTCGCTGGTATGACTGCCCCAC-3, 5 -GGCTGTGAGT GCTAAAAGAATTATGC-3 5 -GTGATCGACGACTCCGAGAAGCAG-3 5 -CGCAGATCTATGACGGAATCTGGAATCGATTTG-3 5 -CGGAGGCCTTTAACTCAATATCCAATGTGTCAGATGC-3 5 -CGCGGATCCGGTATTATGTACATAGAAGATTAGGC-3 5 -CGCGGTACCTCTTTAGTTGAGATTTTTACCAATTG-3 5 -CGCGGTACCCGACAACAAATGAGTCGATATAC-3 5 -CACTCCGCGGCAATATAATCATCGAATGTGTAC-3 5 -CGTGGTGTATACTGTGCAAGTC-3 5 -CAGCTTTCCGGCACACTGC-3 5 -CTTACGAGCGATGTGGACAG-3 5 -CAGAAGATACCGTTACGCTACG-3 5 -GATGGCGAAGTCATGGGATC-3 5 -CGTTAAAGTTTTGATAACGTAAGGGG-3 5 -CATTGCAAAAAGGGTCATCTGACT-3 5 -GAGCCACGCAAACAGCTG-3 5 -TGCGCGTGGAAAATTATTTTGCC-3 5 -GCTCTCTCGCTCTCCATTC-3 5 -GCGAATTCGGCTTGCTCTAGAC-3 5 -GCTTAGCGACGTGTTCACTTTGC-3 5 -ACAGATCCCGGCAGACAGT-3 5 -CAATGCAGTAGTGCGACTGTC-3