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, 22 (17), 1616-21

Drosophila TRPML Is Required for TORC1 Activation

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Drosophila TRPML Is Required for TORC1 Activation

Ching-On Wong et al. Curr Biol.

Abstract

Loss-of-function mutations in TRPML1 (transient receptor potential mucolipin 1) cause the lysosomal storage disorder, mucolipidosis type IV (MLIV). Here, we report that flies lacking the TRPML1 homolog displayed incomplete autophagy and reduced viability during the pupal period--a phase when animals rely on autophagy for nutrients. We show that TRPML was required for fusion of amphisomes with lysosomes, and its absence led to accumulation of vesicles of significantly larger volume and higher luminal Ca(2+). We also found that trpml(1) mutant cells showed decreased TORC1 (target of rapamycin complex 1) signaling and a concomitant upregulation of autophagy induction. Both of these defects in the mutants were reversed by genetically activating TORC1 or by feeding the larvae a high-protein diet. The high-protein diet also reduced the pupal lethality and the increased volume of acidic vesicles. Conversely, further inhibition of TORC1 activity by rapamycin exacerbated the mutant phenotypes. Finally, TORC1 exerted reciprocal control on TRPML function. A high-protein diet caused cortical localization of TRPML, and this effect was blocked by rapamycin. Our findings delineate the interrelationship between the TRPML and TORC1 pathways and raise the intriguing possibility that a high-protein diet might reduce the severity of MLIV.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Increased accumulation of late-endosomes and amphisomes in trpml1 mutants
(A–C) TRPML::MYC (A, green) decorates the periphery of LysoTracker-positive vesicles (B, magenta) in vivo. Confocal images of 3rd instar larval fat bodies dissected from animals expressing UAS-trpml::myc under the control of fat-body specific lsp2-GAL4. The TRPML::MYC localization was detected using anti-MYC. (C) Merged image of (A–B). Scale bar shown in (A) applies to (A–C). (D–F) TRPML::MYC (D, magenta) colocalizes with LAMP::GFP-positive vesicles (E, green) in vivo. Confocal images of 3rd instar larval fat bodies dissected from animals expressing UAS-trpml::myc under the control of fat-body specific cg-GAL4. The TRPML::MYC localization was detected using anti-MYC. (F) Merged image of (D–E). Scale bar shown in (D) applies to (D–F). (G–J) Wild-type (wt) (G and I) and trpml1 (H and J) 3rd instar larval (96 hours old) wing discs stained with: (G and H) anti-Wingless (αWg), (I and J) anti-Hindsight (αHnt), and (N and O) anti-Notch (Notch). The scale bar shown in (G) applies to (G–J). (K) Quantification of increased Wingless and Notch fluorescent intensity in the trpml1 and P[trpml1];trpml1 wing discs normalized to wt. Values represent mean ±SEM; “*” indicates p<0.05, Student’s t-test (n≥3). (L–M) Fat bodies dissected from normally-fed wild-type (wt) (L) and trpml1 (M) wandering 3rd instar larvae expressing UAS-GFP::atg8 under the control of the fat-body driver cg-GAL4. The tissues were stained with anti-GFP (ATG8, green) and LysoTracker (LysoT, magenta). The scale bar shown in L also applies to M. (N) Magnified image of the boxed area from (M) showing autophagosomes (aut, vesicles labeled only by anti-GFP), and amphisomes (amph, vesicles labeled by both anti-GFP and LysoTracker).
Figure 2
Figure 2. Ultrastructural Analyses of vesicle fusion defects in trpml1
(A–B) Representative transmission EM images of normally fed wild-type (wt) (A) and trpml1 (B) Wandering 3rd instar larval fat-bodies. The scale bar shown in (A) also applies to (B). (C) Magnified image of the boxed area shown in (A). Blue line indicates two lysosomes in the process of fusing. Scale bar shown in (C) also applies to (D–F). (D) Box 1 from (B). (E) Box 2 from (B). (F) Magnified image showing two fusion-clamped lysosomes. The structures labeled include lysosomes (lyso) and multivesicular bodies (MVB). The dashed red lines indicate the area of contact between the “fusion-clamped” vesicles.
Figure 3
Figure 3. Elevated lysosomal Ca2+, increased induction of autophagy, and decreased TORC1 signaling in the trpml1 mutants
(A) Graph showing overall changes in F340/F380 ratio in wt (black) and trpml1 (magenta) tissues. Arrow represents point at which bafilomycin A1 was added. Values represent mean ±SEM, n~70 cells of each genotype (7 separate experiments with ~10 cells per experiment). (B) Bar graph showing change in Fura-2 F340/F380 ratio from t=0 sec to t=600 sec. Values represent mean ±SEM. “*” represents p<0.05, Student’s t-test (n=7 experiments per genotype). (C–E) Volumetric 3-dimensional reconstructions of a 400 mm2 area (20 mm × 20 mm) showing LysoTracker (magenta) and DAPI (blue) stained 3rd instar larval fat bodies of the indicated genotypes and feeding status. The scale bar shown in (C) is applicable to (C–E). (F) Volumes of LysoTracker-positive vesicles in fat-bodies dissected from 1st, 2nd and 3rd instar wt and trpml1 larvae (L1-L3 respectively). Values were normalized to wt for each larval stage. Values represent mean ±SEM. “*” represents p<0.05, Student’s t-test (n=5–7 animals of each genotype). (G) Representative Western blot of larval fat-body extracts derived from wild-type (wt) and trpml1 and yeast-fed trpml1 3rd instar larvae probed with antibodies against - phosphorylated S6K (pS6K) and α-tubulin (Tub). (H) Quantification of relative pS6K intensities in fat-body extracts derived from wandering 3rd instar larvae of the indicated genotypes and feeding status. Values were normalized to wt. Values represent mean ±SEM. “*” indicates p<0.05, Student’s t-test (n=5–9 independent Western blots using different samples each time). The term cg refers to cg-GAL4, a fat-body and hemocyte specific driver. (I–J) Volumetric 3-dimensional reconstructions of a 400 mm2 area (20 mm × 20 mm) showing LysoTracker (magenta) and DAPI (blue) stained 3rd instar trpml1 larval fat bodies. (J) shows LysoTracker-positive vesicles after thapsigargin (TG) treatment. The scale bar shown in (I) is applicable to (I–J). (K) Bar graph showing the relative volumes of LysoTracker-positive vesicles in fatbodies dissected from 3rd instar trpml1 larvae that were either untreated or treated with TG. Values were normalized to the untreated samples. Values represent mean ±SEM. “*” represents p<0.05, Student’s t-test (n=9 animals of each genotype).
Figure 4
Figure 4. Activating TORC1 by feeding larvae a protein-rich diet suppresses the toxicity associated with the loss of trpml1
(A) Bar graph showing survival of flies of the indicated genotypes that were reared on either standard food or food containing 20% yeast. Values represent mean ±SEM. “*” indicates p<0.05, Student’s t-test (n≥3 independent fly vials). (B) Bar graph showing survival of flies of the indicated genotypes that were reared on either standard food or food containing the indicated additional feedants: trypt. (tryptone) or suc. (sucrose). Values represent means ±SEM. “*” indicates p<0.05, ANOVA followed by pairwise Student’s t-test with Bonferroni post-hoc corrections (n≥3 independent fly vials). (C) Relative survival of wt and trpml1 animals through the pupal period when reared on food in the presence or absence of 1 µM rapamycin (rap). The values were normalized to the survival of flies reared without rapamycin. Values represent mean ± SEM. “*” indicates p<0.05, Student’s t-test; n.s. indicated no significance (n≥3 independent fly vials). (D) Confocal images showing TRPML::MYC (green) and phalloidin (blue) staining in fat-bodies dissected from 3rd instar larvae expressing UAS-trpml::myc under the control of the cg-GAL4. The tissues dissected from yeast-fed larvae are indicated. The scale bar shown in the top left panel is applicable to all of the panels. (E) Confocal images showing TRPML::MYC staining in fat-bodies dissected from 3rd instar larvae expressing UAS-trpml::myc under the control of cg-GAL4. The tissues dissected from yeast-fed and yeast + rapamycin-fed larvae are indicated. The scale bar shown in the left panel is applicable to both panels. (F) Model of TRPML function in regulating endosomal trafficking and TORC1 activity. PM, plasma membrane; EE, early-endosome; LE, late-endosome; aut, autophagosome; lys, lysosome.

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