(A) DAPI/Annexin V-FITC and DAPI/PI stained brains from 21 day-old flies.
(B) Percentage of early apoptotic and late apoptotic/necrotic cells in brains from 21 day-old flies. n≥3; *, difference from wt, p≤0.01, ANOVA.
(C) Brains from 21 day-old flies viewed at 310 nm to detect DAPI, 488 nm to detect anti-REPO, 568 nm to detect TUNEL and 633 nm to detect anti-ELAV. Arrows indicate glia.
(D) Percentage of pharate adults without the TM3 balancer. n=3–11; *, difference from trpml¹, p≤0.05, ANOVA.
(E) Climbing indices of adult flies. n≥5; *, difference from trpml¹, p≤5×10⁻⁵, t-test.
(F) Brains from 21 day-old flies viewed at 310 nm to detect DAPI, 488 nm to detect anti-REPO, 568 nm to detect TUNEL and 633 nm to detect ELAV. Arrows indicate glia.

(A) Time-course of flies with restored mobility following a 3 min exposure to CO₂. n=3, 10 flies per experiment.
(B) Excitatory junctional current (EJC) from the 3^rd instar NMJ.
(C) Quantification of the EJC amplitudes in wt and trpml¹. n=5 animals; 10 NMJs for each genotype; *, p≤0.001.
(D) NMJ synapses following FM1-43 loading and unloading. Arrows indicate synaptic boutons. Panels below show enlarged magnification of synaptic boutons.
(E) Quantification of FM1-43 fluorescence intensity in NMJ boutons following dye loading and unloading. n=5 animals,10 NMJs for each genotype; *, difference from wt, p≤0.001.
All statistical analyses, t-test.

(A) Ommatidia viewed by the optical neutralization technique ≤24 h post-eclosion.
(B) Time course of photoreceptor degeneration viewed by optical neutralization. n≥3 flies, ≥50 ommatidia/fly; *, difference at each time point, p≥0.05.
(C) Percentage of pharate adults without the TM3 balancer in wt, trpml¹ and trpml¹ with the indicated transgenes, n=7–11. GAL4 expression is indicated (fb, fat bodies).
(D) Climbing indices. n=3–8, 10–20 flies each.
(E) Merged confocal images of brains from 21 day-old flies viewed at 310 nm to detect DAPI and 488 nm to detect Annexin V-FITC.
(F) Percentages of adult brain cells labeled with Annexin V-FITC. n≥3 brains; *, difference from wt, p≤0.05.
All statistical analyses, t-test.

(A) Percentage of pharate adults without the TM3 balancer. n=7–11. GAL4 expression patterns are indicated.
(B) Climbing indices of adult flies. n=3–8 experiments, 10–20 flies each.
(C) Representative EJC traces of larvae.
(D) Quantification of the EJC amplitudes. n=5 animals, 10 NMJs for each genotype; *, p≤10⁻⁴, ANOVA. hem, hemocytes.
(E) Age-progressive decline in climbing index of wt (black) and trpml¹ flies expressing UAS-trpml and the indicated GAL4s. Indices are normalized with 5 day values as 100%. n≥3, 10–20 flies per experiment; *, difference from wt, p≤0.05, ANOVA.

(A and B) Transmission EMs of photoreceptor cells (21 day-old flies): (A) wt, and (B) trpml¹.
(C) Percentages of ommatidia with full set of 7 rhabdomeres. n≥3 animals, ≥30 ommatidia per animal; *, difference from wt, p≤0.01, ANOVA; ¶, p≤0.05, t-test.
(D and E) Confocal images of TUNEL-labeled brains (21 day-old flies): (D) wt, and (E) trpml¹.
(F) Fold increase in TUNEL labeling in trpml¹ normalized to wt mean. n=3; *, p≤5×10⁻⁴, t-test.
(G–I) Confocal images of brains from 21 day-old trpml¹ viewed at 310 nm to detect DAPI (G) and 568 nm to detect TUNEL (H). (I) Merge.
(J–L) Same as (G–I) but brains viewed at 633 nm to detect ELAV (J).
(M–O) Same as (G–I) but brains viewed at 488 nm to detect REPO (M). Arrows indicate glia.
(P–R) Confocal images of 21 day-old trpml¹ brains viewed at 310 nm to detect DAPI (P) and 488 nm to detect Annexin V-FITC (Q). (R) Merge.
(S) Same as (R) but with 21 day-old wt brains.

(A) Alignment of Drosophila and human TRPML proteins. Lines indicate TMDs.
(B) trpml genomic locus. The deletions in trpml¹ and trpml² are shown.
(C) Western blot of extracts from wild-type (wt) and trpml¹ flies probed with anti-TRPML antibodies and reprobed with anti-Rh1 antibodies (see Supplemental Experimental Procedures). The ~75 kDa band (arrowhead) corresponds to TRPML. The lower bands were due to non-specific interactions with anti-TRPML.
(D) Percentage of pharate adults without the TM3 balancer after inter se crosses. n≥4 vials of each genotype; *, p≤5×10⁻⁶, ANOVA. Df1, Df(3L)ED228; Df2, Df(3L)Exel6135; trpml^1/2, trpml¹/trpml²; P[trpml], genomic rescue; trpml^rev, precise excision.
(E) Percentage of dead pupae. n=4 vials; *, p≤0.005, ANOVA.
(F) Climbing indices. Percentages of flies in a 50 ml glass cylinder that climb to the 25 ml mark in 15 s after being tapped down. n≥13, 10–20 flies each; *, p≤×10⁻¹², ANOVA; ¶, p≤5×10⁻⁴, t-test.
(G) Number of crossings of an infra-red beam (24 h period) in an actometer. n=3, 13–14 individual 5 day-old flies each; *, p≤5×10⁻¹¹, t-test.

(A–D) Transmission EMs of 21 day-old photoreceptor cells: (A and B) wt, (C and D) trpml¹. (B and D) Magnifications of the boxed regions in (A and C). The red and yellow arrows in (D) indicate multilamellar and multivesicular bodies respectively.
(E) Fold-increase in autophagosome-like vesicles in trpml¹ photoreceptors. n=5 animals, ≥30 ommatidia per animal; *, p≤10⁻⁴.
(F and G) Brains (21 day-old flies) expressing GFP-ATG8 and viewed at 488 nm: (F) wt, (G) trpml¹.
(H) Fold-increase in GFP-ATG8 fluorescence in trpml¹ brains. n=3; *, p≤5×10⁻⁴.
(I–N) Ommatidia from 21 day-old flies expressing GFP-ATG8: (I–K) control, (L–N) trpml¹. Ommatidia were viewed at 488 nm to detect GFP-ATG8 (I–L) and 568 nm to detect LysoTracker (Lyso; J–M). (K and N) Merges.
(O) Fold-increase in GFP-positive vesicles in trpml¹ ommatidia. n=3 independent experiments; *, p≤5×10⁻⁵.
(P) Rh1 remaining after 6 h exposure to blue light (480 nm) in 5 day-old flies. Means are relative to the starting value (defined as 100%). n=3; *, p≤0.05.
(Q and R) 4 week-old brains stained with anti-ubiquitin antibodies (Supplemental Experimental Procedures): (Q) wt, (R) trpml¹.
(S) Western blot of head extracts (4 week-old flies) probed with anti-ubiquitin antibodies and reprobed with anti-Tubulin antibodies.
(T) Fold increase of ubiquitination in head extracts based on Western blotting. Data were normalized to wt (1.0). n=3; *, p ≤0.05.
(U) Mitochondria per photoreceptor cell in 21 day-old wt flies. n=5 animals; *, p≤0.05.
(V and W) Ommatidia from 21 day-old wt (V) and trpml¹ (W) flies viewed at 310 nm to detect DAPI (blue) and 568 nm to detect MitoTracker-orange CM-H2TMRos (red).
(X) Fold decrease in MitoTracker-orange CM-H2TMRos staining in trpml¹ head extracts, normalized to wt (set at 1.0). n=3; *, p≤5×10⁻⁶.
(Y) Relative H₂O₂ levels in whole fly extracts. Data were normalized to wt (set at 1.0). n≥6, 10 flies/experiment; *, p≤5×10⁻⁶. Dissected brains and ommatidia were viewed by confocal microscopy.
All statistical analyses, t-test.