Fig. 3

Activation of TRPML1 evokes cytosolic Zn²⁺ signals in neurons. a Representative micrographs of primary rat hippocampal neurons co-expressing mCherry-TRPML1^WT (left) and GZnP3 at baseline after ML-SA1 addition, or after TPEN treatment. Pseudocolor shows GZnP3 fluorescent intensity indicated by calibration bar (far right; minimum = white, maximum = black). Scale bar = 20 µm. b Representative traces of neurons co-expressing GZnP3 and mCherry-TRPML1^WT (blue) or mCherry-TRPML1^DDKK (magenta). Neurons were treated with ML-SA1 at 0 s and TPEN at 300 s. c Representative traces of neurons co-expressing GZnP3 and mCherry-TRPML1 pretreated with ML-SI4 at -300 s, ML-SA1 at 0 s, and TPEN at 300 s. d Representative traces of neurons co-expressing GZnP3 and mCherry-TRPML1 pretreated with TPEN at -300 s and ML-SA1 at 0 s. e Representative traces of neurons co-expressing GZnP3 and mCherry-TRPML1 treated with ML-SA1 at 0 sec, 2,2’-bipyridyl (Fe²⁺ chelator) at 180 s, and TPEN at 300 s. f Mean integrated GZnP3 signal (±s.e.m.) within 300 s after ML-SA1 addition for TRPML1^WT (blue, n = 10), TRPML1^DDKK (magenta, n = 7), inhibitor pretreatment (pre-ML-SI4, cyan, n = 4), TPEN pretreatment (pre-TPEN, gray, n = 3), and Fe²⁺ chelation (2,2’-bipyridyl, green, n = 4). One-way ANOVA with Dunnett’s multiple comparison to TRPML1^WT. g Representative traces of neurons co-expressing GZnP3 and NES-mCherry treated with 10–20 µM ML-SA5 at 0 s (gray arrow) and TPEN at 300 s. h Representative traces of neurons co-expressing mCherry-TRPML1 and either GZnP3 (blue) or NES-ZapCV2 (gray) treated as in b. i Mean maximum sensor signal (±s.e.m.) within 300 s after ML-SA1 addition for GZnP3 (blue, n = 10) and NES-ZapCV2 (gray, n = 3). Student’s one-tailed t-test. **p < 0.01, *p < 0.05. n.s., not significant. p-values displayed in italics above corresponding comparisons where applicable. 50 µM ML-SA1 (black arrow), 100 µM TPEN (white arrow), 50 µM ML-SI4 (cyan arrow), 10 µM 2,2’-bipyridyl (green arrow). Source data are provided as a Source Data file