TPC2 defects in vivo require Ca 2+ channel activity in the lysosome and Rab interactivity. (a) Schematic of TPC2-mCherry depicting mutations within the N-terminal Rab binding motif to disrupt Rab interaction (RD) and the S4–S5 linker to disrupt lipid binding (LD). Mutations within the pore (PD) and endo-lysosomal targeting motif (PM) are also highlighted. (b) Visual responses in lamina neurons from transgenic flies expressing mCherry-tagged WT TPC2, the indicated mutants, and TPC1 following disco treatment (mean ± SEM, n = 8–24). Flies expressing the EV were used as controls. Each point represents the response from an individual animal. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA, Dunnett’s test). (c) Exemplar crawling tracks from transgenic flies expressing mCherry-tagged WT TPC2, the indicated mutants, and TPC1. (d) Summary data (mean ± SEM, n = 16–36) quantifying movement of the indicated transgenic larva. Larvae expressing the EV were used as controls. Each point represents the distance traveled in 60 s by an individual animal. **P < 0.01, ****P < 0.0001 (one-way ANOVA, Dunnett’s test). (e–g) Effect of TPC2 agonists on movement. Transgenic flies expressing EV or TPC2-GCaMP6s were fed with 20 μM TPC2-A1-N or TPC2-A1-P (e) or increasing concentrations of TPC2-A1-P (f and g). Data (mean ± SEM) were acquired 5 days after feeding where each point represents the response from an individual animal. *P < 0.05, **P < 0.01 (unpaired t test). WT, wild type; PM, plasma membrane; RD, rab-deaad; LD, lipid-dead; EV, empty vector.
