Figure 5.
Human TPC2 expressed in Drosophila is functional. (a) Schematic depicting the absence and presence of TPC homologs in major insect order genomes within Endopterygota. (b) Synteny analyses in the vicinity of the TPCN gene in the indicated insect. Abbreviations: Cfe, Ctenocephalides felis; Dme, Drosophila melanogaster; Pbr, Pieris brassicae; Cca, Chrysoperla carnea; Tca, Tribolium castaneum; Ame, Apis mellifera. Chromosome assignments are shown to right except for CfeTPC (scaffold, ASM342690v1 CF-g0.4_SCAFFOLD_01886, which is currently unassigned). (c and d) Confocal micrographs of Drosophila S2R+ cells expressing LAMP1-GFP, wild-type or pore-dead TPC2-mCherry and TPC2-GCaMP6s, and TPC2PM-mCherry. In c, cells were stained with DAPI. In d, cells coexpressed LAMP1 or were labeled with BioTracker to mark the plasma membrane. Scale bar: 10 µm. (e) Colocalization analyses of the indicated TPC2 construct with LAMP1 and BioTracker using Pearson’s correlation coefficient (left) and intensity analysis (right) from the images in d. Negative controls for correlations (−) were obtained upon image rotation. (f) Schematic of TPC2 fused at its C terminus with the genetically encoded Ca2+ indicator GCaMP6s. (g) Ca2+ signals (mean ± SEM, n = 3 independent biological replicates) in response to TPC2-A1-N (30 µM) or SGA-10 (30 µM) in combination with TPC2-A1-P (30 µM) in cells expressing wild-type (left) or pore-dead TPC2-GCaMP6s (right). Cells were stimulated in the absence of external Ca2+ and with ionomycin (10 µM) toward the end of the recording using DMSO as a vehicle (Veh.) control. (h) Summary data (mean ± SEM, n = 3 independent biological replicates) quantifying the peak change in the Ca2+ signals from the indicated cells and treatment. Each point represents the mean response from a cell population. **P < 0.01 (two-way ANOVA, Tukey’s test). (i) Schematic of TPC2-mCherry depicting mutations within the N-terminal endo-lysosomal targeting motif to redirect it to the PM. (j) Ca2+ signals (mean ± SEM, n = 3 independent biological replicates) in response to TPC2-A1-N (30 µM) or SGA-10 (30 µM) in combination with TPC2-A1-P (30 µM) in cells expressing TPC2PM (left) or empty vector (right). (k) Summary data (mean ± SEM, n = 3 independent biological replicates) from quantifying the peak change in the Ca2+ signals from the indicated cells and treatment. Each point represents the mean response from a cell population. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA, Tukey’s test). PM, plasma membrane.

Human TPC2 expressed in Drosophila is functional. (a) Schematic depicting the absence and presence of TPC homologs in major insect order genomes within Endopterygota. (b) Synteny analyses in the vicinity of the TPCN gene in the indicated insect. Abbreviations: Cfe, Ctenocephalides felis; Dme, Drosophila melanogaster; Pbr, Pieris brassicae; Cca, Chrysoperla carnea; Tca, Tribolium castaneum; Ame, Apis mellifera. Chromosome assignments are shown to right except for CfeTPC (scaffold, ASM342690v1 CF-g0.4_SCAFFOLD_01886, which is currently unassigned). (c and d) Confocal micrographs of Drosophila S2R+ cells expressing LAMP1-GFP, wild-type or pore-dead TPC2-mCherry and TPC2-GCaMP6s, and TPC2PM-mCherry. In c, cells were stained with DAPI. In d, cells coexpressed LAMP1 or were labeled with BioTracker to mark the plasma membrane. Scale bar: 10 µm. (e) Colocalization analyses of the indicated TPC2 construct with LAMP1 and BioTracker using Pearson’s correlation coefficient (left) and intensity analysis (right) from the images in d. Negative controls for correlations (−) were obtained upon image rotation. (f) Schematic of TPC2 fused at its C terminus with the genetically encoded Ca2+ indicator GCaMP6s. (g) Ca2+ signals (mean ± SEM, n = 3 independent biological replicates) in response to TPC2-A1-N (30 µM) or SGA-10 (30 µM) in combination with TPC2-A1-P (30 µM) in cells expressing wild-type (left) or pore-dead TPC2-GCaMP6s (right). Cells were stimulated in the absence of external Ca2+ and with ionomycin (10 µM) toward the end of the recording using DMSO as a vehicle (Veh.) control. (h) Summary data (mean ± SEM, n = 3 independent biological replicates) quantifying the peak change in the Ca2+ signals from the indicated cells and treatment. Each point represents the mean response from a cell population. **P < 0.01 (two-way ANOVA, Tukey’s test). (i) Schematic of TPC2-mCherry depicting mutations within the N-terminal endo-lysosomal targeting motif to redirect it to the PM. (j) Ca2+ signals (mean ± SEM, n = 3 independent biological replicates) in response to TPC2-A1-N (30 µM) or SGA-10 (30 µM) in combination with TPC2-A1-P (30 µM) in cells expressing TPC2PM (left) or empty vector (right). (k) Summary data (mean ± SEM, n = 3 independent biological replicates) from quantifying the peak change in the Ca2+ signals from the indicated cells and treatment. Each point represents the mean response from a cell population. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA, Tukey’s test). PM, plasma membrane.

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