Figure 7. MSC PIEZO1 imports Ca 2 to... | Rockefeller University Press

Figure 7.

MSC PIEZO1 imports Ca2+to trigger the fusion between lysosomes and the plasma membrane. (A) Representative wide-field epifluorescent images of the local Ca2+ level at FAs detected by RCaMP6-FAT. Bar, 10 µm. Images are inverted to get a better contrast. (B) Quantification results of RCaMP6-FAT punctate size in HeLa cells (mean ± SD; n = 10 images; N = 2 biological replicates; unpaired two-tailed t test; *P ≤ 0.05). (C) Quantification results of RCaMP6-FAT punctate fluorescent intensity in HeLa cells (mean ± SD; n = 10 images; N = 2 biological replicates; unpaired two-tailed t test; **P ≤ 0.01). (D) Representative plasma membrane repair assay results in TRPML1-KO and PIEZO1-KO HeLa cells. (E) Representative results of HEX secretion in TRPML1-KO and PIEZO1-KO HeLa cells. Data are the mean ± SD from three independent experiments (One-way ANOVA with Dunnett correction; ***P ≤ 0.001; ns, P ≥ 0.05). See also Fig. S5 A. (F) Representative CLSM images of WT, TRPML1-KO, and PIEZO1-KO HeLa cells stained with vinculin. Bar, 10 µm. (G) Quantification results of FAs >0.5 µm2 (mean ± SD; n = 10 images; N = 2 biological replicates; One-way ANOVA with Dunnett correction; **P ≤ 0.01; ns, P ≥ 0.05). (H) Representative results of WT U2OS cells transiently transfected with VAMP7-pHluorin before and after treatment with GsMTx4 (5 µM, 5 min). Summary of 30 s TIRF microscopic live imaging. Lysosome-plasma membrane fusion events are represented as magenta dots. Bar, 5 µm. See also Video 7. (I) Representative results of WT U2OS cells transiently transfected with VAMP7-pHluorin before and after treatment with Yoda1 (1.5 µM, 5 min). Summary of 30 s TIRF microscopic live imaging. Lysosome-plasma membrane fusion events are represented as magenta dots. Bar, 5 µm. See also Video 8. (J) Representative results of WT U2OS cells transiently transfected with VAMP7-pHluorin before and after treatment with ML-SI3 (5 µM, 5 min). Summary of 30 s TIRF microscopic live imaging. Lysosome–plasma membrane fusion events are represented as magenta dots. Bar, 5 µm. See also Video 9. (K) Representative results of WT U2OS cells transiently transfected with VAMP7-pHluorin before and after treatment with ML-SA1 (20 µM, 5 min). Summary of 30 s TIRF microscopic live imaging. Lysosome–plasma membrane fusion events are represented as magenta dots. Bar, 5 µm. See also Video 10. (L) Quantification results of Fig. 7, H–K. Note the number of lysosomal exocytosis events varies in untreated cells in different groups, mainly due to the different VAMP7-pHluorin expression level. (n = 15 cells, N = 5 biological replicates for GsMTx4, 13 cells, 4 biological replicates for Yoda1, 10 cells, 4 biological replicates for ML-SI3, and 15 cells, 5 biological replicates for ML-SA1; mean ± SD; paired t test; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001).

MSC PIEZO1 imports Ca ²⁺ to trigger the fusion between lysosomes and the plasma membrane. (A) Representative wide-field epifluorescent images of the local Ca²⁺ level at FAs detected by RCaMP6-FAT. Bar, 10 µm. Images are inverted to get a better contrast. (B) Quantification results of RCaMP6-FAT punctate size in HeLa cells (mean ± SD; n = 10 images; N = 2 biological replicates; unpaired two-tailed t test; *P ≤ 0.05). (C) Quantification results of RCaMP6-FAT punctate fluorescent intensity in HeLa cells (mean ± SD; n = 10 images; N = 2 biological replicates; unpaired two-tailed t test; **P ≤ 0.01). (D) Representative plasma membrane repair assay results in TRPML1-KO and PIEZO1-KO HeLa cells. (E) Representative results of HEX secretion in TRPML1-KO and PIEZO1-KO HeLa cells. Data are the mean ± SD from three independent experiments (One-way ANOVA with Dunnett correction; ***P ≤ 0.001; ns, P ≥ 0.05). See also Fig. S5 A. (F) Representative CLSM images of WT, TRPML1-KO, and PIEZO1-KO HeLa cells stained with vinculin. Bar, 10 µm. (G) Quantification results of FAs >0.5 µm² (mean ± SD; n = 10 images; N = 2 biological replicates; One-way ANOVA with Dunnett correction; **P ≤ 0.01; ns, P ≥ 0.05). (H) Representative results of WT U2OS cells transiently transfected with VAMP7-pHluorin before and after treatment with GsMTx4 (5 µM, 5 min). Summary of 30 s TIRF microscopic live imaging. Lysosome-plasma membrane fusion events are represented as magenta dots. Bar, 5 µm. See also Video 7. (I) Representative results of WT U2OS cells transiently transfected with VAMP7-pHluorin before and after treatment with Yoda1 (1.5 µM, 5 min). Summary of 30 s TIRF microscopic live imaging. Lysosome-plasma membrane fusion events are represented as magenta dots. Bar, 5 µm. See also Video 8. (J) Representative results of WT U2OS cells transiently transfected with VAMP7-pHluorin before and after treatment with ML-SI3 (5 µM, 5 min). Summary of 30 s TIRF microscopic live imaging. Lysosome–plasma membrane fusion events are represented as magenta dots. Bar, 5 µm. See also Video 9. (K) Representative results of WT U2OS cells transiently transfected with VAMP7-pHluorin before and after treatment with ML-SA1 (20 µM, 5 min). Summary of 30 s TIRF microscopic live imaging. Lysosome–plasma membrane fusion events are represented as magenta dots. Bar, 5 µm. See also Video 10. (L) Quantification results of Fig. 7, H–K. Note the number of lysosomal exocytosis events varies in untreated cells in different groups, mainly due to the different VAMP7-pHluorin expression level. (n = 15 cells, N = 5 biological replicates for GsMTx4, 13 cells, 4 biological replicates for Yoda1, 10 cells, 4 biological replicates for ML-SI3, and 15 cells, 5 biological replicates for ML-SA1; mean ± SD; paired t test; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001).

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