![Mitochondrial perturbation does not affect the dynamics of cytosol Ca2+waves in ADF synapses. (A) Representative confocal photomicrographs of day 1 adult animals expressing neuronal PM-GCaMP6f in combination with ADF presynaptic vesicle marker (ythEX762[xbx-1p::PM-GCaMP6f; srh-142p:: Scarlet::rab-3+unc-119(+)]). The imaging used Z-planes. Scale bar, 10 μm. (B) Representative confocal photomicrographs of day 1 adult animals expressing neuronal mito-GCaMP6f in combination with ADF presynaptic vesicle marker (ythEX761[xbx-1p::mtLS-GCaMP6f; srh-142p:: Scarlet::rab-3+unc-119(+)]). The imaging used Z-planes. Scale bar, 10 μm. (C) Representative confocal photomicrographs of day 1 adult animals expressing neuronal PM-GCaMP6f with plasma membrane dye DiD. The imaging used Z-planes. Scale bar, 1 μm. (D) Representative confocal photomicrographs of day 1 adult animals expressing neuronal mito-GCaMP6f in combination with neuronal mitochondria marker (forSi44[rgef-1p::tomm-20::mKate2::HA]). Scale bar, 1 μm. The imaging used Z-planes. Scale bar, 1 μm. (E) Representative fluorescence traces of average background-subtracted fluorescence intensity ΔF/F0 of plasma membrane Ca2+ indicator GCaMP6f of WT and egl-19(ad695) gain of function mutant animals in the 60 s. (F) Quantification of the amplitude of PM-GCaMP6f fluorescence intensity changes in WT and egl-19(ad695) animals. n ≥ 10 worms. (G) Representative fluorescence traces of average background-subtracted fluorescence intensity ΔF/F0 of mitochondrial Ca2+ indicator GCaMP6f of WT and mcu-1(ju1154) mutant animals in the 60 s. (H) Quantification of the amplitude of mito-GCaMP6f fluorescence intensity changes in WT and mcu-1(ju1154) animals. n ≥ 13 worms. (I) Quantitative analysis of Ca2+ Fmin level by the PM-GCaMP6.0/RFP ratio with the presence or absence of neuronal cox-5B KD in WT (yellow) and tmbim-2 (blue) animals. n ≥ 12 worms. (J) Quantitative analysis of Ca2+ Fmin level by the Mito-GCaMP6.0/RFP ratio with the presence or absence of neuronal cox-5B KD in WT (yellow) and tmbim-2 (blue) animals. n ≥ 12 worms. (K) Representative fluorescence traces of average background-subtracted fluorescence intensity ΔF/F0 of plasma membrane Ca2+ indicator GCaMP6f of WT and ADF neuron overexpressing TMBIM-2 (ythIs102[srh-142p::tmbim-2]) animals in 60 s. (L) Quantification of the amplitude of PM-GCaMP6f fluorescence intensity changes in WT and ADF neuron overexpressing TMBIM-2 animals. n ≥ 14 worms. **P < 0.01; *P < 0.05; ns denotes P > 0.05 via unpaired two-tailed Student’s t test in F, H, and L. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test in I and J (**P < 0.01; ***P < 0.001). Error bars, SEM.](https://cdn.rupress.org/rup/content_public/journal/jcb/224/5/10.1083_jcb.202408050/2/jcb_202408050_figs3.png?Expires=1770186720&Signature=ZRyTls5nj6XhX6bC67ur9gJfx3NYOfLKyCZM9bKD1~vWPUCgIeBXp18QO69t6YVcHCsC8Db~rX043LK0UlcaHNF4dfPpyLuE6QYAGkfw3LhGln-Aq~ShuA14bfkEzyorh4JFJggHalXc68u~0csRiq-rsongP9YxdrIOQGWYGJxSpPqie53eaAFVJY~u~inU5MfAH2pI9F3AUPASciX47YMpxMhAn8sH-QDRSXMQTlAtO0LkyuCYO5tQbmPRcXudnYoKizNhjLdGgkmUs8RrmO5dm3EyeEFhF~xojWC8HwsKx2YUkBPqIpfjq8TI2MxVIO2xtlENoT~dKk4S7eC07A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Mitochondrial perturbation does not affect the dynamics of cytosol Ca 2+ waves in ADF synapses. (A) Representative confocal photomicrographs of day 1 adult animals expressing neuronal PM-GCaMP6f in combination with ADF presynaptic vesicle marker (ythEX762[xbx-1p::PM-GCaMP6f; srh-142p:: Scarlet::rab-3+unc-119(+)]). The imaging used Z-planes. Scale bar, 10 μm. (B) Representative confocal photomicrographs of day 1 adult animals expressing neuronal mito-GCaMP6f in combination with ADF presynaptic vesicle marker (ythEX761[xbx-1p::mtLS-GCaMP6f; srh-142p:: Scarlet::rab-3+unc-119(+)]). The imaging used Z-planes. Scale bar, 10 μm. (C) Representative confocal photomicrographs of day 1 adult animals expressing neuronal PM-GCaMP6f with plasma membrane dye DiD. The imaging used Z-planes. Scale bar, 1 μm. (D) Representative confocal photomicrographs of day 1 adult animals expressing neuronal mito-GCaMP6f in combination with neuronal mitochondria marker (forSi44[rgef-1p::tomm-20::mKate2::HA]). Scale bar, 1 μm. The imaging used Z-planes. Scale bar, 1 μm. (E) Representative fluorescence traces of average background-subtracted fluorescence intensity ΔF/F0 of plasma membrane Ca2+ indicator GCaMP6f of WT and egl-19(ad695) gain of function mutant animals in the 60 s. (F) Quantification of the amplitude of PM-GCaMP6f fluorescence intensity changes in WT and egl-19(ad695) animals. n ≥ 10 worms. (G) Representative fluorescence traces of average background-subtracted fluorescence intensity ΔF/F0 of mitochondrial Ca2+ indicator GCaMP6f of WT and mcu-1(ju1154) mutant animals in the 60 s. (H) Quantification of the amplitude of mito-GCaMP6f fluorescence intensity changes in WT and mcu-1(ju1154) animals. n ≥ 13 worms. (I) Quantitative analysis of Ca2+ Fmin level by the PM-GCaMP6.0/RFP ratio with the presence or absence of neuronal cox-5B KD in WT (yellow) and tmbim-2 (blue) animals. n ≥ 12 worms. (J) Quantitative analysis of Ca2+ Fmin level by the Mito-GCaMP6.0/RFP ratio with the presence or absence of neuronal cox-5B KD in WT (yellow) and tmbim-2 (blue) animals. n ≥ 12 worms. (K) Representative fluorescence traces of average background-subtracted fluorescence intensity ΔF/F0 of plasma membrane Ca2+ indicator GCaMP6f of WT and ADF neuron overexpressing TMBIM-2 (ythIs102[srh-142p::tmbim-2]) animals in 60 s. (L) Quantification of the amplitude of PM-GCaMP6f fluorescence intensity changes in WT and ADF neuron overexpressing TMBIM-2 animals. n ≥ 14 worms. **P < 0.01; *P < 0.05; ns denotes P > 0.05 via unpaired two-tailed Student’s t test in F, H, and L. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test in I and J (**P < 0.01; ***P < 0.001). Error bars, SEM.
