Neuronal mitochondrial perturbation triggers spatiotemporal dynamics of Ca ²⁺ waves in ADF synapses in a TMBIM-2-dependent manner. (A) Schematic drawing of ADF neurons and GCaMP6f imaging regions in B–L, and representation illustrating the principle behind the GCaMP6f. (B) Representative fluorescence traces of average background-subtracted fluorescence intensity ΔF/F₀ of plasma membrane Ca²⁺ indicator GCaMP6f in the region of interest (ROI) shown in C of day 1 adult animals expressed neuronal PM-GCaMP6f and ADF presynaptic vesicle marker (ythEX762[xbx-1p::PM-GCaMP6f; srh-142p::Scarlet::rab-3+unc-119(+)]) with WT; neuronal cox-5B knockdown; tmbim-2(yth130); neuronal cox-5B knockdown+tmbim-2(yth130) background, respectively. The GCaMP6f signal was imaged for 60 s. (C) Representative confocal photomicrographs of animals in B. Presynaptic region of ADF neuron was marked with rab-3 (magenta). A–C stand for different points in time shown in B. The imaging used Z-planes. Scale bar, 2 μm. (D) Quantification of the maximal ΔF/F₀ with GCaMP6f signal in ROI shown in B. n ≥ 15 worms. (E) Quantification of the frequency with GCaMP6f fluorescence intensity changes in ROI shown in B. n ≥ 20 worms. (F) Representative fluorescence traces of average background-subtracted fluorescence intensity ΔF/F₀ of mitochondrial Ca²⁺ indicator GCaMP6f in the region of interest (ROI) shown in G of day 1 adult animals expressed neuronal mito-GCaMP6f and ADF presynaptic marker (ythEX761[xbx-1p::mtLS-GCaMP6f; srh-142p::Scarlet::rab-3+unc-119(+)]) with WT; neuronal cox-5B knockdown; tmbim-2(yth130); neuronal cox-5B knockdown+tmbim-2(yth130) background, respectively. The GCaMP6f signal was imaged for 120 s. (G) Representative confocal photomicrographs of animals in F. Presynaptic region of ADF neuron was marked with rab-3 (magenta). A–C stand for different points in time shown in F. The imaging used Z-planes. Scale bar, 2 μm. (H) Quantification of the maximal ΔF/F₀ with GCaMP6f signal in ROI shown in F. n ≥ 15 worms. (I) Quantification of the frequency with GCaMP6f signal in ROI shown in F. n ≥ 20 worms. (J) Representative fluorescence traces of average background-subtracted fluorescence intensity ΔF/F₀ of cytosolic Ca²⁺ indicator GCaMP6f of WT and neuronal cox-5B knockdown animals in the 60 s. (K) Representative confocal photomicrographs of animals in J. Presynaptic region of ADF neuron was marked with rab-3 (magenta). A–C stand for different points in time shown in J. The imaging used Z-planes. Scale bar, 2 μm. (L) Quantification of the maximal ΔF/F₀ with GCaMP6f signal in ROI shown in J. n ≥ 15 worms. **P < 0.01; *P < 0.05; ns denotes P > 0.05 via unpaired two-tailed Student’s t test. Error bars, SEM.