Figure S4.
Overexpression of MCU-1 and loss of mca-3 in neurons partially suppresses neuronal-to-intestinal UPRmtactivation. (A) Representative photomicrographs of dve-1 reporter in WT; pan-neuron overexpressing MCU-1 (rgef-1p::mcu-1); ciliated-sensory neuron overexpressing MCU-1 (osm-6p::mcu-1) animals with neuronal Q40::YFP, respectively. Scale bar, 250 μm. (B) Quantification of dve-1p::dve-1::gfp expression in the entire intestine of animals as depicted in A. n ≥ 15 worms. (C) Representative photomicrographs of hsp-6 reporter in WT and ADF neuron mcu-1 knockdown animals (srh-142p::Cas9+u6p::mcu-1 sgRNA) with neuronal Q40::YFP, respectively. Scale bar, 250 μm. (D) Quantification of hsp-6p::gfp expression in the entire intestine of animals as depicted in C. n ≥ 15 worms. (E) Quantification of PM-GCaMP6 maximal fluorescence intensity changes in WT and tmbim-2; mcu-1(ju1154) animals. n ≥ 20 worms. (F) Quantification of the frequency with GCaMP6f fluorescence intensity changes in WT and tmbim-2; mcu-1(ju1154) animals. n ≥ 20 worms. (G) Representative photomicrographs of hsp-6 reporter in ciliated neuronal knockout of mca-3 with neuronal Q40::YFP worms. Scale bar, 250 μm. (H) Quantification of hsp-6p::gfp expression in the entire intestine of animals as depicted in G. n ≥ 15 worms. (I) Representative DNA gels of T7E1 assay identifying the PCR products amplified from genomic DNA of control worms or worms with mca-3 deletion in ciliated sensory neurons. (J) Quantification of mca-3 mRNA levels in day1 (grey), day4 (yellow) and day7 (blue) WT adult animals. n ≥ 3 biologically independent samples. (K) Quantification of mca-3 mRNA levels in WT (grey), neuronal overexpressing Q40 (yellow) and neuronal cox-5B knockdown (blue) animals. n ≥ 3 biologically independent samples. (L) Representative photomicrographs of hsp-6 reporter in neuronal Q40::YFP animals with PMCA inhibitor Caloxin 2A1, respectively. Scale bar, 250 μm. (M) Quantification of hsp-6p::gfp expression in the entire intestine of animals as depicted in L. n ≥ 10 worms. ***P < 0.001; **P < 0.01; *P < 0.05 via unpaired two-tailed Student’s t test. Error bars, SEM. Source data are available for this figure: SourceData FS4.

Overexpression of MCU-1 and loss of mca-3 in neurons partially suppresses neuronal-to-intestinal UPR mt activation. (A) Representative photomicrographs of dve-1 reporter in WT; pan-neuron overexpressing MCU-1 (rgef-1p::mcu-1); ciliated-sensory neuron overexpressing MCU-1 (osm-6p::mcu-1) animals with neuronal Q40::YFP, respectively. Scale bar, 250 μm. (B) Quantification of dve-1p::dve-1::gfp expression in the entire intestine of animals as depicted in A. n ≥ 15 worms. (C) Representative photomicrographs of hsp-6 reporter in WT and ADF neuron mcu-1 knockdown animals (srh-142p::Cas9+u6p::mcu-1 sgRNA) with neuronal Q40::YFP, respectively. Scale bar, 250 μm. (D) Quantification of hsp-6p::gfp expression in the entire intestine of animals as depicted in C. n ≥ 15 worms. (E) Quantification of PM-GCaMP6 maximal fluorescence intensity changes in WT and tmbim-2; mcu-1(ju1154) animals. n ≥ 20 worms. (F) Quantification of the frequency with GCaMP6f fluorescence intensity changes in WT and tmbim-2; mcu-1(ju1154) animals. n ≥ 20 worms. (G) Representative photomicrographs of hsp-6 reporter in ciliated neuronal knockout of mca-3 with neuronal Q40::YFP worms. Scale bar, 250 μm. (H) Quantification of hsp-6p::gfp expression in the entire intestine of animals as depicted in G. n ≥ 15 worms. (I) Representative DNA gels of T7E1 assay identifying the PCR products amplified from genomic DNA of control worms or worms with mca-3 deletion in ciliated sensory neurons. (J) Quantification of mca-3 mRNA levels in day1 (grey), day4 (yellow) and day7 (blue) WT adult animals. n ≥ 3 biologically independent samples. (K) Quantification of mca-3 mRNA levels in WT (grey), neuronal overexpressing Q40 (yellow) and neuronal cox-5B knockdown (blue) animals. n ≥ 3 biologically independent samples. (L) Representative photomicrographs of hsp-6 reporter in neuronal Q40::YFP animals with PMCA inhibitor Caloxin 2A1, respectively. Scale bar, 250 μm. (M) Quantification of hsp-6p::gfp expression in the entire intestine of animals as depicted in L. n ≥ 10 worms. ***P < 0.001; **P < 0.01; *P < 0.05 via unpaired two-tailed Student’s t test. Error bars, SEM. Source data are available for this figure: SourceData FS4.

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