Reduced ingestion of toxic and fluorescent heme analogs may explain differences observed between wildtype C. elegans and worms lacking HRG-9 and HRG-10 (double knockout; DKO).

A) Survival of N2, DKO, or eat-2 knockout worms exposed to 1, 2, 5, or 10 µM GaPP for 72 hours. Each dot represents the number of offspring laid by one adult worm on one GaPP-treated plate after 24 hours. N=3 independent experiments. * p<0.05, **** p<0.0001 B) Quantification of fluorescent staining in N2, DKO, and eat-2 worms grown on OP50 E. coli plates under fed and starved conditions with or without 40 µM ZnMP treatment. A.U. arbitrary units, N=15 worms analyzed over 3 independent experiments. * p<0.05. C) Representative images of red fluorescence in N2, DKO, and eat-2 worms grown on OP50 E. coli plates. Scale bars represent 50 µM. D) Quantification of fluorescent staining in N2 and DKO cultured in normal heme axenic media with or without 40 µM ZnMP treatment. A.U. arbitrary units, N=7-15 worms analyzed over 3 independent experiments. * p<0.05. E) Representative images of red fluorescence in N2 and DKO worms grown in liquid media. Scale bars represent 50 µM.

DKO nematodes demonstrate lawn avoidance and reduced pharyngeal pumping, brood sizes, motility, and survival.

A) Proportion of N2 or DKO worms present on OP50 lawn (innermost ring), off OP50 lawn (middle ring), or missing or dead from NGM plate (outer ring). N=180 worms over 3 independent experiments B) Number of of pharyngeal pumps in a one-minute period in N2, DKO, and eat-2 knockout worms. N=15 worms over 3 independent experiments C) Number of viable offspring laid by single adult N2 or DKO worms either after 24 hours of egg lay or across total egg-laying period (5 days). N=8 broods for 24-hour counts, N=5 broods for total brood size counts. D) Swimming behavior of N2 and DKO worms over a 20 minute interval. Worms were observed at 4 minute intervals and scored from 0-5 on swimming intensity. Bars represent the proportion of worms at each score. E) Quantification of thrashes after 4 minutes in M9 buffer. F) Longevity of N2 and DKO C. elegans observed from L4 larval stage. N=30 worms over 3 independent experiments. (** p<0.01, *** p<0.001, **** p<0.0001).

RNA-seq and qPCR analysis show that hrg-9 and hrg-10 are not uniquely heme responsive but instead may be preferentially induced under conditions of oxidative stress.

A) Analysis of top 500 genes with differential expression under low heme (2 µM) and high heme (400 µM) conditions. Outline represents 134 relevant genes identified by cluster analysis. R186.1 is the alternative sequence name for hrg-9. B) Gene ontology analysis identified a variety of biological roles for genes within this cluster C) RT-qPCR of hrg-9 and hrg-10 under non-heme stress conditions: 24-hour starvation, 4-hour exposure to 34° C heat, and 25 mM paraquat (pqt). N=3 independent experiments.

Growth and muscle fiber integrity in yeast and zebrafish models of TANGO2 deficiency fail to replicate previously reported phenotypes.

A) Yeast growth curves. Different strains were grown in SC medium or SC medium lacking histidine at 25° C. B) Whole mount phalloidin staining of control and two strains of tango2-/-zebrafish (bwg210 and bwg211). Myofibers in mutants lack the parallel organization observed in controls but do not demonstrate significant myofiber breakdown or damage. Representative images; N=8-10 in each group. Scale bar=5 mm.

Proposed model of TANGO2 and its homologs acting as stress-responsive mediators of mitochondrial dysfunction.

A) Paraquat exposure and heme deficiency each induce reactive oxygen species (ROS) formation, mitochondrial stress, and enrichment of hrg-9 in C. elegans. TANGO2 and its homologs may help mitigate mitochondrial stress under these conditions, though the exact function of these proteins remains unknown. B) Physiological triggers such as fasting or illness also precipitate oxidative stress in the absence of TANGO2. Abnormal lipid profiles have been observed in multiple models of TANGO2 deficiency in the setting of impaired lipid mobilization and reduced fatty acid oxidation. In humans, loss of TANGO2 results in a complex clinical syndrome reminiscent of multiple secondary mitochondrial disorders. In C. elegans, loss of hrg-9 and hrg-10 induces a phenotype previously observed in nematode strains exhibiting mitochondrial dysfunction. Image created with BioRender.com/nsnn9pe.