XBX-6/TMBIM-2 functions in neurons to coordinate neuronal-to-intestinal UPR ^mt activation. (A) Sequence alignment of TMBIM2 proteins across species. (B) Alphafold predicted the protein structure of TMBIM-2. (C) A PAE (Predicted Aligned Error) plot, showing regions of high confidence (dark green) and low confidence (pale green) for the AlphaFold prediction of TMBIM-2 structure. (D) Representative photomicrograph of dve-1 reporter in neuronal EGL-20; tmbim-2 animals with or without human TMBIM2 overexpressing (tmbim-2p::hTmbim2) rescue. (E) Quantification of the number of intestinal nuclei puncta with GFP signal per worm as shown in D. n ≥ 9 biologically independent samples. (F) Representative photomicrographs of dve-1 reporter expression in neuronal EGL-20 overexpressing (ythIs3[rgef-1p::egl-20 + myo-2p::tdtomato]) and intestinal EGL-20 overexpressing (ythIs1[gly-19p::egl-20]) animals in WT or tmbim-2(yth26) background, respectively. The posterior region of the intestine where DVE-1::GFP is induced or suppressed is highlighted. (G) Quantification of the number of intestinal nuclei puncta with GFP signal per worm as shown in F. n ≥ 12 worms. (H) Quantification of tmbim-2 mRNA levels in WT (yellow), tmbim-2 (yth57) (blue), tmbim-2 (yth26) (purple), tmbim-2 (yth130) (grey), and tmbim-2 (yth92) (pink) animals. Error bars, SEM; n ≥ 3 biologically independent samples. (I) Representative photomicrographs of hsp-6 reporter (zcIs3[hsp-6p::gfp]) expression in neuronal EGL-20 overexpressing (ythIs3[rgef-1p::egl-20 + myo-2p::tdtomato]) animals in WT or tmbim-2(yth92) background, respectively. (J) Quantification of hsp-6p::gfp expression in the entire intestine of animals as depicted in I. n ≥ 15 worms. (K) Representative photomicrographs of hsp-6 reporter (zcIs3[hsp-6p::gfp]) expression in neuronal Q40::YFP overexpressing animals in WT or tmbim-2(yth92) background, respectively. (L) Quantification of hsp-6p::gfp expression. The genotypes are as in K. n ≥ 15 worms. (M) qRT-PCR analysis of transcripts (n = 3 biologically independent samples) in neuronal cox-5B knockdown animals in WT or tmbim-2 background. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test (**P < 0.01; *P < 0.05). (N) Representative photomicrographs demonstrating: dve-1 reporter expression in tmbim-2 (yth57) animals expressing neuronal EGL-20 (a); TMBIM-2 rescue (ythIs62[tmbim-2p::tmbim-2::GFP; unc-119(+)]) (b); TMBIM-2 pan-neuron rescue (ythEx226[rgef-1p::tmbim-2::mcherry::HA; pRF4(rol-6)]) (c); TMBIM-2 intestinal rescue (ythEx229[gly-19p::tmbim-2::mcherry::HA; pRF4(rol-6)]) (d). The posterior region of the intestine where DVE-1::GFP is induced or suppressed is highlighted. (O) Quantification of the number of intestinal nuclei puncta with GFP signal per worm as shown in N. (P) Quantification of tmbim-2 mRNA levels in WT, TMBIM-2 overexpression and intestinal TMBIM-2 overexpression animals. Error bars, SEM; n ≥ 3 biologically independent samples. (Q) Representative photomicrograph of dve-1 reporter in neuronal Q40::YFP overexpressing animals with or without ADF neuronal TMBIM-2 knockout (ythEx574[srh-142p::Cas9::u6p::tmbim-2 sgRNA]). (R) Quantification of the number of intestinal nuclei puncta with GFP signal per worm as shown in Q. n ≥ 15 worms. ***P < 0.001, *P < 0.05, ns denotes P > 0.05 via unpaired two-tailed Student’s t test. Error bars, SEM. Scale bar, 250 μm.