Comparative profiling of N-respirasomes predicts aberrant mitochondrial bioenergetics at single-cell resolution

Abstract

Mitochondria sustain the energy demand of the cell. The composition and functional state of the mitochondrial oxidative phosphorylation system are informative indicators of organelle homeostasis and bioenergetic capacity. Here we describe a highly sensitive and reproducible method for single-cell visualization and quantification of mitochondrial respiratory supercomplexes as a novel means of measuring mitochondrial respiratory chain integrity. We apply a proximity ligation assay (PLA) and perform comparative studies of mitochondrial CI, CIII and CIV-containing supercomplexes (or N-respirasomes) in fixed human and mouse brain tissues, tumorigenic cells, iPSCs and iPSC-derived NPCs and neurons. Our optimized approach enables a quantitative in-situ assessment of even subtle mitochondrial lesions associated with aberrant respiration. By combining quantitative proteomics with single cell imaging analysis, we also report the mechanistic contribution of the MICOS complex subunit CHCHD3 in regulating N-respirasomes. Overall, our PLA-based profiling of N-respirasomes establishes a sensitive and complementary technique for detecting cell-type specific mitochondrial perturbations in fixed materials.

SGPL1 stimulates VPS39 recruitment to the mitochondria in MICU1 deficient cells

“…For co-localization indices, Pearson's and Mander's coefficients were determined using the co-localization analysis plugin JACoP. PLA-dots were semi-automatically quantified as previously described [ 32 ].…”

“…PLA experiments were performed using Duolink® PLA fluorescence reagents (Sigma) according to the manufacturer's instructions and as previously described [ 32 ]. Briefly, cells and tissue samples were first permeabilized with 0.25% Triton X-100 in PBS, and then treated with Blocking solution for 1 h at 37 °C.…”

“…S5C–D ). Using a newly validated proximity ligation assay (PLA)-based protocol [ 32 ], we performed in situ analysis of mitochondrial CI–CIV proximity as an indicator of mitochondrial respiratory supercomplexes (RSCs). We observed that MICU1 KO cells exhibited less CI, CIV-containing RSCs ( Supplementary Fig.…”

“…Finally, to demonstrate that these newly identified molecular signatures may be commonly observed in cells carrying mitochondrial lesions, we employed muscle tissues from wt and transgenic mice expressing a disease-causing Aifm1(R200 del) mutation ( Figure 5 H) [ 28 , 33 ]. These knockin mice develop severe myopathy due to apoptosis-inducing factor (AIF) loss and consequent aberrant expression of ETC components [ 32 , 33 , 49 , 96 ]. We ran immunoblot analysis and showed that AIF loss stimulated VPS39 expression in 6-month-old muscles ( Supplementary Fig.…”

AIFM1 beyond cell death: An overview of this OXPHOS-inducing factor in mitochondrial diseases

“…In this regard, it was shown that CHCHD4 can bind the mitochondrial Ca 2+ uptake protein 1 (MICU1) and stimulate the formation of an intermolecular disulfide bond between MICU1 and its paralog MICU2, resulting in aberrant regulation of mitochondrial Ca 2+ uptake. 69 In Drosophila and in mammalian cells, AIF deficiency may compromise IMS import and cristae junction localization of CHCHD3/MI19, 56 , 71 thus disrupting mitochondrial contact site and crista organizing system (MICOS) complex to an extent that phenotypically resembles CHCHD4/MIA40 loss in HeLa cells and yeast. 72 , 73 , 74 The impact of AIF deficiency on MICOS complex stability is not limited to one single subunit, but extends to others, such as coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10).…”