Function of hTim8a in complex IV assembly in neuronal cells provides insight into pathomechanism underlying Mohr-Tranebjærg syndrome
- The University of Melbourne, Australia
- Peter MacCallum Cancer Centre, Australia
- Royal Children’s Hospital, Australia
- University of Melbourne, Australia
- Royal Children's Hospital, Australia
- Monash University, Australia
Figures
Figure 1 with 2 supplements
Cells lacking hTim8a have no defects in the TIM22, TIM23 or carrier biogenesis pathway.
(A–E) Mitochondria were isolated from control, (A) hTim9MUT HEK293 cells, (B) hTim8aKO HEK293 cells, (C) hTim8aMUT SH-SY5Y cells, (D) hTim8bKO HEK293 cells, or (E) hTim8bKO SH-SY5Y cells prior to …
Figure 1—figure supplement 1
Generation of CRISPR/Cas9 genome-edited cell lines.
(A–E) Schematic representation of the CRISPR/Cas9 editing system used to generate (A) hTim9MUT (MUT = mutant) in Flp-In T-REx HEK293 cells, (B) hTim8aKO (KO = knock out) in Flp-In T-REx HEK293 …
Figure 1—figure supplement 2
Loss of hTim8a or hTim8b has no defect on TIM or carrier biogenesis.
(A) Mitochondria isolated from control and hTim9MUT, hTim8aKO, hTim8aMUT SH or hTim8bKO SH cells, were subjected to SDS-PAGE analysis and immunoblotted using the indicated antibodies. (B) Relative …
Figure 2
Loss of hTim8a shows cell-type specific consequences on the mitochondrial proteome.
(A–E) Mitochondria were isolated from control and (A) hTim9MUT HEK293, (B) hTim8aKO HEK293, (C) hTim8aMUT SH-SY5Y, (D) hTim8bKO HEK293, or (E) hTim8bKO SH-SY5Y cells and subjected to label-free …
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Figure 2—source data 1
Label-free quantitiative mass spectrometry on mitochondria isolated from: hTim9MUT, hTim8aKO, hTim8aMUT SH,hTim8bKO and hTim8bKO SH.
- https://cdn.elifesciences.org/articles/48828/elife-48828-fig2-data1-v3.xlsx
Figure 3 with 1 supplement
Lack of hTim8a, but not hTim8b, sensitises cells to apoptotic-cell death.
(A) Mitochondrial lysates from control and hTim8aKO (left panel) or hTim8bKO (right panel) HEK293 cells were analysed by SDS-PAGE and western blotting. Relative protein levels of cytochrome c (Cyt c)…
Figure 3—figure supplement 1
Cells lacking fucntional hTim8a are not sensitised to ferroptosis-induced cell death, but intrinsic cell death.
(A) Relative fold change of mRNA expression of Cyt c, AIF and Bax in hTim8aKO compared to control HEK293 were quantified and tabulated as mean ± SEM (n = 3). *, p<0.05; **, p<0.01. (B) Control and …
Figure 4
Cellular dysfunction in cells lacking hTim8a or hTim8b.
(A) Cell viability of control and hTim8aKO or hTim8bKO HEK293 cells (left panel) or hTim8aMUT SH or hTim8bKO SH cells (right panel) was measured using trypan blue staining. Cells were seeded at the …
Figure 5 with 2 supplements
Metabolic rewiring in hTim8aMUT SH-SY5Y cells.
(A) Relative abundance of significantly affected metabolites in hTim8aMUT compared to control SH-SY5Y cells. Heatmap representing the top 50 most affected metabolites extracted from control and …
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Figure 5—source data 1
Untargeted metabolomics profiling in Control, hTim8aKO and hTim8a MUT SH cells.
- https://cdn.elifesciences.org/articles/48828/elife-48828-fig5-data1-v3.xlsx
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Figure 5—source data 2
13C-glucose and 13C-glutamine labelling of Control SH-SY5Y and hTim8MUT SH cells.
- https://cdn.elifesciences.org/articles/48828/elife-48828-fig5-data2-v3.xlsx
Figure 5—figure supplement 1
Metabolomic profile in hTim8aKO HEK293 cells.
(A) Hierarchical clustering of relative abundance of the top 40 significantly-affected intracellular metabolites depicted as a heat map (p<0.05) in control (n = 6) and hTim8aKO (n = 5) HEK293 cells. …
Figure 5—figure supplement 2
Metabolomic profile in hTim8aMUT SH-SY5Y cells.
(A) Relative abundances of intracellular metabolites in hTim8aMUT compared to control SH-SY5Y cells (n = 6) were mapped onto metabolic networks using VANTED analysis tool. Each pathway is …
Figure 6 with 1 supplement
hTim8a and hTim8b have a cell type specific function in Complex IV biogenesis.
(A) Data from label-free quantitative mass spectrometry shown in Figure 2 was used to create volcano plots showing relative levels of proteins in CRISPR-edited cells compared to control cells. n = 3 …
Figure 6—figure supplement 1
Loss of hTim8a or hTim8b in SH-SY5Y cells leads to altered levels of multiple Complex IV subunits or assembly factors.
(A) Mitochondria isolated from control or hTim8bKO HEK293 or hTim8aMUT SH-SY5Y cells were analysed using SDS-PAGE and immunoblotted with indicated antibodies. Relative abundance of Complex IV …
Figure 7
hTim8a functions in the early stage of assembly of Complex IV in SH-SY5Y cells.
(A) Mitochondria isolated from control and hTim8aMUT SH cells were separated using SDS-PAGE and immunoblotted using antibodies against hTim8a, COX17 and COX4. Relative level of COX17 proteins in …
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Figure 7—source data 1
Interacting partners of hTim8b in SH-SY5Y cells in glucose versus galactose media.
- https://cdn.elifesciences.org/articles/48828/elife-48828-fig7-data1-v3.xlsx
Figure 8 with 1 supplement
Elevated oxidative stress in hTim8aMUT SH-SY5Y cells sensitises cells to apoptosis.
(A) Reactive H2O2 species present in untreated or menadione-pretreated (2 hr) control and hTim8aMUT SH-SY5Y cells were quantified using ROS-Glo H2O2 Assay. Data are shown as mean ± SD (n = 3, …
Figure 8—figure supplement 1
Apoptotic sensitivity in the absence of hTim8a or hTim8b and the effects of Vitamin E treatment.
(A) Control, hTim8aKO and hTim8bKO HEK293 cells were treated with Menadione (10 μM) for 0, 1.5 or 3 hr. Rate of apoptosis was calculated by measuring phosphatidylserine (PS) exposure to the outer …
Figure 9
Loss of COX17 in SH-SY5Y cells leads to Complex IV defects and primes cells to apoptotic-cell death.
(A) Cell lysate from SH-SY5Y cells depleted of COX17 (COX17KD SH) using siRNA were analysed using SDS-PAGE and immunoblotted with the indicated antibodies. Relative protein levels of COX17 and …
