slp2  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 is identified as an interacting partner of MIC13 (A) Interactome of MIC13 with co-IP (co-immunoprecipitation) coupled mass spectrometry revealed SLP2 as an interactor of MIC13. (B) The interaction between SLP2 and MIC13 was confirmed by co-IP using FLAG antibody in isolated mitochondria from MIC13 KO cells stably expressing MIC13-FLAG or empty vector (EV) pMSCVpuro as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from anti-Flag M2 beads, ∗ non-specific IgG bands. (C) Co-IP was used to detect the SLP2-MICOS interaction using isolated mitochondria from SLP2 KO stably expressing pMSCVpuro EV (background control) or SLP2-MYC. Co-IP was performed using MYC-Trap agarose beads. I: Input fraction (10% of total lysate), E: Eluate fraction. YME1L was used as a positive interactor of SLP2 whereas Mt-CO2 and HSP60 served as non-interactors. All the MICOS subunits were detected in the elution fraction from SLP2-MYC co-IP. (D) Proximity ligation assay (PLA) in HeLa cells with antibodies against MICOS subunits and SLP2. PLA signals are shown as red spots indicating respective protein interactions. SLP2 alone and Mt-CO2 & SLP2 antibodies were probed as negative controls. Scale bar 20 μm. (E) BN-PAGE with isolated mitochondria from WT cells revealed a co-migration pattern of SLP2 with higher molecular weight MICOS complex. (F) A heatmap and graph represent the normalized occurrence of SLP2 and MICOS subunits in complexome profiling data obtained from HEK293 cells studied previously. SLP2 co-clustered with high molecular weight MICOS complex at around 2000 kDa. (G) Scaffolding model depicting interaction of SLP2 with MICOS subunits shows that SLP2 provides a scaffold for interaction of MICOS subunits.

Article Snippet: Human SLP2-HA ORF , Sino Biologicals , HG16147-CY.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Mass Spectrometry, Isolation, Stable Transfection, Expressing, Plasmid Preparation, Control, Proximity Ligation Assay, Migration, Molecular Weight, Scaffolding

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Loss of SLP2 leads to aberrant cristae structure and reduced MIC26 levels (A) Steady state levels of MICOS proteins with western blot analysis from WT, SLP2 KO and SLP2 KO cells stably expressing pMSCVpuro EV or SLP2-MYC. Tubulin serves as a loading control for western blots. (B) Western blot quantification depicting relative protein levels. SLP2 KO was normalized to WT and SLP2 KO + SLP2 MYC was normalized to SLP2 KO + EV samples. Data is represented as mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗∗ p -value ≤0.001. MIC26 levels were reduced in SLP2 KO. Notably, MIC27 levels showed a slight increase in SLP2 KO. (C) Western blot analysis of steady state levels of MICOS proteins from WT and SLP2 KO cells stably expressing pGIPZ-control shRNA or YME1L shRNA. The results are depicted by a model illustrating the role of SLP2 in stabilizing MIC26 through the regulation of YME1L-mediated proteolysis. HSP60 acts as an internal protein loading control. (D) TEM images from WT, SLP2 KO and MIC26 KO cells. SLP2 KO shows loss of cristae and CJs with accumulation of swollen cristae, while MIC26 KO shows slight cristae branching. Scale bar represents 500 nm. The skeletonization of the TEM image is depicted on right side. (E) Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, p -value >0.05. (F) Assessment of mitochondrial morphologies from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells untreated or post treatment with 10 μM cycloheximide for 2 h. Scale bar represented as 15 μm. (G) Percentage of cells displaying tubular, intermediate, fragmented or hyperfused mitochondria ( n = 3). Statistical analysis was performed using Student’s t test. ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001. Data represented as mean ± standard error of mean.

Article Snippet: Human SLP2-HA ORF , Sino Biologicals , HG16147-CY.

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 and MIC13 synergistically modulate assembly and nanoscale distribution of MIC60 (A) Assessment of steady state levels of MICOS proteins with western blot from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells. HSP60 serves as a loading control. (B) BN-PAGE of isolated mitochondria from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells to assess MICOS assembly. MIC13-SLP2 DKO showed reduced MIC60 assembly in MICOS complex compared to any single KO. OXPHOS cocktail antibody is used as a control in BN-PAGE. (C) BN-PAGE quantification depicting relative protein assembly levels normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean, MIC60 ( n = 5), MIC10 ( n = 4), OXPHOS ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, p -value >0.05. (D) STED nanoscopy images from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells displaying MIC60 punctae. Images on the left side show individual mitochondria that are delineated by dotted lines. Images on the right-side zooms into the boxed mitochondria region. Blue arrows indicate individual MIC60 punctae in a rail-like arrangement in WT cells. Green arrow represents perturbed MIC60 puncta in MIC13 KO and SLP2 KO that are not arranged in rail-like arrangement. Purple arrow shows evenly spread (diffuse) MIC60 puncta in MIC13-SLP2 DKO cells suggesting an altered pattern compared to WT. Scale bars represent the 500 nm. (E) Percentage of individual mitochondria displaying MIC60 puncta predominately arranged as rail-like pattern, non-rail-like pattern, or diffuse pattern is presented in a pie-chart format. (F) A model depicting that MIC60-subcomplex and MIB assembly is dependent on SLP2-MIC13.

Article Snippet: Human SLP2-HA ORF , Sino Biologicals , HG16147-CY.

Techniques: Western Blot, Control, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 specifically regulates assembly kinetics of MIC60 (A) WT cells stably expressing pLIX403 EV and MIC13 KO, MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG were treated with 1 μg/mL of doxycycline (Dox) for indicated time points and western blot analysis depicting steady state levels of MICOS proteins upon induction of MIC13-FLAG are shown. HSP60 serves as a loading control. (B) BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/mL of doxycycline (Dox) for indicated time points showing stable incorporation of MIC13-FLAG in MICOS complex. (C) BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/mL of Dox for indicated time points was probed for MIC10, MIC27 and MIC60 antibody. The green arrow (in the MIC13 KO lane) and red arrow (in the MIC13-SLP2 DKO lane) highlight the delay in assembly of MIC60 at the 8-h timepoint. The assembly kinetics of the MIC60-subcomplex, rather than the MIC10-subcomplex, is dependent on SLP2. (D) BN-PAGE quantification depicting relative protein assembly levels of MIC60 at 8 h normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean ( n = 3). The quantification reveals delayed MIC60 assembly in the absence of SLP2. Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05. (E) A model depicting the assembly kinetics of MIC60 in MICOS complex depends on SLP2. Slower incorporation of MIC60 is observed in the absence of SLP2 upon reintroduction of MIC13 in MIC13-SLP2 DKO .

Article Snippet: Human SLP2-HA ORF , Sino Biologicals , HG16147-CY.

Techniques: Stable Transfection, Expressing, Western Blot, Control, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Stabilized MIC10-subcomplex for MIC60 seeding and CJ formation (A) Western blot analysis of WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (KD) to assess steady state levels of MICOS proteins. The steady state levels of MIC60 remain unaltered across different cell lines. HSP60 serves as a loading control. (B) BN-PAGE with isolated mitochondria from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ- YME1L shRNA. The stabilized MIC10-subcomplex upon YME1L depletion could partially rescue the incorporation of MIC60 and MTX assembly in MIC13-SLP2 DKO. This shows that MIC10-subcomplex provides a docking site for assembly of MIC60. (C) BN-PAGE quantification depicting relative protein assembly levels normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ns = non-significant, p -value >0.05. (D) Mitochondrial cristae morphology accessed using TEM from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. Scale bar represents 0.5 μm. The skeletonization of the TEM image is depicted below the image. Mitochondria in MIC13 KO, SLP2 KO, MIC13-SLP2 DKO display loss of cristae and CJs, with cristae arranged as either stacks or concentric rings. SLP2 KO additionally display swollen cristae. YME1L depletion showed beneficial consequences on cristae morphology with presence of nascent CJs (red arrows). This shows that loss of CJs in MIC13 KO is attributed to MIC10 loss, and swelling of SLP2 KO cristae is visibly restored upon YME1L depletion. (E) Quantification of crista and CJs per mitochondrial section. Outliers were removed with Grubbs’ method and statistical significance was analyzed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, P-value >0.05.

Article Snippet: Human SLP2-HA ORF , Sino Biologicals , HG16147-CY.

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Schematic illustration of synergistic role of SLP2 and MIC13 in MICOS assembly and crista junction formation The schematic model illustrates the quality control processes involved in cristae junction (CJ) morphogenesis and MICOS homeostasis, mediated by the MIC13-YME1L and SLP2-YME1L, which differentially stabilize components of the MIC10-subcomplex. This process facilitates the formation of the “seeder complex”, which consists of SLP2 and the stabilized MIC10-subcomplex. The simultaneous depletion of MIC13 and SLP2 disrupts the nanoscale distribution of MIC60 and its integration into the MICOS complex. The proposed “seeder model” suggests that the formation of the seeder complex is crucial for organizing the punctate distribution of MIC60 and facilitating its assembly into the MICOS-MIB complex. This process is essential for the formation of nascent CJs and establishing contact sites between IM and OM. In addition to identifying SLP2 as a regulator of cristae morphology, this model provides key insights into the intricate and partially overlapping quality control pathways governing MICOS regulation, elucidates specific functions attributed to MIC13, and highlights the interdependency between the MIC10- and MIC60-subcomplexes in MICOS-MIB complex assembly.

Article Snippet: Human SLP2-HA ORF , Sino Biologicals , HG16147-CY.

Techniques: Control

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet:

Article Snippet: Human SLP2-HA ORF , Sino Biologicals , HG16147-CY.

Techniques: Virus, Recombinant, Protease Inhibitor, In Situ, Clone Assay, Transfection, Mass Spectrometry, Knock-Out, Double Knockout, Stable Transfection, Expressing, Plasmid Preparation, Control, shRNA, CRISPR, Software

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 is identified as an interacting partner of MIC13 (A) Interactome of MIC13 with co-IP (co-immunoprecipitation) coupled mass spectrometry revealed SLP2 as an interactor of MIC13. (B) The interaction between SLP2 and MIC13 was confirmed by co-IP using FLAG antibody in isolated mitochondria from MIC13 KO cells stably expressing MIC13-FLAG or empty vector (EV) pMSCVpuro as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from anti-Flag M2 beads, ∗ non-specific IgG bands. (C) Co-IP was used to detect the SLP2-MICOS interaction using isolated mitochondria from SLP2 KO stably expressing pMSCVpuro EV (background control) or SLP2-MYC. Co-IP was performed using MYC-Trap agarose beads. I: Input fraction (10% of total lysate), E: Eluate fraction. YME1L was used as a positive interactor of SLP2 whereas Mt-CO2 and HSP60 served as non-interactors. All the MICOS subunits were detected in the elution fraction from SLP2-MYC co-IP. (D) Proximity ligation assay (PLA) in HeLa cells with antibodies against MICOS subunits and SLP2. PLA signals are shown as red spots indicating respective protein interactions. SLP2 alone and Mt-CO2 & SLP2 antibodies were probed as negative controls. Scale bar 20 μm. (E) BN-PAGE with isolated mitochondria from WT cells revealed a co-migration pattern of SLP2 with higher molecular weight MICOS complex. (F) A heatmap and graph represent the normalized occurrence of SLP2 and MICOS subunits in complexome profiling data obtained from HEK293 cells studied previously. SLP2 co-clustered with high molecular weight MICOS complex at around 2000 kDa. (G) Scaffolding model depicting interaction of SLP2 with MICOS subunits shows that SLP2 provides a scaffold for interaction of MICOS subunits.

Article Snippet: SLP2 (mouse) , OriGene , Cat# TA808240; RRID: AB_2628756.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Mass Spectrometry, Isolation, Stable Transfection, Expressing, Plasmid Preparation, Control, Proximity Ligation Assay, Migration, Molecular Weight, Scaffolding

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Loss of SLP2 leads to aberrant cristae structure and reduced MIC26 levels (A) Steady state levels of MICOS proteins with western blot analysis from WT, SLP2 KO and SLP2 KO cells stably expressing pMSCVpuro EV or SLP2-MYC. Tubulin serves as a loading control for western blots. (B) Western blot quantification depicting relative protein levels. SLP2 KO was normalized to WT and SLP2 KO + SLP2 MYC was normalized to SLP2 KO + EV samples. Data is represented as mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗∗ p -value ≤0.001. MIC26 levels were reduced in SLP2 KO. Notably, MIC27 levels showed a slight increase in SLP2 KO. (C) Western blot analysis of steady state levels of MICOS proteins from WT and SLP2 KO cells stably expressing pGIPZ-control shRNA or YME1L shRNA. The results are depicted by a model illustrating the role of SLP2 in stabilizing MIC26 through the regulation of YME1L-mediated proteolysis. HSP60 acts as an internal protein loading control. (D) TEM images from WT, SLP2 KO and MIC26 KO cells. SLP2 KO shows loss of cristae and CJs with accumulation of swollen cristae, while MIC26 KO shows slight cristae branching. Scale bar represents 500 nm. The skeletonization of the TEM image is depicted on right side. (E) Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, p -value >0.05. (F) Assessment of mitochondrial morphologies from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells untreated or post treatment with 10 μM cycloheximide for 2 h. Scale bar represented as 15 μm. (G) Percentage of cells displaying tubular, intermediate, fragmented or hyperfused mitochondria ( n = 3). Statistical analysis was performed using Student’s t test. ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001. Data represented as mean ± standard error of mean.

Article Snippet: SLP2 (mouse) , OriGene , Cat# TA808240; RRID: AB_2628756.

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 and MIC13 synergistically modulate assembly and nanoscale distribution of MIC60 (A) Assessment of steady state levels of MICOS proteins with western blot from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells. HSP60 serves as a loading control. (B) BN-PAGE of isolated mitochondria from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells to assess MICOS assembly. MIC13-SLP2 DKO showed reduced MIC60 assembly in MICOS complex compared to any single KO. OXPHOS cocktail antibody is used as a control in BN-PAGE. (C) BN-PAGE quantification depicting relative protein assembly levels normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean, MIC60 ( n = 5), MIC10 ( n = 4), OXPHOS ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, p -value >0.05. (D) STED nanoscopy images from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells displaying MIC60 punctae. Images on the left side show individual mitochondria that are delineated by dotted lines. Images on the right-side zooms into the boxed mitochondria region. Blue arrows indicate individual MIC60 punctae in a rail-like arrangement in WT cells. Green arrow represents perturbed MIC60 puncta in MIC13 KO and SLP2 KO that are not arranged in rail-like arrangement. Purple arrow shows evenly spread (diffuse) MIC60 puncta in MIC13-SLP2 DKO cells suggesting an altered pattern compared to WT. Scale bars represent the 500 nm. (E) Percentage of individual mitochondria displaying MIC60 puncta predominately arranged as rail-like pattern, non-rail-like pattern, or diffuse pattern is presented in a pie-chart format. (F) A model depicting that MIC60-subcomplex and MIB assembly is dependent on SLP2-MIC13.

Article Snippet: SLP2 (mouse) , OriGene , Cat# TA808240; RRID: AB_2628756.

Techniques: Western Blot, Control, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 specifically regulates assembly kinetics of MIC60 (A) WT cells stably expressing pLIX403 EV and MIC13 KO, MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG were treated with 1 μg/mL of doxycycline (Dox) for indicated time points and western blot analysis depicting steady state levels of MICOS proteins upon induction of MIC13-FLAG are shown. HSP60 serves as a loading control. (B) BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/mL of doxycycline (Dox) for indicated time points showing stable incorporation of MIC13-FLAG in MICOS complex. (C) BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/mL of Dox for indicated time points was probed for MIC10, MIC27 and MIC60 antibody. The green arrow (in the MIC13 KO lane) and red arrow (in the MIC13-SLP2 DKO lane) highlight the delay in assembly of MIC60 at the 8-h timepoint. The assembly kinetics of the MIC60-subcomplex, rather than the MIC10-subcomplex, is dependent on SLP2. (D) BN-PAGE quantification depicting relative protein assembly levels of MIC60 at 8 h normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean ( n = 3). The quantification reveals delayed MIC60 assembly in the absence of SLP2. Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05. (E) A model depicting the assembly kinetics of MIC60 in MICOS complex depends on SLP2. Slower incorporation of MIC60 is observed in the absence of SLP2 upon reintroduction of MIC13 in MIC13-SLP2 DKO .

Article Snippet: SLP2 (mouse) , OriGene , Cat# TA808240; RRID: AB_2628756.

Techniques: Stable Transfection, Expressing, Western Blot, Control, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Stabilized MIC10-subcomplex for MIC60 seeding and CJ formation (A) Western blot analysis of WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (KD) to assess steady state levels of MICOS proteins. The steady state levels of MIC60 remain unaltered across different cell lines. HSP60 serves as a loading control. (B) BN-PAGE with isolated mitochondria from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ- YME1L shRNA. The stabilized MIC10-subcomplex upon YME1L depletion could partially rescue the incorporation of MIC60 and MTX assembly in MIC13-SLP2 DKO. This shows that MIC10-subcomplex provides a docking site for assembly of MIC60. (C) BN-PAGE quantification depicting relative protein assembly levels normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ns = non-significant, p -value >0.05. (D) Mitochondrial cristae morphology accessed using TEM from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. Scale bar represents 0.5 μm. The skeletonization of the TEM image is depicted below the image. Mitochondria in MIC13 KO, SLP2 KO, MIC13-SLP2 DKO display loss of cristae and CJs, with cristae arranged as either stacks or concentric rings. SLP2 KO additionally display swollen cristae. YME1L depletion showed beneficial consequences on cristae morphology with presence of nascent CJs (red arrows). This shows that loss of CJs in MIC13 KO is attributed to MIC10 loss, and swelling of SLP2 KO cristae is visibly restored upon YME1L depletion. (E) Quantification of crista and CJs per mitochondrial section. Outliers were removed with Grubbs’ method and statistical significance was analyzed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, P-value >0.05.

Article Snippet: SLP2 (mouse) , OriGene , Cat# TA808240; RRID: AB_2628756.

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Schematic illustration of synergistic role of SLP2 and MIC13 in MICOS assembly and crista junction formation The schematic model illustrates the quality control processes involved in cristae junction (CJ) morphogenesis and MICOS homeostasis, mediated by the MIC13-YME1L and SLP2-YME1L, which differentially stabilize components of the MIC10-subcomplex. This process facilitates the formation of the “seeder complex”, which consists of SLP2 and the stabilized MIC10-subcomplex. The simultaneous depletion of MIC13 and SLP2 disrupts the nanoscale distribution of MIC60 and its integration into the MICOS complex. The proposed “seeder model” suggests that the formation of the seeder complex is crucial for organizing the punctate distribution of MIC60 and facilitating its assembly into the MICOS-MIB complex. This process is essential for the formation of nascent CJs and establishing contact sites between IM and OM. In addition to identifying SLP2 as a regulator of cristae morphology, this model provides key insights into the intricate and partially overlapping quality control pathways governing MICOS regulation, elucidates specific functions attributed to MIC13, and highlights the interdependency between the MIC10- and MIC60-subcomplexes in MICOS-MIB complex assembly.

Article Snippet: SLP2 (mouse) , OriGene , Cat# TA808240; RRID: AB_2628756.

Techniques: Control

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet:

Article Snippet: SLP2 (mouse) , OriGene , Cat# TA808240; RRID: AB_2628756.

Techniques: Virus, Recombinant, Protease Inhibitor, In Situ, Clone Assay, Transfection, Mass Spectrometry, Knock-Out, Double Knockout, Stable Transfection, Expressing, Plasmid Preparation, Control, shRNA, CRISPR, Software

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 is identified as an interacting partner of MIC13 (A) Interactome of MIC13 with co-IP (co-immunoprecipitation) coupled mass spectrometry revealed SLP2 as an interactor of MIC13. (B) The interaction between SLP2 and MIC13 was confirmed by co-IP using FLAG antibody in isolated mitochondria from MIC13 KO cells stably expressing MIC13-FLAG or empty vector (EV) pMSCVpuro as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from anti-Flag M2 beads, ∗ non-specific IgG bands. (C) Co-IP was used to detect the SLP2-MICOS interaction using isolated mitochondria from SLP2 KO stably expressing pMSCVpuro EV (background control) or SLP2-MYC. Co-IP was performed using MYC-Trap agarose beads. I: Input fraction (10% of total lysate), E: Eluate fraction. YME1L was used as a positive interactor of SLP2 whereas Mt-CO2 and HSP60 served as non-interactors. All the MICOS subunits were detected in the elution fraction from SLP2-MYC co-IP. (D) Proximity ligation assay (PLA) in HeLa cells with antibodies against MICOS subunits and SLP2. PLA signals are shown as red spots indicating respective protein interactions. SLP2 alone and Mt-CO2 & SLP2 antibodies were probed as negative controls. Scale bar 20 μm. (E) BN-PAGE with isolated mitochondria from WT cells revealed a co-migration pattern of SLP2 with higher molecular weight MICOS complex. (F) A heatmap and graph represent the normalized occurrence of SLP2 and MICOS subunits in complexome profiling data obtained from HEK293 cells studied previously. SLP2 co-clustered with high molecular weight MICOS complex at around 2000 kDa. (G) Scaffolding model depicting interaction of SLP2 with MICOS subunits shows that SLP2 provides a scaffold for interaction of MICOS subunits.

Article Snippet: CRISPR-Cas9 double nickase plasmid: SLP2 , Santa Cruz Biotechnology , sc-403638-NIC.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Mass Spectrometry, Isolation, Stable Transfection, Expressing, Plasmid Preparation, Control, Proximity Ligation Assay, Migration, Molecular Weight, High Molecular Weight, Scaffolding

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Loss of SLP2 leads to aberrant cristae structure and reduced MIC26 levels (A) Steady state levels of MICOS proteins with western blot analysis from WT, SLP2 KO and SLP2 KO cells stably expressing pMSCVpuro EV or SLP2-MYC. Tubulin serves as a loading control for western blots. (B) Western blot quantification depicting relative protein levels. SLP2 KO was normalized to WT and SLP2 KO + SLP2 MYC was normalized to SLP2 KO + EV samples. Data is represented as mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗∗ p -value ≤0.001. MIC26 levels were reduced in SLP2 KO. Notably, MIC27 levels showed a slight increase in SLP2 KO. (C) Western blot analysis of steady state levels of MICOS proteins from WT and SLP2 KO cells stably expressing pGIPZ-control shRNA or YME1L shRNA. The results are depicted by a model illustrating the role of SLP2 in stabilizing MIC26 through the regulation of YME1L-mediated proteolysis. HSP60 acts as an internal protein loading control. (D) TEM images from WT, SLP2 KO and MIC26 KO cells. SLP2 KO shows loss of cristae and CJs with accumulation of swollen cristae, while MIC26 KO shows slight cristae branching. Scale bar represents 500 nm. The skeletonization of the TEM image is depicted on right side. (E) Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, p -value >0.05. (F) Assessment of mitochondrial morphologies from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells untreated or post treatment with 10 μM cycloheximide for 2 h. Scale bar represented as 15 μm. (G) Percentage of cells displaying tubular, intermediate, fragmented or hyperfused mitochondria ( n = 3). Statistical analysis was performed using Student’s t test. ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001. Data represented as mean ± standard error of mean.

Article Snippet: CRISPR-Cas9 double nickase plasmid: SLP2 , Santa Cruz Biotechnology , sc-403638-NIC.

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 and MIC13 synergistically modulate assembly and nanoscale distribution of MIC60 (A) Assessment of steady state levels of MICOS proteins with western blot from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells. HSP60 serves as a loading control. (B) BN-PAGE of isolated mitochondria from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells to assess MICOS assembly. MIC13-SLP2 DKO showed reduced MIC60 assembly in MICOS complex compared to any single KO. OXPHOS cocktail antibody is used as a control in BN-PAGE. (C) BN-PAGE quantification depicting relative protein assembly levels normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean, MIC60 ( n = 5), MIC10 ( n = 4), OXPHOS ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, p -value >0.05. (D) STED nanoscopy images from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells displaying MIC60 punctae. Images on the left side show individual mitochondria that are delineated by dotted lines. Images on the right-side zooms into the boxed mitochondria region. Blue arrows indicate individual MIC60 punctae in a rail-like arrangement in WT cells. Green arrow represents perturbed MIC60 puncta in MIC13 KO and SLP2 KO that are not arranged in rail-like arrangement. Purple arrow shows evenly spread (diffuse) MIC60 puncta in MIC13-SLP2 DKO cells suggesting an altered pattern compared to WT. Scale bars represent the 500 nm. (E) Percentage of individual mitochondria displaying MIC60 puncta predominately arranged as rail-like pattern, non-rail-like pattern, or diffuse pattern is presented in a pie-chart format. (F) A model depicting that MIC60-subcomplex and MIB assembly is dependent on SLP2-MIC13.

Article Snippet: CRISPR-Cas9 double nickase plasmid: SLP2 , Santa Cruz Biotechnology , sc-403638-NIC.

Techniques: Western Blot, Control, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 specifically regulates assembly kinetics of MIC60 (A) WT cells stably expressing pLIX403 EV and MIC13 KO, MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG were treated with 1 μg/mL of doxycycline (Dox) for indicated time points and western blot analysis depicting steady state levels of MICOS proteins upon induction of MIC13-FLAG are shown. HSP60 serves as a loading control. (B) BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/mL of doxycycline (Dox) for indicated time points showing stable incorporation of MIC13-FLAG in MICOS complex. (C) BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/mL of Dox for indicated time points was probed for MIC10, MIC27 and MIC60 antibody. The green arrow (in the MIC13 KO lane) and red arrow (in the MIC13-SLP2 DKO lane) highlight the delay in assembly of MIC60 at the 8-h timepoint. The assembly kinetics of the MIC60-subcomplex, rather than the MIC10-subcomplex, is dependent on SLP2. (D) BN-PAGE quantification depicting relative protein assembly levels of MIC60 at 8 h normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean ( n = 3). The quantification reveals delayed MIC60 assembly in the absence of SLP2. Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05. (E) A model depicting the assembly kinetics of MIC60 in MICOS complex depends on SLP2. Slower incorporation of MIC60 is observed in the absence of SLP2 upon reintroduction of MIC13 in MIC13-SLP2 DKO .

Article Snippet: CRISPR-Cas9 double nickase plasmid: SLP2 , Santa Cruz Biotechnology , sc-403638-NIC.

Techniques: Stable Transfection, Expressing, Western Blot, Control, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Stabilized MIC10-subcomplex for MIC60 seeding and CJ formation (A) Western blot analysis of WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (KD) to assess steady state levels of MICOS proteins. The steady state levels of MIC60 remain unaltered across different cell lines. HSP60 serves as a loading control. (B) BN-PAGE with isolated mitochondria from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ- YME1L shRNA. The stabilized MIC10-subcomplex upon YME1L depletion could partially rescue the incorporation of MIC60 and MTX assembly in MIC13-SLP2 DKO. This shows that MIC10-subcomplex provides a docking site for assembly of MIC60. (C) BN-PAGE quantification depicting relative protein assembly levels normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ns = non-significant, p -value >0.05. (D) Mitochondrial cristae morphology accessed using TEM from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. Scale bar represents 0.5 μm. The skeletonization of the TEM image is depicted below the image. Mitochondria in MIC13 KO, SLP2 KO, MIC13-SLP2 DKO display loss of cristae and CJs, with cristae arranged as either stacks or concentric rings. SLP2 KO additionally display swollen cristae. YME1L depletion showed beneficial consequences on cristae morphology with presence of nascent CJs (red arrows). This shows that loss of CJs in MIC13 KO is attributed to MIC10 loss, and swelling of SLP2 KO cristae is visibly restored upon YME1L depletion. (E) Quantification of crista and CJs per mitochondrial section. Outliers were removed with Grubbs’ method and statistical significance was analyzed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, P-value >0.05.

Article Snippet: CRISPR-Cas9 double nickase plasmid: SLP2 , Santa Cruz Biotechnology , sc-403638-NIC.

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Schematic illustration of synergistic role of SLP2 and MIC13 in MICOS assembly and crista junction formation The schematic model illustrates the quality control processes involved in cristae junction (CJ) morphogenesis and MICOS homeostasis, mediated by the MIC13-YME1L and SLP2-YME1L, which differentially stabilize components of the MIC10-subcomplex. This process facilitates the formation of the “seeder complex”, which consists of SLP2 and the stabilized MIC10-subcomplex. The simultaneous depletion of MIC13 and SLP2 disrupts the nanoscale distribution of MIC60 and its integration into the MICOS complex. The proposed “seeder model” suggests that the formation of the seeder complex is crucial for organizing the punctate distribution of MIC60 and facilitating its assembly into the MICOS-MIB complex. This process is essential for the formation of nascent CJs and establishing contact sites between IM and OM. In addition to identifying SLP2 as a regulator of cristae morphology, this model provides key insights into the intricate and partially overlapping quality control pathways governing MICOS regulation, elucidates specific functions attributed to MIC13, and highlights the interdependency between the MIC10- and MIC60-subcomplexes in MICOS-MIB complex assembly.

Article Snippet: CRISPR-Cas9 double nickase plasmid: SLP2 , Santa Cruz Biotechnology , sc-403638-NIC.

Techniques: Control

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet:

Article Snippet: CRISPR-Cas9 double nickase plasmid: SLP2 , Santa Cruz Biotechnology , sc-403638-NIC.

Techniques: Virus, Recombinant, Protease Inhibitor, In Situ, Cloning, Transfection, Mass Spectrometry, Knock-Out, Double Knockout, Stable Transfection, Expressing, Plasmid Preparation, Control, shRNA, CRISPR, Software

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 is identified as an interacting partner of MIC13 (A) Interactome of MIC13 with co-IP (co-immunoprecipitation) coupled mass spectrometry revealed SLP2 as an interactor of MIC13. (B) The interaction between SLP2 and MIC13 was confirmed by co-IP using FLAG antibody in isolated mitochondria from MIC13 KO cells stably expressing MIC13-FLAG or empty vector (EV) pMSCVpuro as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from anti-Flag M2 beads, ∗ non-specific IgG bands. (C) Co-IP was used to detect the SLP2-MICOS interaction using isolated mitochondria from SLP2 KO stably expressing pMSCVpuro EV (background control) or SLP2-MYC. Co-IP was performed using MYC-Trap agarose beads. I: Input fraction (10% of total lysate), E: Eluate fraction. YME1L was used as a positive interactor of SLP2 whereas Mt-CO2 and HSP60 served as non-interactors. All the MICOS subunits were detected in the elution fraction from SLP2-MYC co-IP. (D) Proximity ligation assay (PLA) in HeLa cells with antibodies against MICOS subunits and SLP2. PLA signals are shown as red spots indicating respective protein interactions. SLP2 alone and Mt-CO2 & SLP2 antibodies were probed as negative controls. Scale bar 20 μm. (E) BN-PAGE with isolated mitochondria from WT cells revealed a co-migration pattern of SLP2 with higher molecular weight MICOS complex. (F) A heatmap and graph represent the normalized occurrence of SLP2 and MICOS subunits in complexome profiling data obtained from HEK293 cells studied previously. SLP2 co-clustered with high molecular weight MICOS complex at around 2000 kDa. (G) Scaffolding model depicting interaction of SLP2 with MICOS subunits shows that SLP2 provides a scaffold for interaction of MICOS subunits.

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Mass Spectrometry, Isolation, Stable Transfection, Expressing, Plasmid Preparation, Control, Proximity Ligation Assay, Migration, Molecular Weight, Scaffolding

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Loss of SLP2 leads to aberrant cristae structure and reduced MIC26 levels (A) Steady state levels of MICOS proteins with western blot analysis from WT, SLP2 KO and SLP2 KO cells stably expressing pMSCVpuro EV or SLP2-MYC. Tubulin serves as a loading control for western blots. (B) Western blot quantification depicting relative protein levels. SLP2 KO was normalized to WT and SLP2 KO + SLP2 MYC was normalized to SLP2 KO + EV samples. Data is represented as mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗∗ p -value ≤0.001. MIC26 levels were reduced in SLP2 KO. Notably, MIC27 levels showed a slight increase in SLP2 KO. (C) Western blot analysis of steady state levels of MICOS proteins from WT and SLP2 KO cells stably expressing pGIPZ-control shRNA or YME1L shRNA. The results are depicted by a model illustrating the role of SLP2 in stabilizing MIC26 through the regulation of YME1L-mediated proteolysis. HSP60 acts as an internal protein loading control. (D) TEM images from WT, SLP2 KO and MIC26 KO cells. SLP2 KO shows loss of cristae and CJs with accumulation of swollen cristae, while MIC26 KO shows slight cristae branching. Scale bar represents 500 nm. The skeletonization of the TEM image is depicted on right side. (E) Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, p -value >0.05. (F) Assessment of mitochondrial morphologies from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells untreated or post treatment with 10 μM cycloheximide for 2 h. Scale bar represented as 15 μm. (G) Percentage of cells displaying tubular, intermediate, fragmented or hyperfused mitochondria ( n = 3). Statistical analysis was performed using Student’s t test. ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001. Data represented as mean ± standard error of mean.

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 and MIC13 synergistically modulate assembly and nanoscale distribution of MIC60 (A) Assessment of steady state levels of MICOS proteins with western blot from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells. HSP60 serves as a loading control. (B) BN-PAGE of isolated mitochondria from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells to assess MICOS assembly. MIC13-SLP2 DKO showed reduced MIC60 assembly in MICOS complex compared to any single KO. OXPHOS cocktail antibody is used as a control in BN-PAGE. (C) BN-PAGE quantification depicting relative protein assembly levels normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean, MIC60 ( n = 5), MIC10 ( n = 4), OXPHOS ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, p -value >0.05. (D) STED nanoscopy images from WT, MIC13 KO, SLP2 KO and MIC13-SLP2 DKO cells displaying MIC60 punctae. Images on the left side show individual mitochondria that are delineated by dotted lines. Images on the right-side zooms into the boxed mitochondria region. Blue arrows indicate individual MIC60 punctae in a rail-like arrangement in WT cells. Green arrow represents perturbed MIC60 puncta in MIC13 KO and SLP2 KO that are not arranged in rail-like arrangement. Purple arrow shows evenly spread (diffuse) MIC60 puncta in MIC13-SLP2 DKO cells suggesting an altered pattern compared to WT. Scale bars represent the 500 nm. (E) Percentage of individual mitochondria displaying MIC60 puncta predominately arranged as rail-like pattern, non-rail-like pattern, or diffuse pattern is presented in a pie-chart format. (F) A model depicting that MIC60-subcomplex and MIB assembly is dependent on SLP2-MIC13.

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Western Blot, Control, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: SLP2 specifically regulates assembly kinetics of MIC60 (A) WT cells stably expressing pLIX403 EV and MIC13 KO, MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG were treated with 1 μg/mL of doxycycline (Dox) for indicated time points and western blot analysis depicting steady state levels of MICOS proteins upon induction of MIC13-FLAG are shown. HSP60 serves as a loading control. (B) BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/mL of doxycycline (Dox) for indicated time points showing stable incorporation of MIC13-FLAG in MICOS complex. (C) BN-PAGE with isolated mitochondria from WT cells stably expressing pLIX403 EV, and MIC13 KO and MIC13-SLP2 DKO cells stably expressing pLIX403-MIC13-FLAG treated with 1 μg/mL of Dox for indicated time points was probed for MIC10, MIC27 and MIC60 antibody. The green arrow (in the MIC13 KO lane) and red arrow (in the MIC13-SLP2 DKO lane) highlight the delay in assembly of MIC60 at the 8-h timepoint. The assembly kinetics of the MIC60-subcomplex, rather than the MIC10-subcomplex, is dependent on SLP2. (D) BN-PAGE quantification depicting relative protein assembly levels of MIC60 at 8 h normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean ( n = 3). The quantification reveals delayed MIC60 assembly in the absence of SLP2. Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05. (E) A model depicting the assembly kinetics of MIC60 in MICOS complex depends on SLP2. Slower incorporation of MIC60 is observed in the absence of SLP2 upon reintroduction of MIC13 in MIC13-SLP2 DKO .

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Stable Transfection, Expressing, Western Blot, Control, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Stabilized MIC10-subcomplex for MIC60 seeding and CJ formation (A) Western blot analysis of WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (KD) to assess steady state levels of MICOS proteins. The steady state levels of MIC60 remain unaltered across different cell lines. HSP60 serves as a loading control. (B) BN-PAGE with isolated mitochondria from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ- YME1L shRNA. The stabilized MIC10-subcomplex upon YME1L depletion could partially rescue the incorporation of MIC60 and MTX assembly in MIC13-SLP2 DKO. This shows that MIC10-subcomplex provides a docking site for assembly of MIC60. (C) BN-PAGE quantification depicting relative protein assembly levels normalized to Coomassie. Normalized intensities of MIC60 antibody signal throughout gel were used to calculate fold change relative to WT. Data is represented as mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ns = non-significant, p -value >0.05. (D) Mitochondrial cristae morphology accessed using TEM from WT, MIC13 KO, SLP2 KO, MIC13-SLP2 DKO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. Scale bar represents 0.5 μm. The skeletonization of the TEM image is depicted below the image. Mitochondria in MIC13 KO, SLP2 KO, MIC13-SLP2 DKO display loss of cristae and CJs, with cristae arranged as either stacks or concentric rings. SLP2 KO additionally display swollen cristae. YME1L depletion showed beneficial consequences on cristae morphology with presence of nascent CJs (red arrows). This shows that loss of CJs in MIC13 KO is attributed to MIC10 loss, and swelling of SLP2 KO cristae is visibly restored upon YME1L depletion. (E) Quantification of crista and CJs per mitochondrial section. Outliers were removed with Grubbs’ method and statistical significance was analyzed using Student’s t test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p -value ≤0.0001, ns = non-significant, P-value >0.05.

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA, Isolation

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet: Schematic illustration of synergistic role of SLP2 and MIC13 in MICOS assembly and crista junction formation The schematic model illustrates the quality control processes involved in cristae junction (CJ) morphogenesis and MICOS homeostasis, mediated by the MIC13-YME1L and SLP2-YME1L, which differentially stabilize components of the MIC10-subcomplex. This process facilitates the formation of the “seeder complex”, which consists of SLP2 and the stabilized MIC10-subcomplex. The simultaneous depletion of MIC13 and SLP2 disrupts the nanoscale distribution of MIC60 and its integration into the MICOS complex. The proposed “seeder model” suggests that the formation of the seeder complex is crucial for organizing the punctate distribution of MIC60 and facilitating its assembly into the MICOS-MIB complex. This process is essential for the formation of nascent CJs and establishing contact sites between IM and OM. In addition to identifying SLP2 as a regulator of cristae morphology, this model provides key insights into the intricate and partially overlapping quality control pathways governing MICOS regulation, elucidates specific functions attributed to MIC13, and highlights the interdependency between the MIC10- and MIC60-subcomplexes in MICOS-MIB complex assembly.

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Control

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet:

Article Snippet: Blocking solution was removed and primary antibodies with 1:100 dilution ratio was added to the samples and incubation was carried out at 37°C for 2 h. The following primary antibodies were used: MIC10 (Abcam, 84969), MIC13 (custom made by Pineda (Berlin) against human MIC13 peptide CKAREYSKEGWEYVKARTK), MIC19 (Proteintech, 25625-1-AP), MIC25 (Proteintech, 20639-1-AP), MIC26 (Thermofisher Scientific, MA5-15493), MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), SLP2 (OriGene, TA808240), Mt-CO2 (Abcam, ab110258)), OPA1 (custom-made, Pineda against human OPA1 using peptide CDLKKVREIQEKLDAFIEALHQEK, Vinculin (Sigma-Aldrich, V9131).

Techniques: Virus, Recombinant, Protease Inhibitor, In Situ, Clone Assay, Transfection, Mass Spectrometry, Knock-Out, Double Knockout, Stable Transfection, Expressing, Plasmid Preparation, Control, shRNA, CRISPR, Software