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Proteintech mic13

<t>MIC13</t> primarily stabilizes MIC10-subcomplex to promote CJ formation (A) Western blot analysis of WT and MIC13 KO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (knockdown represented as YME1L KD) to assess steady state levels of MICOS proteins. MIC13 KO leads to loss of MIC10, MIC26 and MIC27, which are restored upon YME1L KD, indicating their dependency on YME1L-mediated proteolysis. HSP60 serves as a loading control. The red arrow highlights the lower band of MIC27 that appears specifically in the MIC13 KO. Although YME1L knockdown in MIC13 KO cells restores the steady-state levels of MIC27, the persistence of the lower band suggest that this alteration is specifically linked to absence of MIC13. (B) Western blot quantification shows relative protein levels, with values normalized to those of the WT. Data is represented as the mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test or one sample t -test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ns = non-significant, p -value >0.05. (C) The interaction between MIC60 and MIC10 was validated by co-IP using MIC60 antibody conjugated Protein A Sepharose beads in isolated mitochondria from WT and MIC13 KO cells stably expressing pGIPZ YME1L shRNA or empty vector (EV) pGIPZ as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from beads. MIC10 and MIC27, which are restored upon YME1L depletion, could still interact with MIC60 even in the absence of MIC13 (indicated by red arrow). (D) Mitochondrial cristae morphology accessed using TEM from WT and MIC13 KO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. The skeletonization of the TEM image is shown below each corresponding image. Scale bar represents 0.5 μm. YME1L depletion showed beneficial consequences on cristae morphology with presence of nascent CJs (red arrows). This indicates that loss of CJs in MIC13 KO is attributed to MIC10 loss. (E) Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed using Student’s t test. ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001. (F) Scheme depicting the role of MIC13 in protecting the MIC10-subcomplex from YME1L-mediated proteolysis. In absence of MIC13, the stabilized MIC10-subcomplex could still facilitate the formation of crista junctions and maintain the interaction between MIC60-subcomplex and remaining MIC10-subcomplex.

Mic13, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mic13 - by Bioz Stars, 2026-03

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1) Product Images from "SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation"

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

Journal: iScience

doi: 10.1016/j.isci.2024.111467

MIC13 primarily stabilizes MIC10-subcomplex to promote CJ formation (A) Western blot analysis of WT and MIC13 KO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (knockdown represented as YME1L KD) to assess steady state levels of MICOS proteins. MIC13 KO leads to loss of MIC10, MIC26 and MIC27, which are restored upon YME1L KD, indicating their dependency on YME1L-mediated proteolysis. HSP60 serves as a loading control. The red arrow highlights the lower band of MIC27 that appears specifically in the MIC13 KO. Although YME1L knockdown in MIC13 KO cells restores the steady-state levels of MIC27, the persistence of the lower band suggest that this alteration is specifically linked to absence of MIC13. (B) Western blot quantification shows relative protein levels, with values normalized to those of the WT. Data is represented as the mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test or one sample t -test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ns = non-significant, p -value >0.05. (C) The interaction between MIC60 and MIC10 was validated by co-IP using MIC60 antibody conjugated Protein A Sepharose beads in isolated mitochondria from WT and MIC13 KO cells stably expressing pGIPZ YME1L shRNA or empty vector (EV) pGIPZ as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from beads. MIC10 and MIC27, which are restored upon YME1L depletion, could still interact with MIC60 even in the absence of MIC13 (indicated by red arrow). (D) Mitochondrial cristae morphology accessed using TEM from WT and MIC13 KO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. The skeletonization of the TEM image is shown below each corresponding image. Scale bar represents 0.5 μm. YME1L depletion showed beneficial consequences on cristae morphology with presence of nascent CJs (red arrows). This indicates that loss of CJs in MIC13 KO is attributed to MIC10 loss. (E) Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed using Student’s t test. ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001. (F) Scheme depicting the role of MIC13 in protecting the MIC10-subcomplex from YME1L-mediated proteolysis. In absence of MIC13, the stabilized MIC10-subcomplex could still facilitate the formation of crista junctions and maintain the interaction between MIC60-subcomplex and remaining MIC10-subcomplex.

Figure Legend Snippet: MIC13 primarily stabilizes MIC10-subcomplex to promote CJ formation (A) Western blot analysis of WT and MIC13 KO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (knockdown represented as YME1L KD) to assess steady state levels of MICOS proteins. MIC13 KO leads to loss of MIC10, MIC26 and MIC27, which are restored upon YME1L KD, indicating their dependency on YME1L-mediated proteolysis. HSP60 serves as a loading control. The red arrow highlights the lower band of MIC27 that appears specifically in the MIC13 KO. Although YME1L knockdown in MIC13 KO cells restores the steady-state levels of MIC27, the persistence of the lower band suggest that this alteration is specifically linked to absence of MIC13. (B) Western blot quantification shows relative protein levels, with values normalized to those of the WT. Data is represented as the mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test or one sample t -test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ns = non-significant, p -value >0.05. (C) The interaction between MIC60 and MIC10 was validated by co-IP using MIC60 antibody conjugated Protein A Sepharose beads in isolated mitochondria from WT and MIC13 KO cells stably expressing pGIPZ YME1L shRNA or empty vector (EV) pGIPZ as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from beads. MIC10 and MIC27, which are restored upon YME1L depletion, could still interact with MIC60 even in the absence of MIC13 (indicated by red arrow). (D) Mitochondrial cristae morphology accessed using TEM from WT and MIC13 KO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. The skeletonization of the TEM image is shown below each corresponding image. Scale bar represents 0.5 μm. YME1L depletion showed beneficial consequences on cristae morphology with presence of nascent CJs (red arrows). This indicates that loss of CJs in MIC13 KO is attributed to MIC10 loss. (E) Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed using Student’s t test. ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001. (F) Scheme depicting the role of MIC13 in protecting the MIC10-subcomplex from YME1L-mediated proteolysis. In absence of MIC13, the stabilized MIC10-subcomplex could still facilitate the formation of crista junctions and maintain the interaction between MIC60-subcomplex and remaining MIC10-subcomplex.

Techniques Used: Western Blot, Stable Transfection, Expressing, Control, shRNA, Knockdown, Co-Immunoprecipitation Assay, Isolation, Plasmid Preparation

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

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

Techniques Used: Western Blot, Control, Isolation

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 .

Figure Legend 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 .

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

Techniques Used: Control

Figure Legend Snippet:

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

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(A – D) Blue Native (BN)-PAGE analyses of four cell lines stably expressing MICOS-APEX2 fusion proteins in the corresponding MICOS KO cell lines along with WT HEK293 cells expressing an empty vector (EV) or IM-APEX2 fusion protein used as control cell line. Efficient incorporation of (A) MIC10-, (B) MIC13-, (C) MIC26- and (D) MIC27-APEX2 fusion proteins (enclosed by solid rectangles) into the MICOS complex is observed using the respective antibodies. The dotted rectangles represent loss of various MICOS proteins in the respective KO cell lines. Coomassie stain is used as loading control comprising regions between 500 and 800 kDa. (E) Transmission electron microscopy (TEM) images demonstrating the MICOS-APEX2, IM-APEX2 and matrix-APEX2 submitochondrial localization in various cell lines. DAB oxidation catalysed by APEX2 results in local deposition of DAB polymer. All the MICOS-APEX2 fusion proteins as well as the IM control reveal the DAB staining in the intermembrane space (IMS) as well as intracristal space confirming the orientation of APEX2 towards the IMS. The matrix-APEX2 reveals matrix DAB staining as expected. Scale bar 500 nm. (F) Images acquired by STED super-resolution nanoscopy demonstrate the pattern of biotinylation in individual mitochondria resulting from MICOS-APEX2 fusion proteins. Biotinylation is prevalent at the rim of mitochondria as indicated by white arrows. The IM-APEX2 reveals similar biotinylation pattern to MICOS-APEX2 different to matrix-APEX2. The MICOS complex, enriched at the CJs, is marked using an anti-MIC60 antibody. Scale bar 500 nm.

Journal: bioRxiv

Article Title: MINDNet: Proximity interactome of the MICOS complex revealing a multifaceted network orchestrating mitochondrial biogenesis

doi: 10.1101/2025.05.20.655052

Figure Lengend Snippet: (A – D) Blue Native (BN)-PAGE analyses of four cell lines stably expressing MICOS-APEX2 fusion proteins in the corresponding MICOS KO cell lines along with WT HEK293 cells expressing an empty vector (EV) or IM-APEX2 fusion protein used as control cell line. Efficient incorporation of (A) MIC10-, (B) MIC13-, (C) MIC26- and (D) MIC27-APEX2 fusion proteins (enclosed by solid rectangles) into the MICOS complex is observed using the respective antibodies. The dotted rectangles represent loss of various MICOS proteins in the respective KO cell lines. Coomassie stain is used as loading control comprising regions between 500 and 800 kDa. (E) Transmission electron microscopy (TEM) images demonstrating the MICOS-APEX2, IM-APEX2 and matrix-APEX2 submitochondrial localization in various cell lines. DAB oxidation catalysed by APEX2 results in local deposition of DAB polymer. All the MICOS-APEX2 fusion proteins as well as the IM control reveal the DAB staining in the intermembrane space (IMS) as well as intracristal space confirming the orientation of APEX2 towards the IMS. The matrix-APEX2 reveals matrix DAB staining as expected. Scale bar 500 nm. (F) Images acquired by STED super-resolution nanoscopy demonstrate the pattern of biotinylation in individual mitochondria resulting from MICOS-APEX2 fusion proteins. Biotinylation is prevalent at the rim of mitochondria as indicated by white arrows. The IM-APEX2 reveals similar biotinylation pattern to MICOS-APEX2 different to matrix-APEX2. The MICOS complex, enriched at the CJs, is marked using an anti-MIC60 antibody. Scale bar 500 nm.

Article Snippet: For lentiviral transduction, HEK293FT cells were transfected with 1 µg of pLIX403 EV or pLIX403-MIC13-Flag, pLIX403-MIC10 or pLIX403-MIC60 along with 1 µg of psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) using GeneJuice transfection reagent.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Control, Staining, Transmission Assay, Electron Microscopy, Polymer

(A) Schematic representation of the experimental setup and the analysis pipeline. The MICOS interactors were obtained after normalization over the matrix-APEX2 and IM-APEX2 controls separately upon MitoCarta3.0 filtering. 95 to 117 MICOS interactors (FOM ( F ound O nly in respective M ICOS) proteins absent in the control, represented using a hexagon) and 36 to 45 MICOS interactors (log 2 FC enriched proteins over control condition, represented using circle) were uncovered upon normalization over matrix-APEX2 control whereas 7 to 18 (FOM category, represented using square diamond) and 10 to 20 MICOS interactors (log 2 FC enrichment, represented using square) were uncovered upon using IM-APEX2 as control. (B – D) Histograms depicting the percentage of proteins possessing the respective submitochondrial localization assigned according to MitoCarta3.0 compared to the interactome of various MICOS subunits. Histograms representing the mitochondrial localization of the total sum of interactors obtained upon combining interactomes normalized to both controls (B), matrix-APEX2 control (C) and IM-APEX2 control (D). Annotated mitochondrial subcompartments include outer membrane (OM), intermembrane space (IMS), inner membrane (IM), matrix, membrane, unknown and miscellaneous compartment and are shown using different colour-codes. (E – G) Histograms depicting the percentage of proteins possessing the respective mitochondrial functions assigned according to MitoCarta3.0 compared to the interactome of various MICOS subunits. Histograms representing the mitochondrial functions of the sum total of interactors obtained upon combining interactomes normalized to both controls (E), matrix-APEX2 control (F) and IM-APEX2 control (G). Various mitochondrial functions are shown using different colour-codes and comprise the following categories: metabolism, protein import, central dogma, dynamics and surveillance, OXPHOS, Signaling, small molecule transport and unknown. (H and I) Venn diagram depicting the number of common enriched proteins among the MIC10/MIC13/MIC26/MIC27 interactome in different combinations obtained using matrix-APEX2 (H) and IM-APEX2 (I) as controls, respectively. The category of FOM proteins ( F ound O nly in M ICOS and absent in the control) is represented using a hexagon and square diamond obtained upon using matrix-APEX2 and IM-APEX2 controls, respectively. Log 2 FC enriched proteins over control, are represented using circle and square upon using matrix-APEX2 and IM-APEX2 control, respectively.

Journal: bioRxiv

Article Title: MINDNet: Proximity interactome of the MICOS complex revealing a multifaceted network orchestrating mitochondrial biogenesis

doi: 10.1101/2025.05.20.655052

Figure Lengend Snippet: (A) Schematic representation of the experimental setup and the analysis pipeline. The MICOS interactors were obtained after normalization over the matrix-APEX2 and IM-APEX2 controls separately upon MitoCarta3.0 filtering. 95 to 117 MICOS interactors (FOM ( F ound O nly in respective M ICOS) proteins absent in the control, represented using a hexagon) and 36 to 45 MICOS interactors (log 2 FC enriched proteins over control condition, represented using circle) were uncovered upon normalization over matrix-APEX2 control whereas 7 to 18 (FOM category, represented using square diamond) and 10 to 20 MICOS interactors (log 2 FC enrichment, represented using square) were uncovered upon using IM-APEX2 as control. (B – D) Histograms depicting the percentage of proteins possessing the respective submitochondrial localization assigned according to MitoCarta3.0 compared to the interactome of various MICOS subunits. Histograms representing the mitochondrial localization of the total sum of interactors obtained upon combining interactomes normalized to both controls (B), matrix-APEX2 control (C) and IM-APEX2 control (D). Annotated mitochondrial subcompartments include outer membrane (OM), intermembrane space (IMS), inner membrane (IM), matrix, membrane, unknown and miscellaneous compartment and are shown using different colour-codes. (E – G) Histograms depicting the percentage of proteins possessing the respective mitochondrial functions assigned according to MitoCarta3.0 compared to the interactome of various MICOS subunits. Histograms representing the mitochondrial functions of the sum total of interactors obtained upon combining interactomes normalized to both controls (E), matrix-APEX2 control (F) and IM-APEX2 control (G). Various mitochondrial functions are shown using different colour-codes and comprise the following categories: metabolism, protein import, central dogma, dynamics and surveillance, OXPHOS, Signaling, small molecule transport and unknown. (H and I) Venn diagram depicting the number of common enriched proteins among the MIC10/MIC13/MIC26/MIC27 interactome in different combinations obtained using matrix-APEX2 (H) and IM-APEX2 (I) as controls, respectively. The category of FOM proteins ( F ound O nly in M ICOS and absent in the control) is represented using a hexagon and square diamond obtained upon using matrix-APEX2 and IM-APEX2 controls, respectively. Log 2 FC enriched proteins over control, are represented using circle and square upon using matrix-APEX2 and IM-APEX2 control, respectively.

Techniques: Control, Membrane

(A) Pie charts representing the percentage of enriched hits among the various combinations of MICOS-APEX2 groups. The majority of enriched proteins (68.2 %) are common for all MICOS-APEX2 fusion proteins obtained upon using matrix-APEX2 control. Conversely, a majority of the enriched proteins are unique interactors (55.1 %) for MIC10-, MIC13-, MIC26- and MIC27-APEX2 fusion proteins when compared to the IM-APEX2 control. (B and C) Proximity proteome representation of enriched hits shared between any three (B) or any two (C) MICOS-APEX2 groups upon using the respective APEX2 control. (D) Unique interactors of the respective MICOS-APEX2 fusion proteins upon using the respective APEX2 control are shown. The proteins were colour-coded in accordance to the functions assigned in MitoCarta3.0 and the node shapes represent the affiliation to Log 2 FC enriched or FOM category as in previous figures. The interactions are shown by lines connecting the different nodes where the gene names of the interactors are used. MICOS subunits present in yellow represent the preys, whereas MICOS subunits in orange represent the employed APEX2-fused baits.

Journal: bioRxiv

Article Title: MINDNet: Proximity interactome of the MICOS complex revealing a multifaceted network orchestrating mitochondrial biogenesis

doi: 10.1101/2025.05.20.655052

Figure Lengend Snippet: (A) Pie charts representing the percentage of enriched hits among the various combinations of MICOS-APEX2 groups. The majority of enriched proteins (68.2 %) are common for all MICOS-APEX2 fusion proteins obtained upon using matrix-APEX2 control. Conversely, a majority of the enriched proteins are unique interactors (55.1 %) for MIC10-, MIC13-, MIC26- and MIC27-APEX2 fusion proteins when compared to the IM-APEX2 control. (B and C) Proximity proteome representation of enriched hits shared between any three (B) or any two (C) MICOS-APEX2 groups upon using the respective APEX2 control. (D) Unique interactors of the respective MICOS-APEX2 fusion proteins upon using the respective APEX2 control are shown. The proteins were colour-coded in accordance to the functions assigned in MitoCarta3.0 and the node shapes represent the affiliation to Log 2 FC enriched or FOM category as in previous figures. The interactions are shown by lines connecting the different nodes where the gene names of the interactors are used. MICOS subunits present in yellow represent the preys, whereas MICOS subunits in orange represent the employed APEX2-fused baits.

Techniques: Control

(A and B) Heatmap representing the MIC10-, MIC13-, MIC26- and MIC27-APEX2 interactomes obtained using matrix-APEX2 (A) and IM-APEX2 (B) as controls. The heatmaps of the corresponding interactomes are distributed into mitochondrial subcompartments and functions. Missing proteins are crossed out.

Journal: bioRxiv

Article Title: MINDNet: Proximity interactome of the MICOS complex revealing a multifaceted network orchestrating mitochondrial biogenesis

doi: 10.1101/2025.05.20.655052

Figure Lengend Snippet: (A and B) Heatmap representing the MIC10-, MIC13-, MIC26- and MIC27-APEX2 interactomes obtained using matrix-APEX2 (A) and IM-APEX2 (B) as controls. The heatmaps of the corresponding interactomes are distributed into mitochondrial subcompartments and functions. Missing proteins are crossed out.

Techniques:

(A – D) BN-PAGE analyses reveals a consistent reduction of OXPHOS complex I and IV assembly in MIC10 and MIC60 KO cells among all MICOS KO cells (indicated by dotted rectangles). Coomassie stain is used as loading control comprising regions between 500 and 800 kDa. (E – G) Representative TEM images demonstrating the mitochondrial ultrastructure of individual MICOS KO cells (E). Quantification reveals a significant reduction in the number of crista junctions (CJs) per crista in MIC10 KO, MIC13 KO, MIC19 KO and MIC60 KO cells (F) and cristae number per unit length (µm) in MIC10 KO, MIC13 KO and MIC60 KO cells (G), (n = 36-79) (N = 2). (H and I) Representative mitochondrial stress test assessed with Seahorse XF analyzer using sequential injection of oligomycin, FCCP and rotenone/antimycin a (n = 8-9) (H). Quantification from various biological replicates shows a significant decrease in mitochondrial coupling efficiency in MIC10 KO and MIC60 KO cells (N = 4) (I). (J and K) Representative individual complex feeding run as assessed in permeabilized cells with Seahorse XF analyzer by sequential injections of rotenone, succinate, antimycin a and ascorbate/TMPD (n = 8-9) (J). Quantification of different biological replicates reveals a significant decrease of complex II and complex IV activities in MIC10 KO, MIC13 KO and MIC60 KO, whereas complex I activity is significantly decreased only in MIC10 KO and MIC60 KO cells (K) (N = 6). Data are represented as mean ± SEM (H – K). Statistical analysis was performed using one sample t -test with * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (I (Basal OCR) and K). Statistical analysis was performed using one-way ANOVA with * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (F, G, I (SRC and coupling efficiency)). N represents the number of biological replicates.

Journal: bioRxiv

Article Title: MINDNet: Proximity interactome of the MICOS complex revealing a multifaceted network orchestrating mitochondrial biogenesis

doi: 10.1101/2025.05.20.655052

Figure Lengend Snippet: (A – D) BN-PAGE analyses reveals a consistent reduction of OXPHOS complex I and IV assembly in MIC10 and MIC60 KO cells among all MICOS KO cells (indicated by dotted rectangles). Coomassie stain is used as loading control comprising regions between 500 and 800 kDa. (E – G) Representative TEM images demonstrating the mitochondrial ultrastructure of individual MICOS KO cells (E). Quantification reveals a significant reduction in the number of crista junctions (CJs) per crista in MIC10 KO, MIC13 KO, MIC19 KO and MIC60 KO cells (F) and cristae number per unit length (µm) in MIC10 KO, MIC13 KO and MIC60 KO cells (G), (n = 36-79) (N = 2). (H and I) Representative mitochondrial stress test assessed with Seahorse XF analyzer using sequential injection of oligomycin, FCCP and rotenone/antimycin a (n = 8-9) (H). Quantification from various biological replicates shows a significant decrease in mitochondrial coupling efficiency in MIC10 KO and MIC60 KO cells (N = 4) (I). (J and K) Representative individual complex feeding run as assessed in permeabilized cells with Seahorse XF analyzer by sequential injections of rotenone, succinate, antimycin a and ascorbate/TMPD (n = 8-9) (J). Quantification of different biological replicates reveals a significant decrease of complex II and complex IV activities in MIC10 KO, MIC13 KO and MIC60 KO, whereas complex I activity is significantly decreased only in MIC10 KO and MIC60 KO cells (K) (N = 6). Data are represented as mean ± SEM (H – K). Statistical analysis was performed using one sample t -test with * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (I (Basal OCR) and K). Statistical analysis was performed using one-way ANOVA with * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (F, G, I (SRC and coupling efficiency)). N represents the number of biological replicates.

Techniques: Staining, Control, Injection, Activity Assay

MICOS proteins and their possible functions.

Journal: International Journal of Molecular Sciences

Article Title: A New Perspective on the Role of Alterations in Mitochondrial Proteins Involved in ATP Synthesis and Mobilization in Cardiomyopathies

doi: 10.3390/ijms26062768

Figure Lengend Snippet: MICOS proteins and their possible functions.

Article Snippet: Bridging , Mic13 , QIL1 , Bridges and stabilizes the Mic60 and Mic10 subcomplexes to form the mature MICOS , QIl1, C19orf70, MICOS13 , [ , ] .

Techniques:

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: MIC13 primarily stabilizes MIC10-subcomplex to promote CJ formation (A) Western blot analysis of WT and MIC13 KO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA (knockdown represented as YME1L KD) to assess steady state levels of MICOS proteins. MIC13 KO leads to loss of MIC10, MIC26 and MIC27, which are restored upon YME1L KD, indicating their dependency on YME1L-mediated proteolysis. HSP60 serves as a loading control. The red arrow highlights the lower band of MIC27 that appears specifically in the MIC13 KO. Although YME1L knockdown in MIC13 KO cells restores the steady-state levels of MIC27, the persistence of the lower band suggest that this alteration is specifically linked to absence of MIC13. (B) Western blot quantification shows relative protein levels, with values normalized to those of the WT. Data is represented as the mean ± standard error of mean ( n = 3). Statistical analysis was performed using Student’s t test or one sample t -test. ∗ p -value ≤0.05, ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001, ns = non-significant, p -value >0.05. (C) The interaction between MIC60 and MIC10 was validated by co-IP using MIC60 antibody conjugated Protein A Sepharose beads in isolated mitochondria from WT and MIC13 KO cells stably expressing pGIPZ YME1L shRNA or empty vector (EV) pGIPZ as background control. I: input lanes represent loading of 10% of total lysates, E: eluate represent proteins eluted from beads. MIC10 and MIC27, which are restored upon YME1L depletion, could still interact with MIC60 even in the absence of MIC13 (indicated by red arrow). (D) Mitochondrial cristae morphology accessed using TEM from WT and MIC13 KO stably expressing pGIPZ-Control shRNA or pGIPZ-YME1L shRNA. The skeletonization of the TEM image is shown below each corresponding image. Scale bar represents 0.5 μm. YME1L depletion showed beneficial consequences on cristae morphology with presence of nascent CJs (red arrows). This indicates that loss of CJs in MIC13 KO is attributed to MIC10 loss. (E) Cristae number and CJs per mitochondrial section quantified from TEM images. Statistical analysis was performed using Student’s t test. ∗∗ p -value ≤0.01, ∗∗∗ p -value ≤0.001. (F) Scheme depicting the role of MIC13 in protecting the MIC10-subcomplex from YME1L-mediated proteolysis. In absence of MIC13, the stabilized MIC10-subcomplex could still facilitate the formation of crista junctions and maintain the interaction between MIC60-subcomplex and remaining MIC10-subcomplex.

Article Snippet: Membranes were blocked in 5% skimmed milk for 1 h and incubation was carried out overnight in 4°C under shaking conditions with primary antibodies: 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, MIC27 (Sigma-Aldrich, HPA000612-100UL), MIC60 (Abcam, ab110329), SLP2 (Abcam, ab102051), MTX1 (Abcam, ab233205).

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA, Knockdown, Co-Immunoprecipitation Assay, Isolation, Plasmid Preparation

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.

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.

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.

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 .

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.

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.

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:

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

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA, Knockdown, Co-Immunoprecipitation Assay, Isolation, Plasmid Preparation

Journal: iScience

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

doi: 10.1016/j.isci.2024.111467

Journal: iScience

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

doi: 10.1016/j.isci.2024.111467

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

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

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

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

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:

Journal: iScience

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

doi: 10.1016/j.isci.2024.111467

Article Snippet: The re-established interaction of MIC60 and replenished MIC10 in the absence of MIC13 prompted us to further investigate a specific role of MIC13 in MICOS assembly.

Techniques: Western Blot, Stable Transfection, Expressing, Control, shRNA, Knockdown, Co-Immunoprecipitation Assay, Isolation, Plasmid Preparation

Journal: iScience

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

doi: 10.1016/j.isci.2024.111467

Article Snippet: The re-established interaction of MIC60 and replenished MIC10 in the absence of MIC13 prompted us to further investigate a specific role of MIC13 in MICOS assembly.

Journal: iScience

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

doi: 10.1016/j.isci.2024.111467

Article Snippet: The re-established interaction of MIC60 and replenished MIC10 in the absence of MIC13 prompted us to further investigate a specific role of MIC13 in MICOS assembly.

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

Article Snippet: The re-established interaction of MIC60 and replenished MIC10 in the absence of MIC13 prompted us to further investigate a specific role of MIC13 in MICOS assembly.

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

Article Snippet: The re-established interaction of MIC60 and replenished MIC10 in the absence of MIC13 prompted us to further investigate a specific role of MIC13 in MICOS assembly.

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

Article Snippet: The re-established interaction of MIC60 and replenished MIC10 in the absence of MIC13 prompted us to further investigate a specific role of MIC13 in MICOS assembly.

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

Article Snippet: The re-established interaction of MIC60 and replenished MIC10 in the absence of MIC13 prompted us to further investigate a specific role of MIC13 in MICOS assembly.

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: The re-established interaction of MIC60 and replenished MIC10 in the absence of MIC13 prompted us to further investigate a specific role of MIC13 in MICOS assembly.

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