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plix403 mic60  (Addgene inc)


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    Addgene inc plix403 mic60
    (A) Schematic overview of various MICOS-APEX2-Myc fusion proteins (denoted as MICOS-APEX2 for simplicity from hereon) generated along with IM-APEX2-Myc (denoted as IM-APEX2 for simplicity from hereon) control fusion protein. Matrix-APEX2 was used as the second control condition. The amino acid linker sequences between respective MICOS proteins or IM transmembrane (TMD) domain and APEX2 are shown. AlphaFold 3 structure predictions, without cleavable mitochondrial targeting sequence, are represented using PyMol. Different MICOS subunits are depicted in blue colour. APEX2 and Myc tag are shown in red and orange (Myc tag additionally shown with arrows) respectively. The transmembrane domain of IM-APEX2 is depicted in light grey. The MICOS-APEX2 and IM-APEX2 fusion proteins were stably expressed in the background of respective MICOS KO and WT HEK293 cells, respectively. WT HEK293 cells, stably expressing empty vector (EV) were used as background control and, were employed for transient expression of matrix-APEX2. (B) Schematic representation of the APEX2 tagged to the respective MICOS subunit, IM-TMD and matrix controls. APEX2 catalyzes the biotin-phenoxyl radical production in the presence of biotin-phenol (BP) and H 2 O 2 to initiate proteome tagging in the molecular neighborhood depicted in red rings. (C) Western blot (WB) analyses of various MICOS-APEX2 fusion proteins and IM-APEX2 control stably expressed in the corresponding MICOS KO background and WT cells, respectively. Stable expression of different MICOS-APEX2 fusion proteins results in restoration of the expected MICOS proteins (enclosed by solid rectangles) compared to corresponding KO cell lines (enclosed by dotted rectangles). All the MICOS proteins in cells expressing the respective MICOS-APEX2 along with Myc displayed increased molecular weight compared to the endogenous MICOS proteins as expected. Therefore, the rescue can only be seen using the anti-Myc antibody. Their expression is shown using the Myc antibody and the band at the expected molecular weight is additionally indicated with solid rectangles. (D) Fluorescence microscopy demonstrating the biotinylation activity of APEX2-constructs, using streptavidin conjugated to DyLight 488 within mitochondria marked using <t>anti-MIC60</t> antibody in various cell lines mentioned. Nucleus is stained with Hoechst and shown in blue. Scale bar 10 µm.
    Plix403 Mic60, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plix403 mic60/product/Addgene inc
    Average 96 stars, based on 107 article reviews
    plix403 mic60 - by Bioz Stars, 2026-03
    96/100 stars

    Images

    1) Product Images from "MINDNet: Proximity interactome of the MICOS complex revealing a multifaceted network orchestrating mitochondrial biogenesis"

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

    Journal: bioRxiv

    doi: 10.1101/2025.05.20.655052

    (A) Schematic overview of various MICOS-APEX2-Myc fusion proteins (denoted as MICOS-APEX2 for simplicity from hereon) generated along with IM-APEX2-Myc (denoted as IM-APEX2 for simplicity from hereon) control fusion protein. Matrix-APEX2 was used as the second control condition. The amino acid linker sequences between respective MICOS proteins or IM transmembrane (TMD) domain and APEX2 are shown. AlphaFold 3 structure predictions, without cleavable mitochondrial targeting sequence, are represented using PyMol. Different MICOS subunits are depicted in blue colour. APEX2 and Myc tag are shown in red and orange (Myc tag additionally shown with arrows) respectively. The transmembrane domain of IM-APEX2 is depicted in light grey. The MICOS-APEX2 and IM-APEX2 fusion proteins were stably expressed in the background of respective MICOS KO and WT HEK293 cells, respectively. WT HEK293 cells, stably expressing empty vector (EV) were used as background control and, were employed for transient expression of matrix-APEX2. (B) Schematic representation of the APEX2 tagged to the respective MICOS subunit, IM-TMD and matrix controls. APEX2 catalyzes the biotin-phenoxyl radical production in the presence of biotin-phenol (BP) and H 2 O 2 to initiate proteome tagging in the molecular neighborhood depicted in red rings. (C) Western blot (WB) analyses of various MICOS-APEX2 fusion proteins and IM-APEX2 control stably expressed in the corresponding MICOS KO background and WT cells, respectively. Stable expression of different MICOS-APEX2 fusion proteins results in restoration of the expected MICOS proteins (enclosed by solid rectangles) compared to corresponding KO cell lines (enclosed by dotted rectangles). All the MICOS proteins in cells expressing the respective MICOS-APEX2 along with Myc displayed increased molecular weight compared to the endogenous MICOS proteins as expected. Therefore, the rescue can only be seen using the anti-Myc antibody. Their expression is shown using the Myc antibody and the band at the expected molecular weight is additionally indicated with solid rectangles. (D) Fluorescence microscopy demonstrating the biotinylation activity of APEX2-constructs, using streptavidin conjugated to DyLight 488 within mitochondria marked using anti-MIC60 antibody in various cell lines mentioned. Nucleus is stained with Hoechst and shown in blue. Scale bar 10 µm.
    Figure Legend Snippet: (A) Schematic overview of various MICOS-APEX2-Myc fusion proteins (denoted as MICOS-APEX2 for simplicity from hereon) generated along with IM-APEX2-Myc (denoted as IM-APEX2 for simplicity from hereon) control fusion protein. Matrix-APEX2 was used as the second control condition. The amino acid linker sequences between respective MICOS proteins or IM transmembrane (TMD) domain and APEX2 are shown. AlphaFold 3 structure predictions, without cleavable mitochondrial targeting sequence, are represented using PyMol. Different MICOS subunits are depicted in blue colour. APEX2 and Myc tag are shown in red and orange (Myc tag additionally shown with arrows) respectively. The transmembrane domain of IM-APEX2 is depicted in light grey. The MICOS-APEX2 and IM-APEX2 fusion proteins were stably expressed in the background of respective MICOS KO and WT HEK293 cells, respectively. WT HEK293 cells, stably expressing empty vector (EV) were used as background control and, were employed for transient expression of matrix-APEX2. (B) Schematic representation of the APEX2 tagged to the respective MICOS subunit, IM-TMD and matrix controls. APEX2 catalyzes the biotin-phenoxyl radical production in the presence of biotin-phenol (BP) and H 2 O 2 to initiate proteome tagging in the molecular neighborhood depicted in red rings. (C) Western blot (WB) analyses of various MICOS-APEX2 fusion proteins and IM-APEX2 control stably expressed in the corresponding MICOS KO background and WT cells, respectively. Stable expression of different MICOS-APEX2 fusion proteins results in restoration of the expected MICOS proteins (enclosed by solid rectangles) compared to corresponding KO cell lines (enclosed by dotted rectangles). All the MICOS proteins in cells expressing the respective MICOS-APEX2 along with Myc displayed increased molecular weight compared to the endogenous MICOS proteins as expected. Therefore, the rescue can only be seen using the anti-Myc antibody. Their expression is shown using the Myc antibody and the band at the expected molecular weight is additionally indicated with solid rectangles. (D) Fluorescence microscopy demonstrating the biotinylation activity of APEX2-constructs, using streptavidin conjugated to DyLight 488 within mitochondria marked using anti-MIC60 antibody in various cell lines mentioned. Nucleus is stained with Hoechst and shown in blue. Scale bar 10 µm.

    Techniques Used: Generated, Control, Sequencing, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Molecular Weight, Fluorescence, Microscopy, Activity Assay, Construct, Staining

    (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.
    Figure Legend 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.

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

    (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.
    Figure Legend 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 Used: Staining, Control, Injection, Activity Assay



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    plix403 mic60  (Addgene inc)
    96
    Addgene inc plix403 mic60
    (A) Schematic overview of various MICOS-APEX2-Myc fusion proteins (denoted as MICOS-APEX2 for simplicity from hereon) generated along with IM-APEX2-Myc (denoted as IM-APEX2 for simplicity from hereon) control fusion protein. Matrix-APEX2 was used as the second control condition. The amino acid linker sequences between respective MICOS proteins or IM transmembrane (TMD) domain and APEX2 are shown. AlphaFold 3 structure predictions, without cleavable mitochondrial targeting sequence, are represented using PyMol. Different MICOS subunits are depicted in blue colour. APEX2 and Myc tag are shown in red and orange (Myc tag additionally shown with arrows) respectively. The transmembrane domain of IM-APEX2 is depicted in light grey. The MICOS-APEX2 and IM-APEX2 fusion proteins were stably expressed in the background of respective MICOS KO and WT HEK293 cells, respectively. WT HEK293 cells, stably expressing empty vector (EV) were used as background control and, were employed for transient expression of matrix-APEX2. (B) Schematic representation of the APEX2 tagged to the respective MICOS subunit, IM-TMD and matrix controls. APEX2 catalyzes the biotin-phenoxyl radical production in the presence of biotin-phenol (BP) and H 2 O 2 to initiate proteome tagging in the molecular neighborhood depicted in red rings. (C) Western blot (WB) analyses of various MICOS-APEX2 fusion proteins and IM-APEX2 control stably expressed in the corresponding MICOS KO background and WT cells, respectively. Stable expression of different MICOS-APEX2 fusion proteins results in restoration of the expected MICOS proteins (enclosed by solid rectangles) compared to corresponding KO cell lines (enclosed by dotted rectangles). All the MICOS proteins in cells expressing the respective MICOS-APEX2 along with Myc displayed increased molecular weight compared to the endogenous MICOS proteins as expected. Therefore, the rescue can only be seen using the anti-Myc antibody. Their expression is shown using the Myc antibody and the band at the expected molecular weight is additionally indicated with solid rectangles. (D) Fluorescence microscopy demonstrating the biotinylation activity of APEX2-constructs, using streptavidin conjugated to DyLight 488 within mitochondria marked using <t>anti-MIC60</t> antibody in various cell lines mentioned. Nucleus is stained with Hoechst and shown in blue. Scale bar 10 µm.
    Plix403 Mic60, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plix403 mic60/product/Addgene inc
    Average 96 stars, based on 1 article reviews
    plix403 mic60 - by Bioz Stars, 2026-03
    96/100 stars
    		  Buy from Supplier

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    (A) Schematic overview of various MICOS-APEX2-Myc fusion proteins (denoted as MICOS-APEX2 for simplicity from hereon) generated along with IM-APEX2-Myc (denoted as IM-APEX2 for simplicity from hereon) control fusion protein. Matrix-APEX2 was used as the second control condition. The amino acid linker sequences between respective MICOS proteins or IM transmembrane (TMD) domain and APEX2 are shown. AlphaFold 3 structure predictions, without cleavable mitochondrial targeting sequence, are represented using PyMol. Different MICOS subunits are depicted in blue colour. APEX2 and Myc tag are shown in red and orange (Myc tag additionally shown with arrows) respectively. The transmembrane domain of IM-APEX2 is depicted in light grey. The MICOS-APEX2 and IM-APEX2 fusion proteins were stably expressed in the background of respective MICOS KO and WT HEK293 cells, respectively. WT HEK293 cells, stably expressing empty vector (EV) were used as background control and, were employed for transient expression of matrix-APEX2. (B) Schematic representation of the APEX2 tagged to the respective MICOS subunit, IM-TMD and matrix controls. APEX2 catalyzes the biotin-phenoxyl radical production in the presence of biotin-phenol (BP) and H 2 O 2 to initiate proteome tagging in the molecular neighborhood depicted in red rings. (C) Western blot (WB) analyses of various MICOS-APEX2 fusion proteins and IM-APEX2 control stably expressed in the corresponding MICOS KO background and WT cells, respectively. Stable expression of different MICOS-APEX2 fusion proteins results in restoration of the expected MICOS proteins (enclosed by solid rectangles) compared to corresponding KO cell lines (enclosed by dotted rectangles). All the MICOS proteins in cells expressing the respective MICOS-APEX2 along with Myc displayed increased molecular weight compared to the endogenous MICOS proteins as expected. Therefore, the rescue can only be seen using the anti-Myc antibody. Their expression is shown using the Myc antibody and the band at the expected molecular weight is additionally indicated with solid rectangles. (D) Fluorescence microscopy demonstrating the biotinylation activity of APEX2-constructs, using streptavidin conjugated to DyLight 488 within mitochondria marked using anti-MIC60 antibody in various cell lines mentioned. Nucleus is stained with Hoechst and shown in blue. Scale bar 10 µm.

    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 overview of various MICOS-APEX2-Myc fusion proteins (denoted as MICOS-APEX2 for simplicity from hereon) generated along with IM-APEX2-Myc (denoted as IM-APEX2 for simplicity from hereon) control fusion protein. Matrix-APEX2 was used as the second control condition. The amino acid linker sequences between respective MICOS proteins or IM transmembrane (TMD) domain and APEX2 are shown. AlphaFold 3 structure predictions, without cleavable mitochondrial targeting sequence, are represented using PyMol. Different MICOS subunits are depicted in blue colour. APEX2 and Myc tag are shown in red and orange (Myc tag additionally shown with arrows) respectively. The transmembrane domain of IM-APEX2 is depicted in light grey. The MICOS-APEX2 and IM-APEX2 fusion proteins were stably expressed in the background of respective MICOS KO and WT HEK293 cells, respectively. WT HEK293 cells, stably expressing empty vector (EV) were used as background control and, were employed for transient expression of matrix-APEX2. (B) Schematic representation of the APEX2 tagged to the respective MICOS subunit, IM-TMD and matrix controls. APEX2 catalyzes the biotin-phenoxyl radical production in the presence of biotin-phenol (BP) and H 2 O 2 to initiate proteome tagging in the molecular neighborhood depicted in red rings. (C) Western blot (WB) analyses of various MICOS-APEX2 fusion proteins and IM-APEX2 control stably expressed in the corresponding MICOS KO background and WT cells, respectively. Stable expression of different MICOS-APEX2 fusion proteins results in restoration of the expected MICOS proteins (enclosed by solid rectangles) compared to corresponding KO cell lines (enclosed by dotted rectangles). All the MICOS proteins in cells expressing the respective MICOS-APEX2 along with Myc displayed increased molecular weight compared to the endogenous MICOS proteins as expected. Therefore, the rescue can only be seen using the anti-Myc antibody. Their expression is shown using the Myc antibody and the band at the expected molecular weight is additionally indicated with solid rectangles. (D) Fluorescence microscopy demonstrating the biotinylation activity of APEX2-constructs, using streptavidin conjugated to DyLight 488 within mitochondria marked using anti-MIC60 antibody in various cell lines mentioned. Nucleus is stained with Hoechst and shown in blue. Scale bar 10 µm.

    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: Generated, Control, Sequencing, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Molecular Weight, Fluorescence, Microscopy, Activity Assay, Construct, Staining

    (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 – 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.

    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: Staining, Control, Injection, Activity Assay