crispr cas9 nickases  (Addgene inc)

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: Individual transfection of the respective CRISPR-Cas9 double nickase plasmids (Santa Cruz Biotechnology, MIC26: sc-413137-NIC, MIC60: sc-403617-NIC, MIC25: sc-413621-NIC, MIC19: sc-408682-NIC, MIC10: sc-417564-NIC, MIC27: sc-414464-NIC) was performed with GeneJuice (Sigma-Aldrich, 70967-3) in Flp-In T-REx HEK293 WT cell line to generate the corresponding MICOS KO cell lines.

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

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: Individual transfection of the respective CRISPR-Cas9 double nickase plasmids (Santa Cruz Biotechnology, MIC26: sc-413137-NIC, MIC60: sc-403617-NIC, MIC25: sc-413621-NIC, MIC19: sc-408682-NIC, MIC10: sc-417564-NIC, MIC27: sc-414464-NIC) was performed with GeneJuice (Sigma-Aldrich, 70967-3) in Flp-In T-REx HEK293 WT cell line to generate the corresponding MICOS KO cell lines.

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

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.

Article Snippet: Individual transfection of the respective CRISPR-Cas9 double nickase plasmids (Santa Cruz Biotechnology, MIC26: sc-413137-NIC, MIC60: sc-403617-NIC, MIC25: sc-413621-NIC, MIC19: sc-408682-NIC, MIC10: sc-417564-NIC, MIC27: sc-414464-NIC) was performed with GeneJuice (Sigma-Aldrich, 70967-3) in Flp-In T-REx HEK293 WT cell line to generate the corresponding MICOS KO cell lines.

Techniques: Control, Membrane

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.

Article Snippet: Individual transfection of the respective CRISPR-Cas9 double nickase plasmids (Santa Cruz Biotechnology, MIC26: sc-413137-NIC, MIC60: sc-403617-NIC, MIC25: sc-413621-NIC, MIC19: sc-408682-NIC, MIC10: sc-417564-NIC, MIC27: sc-414464-NIC) was performed with GeneJuice (Sigma-Aldrich, 70967-3) in Flp-In T-REx HEK293 WT cell line to generate the corresponding MICOS KO cell lines.

Techniques: Control

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.

Article Snippet: Individual transfection of the respective CRISPR-Cas9 double nickase plasmids (Santa Cruz Biotechnology, MIC26: sc-413137-NIC, MIC60: sc-403617-NIC, MIC25: sc-413621-NIC, MIC19: sc-408682-NIC, MIC10: sc-417564-NIC, MIC27: sc-414464-NIC) was performed with GeneJuice (Sigma-Aldrich, 70967-3) in Flp-In T-REx HEK293 WT cell line to generate the corresponding MICOS KO cell lines.

Techniques:

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: Individual transfection of the respective CRISPR-Cas9 double nickase plasmids (Santa Cruz Biotechnology, MIC26: sc-413137-NIC, MIC60: sc-403617-NIC, MIC25: sc-413621-NIC, MIC19: sc-408682-NIC, MIC10: sc-417564-NIC, MIC27: sc-414464-NIC) was performed with GeneJuice (Sigma-Aldrich, 70967-3) in Flp-In T-REx HEK293 WT cell line to generate the corresponding MICOS KO cell lines.

Techniques: Staining, Control, Injection, Activity Assay