mito snap  (Addgene inc)

Journal: Cell Reports

Article Title: OCIAD1 and prohibitins regulate the stability of the TIM23 protein translocase

doi: 10.1016/j.celrep.2024.115038

Figure Lengend Snippet: OCIAD1 localizes predominantly in the outer membrane of mitochondria (A) Subcellular fractionation of HEK293 cells that expressed OCIAD1 FLAG and an empty vector. T, total; C, cytosol; M, mitochondria; US, ultracentrifugation supernatant; V, light membranes. (B) Extraction of proteins by sodium carbonate. Samples were analyzed by SDS-PAGE and western blot. Membr., membranes. (C) Localization of mitochondrial proteins, analyzed by limited degradation with proteinase K in intact mitochondria (250 mM sucrose), mitoplasts (5 mM sucrose), and mitochondrial lysates (1% Triton X-100). The samples were analyzed by SDS-PAGE and western blot. Mitos, mitochondria; Mitopl, mitoplasts; Sup, supernatant; OM, outer membrane; IM, inner membrane; IMS, intermembrane space. (D and E) Mitochondria isolated from HEK293 and HeLa (D) or U-2 OS (E) cells were treated with increasing concentrations of proteinase K. The samples were then analyzed with SDS-PAGE followed by western blotting. (F and G) Submitochondrial localization of OCIAD1-SNAP in HeLa cells visualized by live-cell 2D STED microscopy. Cells were transfected with a plasmid encoding OCIAD-SNAP and labeled with SNAP-cell 647-SiR and the inner membrane (IM) marker PK Mito Orange (PKMO). The image shows a 2D projection of the mitochondrial tubules. (F) Representative dual-color STED recording. (G) Fluorescence intensity line profiles were measured at the sites indicated by arrowheads in the composite view. The fluorescence intensity was estimated along the transparent dashed lines, normalized, and plotted. Scale bar: 1 μm. See also Figures S1 and .

Article Snippet: The cells were stained with 200 nM PK Mito Orange and 1 μM SNAP-cell 647-SiR (NEB) as described previously.

Techniques: Membrane, Fractionation, Plasmid Preparation, Extraction, SDS Page, Western Blot, Isolation, Microscopy, Transfection, Labeling, Marker, Fluorescence

Journal: Nature Communications

Article Title: Dynamic actin cycling through mitochondrial subpopulations locally regulates the fission–fusion balance within mitochondrial networks

doi: 10.1038/ncomms12886

Figure Lengend Snippet: ( a ) Confocal image of GFP-actin (red) recruitment to elongated, Mito-SNAP-labelled mitochondria (blue) in a HeLa cell. Actin assembly is enriched at sites of ER tubule (DsRed2-ER, green) overlap with mitochondria. Arrows indicate regions of Actin/ER co-localization at prospective sites of mitochondrial fission. ( b ) Enlarged image of Box B (+4 min), demonstrating co-localization of actin and ER at the site of mitochondrial fission. ( c ) Line scan indicating overlapping peak actin and ER intensity at site of mitochondrial constriction in ( b ). ( d ) Three-dimensional rendering of F-actin (LifeAct-GFP) and ER-tubules (DsRed2-ER) assembled on a constricted HeLa cell mitochondrion (Mito-SNAP). Scale bars, 1 μm ( a ), 0.5 μm ( b ) and 0.75 μm per unit ( d ).

Article Snippet: Cells expressing Mito-SNAP were incubated with 2.5 μM SNAP-cell 647-SiR (S9102S, NEB) for 30 min and washed 2 × before imaging.

Techniques: