Journal: Journal of Cell Science
Article Title: Regulation of mitochondrial cristae organization by Myo19, Miro1 and Miro2, and metaxin 3
doi: 10.1242/jcs.263637
Figure Lengend Snippet: Membrane anchor mutants of Miro1/2 rescue Miro DKO. (A) Scheme of Miro1 and Miro2 residues that were exchanged with the corresponding residues of VAMP1B and Bcl-xL, respectively. Residues highlighted in green and blue are part of the transmembrane regions and those in red of the intermembrane space (IMS). Residues in light blue color are derived from VAMP1B and face the cytosol. Dm: Drosophila melanogaster , Hs: Homo sapiens . (B) Western blot analysis of protein levels of Miro, Halo–Miro, Myo19 and β-actin in WT, Miro DKO and Miro DKO cells expressing different Miro constructs as indicated. (C) Quantification of Myo19 protein levels in the different cell lines as indicated (normalized to β-actin levels). Results are mean±s.e.m. ( n =3). * P <0.05; ** P <0.01; *** P <0.001 (two-sample unpaired t -test). (D,E) Confocal images of Miro DKO cells rescued with Halo-tagged Miro constructs (D) and Halo-tagged mutant Miro constructs (E) that were transfected with GFP–Myo19. Mitochondria were stained with Mitotracker Orange. n =20–30 cells per experiment ( n =3 experiments). Scale bars, 10 µm. (F) Miro DKO cells rescued with either Halo–Miro1, Halo–Miro2, Halo–Miro1mutant or Halo–Miro2mutant were lysed. The lysates were subjected to Halo–Trap agarose pull-downs. Western blot analysis of inputs and eluates for Miro, Myo19, Mic60, Sam50, Mtx3 and GAPDH are shown. Images in F are representative of four repeats.
Article Snippet: A human Miro2 target specific CRISPR-Cas9 knockout plasmid was purchased from Santa Cruz Biotechnology (sc-406496).
Techniques: Membrane, Derivative Assay, Western Blot, Expressing, Construct, Mutagenesis, Transfection, Staining
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