Extended Data Fig. 10: SMEM arise from collapsed and condensed inner mitochondrial membranes through MISO oligomerization.
From: MISO regulates mitochondrial dynamics and mtDNA homeostasis by establishing membrane subdomains
a. Representative TEM images of mitochondria in U2OS cells expressing APEX2-MISO. Scale bar: 500 nm. b. Representative images of U2OS cells expressing PHB1-mCherry and MISO–Flag, incubated with nonyl acridine orange (NAO). White arrows indicate subdomains. Scale bars: main panels 10 μm, magnified insets 2 μm. c-d. Representative immunoblots (c) and corresponding quantification (d) of proteins involved in the maintenance of mitochondrial cristae structure and morphology from U2OS cells expressing an empty vector (pLenti) or MISO–Flag. n = three biological replicates. e. Crosslinking analysis of the oligomerization of two MISO isoforms in U2OS cells using disuccinimidyl glutarate (DSG). Arrowheads point to the MISO dimers. Asterisks denote non-specific bands. f. Crosslinking analysis of MISO oligomerization using DSG in U2OS cells expressing MISO–Flag and treated with the indicated shRNAs. Red arrowheads point to the MISO dimers and trimers. g. Crosslinking analysis of oligomerization of MISO and corresponding truncation variants in U2OS cells. h-i. Representative images (h) and corresponding quantification (i) of mitochondrial morphology in HEK293T cells expressing OMM-GFP or OMM-MISO. Scale bars: main panels 10 μm, magnified insets 2 μm. n = three biological replicates. j-k. Representative images (j) and corresponding quantification (k) of mitochondrial morphology in U2OS cells expressing OMM-MISO truncation mutants. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. l. Representative images showing the localization of IMM-MISO and IMM-MISO-M in U2OS cells. m-n. Representative images (m) and corresponding quantification (n) of mitochondrial morphology in U2OS cells expressing IMM-MISO truncation mutants. Scale bars: main panels 10 μm, magnified insets 5 μm. n = three biological replicates. Data are presented as mean ± SD. (d) and (i): two-tailed unpaired Student’s t-test; (k) and (n): ordinary one-way with Tukey’s multiple comparisons.
