![Figure 4. INF2-mediated actin polymerization is required for stimulation of ER–mitochondrial contact. (A) Change in calcium levels at the cytoplasmic face of the OMM upon 4 µM ionomycin stimulation in U2OS-WT and INF2-KO cells. Cells were transfected with the Mass70-LAR-GECO1.2 construct, a low-affinity calcium probe (Kd of 12 µM) tethered to the cytoplasmic face of the OMM. For rescue, INF2-KO cells were transfected with plasmid expressing GFP-INF2-CAAX or GFP-INF2-nonCAAX. Ionomycin was added at 0 s in n = 24 cells (WT), 19 cells (INF2-KO), 22 cells (INF2-CAAX), and 16 cells (INF2-non CAAX). Error bars represent SEM. (B) Electron micrographs showing examples of the relationship between the ER and mitochondria in WT cells (top images) and INF2-KO cells (bottom images) in either the unstimulated condition [(−) ionomycin, left images] or after 60 s stimulation with 4 µM ionomycin [(+) ionomycin, right images]. Two zooms shown for each panel (regions indicated by numbered arrows on the main panels). Examples of ER–mitochondrial contacts of <30 nm are indicated by red arrows, whereas examples of more distant contacts indicated by black arrows. Bars: (main micrographs) 500 nm; (zooms) 100 nm. (C) Quantification from electron micrographs of the percentage of mitochondria with close ER contacts in WT cells (n = 244 mitochondria), WT cells stimulated with ionomycin (n = 176 mitochondria), INF2-KO cells (n = 245 mitochondria), and INF2-KO cells stimulated with ionomycin (n = 204 mitochondria). Values represent mean ± SEM; ***, P < 0.001 (unpaired Student’s t test). Each point represents one imaged field. (D) Rescue of mitochondrial calcium response in INF2-KO cells by overexpression of ER–mitochondrial tethers. INF2-KO U2OS cells were transfected with a mitochondrial matrix calcium probe (Mito-R-GECO) along with either CFP-VAPB or GFP-PTPIP51 and then stimulated with 4 µM ionomycin. The effects of either GFP-INF2-CAAX or GFP-INF2-nonCAAX reexpression are shown for comparison; n = 10 cells (WT), 8 cells (INF2-KO), 11 cells (INF2 non-CAAX), 15 cells (INF2-CAAX), 10 cells (PTPIP51), or 14 cells (VAPB). Error bars represent SEM.](https://cdn.rupress.org/rup/content_public/journal/jcb/217/1/10.1083_jcb.201709111/14/jcb_201709111_fig4.jpeg?Expires=1773492524&Signature=hsYaPTvY1R6I9v3Vo7CIR7OVpi3MUfeZGmdvNPRxhZjpcuO6W1QUuWJXfIDuSdfxIjih1~2TKVt039JbuFdQA1GZ-v7reCeaaJ6ZBWzMrDlaqxvEytkMKUD2j50yEFaynMtOKQyEC21u4eYvUu3WEKJopKmOkXPv-yatWr-Jx9UJTgH0-hV5sWSNxtWBmvq5tXxzEEyQjZ88mQFHyDsLG8IJLWbYRrrD4VerY1uPIK57sxb5iu8KlZ2iXUfaUSKP2OII3ZPFH5paMpVK2rnb7QZDU8SxeFfZDUTw9TvZprgKXycYsNW393HpGwpKUbDZ4~90FOlG4T9HI9oKNflD9Q__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
INF2-mediated actin polymerization is required for stimulation of ER–mitochondrial contact. (A) Change in calcium levels at the cytoplasmic face of the OMM upon 4 µM ionomycin stimulation in U2OS-WT and INF2-KO cells. Cells were transfected with the Mass70-LAR-GECO1.2 construct, a low-affinity calcium probe (Kd of 12 µM) tethered to the cytoplasmic face of the OMM. For rescue, INF2-KO cells were transfected with plasmid expressing GFP-INF2-CAAX or GFP-INF2-nonCAAX. Ionomycin was added at 0 s in n = 24 cells (WT), 19 cells (INF2-KO), 22 cells (INF2-CAAX), and 16 cells (INF2-non CAAX). Error bars represent SEM. (B) Electron micrographs showing examples of the relationship between the ER and mitochondria in WT cells (top images) and INF2-KO cells (bottom images) in either the unstimulated condition [(−) ionomycin, left images] or after 60 s stimulation with 4 µM ionomycin [(+) ionomycin, right images]. Two zooms shown for each panel (regions indicated by numbered arrows on the main panels). Examples of ER–mitochondrial contacts of <30 nm are indicated by red arrows, whereas examples of more distant contacts indicated by black arrows. Bars: (main micrographs) 500 nm; (zooms) 100 nm. (C) Quantification from electron micrographs of the percentage of mitochondria with close ER contacts in WT cells (n = 244 mitochondria), WT cells stimulated with ionomycin (n = 176 mitochondria), INF2-KO cells (n = 245 mitochondria), and INF2-KO cells stimulated with ionomycin (n = 204 mitochondria). Values represent mean ± SEM; ***, P < 0.001 (unpaired Student’s t test). Each point represents one imaged field. (D) Rescue of mitochondrial calcium response in INF2-KO cells by overexpression of ER–mitochondrial tethers. INF2-KO U2OS cells were transfected with a mitochondrial matrix calcium probe (Mito-R-GECO) along with either CFP-VAPB or GFP-PTPIP51 and then stimulated with 4 µM ionomycin. The effects of either GFP-INF2-CAAX or GFP-INF2-nonCAAX reexpression are shown for comparison; n = 10 cells (WT), 8 cells (INF2-KO), 11 cells (INF2 non-CAAX), 15 cells (INF2-CAAX), 10 cells (PTPIP51), or 14 cells (VAPB). Error bars represent SEM.
