Extended Data Fig. 9: Mitochondria- and ER-associated actin accumulates during Drp1-mediated mitochondrial fission.

a) AC-ER accumulates prior to Drp1-mediated fission. AC-ER accumulation is evident prior to Drp1 recruitment and mitochondrial fission. Hollow arrows mark the location of mitochondrial fission. Yellow lines in the left column mark the area used to generate the line scans. Scale bar: 1 µm. These results were reproducible across 8 independent experiments. b) AC-mito accumulates prior to Drp1-mediated fission. Same as A with cells expressing AC-mito instead of AC-ER. c) Expression of AC probes does not alter mitochondrial membrane potential, mitochondrial fission rate, or ER-mitochondria contact sites. Top graphs: the pixel intensity of MitoTracker Deep Red (MTDR) was measured in cells that were not transfected vs. neighboring cells expressing AC-mito/ER and normalized as described under Methods. Left graph: non-transfected: n = 23 cells, AC-ER: n = 19 cells. Right graph: non-transfected: n = 14 cells, AC-mito: n = 9 cells. P-values determined by two-tailed Welch’s t-test. Bottom left graph: the number of fission events were counted in cells that were not transfected or in cells expressing AC-mito/ER. Non-transfected: n = 17 cells, AC-ER: n = 8 cells, AC-mito: n = 12 cells. P-values determined by two-tailed Welch’s t-test. Bottom right graph: HeLa cells were transfected with mCherry-ER alone “No AC Probe”, AC-ER+mCherry-ER “AC-ER”, AC-mito+mCherry-ER “AC-mito, or VAPB1-Halo+mCherry-ER “VAPB1” (positive control for changing ER-mitochondria contacts) and counterstained with MitoTracker. A region of the cell was selected as described under Methods and Pearson’s correlation coefficient between MitoTracker and mCherry-ER was determined. N = 10 cells per condition. P-values determined by two-tailed Welch’s t-test.

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