Figure 3: Mitochondria- and ER-associated actin accumulates prior to mitochondrial fission.

A) AC-mito accumulates at mitochondrial fission sites. AC-mito accumulation is evident prior to mitochondrial fission. Hollow arrows mark the site of mitochondrial fission. Green arrows mark the location of AC-mito accumulation. Yellow lines indicate the area used to generate the line scan. Scale bar: 1 µm. These results were reproducible across 21 independent experiments. B) AC-ER accumulates at mitochondrial fission sites. Similar to A except cells are co-expressing BFP-mito and AC-ER. These results were reproducible across 21 independent experiments. C) Examples of AC probe areas used to determine that accumulation at mitochondrial fission sites is not due to chance. Images show the mitochondria and AC probe areas generated by the images from the first timepoints in A and B. The frequency of the AC probes observed at fission sites (AC-mito: 32/33 fission events, AC-ER: 31/31 fission events) is compared to the frequency that the probe would be expected at fission sites due to chance as detailed under Methods. P-values determined by Fisher’s exact test. D) AC-mito accumulates at mitochondrial fission sites prior to AC-ER accumulation. The first timepoint shows AC-mito accumulation in the absence of AC-ER accumulation. Subsequently, AC-ER accumulates and mitochondrial fission occurs. Hollow arrows mark the site of mitochondrial fission. Yellow arrows mark AC-mito accumulation and magenta arrows mark AC-ER accumulation. The red lines in the left column mark the area used to the generate the line scan. Scale bar: 1 µm. This order of events is consistently observed and quantified in the scatter plot. Each dot color corresponds to an individual fission event (n = 17 fission events). P-values determined by two-tailed ratio paired t-test. These results were reproducible across 7 independent experiments.