Actin-dependent oligomerization of ER-associated Drp1 punctae. (A) Left: Merged image of a live GFP-Drp1-KI cell before ionomycin treatment, transiently expressing mPlum-mito3 (gray), eBFP2-peroxisome (blue), and ER-tagRFP (ER, red). Drp1 in green. Right: Inset from boxed region before (top) and after (bottom) ionomycin treatment (4 µM, 10 min). Yellow arrows denote independent Drp1 maturing upon ionomycin treatment. See also Video 9. (B) Similar experiment as in A, except cells were pretreated for 10 min with 1 µM LatA. See also Video 10. (C) Quantification of independent Drp1 punctae number in response to vehicle treatment (DMSO), ionomycin treatment, and LatA pretreatment followed by ionomycin treatment. Six ROIs from six DMSO-treated cells, 16 ROIs from 14 ionomycin-treated cells, and eight ROIs from six LatA-pretreated/ionomycin-treated cells. Punctae per ROI normalized to 1 at time of ionomycin addition. Error bar, SEM. Arrow indicates time point where ionomycin was added during imaging (time 0). Bars: (left) 10 µm; (right) 2 µm. Time in seconds.