A population of Drp1 associates with ER independently of mitochondria or peroxisomes. (A) Drp1 distribution in GFP-Drp1-KI cells. Left: Merged image of a live Drp1 KI cell transiently expressing mCherry-mito7 (red) and eBFP2-peroxisome (blue). Drp1 in green. Right: Insets from boxed region at three time points. Yellow arrow, independent Drp1 puncta; blue arrow, peroxisome-associated Drp1; red arrow, mitochondrially associated Drp1; white arrowhead, example of Drp1 puncta localizing at the interface of mitochondrion and peroxisome. (B) Venn diagram of Drp1 distribution in GFP-Drp1-KI cells expressing mitochondrial and peroxisomal markers. Black circles, mitochondrially associated Drp1 punctae; blue circles, peroxisomal associated Drp1 punctae (Pex); red circles, independent Drp1 punctae (Ind). The percentage of Drp1 punctae in each category is the mean from 10 consecutive frames with 12-s time intervals from whole-cell videos. Five cells measured (10,761 punctae). (C) Four-color imaging of a live Drp1 KI cell expressing mPlum-mito3 (Mito, gray); eBFP2-peroxisome (Peroxisome, blue); and ER-tagRFP (ER, red); Drp1 in green. Yellow arrows denote independent Drp1 puncta stably associating with ER. See also Video 1. (D) Time-lapse montage showing de novo assembly of an independent Drp1 punctum (yellow arrow) on an ER tubule. Imaging as in C. (E) Graph depicting the degree of association between independent Drp1 punctae and ER during 2.5-min videos imaged every 1.6 s. 30 ROIs from 25 GFP-Drp1-KI cells analyzed (1,003 punctae). Mean values from ROIs: 76.7 ± 11.7%, stable association between Drp1 punctae and ER (no apparent dissociation from ER in any frame); 8.9 ± 9.5%, partial association; 14.4 ± 8.0%, no association. Bars: (A, whole-cell image) 10 µm; (A, inset; C; and D) 2 µm. Time in seconds.