A subpopulation of Fis1 localizes to ER. (A) Endogenous Fis1 localization in fixed U2OS cells by immunofluorescence. Cells labeled with anti-Tom20 (mitochondria, blue), anti-PMP70 (peroxisomes, gray), and anti-Fis1 (green) and transfected with ER-TagRFP (ER, red). Left, scrambled siRNA; right, Fis1 siRNA. Yellow arrows, independent punctae; blue arrow, peroxisome-associated puncta; white arrow, mitochondrially associated Fis1. (B) Graph depicting the percentage of colocalization between independent Fis1 punctae and ER in U2OS cells by immunofluorescence (endogenous Fis1). 117 independent Fis1 punctae counted from nine ROIs from four cells. Mean values from ROIs: 79.9 ± 11.3%, colocalized Fis1 punctae with ER; 6.0 ± 7.4%, not colocalized; 14.1 ± 10.2%, unclear localization. (C) Live-cell time-lapse of GFP-Fis1 in U2OS cell also expressing mCherry-mito3 (gray), eBFP2-peroxisome (blue), and E2-Crimson-ER (red). Right: Individual frames from the time course of boxed region, showing independent Fis1 punctae associated with ER (yellow arrow) next to a peroxisome that is positive for Fis1 (blue arrow). (D) Graph depicting the degree of association between independent GFP-Fis1 punctae and ER from live-cell videos as in C (2.5-min videos imaged every 1.7 s). 16 ROIs from 15 U2OS cells (100 independent Fis1 punctae) analyzed. Mean values from ROIs: 78.8% ± 26.9%, stably associated Fis1 punctae with ER; 11.9 ± 18.2%, partially associated; 9.2 ± 14.8%, not associated. Bars: (A and C, whole-cell images) 10 µm; (A, inset) 5 µm; (C, inset) 2 µm inset. Time in seconds.