Fig. 8
From: Dopey1-Mon2 complex binds to dual-lipids and recruits kinesin-1 for membrane trafficking
Dopey1–Mon2 complex inhibits the ER-to-Golgi but promotes the reverse trafficking. a–c The ER-to-Golgi trafficking requires Dopey1 and Mon2. siRNA-treated cells expressing ManII-SBP-GFP or VSVGtso45-GFP were chased in biotin or by temperature shift followed by staining of GM130. Deconvolved images are shown in (a). Dotted white lines indicate cell contours. Golgi fractions are quantified in (b, c). Plots are representatives of three experiments; n indicates the number of cells analyzed. d, e Dopey1 and Mon2 can be found on mCherry-GPI- and ManII-positive carriers moving from the ER to the Golgi. Cells co-expressing indicated reporters were treated with biotin and the time lapse was acquired under a spinning disk confocal microscope. White and yellow arrows indicate positions of mobile carriers at time t1 and t2, respectively. Arrow heads show colocalization at immobile puncta. f–j Particle analysis of single carriers involved in the ER-to-Golgi trafficking. siRNA-treated cells transiently expressing ManII-SBP-GFP were chased with biotin during the time lapse imaging. See Supplementary Movie 4, for an example of the control. Segment speed, time, frequency, distance and their corresponding centrifugal biases are shown in (f–i). j The statistical analysis of single carrier mean velocity. n the number of segments (f, g, i) or tracks (h, j). Tracks were identified from time lapses of 9, 7, and 9 cells in 3 experiments, respectively. k, l The Golgi-to-ER trafficking requires Dopey1 and Mon2. siRNA-treated cells expressing GFP-ERGIC53 were subjected to FRAP (see Methods). See Supplementary Movie 5, for an example of the control. Normalized ER intensity traces are in (k) and the FRAP t1/2 of each experiment is shown in (l). Scale bar, 10 µm; error bar, mean ± s.e.m. P values are from the t test (unpaired and two-tailed) comparison of indicated pairs (f–j, l) or between the knockdown and corresponding control (b, c). N.S. not significant, *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.00005, *****P ≤ 0.000005. Source data are provided as a Source Data file
