Procollagen-carrying vesicles exhibit movement. (A) Confocal microscopy imaging on KI6 cells treated with doxycycline for 7.5 h were labeled with SEC16A and SEC31A antibodies. Open arrow in merged image points to a colocalized spot; closed arrow points to a large SEC31A-labeled vesicle not colocalizing with SEC16A. (B) Two-color STORM imaging was performed on KI6 cells induced for KLHL12 overexpression for 7.5 h and then labeled with KDEL and SEC31A antibodies. Panels show magnified xy view of a SEC31A (green)-labeled large COPII cage (i); KDEL marker labeling ER (magenta; ii); and merged image of the two channels (iii). (C) KI6 cells were transfected with YFP-tagged SEC31A and induced for KLHL12 expression for 7.5 h. (i–iii) Time lapse imaging of SEC31A-YFP puncta in cells not treated with nocodazole. Arrowheads indicate a puncta exhibiting long-range transport (>10 µm distance). n = 3. (iv–vi) Time-lapse images of SEC31A-YFP puncta after the treatment of 5 µM nocodazole for 30 min. (D) Images shown are the first frame of the time-lapse movie overlaid with the trajectories of SEC31A-YFP puncta in the absence (i) and presence (ii) of nocodazole. Trajectories in time (0–120 s) represented by color according to the color scale in D. n = 3. (E) Time-lapse imaging of SEC31A-YFP– and PC1-CFP–positive puncta in KI6 cells. Arrowheads point to a vesicle that displays a spatial displacement in time. (F) Overview of the cell imaged in E; overlay of merged fluorescent image over DIC image shows PC1-CFP in green and SEC31A-YFP in magenta. Bars: (A) 5 µm; (B) 500 nm; (C)10 µm; (D–F) 5 µm.