In U2OS cells, LC3 and JMY vesicles move on polarized actin network. (A) A subset of punctate JMY structures (red) are propelled through the cytoplasm (arrowheads) by actin comet tails (green) in fed U2OS cells. Enlarged boxes show one motile JMY focus with an associated actin comet tail (left) and one nonmotile JMY focus with no actin network (right). (B) Raw distance versus time plots of JMY-positive foci propelled by actin comet tails (63 puncta, 9 cells, n = 3 independent experiments). (C) Polarized actin networks (labeled by phalloidin, green) attach to endogenous JMY (JMY antibody, red) puncta in immunofluorescent staining in fed cells. (D) JMY- and LC3-positive vesicles are propelled by polarized actin networks in cells starved in HBSS. Enlarged boxes show actin networks (magenta) pushing JMY (red) and LC3 (green) positive vesicles (arrowhead) toward plasma membrane (1), toward nucleus (2), or in a more complex trajectory (“V” shaped movement; 3). (E) Quantification of migration speed of JMY-positive vesicles in cells expressing various sets of two proteins: JMY and F-tractin, JMY and LC3 (with or without CK666), JMY and STRAP (8∼11 cells, **, P < 0.01, Student’s t test). (F) Histograms show decrease in migration speed of LC3-positive vesicles in JMY knockout cell line (bottom) compared with WT U2OS cells (top). (G) The migration speed of LC3 vesicles decreases when U2OS cells overexpress STRAP (20 ~ 25 cells, n = 3 independent experiments, **, P < 0.01, Student’s t test). Temperature: 37°C. Scale bars: whole cell, 5 µm; zoom-in box, 2 µm.