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. 2019 Jun 1;30(12):1477–1489. doi: 10.1091/mbc.E19-01-0022

FIGURE 4:

FIGURE 4:

Dynein motor activity is required for macropinocytosis. (A) HT1080 cells were imunolabeled with antibody to cytosolic dynein. (B) HT1080 cells were pretreated with either 50 μM Ciliobrevin D (CBD) or DMSO vehicle for 30 min before addition of dextran-594 and incubated for 20 min. Images of cells under each condition are shown, cells are outlined with white dotted lines. HT1080 cell expressing EMTB-3xGFP and treated with CBD is shown, revealing intact microtubules throughout the cell in the presence of this drug. Bar graphs show the average (± SEM) dextran fluorescence from 150 individual cells per condition in a single matched experiment. Experiments were repeated three times with similar phenotypes. An unpaired t test with Welch’s correction was used for comparison of conditions. ****p value < 0.0001. (C) HT1080 cells expressing farnesyl-RFP to label the cell surface were pretreated with 50 μM Ciliobrevin D (CBD) or DMSO for 30 min and then imaged live; stills from that movie are shown. Nascent macropinosomes can be seen forming in DMSO treated cells, while CBD-treated cells have ruffling membranes but are impaired at forming macropinosomes. (D) HT1080 cells were pretreated with 40 μM Dynarrestin (Dyn) or DMSO for 1 h prior to addition of dextran-594 and incubated for 20 min. Images of cells under each condition are shown; cells are outlined with white dotted lines. HT1080 cell expressing EMTB-3xGFP and treated with Dyn is shown, revealing intact microtubules throughout the cell in the presence of this drug. HT1080 cells were treated as above but incubated with dextran-647 for 20 min prior to measuring dextran uptake by flow cytometry. Bar graphs show the geometric mean dextran fluorescence, reported as a fraction of DMSO control, from at least three experiments (100,000 cells were counted per experiment). Error bars represent ± SD. An unpaired t test with Welch’s correction was used for comparison of conditions. ****p < 0.0001. (E) HT1080 cells expressing GLUT4-mCherry to label the cell surface were pretreated with 40 μM Dynarrestin (Dyn) or DMSO for 1 h and then imaged live; stills from that movie are shown. Nascent macropinosomes can be seen forming in DMSO-treated cells, while Dyn-treated cells have ruffling membranes but are impaired at forming macropinosomes. Bars, 5 μm.