Actin severing by villin is required to maintain intracellular actin dynamics in response to CPT treatment. (a) F-actin levels in VIL/NULL and VIL/WT cells treated with CPT (20 μM, 0–5 h). CPT treatment significantly increased total cellular F-actin concentration in VIL/NULL cells (*P<0.001, n=6). F-actin concentrations in CPT-treated VIL/NULL cells were significantly higher than F-actin in CPT-treated VIL/WT cells (#
P<0.001, n=6). Bar, 20 μm. (b) Total cellular F- and G-actin levels were measured in VIL/NULL and VIL/WT cells treated with CPT (20 μM, 0–5 h). Experiments were performed in triplicate. In response to CPT treatment there was a significant increase in F-actin (*P<0.01, n=3) and decrease in G-actin (*P<0.001, n=3) levels in VIL/NULL cells. (c) Free barbed ends were visualized as described in Materials and Methods. Alexa 488-labeled actin incorporation was measured using confocal laser scanning microscopy. Experiments were performed in triplicate. Cytochalasin D that binds to the barbed ends of actin filaments was used as a control. Untreated VIL/WT cells had significantly higher numbers of free barbed ends compared with untreated VIL/NULL cells ($P<0.05, n=3). CPT treatment increased the number of free barbed ends in VIL/WT cells compared with untreated VIL/WT cells (#P<0.01, n=3). In contrast CPT treatment significantly reduced the number of free barbed ends in VIL/NULL cells compared with untreated VIL/NULL cells (*P<0.01, n=3). Bar, 20 μm