Figure 3.

Actin severing by gelsolin is required to maintain intracellular actin dynamics in response to CPT treatment. (a) Western analysis of full-length human cytoplasmic gelsolin expressed in MDCK Tet-Off cells. Western blot with anti-actin antibody was performed in parallel as a quantitative control. Data are representative of six experiments with similar results. (b) GSN/NULL and GSN/WT cells were treated with CPT (20 μM, 0–5 h) and apoptosis measured as described in the Methods. Apoptotic cells were visualized by phase-contrast microscopy and identified using Hoechst 33258 staining. All experiments were performed in triplicate, with similar results, n=6. Bar, 50 μm. PI permeability was analyzed by flow cytometry as a quantitative measure of cell viability. There was a significant increase in PI positive GSN/NULL cells in response to CPT treatment (^*P<0.01, n=6). (c) Total cellular F- and G-actin levels were measured in GSN/WT cells treated with CPT (20 μM, 0–5 h). There was a significant increase in F-actin (^*P<0.01, n=3) and decrease in G-actin (^*P<0.01, n=3) in GSN/NULL cells in response to CPT treatment. Experiments were performed in triplicate. (d) Free barbed ends were localized as described in Methods. Alexa 488-labeled actin incorporation was assessed by confocal laser scanning microscopy. Experiments were performed in triplicate. Untreated GSN/WT cells had significantly higher numbers of free barbed ends compared with GSN/NULL cells (^$P<0.05, n=3). CPT treatment increased the number of free barbed ends in GSN/WT cells compared with untreated GSN/WT cells (^#P<0.01, n=3). In contrast CPT treatment reduced the number of free barbed ends in GSN/NULL cells compared with untreated GSN/NULL cells (^*P<0.05, n=3). Cytochalasin D prevented the incorporation of Alexa 488-labeled actin into both GSN/NULL and GSN/WT cells. Bar, 20 μm