Figure 3.
The formation of WAVE complex rings is dependent on upstream signals. (A) Potential inputs into the formation of WAVE complex rings. (B) WAVE complex rings form upon stimulation of additional chemoattractant. Additional WAVE complex (Hem1-EGFP) is recruited to the plasma membrane upon additional chemoattractant stimulation (100 nM fMLP, time point 0 s) in F-actin–inhibited (500 nM latrunculin B) cells. TIRF-SIM imaging; scale bar: 1 µm. Graph shows normalized integrated intensities of the WAVE complex before (−) and after (+) additional chemoattractant stimulation of F-actin–inhibited cells; n = 10 cells pooled from at least three independent experiments of TIRF-SIM imaging; paired t test, two-tailed; **, P = 0.0053 < 0.01. (C) dHL60s expressing WAVE complex (Hem1-EGFP; cyan) and membrane (CAAX-tagBFP; magenta) treated with (+) or without (−) C. difficile toxin B for at least 4 h and acutely treated with latrunculin B (500 nM final). Scale bar: 5 µm. Graph comparing the mean ± SEM number of the WAVE complex rings in cells treated with (+) and without (−) toxin B; (−) toxin B, n = 37 cells; (+) toxin B, n = 66 cells; cells pooled from the same three independent experiments; unpaired t test, two-tailed; ****, P < 0.0001. (D) Colocalization of Pak-PBD-mCherry (red) and Hem1-EGFP (green) in dHL60s treated with chemoattractant (10 nM fMLP final; top) and latrunculin B (500 nM final; bottom). Median filter applied to inset images. Scale bars: 5 µm and 1 µm (inset). (E) Distribution of endogenously labeled split-mNeonGreen clathrin light chain A (CLTA; red) and Hem1-mCherry (green) dHL60s treated with latrunculin B (500 nM final). Insets (i and ii) shown on right. Scale bars: 5 µm and 1 µm (insets). (F) Distribution of SEPT7 (immunofluorescence; red) in Hem1-EGFP (green) dHL60 cell line treated with latrunculin B (500 nM final). Left image shows WAVE complex rings (inset i) and septin rings (inset ii). Right image shows complementary localization of Hem1 and SEPT7 along the cell periphery. Scale bars: 5 µm and 0.5 µm (insets).

The formation of WAVE complex rings is dependent on upstream signals. (A) Potential inputs into the formation of WAVE complex rings. (B) WAVE complex rings form upon stimulation of additional chemoattractant. Additional WAVE complex (Hem1-EGFP) is recruited to the plasma membrane upon additional chemoattractant stimulation (100 nM fMLP, time point 0 s) in F-actin–inhibited (500 nM latrunculin B) cells. TIRF-SIM imaging; scale bar: 1 µm. Graph shows normalized integrated intensities of the WAVE complex before (−) and after (+) additional chemoattractant stimulation of F-actin–inhibited cells; n = 10 cells pooled from at least three independent experiments of TIRF-SIM imaging; paired t test, two-tailed; **, P = 0.0053 < 0.01. (C) dHL60s expressing WAVE complex (Hem1-EGFP; cyan) and membrane (CAAX-tagBFP; magenta) treated with (+) or without (−) C. difficile toxin B for at least 4 h and acutely treated with latrunculin B (500 nM final). Scale bar: 5 µm. Graph comparing the mean ± SEM number of the WAVE complex rings in cells treated with (+) and without (−) toxin B; (−) toxin B, n = 37 cells; (+) toxin B, n = 66 cells; cells pooled from the same three independent experiments; unpaired t test, two-tailed; ****, P < 0.0001. (D) Colocalization of Pak-PBD-mCherry (red) and Hem1-EGFP (green) in dHL60s treated with chemoattractant (10 nM fMLP final; top) and latrunculin B (500 nM final; bottom). Median filter applied to inset images. Scale bars: 5 µm and 1 µm (inset). (E) Distribution of endogenously labeled split-mNeonGreen clathrin light chain A (CLTA; red) and Hem1-mCherry (green) dHL60s treated with latrunculin B (500 nM final). Insets (i and ii) shown on right. Scale bars: 5 µm and 1 µm (insets). (F) Distribution of SEPT7 (immunofluorescence; red) in Hem1-EGFP (green) dHL60 cell line treated with latrunculin B (500 nM final). Left image shows WAVE complex rings (inset i) and septin rings (inset ii). Right image shows complementary localization of Hem1 and SEPT7 along the cell periphery. Scale bars: 5 µm and 0.5 µm (insets).

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