Figure 7.
The WAVE complex, but not the Arp2/3 complex, is required for the formation of sheet-like protrusions. (A) Arp2/3 complex-disrupted dHL60 cells form sheet-like protrusions. IRSp53, a marker of both filopodial and lamellipodial protrusions, was imaged in chemoattractant-stimulated wild type (top row), WAVE complex-null (middle row), and ARP2-null (bottom row) cells. Middle column panel is 60 s after chemoattractant stimulation of the corresponding left column panel. Right column panel displays the difference (red) to highlight regions of protrusions. TIRF-SIM imaging; scale bar: 5 µm. See Video 7. Western blots for HEM1 and ARP2 with GAPDH as a loading control are shown for wild type and CRISPR/Cas9-edited cells. (B) Graphs comparing the protrusions across cells. Top graph: Mean ± SEM of the average protrusion widths (microns) after 1 min of chemoattractant stimulation per cell; wild type n = 10 cells, WAVE complex-null n = 10 cells, and ARP2-null n = 11 cells; cells pooled from at least three independent experiments per condition; Kruskal–Wallis test; P < 0.0001 with Dunn’s multiple comparisons follow up tests; ****, P < 0.0001; *, P = 0.0141; ns, not significant; P = 0.0717 > 0.05. Bottom graph: Mean ± SEM of the average protrusion speed (micron per minute) after chemoattractant stimulation per cell; wild type n = 10 cells, WAVE complex-null n = 10 cells, and ARP2-null n = 11 cells; cells pooled from at least three independent experiments per condition; Kruskal–Wallis test; P < 0.0001 with Dunn’s multiple comparisons follow-up tests; ****, P < 0.0001; **, P = 0.0029; ns, not significant; P > 0.99. (C)Arpc2-disrupted primary mouse bone marrow macrophages form sheet-like protrusions. IRSp53-EGFP was imaged in PMA-stimulated (100 nM) wild type ([−] 4-OHT) and Arpc2−/− ([+] 4-OHT) cells. Panels displayed similarly to A. TIRF-SIM imaging; scale bar: 5 µm. See Video 8. Western blot for Arpc2 and GAPDH as a loading control is shown. (D) Graphs comparing the protrusions across macrophages. Top graph: Mean ± SEM of the average protrusion widths (microns) after 1 min of PMA stimulation per cell; wild type ([−] 4-OHT) n = 10 cells and Arpc2−/− ([+] 4-OHT) n = 10 cells; cells pooled from two experiments per condition; Mann–Whitney test; **, P = 0.0052. Bottom graph: Mean ± SEM of the average protrusion speed (micron per minute) after PMA stimulation per cell; wild type ([−] 4-OHT) n = 10 cells and Arpc2−/− ([+] 4-OHT) n = 10 cells; cells pooled from two experiments per condition; Mann–Whitney test; ***, P = 0.0002. (E) The WAVE complex in ARP2-null dHL60 cells. The WAVE complex (Hem1-EGFP) localizes to sheet-like protrusions in chemoattractant-stimulated ARP2-null cells (10 nM fMLP; top) and as rings in F-actin–inhibited ARP2-null cells (500 nM latrunculin A; bottom). TIRF-SIM imaging; scale bars: 5 µm and 1 µm (insets). See Video 9. Violin plot of the WAVE complex ring diameters in wild type and ARP2-null cells; wild type n = 210 from 10 cells, ARP2-null n = 179 from 10 cells; cells pooled from at least three independent experiments per condition. Diameters measured in the same fashion shown in Fig. 2 D. (F) F-actin behind the WAVE complex in ARP2-null sheet-like protrusions. ARP2-null dHL60 cell expressing Hem1-EGFP (green) treated with chemoattractant (100 nM fMLP) and stained with phalloidin (red). TIRF imaging; scale bar: 5 µm.

The WAVE complex, but not the Arp2/3 complex, is required for the formation of sheet-like protrusions. (A) Arp2/3 complex-disrupted dHL60 cells form sheet-like protrusions. IRSp53, a marker of both filopodial and lamellipodial protrusions, was imaged in chemoattractant-stimulated wild type (top row), WAVE complex-null (middle row), and ARP2-null (bottom row) cells. Middle column panel is 60 s after chemoattractant stimulation of the corresponding left column panel. Right column panel displays the difference (red) to highlight regions of protrusions. TIRF-SIM imaging; scale bar: 5 µm. See Video 7. Western blots for HEM1 and ARP2 with GAPDH as a loading control are shown for wild type and CRISPR/Cas9-edited cells. (B) Graphs comparing the protrusions across cells. Top graph: Mean ± SEM of the average protrusion widths (microns) after 1 min of chemoattractant stimulation per cell; wild type n = 10 cells, WAVE complex-null n = 10 cells, and ARP2-null n = 11 cells; cells pooled from at least three independent experiments per condition; Kruskal–Wallis test; P < 0.0001 with Dunn’s multiple comparisons follow up tests; ****, P < 0.0001; *, P = 0.0141; ns, not significant; P = 0.0717 > 0.05. Bottom graph: Mean ± SEM of the average protrusion speed (micron per minute) after chemoattractant stimulation per cell; wild type n = 10 cells, WAVE complex-null n = 10 cells, and ARP2-null n = 11 cells; cells pooled from at least three independent experiments per condition; Kruskal–Wallis test; P < 0.0001 with Dunn’s multiple comparisons follow-up tests; ****, P < 0.0001; **, P = 0.0029; ns, not significant; P > 0.99. (C)Arpc2-disrupted primary mouse bone marrow macrophages form sheet-like protrusions. IRSp53-EGFP was imaged in PMA-stimulated (100 nM) wild type ([−] 4-OHT) and Arpc2−/− ([+] 4-OHT) cells. Panels displayed similarly to A. TIRF-SIM imaging; scale bar: 5 µm. See Video 8. Western blot for Arpc2 and GAPDH as a loading control is shown. (D) Graphs comparing the protrusions across macrophages. Top graph: Mean ± SEM of the average protrusion widths (microns) after 1 min of PMA stimulation per cell; wild type ([−] 4-OHT) n = 10 cells and Arpc2−/− ([+] 4-OHT) n = 10 cells; cells pooled from two experiments per condition; Mann–Whitney test; **, P = 0.0052. Bottom graph: Mean ± SEM of the average protrusion speed (micron per minute) after PMA stimulation per cell; wild type ([−] 4-OHT) n = 10 cells and Arpc2−/− ([+] 4-OHT) n = 10 cells; cells pooled from two experiments per condition; Mann–Whitney test; ***, P = 0.0002. (E) The WAVE complex in ARP2-null dHL60 cells. The WAVE complex (Hem1-EGFP) localizes to sheet-like protrusions in chemoattractant-stimulated ARP2-null cells (10 nM fMLP; top) and as rings in F-actin–inhibited ARP2-null cells (500 nM latrunculin A; bottom). TIRF-SIM imaging; scale bars: 5 µm and 1 µm (insets). See Video 9. Violin plot of the WAVE complex ring diameters in wild type and ARP2-null cells; wild type n = 210 from 10 cells, ARP2-null n = 179 from 10 cells; cells pooled from at least three independent experiments per condition. Diameters measured in the same fashion shown in Fig. 2 D. (F) F-actin behind the WAVE complex in ARP2-null sheet-like protrusions. ARP2-null dHL60 cell expressing Hem1-EGFP (green) treated with chemoattractant (100 nM fMLP) and stained with phalloidin (red). TIRF imaging; scale bar: 5 µm.

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