Fig 2: Cell compression restores polarization in cells lacking WAVE or Arp2/3 complex.(A) Schematic for cell confinement experiments. The height of the chamber was set using a vacuum regulator. (B) dHL-60s expressing the Rac biosensor PakPBD were plated on fibronectin-coated glass in media containing 10 µM caged fMLP and imaged every 10 s by confocal microscopy. Chamber height was set as shown in 5A. Values in cyan indicate the degree of Rac activity polarization, as described in 2C, for the topmost cell fully inside each panel. (C) Quantification of Rac polarity as described in 2C for cells prepared as in 5B. Violet bar indicates when UV was used to photo-uncage caged fMLP (0–20 s). Dashed lines, mean polarity of Rac activity. Shaded regions, ±95% CI of the mean Rac polarity. N = 171 wild-type cells pooled from 4 independent experiments, 101 WAVE-null cells pooled from 4 independent experiments, and 108 ARP2-null cells pooled from 3 independent experiments. The underlying data for 5C can be found in S1 Data. Arp2/3, actin-related protein 2/3; dHL-60, differentiated HL-60 cell; fMLP, N-Formylmethionine-leucyl-phenylalanine; PakPBD, p21-activated kinase binding domain; Rac, Ras-related C3 botulinum toxin substrate; WAVE, WASP-family verprolin homologous protein.

Fig 3: How does the WAVE complex achieve the proper spatial organization for lamellipodial generation? (A) Homologous actin NPFs organize different cell morphologies. Given that the homologous NPFs N-WASP and the WAVE complex are both activated by homologous GTPases, associated with homologous BAR domains, stimulated Arp2/3 complex activation, and stripped off the membrane as a function of this actin polymerization, how do they organize different cellular morphologies? N-WASP canonically associates with filopodia, invadopodia, and endocytosis, whereas the WAVE complex associates with sheet-like lamellipodia. Left: Chick cranial neural crest cell with multiple filopodia from Genuth et al. (2018). Right: Head-on view of a neutrophil-like dHL60 lamellipodium (ChimeraX rendering of a confocal z-stack). Scale bars: 10 µm (left) and 5 µm (right). (B) Schematic of how NPF spatial organization could instruct the resulting actin morphologies. N-WASP’s positive feedback results in the focal organization expected for one-dimensional, finger-like actin structures (Banjade and Rosen, 2014). To build lamellipodia, does the WAVE complex’s positive feedback result in a linear organization to template 2D, sheet-like actin structures? (C) Hem1-EGFP, a fluorescently tagged subunit of the WAVE complex, has a linear organization at tips of lamellipodia in chemoattractant-stimulated (10 nM fMLP) dHL60 cells. Top: 3D imaging of the WAVE complex at tips of extending lamellipodia; left, widefield 3D reconstruction; right, lattice light sheet reconstruction of ruffles from a head-on view. Bottom: WAVE complex’s linear organization viewed from the ventral plasma membrane; simultaneous TIRF imaging of Hem1-EGFP (green) and membrane CellMask DeepRed dye (gray). Scale bars: 5 µm and 2 µm (bottom right). See Video 1.

Fig 4: 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.