Fig. 8: NHSL1 negatively regulates Arp2/3 and F-actin content in the lamellipodium. | Nature Communications

Fig. 8: NHSL1 negatively regulates Arp2/3 and F-actin content in the lamellipodium.

From: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

Fig. 8

a First frame of a movie of LifeAct-EGFP expressing B16-F1 cells showing how length, width and area was quantified (see ‘Methods’ section) in (b; S17a–d): red line: area; blue lines: width of lamellipodium. Bar: 20 μm. b Ratio of lamellipodia length/total cell area. n = 15 cells each. One-way ANOVA, Dunnett’s test; ****P < 0.0001; **P = 0.0059; F(2,42) = 10.84. c Total cellular F-actin (Alexa568-phalloidin) intensity/area ratio in wild-type B16-F1 cells expressing Myc-tag (WT Myc-Control, black circles) or CRISPR 2 cells expressing the NHSL1 mutant in the Scar/WAVE complex binding sites (Myc-NHSL1 SW Mut, blue diamonds) or NHSL1 (Myc-NHSL1 WT, red crosses) or Myc-tag as control (pink squares) plated on laminin. One-way ANOVA: P = 0.0009; Kruskal–Wallis statistics 16.44; ***P = 0.0003; **P = 0.0083; nsP = 0.1518; outliers removed: ROUT method (Q = 1%) (n analysed/n outliers = no removed); WT control: n = 67 cells/no = 0; NHSL1 CRISPR2: n = 71 cells/no = 3; NHSL1 CRISPR2 + Myc-NHSL1 SW Mut Rescue: n = 76 cells/no = 5; NHSL1 CRISPR2 + Rescue Myc-NHSL1 WT: n = 82 cells/no = 4. d Relative lamellipodial Arp2/3 intensity: Arp2/3 intensity in the lamellipodium normalised cell-by-cell against whole-cell Arp2/3 intensity. Kruskal–Wallis test (P = 0.0514), Dunn’s test: *P = 0.0353; nsP > 0.9999; wild type (n = 40 cells), NHSL1 CRISPR 2 (n = 38 cells) and NHSL1 CRISPR 21 (n = 33 cells) B16-F1 cells on laminin, stained with anti-ARPC2 antibodies. e Lamellipodial F-actin (Alexa488-phalloidin) intensity/area ratio in B16-F1 wild-type control cells or NHSL1 CRISPR 2 and NHSL1 CRISPR 21 B16-F1 cells plated on laminin. n = control (61), CRISPR 2 (61), CRISPR 21 (43) cells; one-way ANOVA: P = 0.0127; F(2,162) = 4.486; Dunnett’s multiple comparisons test: **P = 0.0099. f Lamellipodial F-actin (LifeAct-EGFP) intensity/area ratio in B16-F1 cells transfected with a tri-cistronic plasmid expressing Myc-tagged NHSL1 (wild type or Scar/WAVE binding mutant), puromycin resistance and LifeAct-EGFP or empty plasmid (Myc-IRES-Puro-T2A-LifeAct-EGFP) after selection with puromycin plated on laminin. n = control (14), WT (12), NHSL1-SW mut (13) cells. One-way ANOVA: P = 0.0074; F(2,36) = 5.636; Dunnett’s multiple comparisons test: **P = 0.0036; nsP = 0.1810. b–f Results are mean ± SEM (error bars) from three (b, d) or four (c, f) or five (e) biologically independent experiments. Source data are provided as a Source Data file.

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