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NHSL1 reduces the stability of lamellipodia protrusion. a, b Movie stills showing protrusion (blue) and retraction (red) dynamics of wild-type B16-F1, NHSL1 CRISPR 2 and B16-F1 cells overexpressing Myc-tagged NHSL1. Scale bar: 20 μm. c, d NHSL1 reduces the length of the longest uninterrupted lamellipodium. Each data point represents the mean length of the longest uninterrupted lamellipodium quantified from all frames of a movie of one LifeAct-EGFP expressing B16-F1 cell (5 s intervals, 10 min duration) (see 'Methods' for details). Control (black circles), NHSL1 CRISPR 2 (magenta squares), 21 (green triangles) (c) and control (black circles) and Myc-NHSL1 overexpression (red crosses) (d). c Control: n = 13; NHSL1 CRISPR 2: n = 12; NHSL1 CRISPR 21: n = 13 cells. One-way ANOVA: P = 0.0111; F(2,35) = 5.133; Dunnett's test *P = 0.0144, control versus CRISPR 21: not significant: ns P = 0.9949. d control n =10; Myc-NHSL1 n = 9 cells. ***P = 0.006, Mann-Whitney test. e, f Quantification (see 'Methods' section) of lamellipodia protrusion speed (e) and lamellipodia stability (f) of randomly migrating wild-type B16-F1 cells or CRISPR 2 cells expressing either NHSL1 (NHSL1 WT) or the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut) or Myc alone (control) plated on laminin. WT Control: n = 13, NHSL1 CRISPR 2: n = 12, Rescue Myc-NHSL1 SW Mut: n = 11, Rescue Myc-NHSL1 SW WT: n = 15 cells. One-way ANOVA: P = 0.2097, F(3,47) = 1.568; Dunnett's test: NHSL1 CRISPR 2 vs Rescue SW mut: ns P = 0.2043; NHSL1 CRISPR 2 vs Rescue SW WT: ns P = 0.1573; NHSL1 CRISPR 2 vs WT control: ns P =0.7639. f The standard deviation of the cone speed shows the fluctuation of speeds along the edge and serves as a measure for the stability of lamellipodial protrusions. WT control: n = 13, NHSL1 CRISPR 2: n = 12, Rescue Myc-NHSL1 SW Mut: n = 11, Rescue Myc-NHSL1 SW WT: n = 15 cells. One-way ANOVA: P = 0.0412, F(3,47) = 2.971; Dunnett's test: NHSL1 CRISPR 2 vs Rescue SW mut: ns P = 0.8865; NHSL1 CRISPR 2 vs Rescue SW WT: *P = 0.0299; NHSL1 CRISPR 2 vs WT control: ns P = 0.1173. c-f Results are mean ± SEM (error bars), four independent biological repeats. Source data are provided as a Source Data file.

NHSL1 reduces the stability of lamellipodia protrusion. a, b Movie stills showing protrusion (blue) and retraction (red) dynamics of wild-type B16-F1, NHSL1 CRISPR 2 and B16-F1 cells overexpressing Myc-tagged NHSL1. Scale bar: 20 μm. c, d NHSL1 reduces the length of the longest uninterrupted lamellipodium. Each data point represents the mean length of the longest uninterrupted lamellipodium quantified from all frames of a movie of one LifeAct-EGFP expressing B16-F1 cell (5 s intervals, 10 min duration) (see 'Methods' for details). Control (black circles), NHSL1 CRISPR 2 (magenta squares), 21 (green triangles) (c) and control (black circles) and Myc-NHSL1 overexpression (red crosses) (d). c Control: n = 13; NHSL1 CRISPR 2: n = 12; NHSL1 CRISPR 21: n = 13 cells. One-way ANOVA: P = 0.0111; F(2,35) = 5.133; Dunnett's test *P = 0.0144, control versus CRISPR 21: not significant: ns P = 0.9949. d control n =10; Myc-NHSL1 n = 9 cells. ***P = 0.006, Mann-Whitney test. e, f Quantification (see 'Methods' section) of lamellipodia protrusion speed (e) and lamellipodia stability (f) of randomly migrating wild-type B16-F1 cells or CRISPR 2 cells expressing either NHSL1 (NHSL1 WT) or the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut) or Myc alone (control) plated on laminin. WT Control: n = 13, NHSL1 CRISPR 2: n = 12, Rescue Myc-NHSL1 SW Mut: n = 11, Rescue Myc-NHSL1 SW WT: n = 15 cells. One-way ANOVA: P = 0.2097, F(3,47) = 1.568; Dunnett's test: NHSL1 CRISPR 2 vs Rescue SW mut: ns P = 0.2043; NHSL1 CRISPR 2 vs Rescue SW WT: ns P = 0.1573; NHSL1 CRISPR 2 vs WT control: ns P =0.7639. f The standard deviation of the cone speed shows the fluctuation of speeds along the edge and serves as a measure for the stability of lamellipodial protrusions. WT control: n = 13, NHSL1 CRISPR 2: n = 12, Rescue Myc-NHSL1 SW Mut: n = 11, Rescue Myc-NHSL1 SW WT: n = 15 cells. One-way ANOVA: P = 0.0412, F(3,47) = 2.971; Dunnett's test: NHSL1 CRISPR 2 vs Rescue SW mut: ns P = 0.8865; NHSL1 CRISPR 2 vs Rescue SW WT: *P = 0.0299; NHSL1 CRISPR 2 vs WT control: ns P = 0.1173. c-f Results are mean ± SEM (error bars), four independent biological repeats. Source data are provided as a Source Data file.

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Article
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Cell migration is important for development and its aberrant regulation contributes to many diseases. The Scar/WAVE complex is essential for Arp2/3 mediated lamellipodia formation during mesenchymal cell migration and several coinciding signals activate it. However, so far, no direct negative regulators are known. Here we identify Nance-Horan Syndr...

Citations

... Moreover, NHSL1 can also interact with active RAC, which indicates that NHSL1 acts as a negative regulator in the RAC-WRC-ARP2/3 pathway. [293]. ...
... A recent study also found that the NHSL1 protein acts as an inhibitor of directional cell migration via interacting with the canonical WRC [293]. In this research, by studying a specific NHSL1 isoform without the WHD domain, two SH3 domain binding sites were identified in NHSL1 and shown to be important for the interaction of NHSL1 with ABI1. ...
Thesis
During cell migration, the RAC1-WAVE-ARP2/3 signaling pathway induces the network of branched actin, that serves as a motor for lamellipodia protrusion. This pathway is finely regulated by numerous feed-back and feed-forward signals that control the protrusion lifetime and migration persistence. We screened in MCF10A human breast epithelial cells for proteins that associate with the WAVE complex during persistent migration, but whose association with WAVE is modulated when the downstream production of branched actin is inhibited. The differential proteomics screen identified PPP2R1A (a regulatory subunit of the PP2A trimeric phosphatase) as the strongest hit and a novel WAVE-associated factor required for migration persistence in normal and cancer human cells, in various conditions. The differential proteomics screen identified PPP2R1A (a regulatory subunit of the PP2A trimeric phosphatase) as the strongest hit and a novel WAVE-associated factor required for migration persistence in normal and cancer human cells, in various conditions. Our observation that PPP2R1A interacts with four WAVE complex subunits, but not with WAVE/WASF, led to a purification and characterization of a “WAVE shell complex (WSC)”, a novel variant of WAVE containing the migration regulatory protein NHSL1 that turned out to be necessary for the existence of WSC. Interestingly, PPP2R1A is mutated on hotspots in different cancer types, and these mutations abolish its interaction with NHSL1 and WSC, suggesting a critical role of for this pathway not only in normal cells, but also in cancer progression.
... Cell migration is finely regulated at all molecular levels. Each positive component required to generate cortical branched actin, RAC1, WRC and Arp2/3, appears to be counteracted by inhibitory proteins, CYRI 14,15 , NHSL1 16 and ARPIN 17,18 , respectively. NHSL1 belongs to the family of Nance-Horan Syndrome (NHS) proteins, which contain an N-terminal WAVE Homology Domain (WHD), as in WAVE proteins 19 . ...
... This intriguing possibility, however, has never been reported until now. Instead NHSL1 has been shown to interact with WRC through the C-terminal SH3 domain of the WRC subunit ABI1 16 . ...
... The WRC subunits did not vary considerably in the different conditions, 20 % at most (Fig.1b). Among the hits, we recognized several described functional partners of the WRC such as SRGAP3, also known as WRP 30 , the Nance-Horan Syndrome family proteins NHS and NHSL1 16,19 , IRSp53 31 and lamellipodin, which was 3-fold more associated with WRC when RAC1 was activated, as previously reported 32 . ...
Preprint
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Proteins of the Wiskott-Aldrich syndrome protein (WASP) family play a central role in regulating actin cytoskeletal dynamics in a wide range of cellular processes. Genetic mutations or misregulation of these proteins are tightly associated with many diseases. The WASP-family proteins act by transmitting various upstream signals to their conserved WH2-Central-Acidic (WCA) peptide sequence at the C-terminus, which in turn binds to the Arp2/3 complex to stimulate the formation of branched actin networks at membranes. Despite this common feature, the regulatory mechanisms and cellular functions of distinct WASP-family proteins are very different. Here, we summarize and clarify our current understanding of WASP-family proteins and how disruption of their functions is related to human disease.
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Actin filaments generate mechanical forces that drive membrane movements during trafficking, endocytosis and cell migration. Reciprocally, adaptations of actin networks to forces regulate their assembly and architecture. Yet, a demonstration of forces acting on actin regulators at actin assembly sites in cells is missing. Here we show that local forces arising from actin filament elongation mechanically control WAVE regulatory complex (WRC) dynamics and function, that is, Arp2/3 complex activation in the lamellipodium. Single-protein tracking revealed WRC lateral movements along the lamellipodium tip, driven by elongation of actin filaments and correlating with WRC turnover. The use of optical tweezers to mechanically manipulate functional WRC showed that piconewton forces, as generated by single-filament elongation, dissociated WRC from the lamellipodium tip. WRC activation correlated with its trapping, dwell time and the binding strength at the lamellipodium tip. WRC crosslinking, hindering its mechanical dissociation, increased WRC dwell time and Arp2/3-dependent membrane protrusion. Thus, forces generated by individual actin filaments on their regulators can mechanically tune their turnover and hence activity during cell migration. Mehidi et al. show that piconewton forces exerted by the polymerization of individual actin filaments displace the WAVE regulatory complex from lamellipodial tips, thereby regulating WAVE complex activity during cell migration.