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NHSL1 negatively regulates cell migration via the Scar/WAVE complex. a Speed of randomly migrating B16-F1 cells overexpressing either wildtype NHSL1 (NHSL1 WT, black circles) or the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut, blue diamonds) or EGFP alone (red crosses) as control plated on fibronectin after selection using a bicistronic puromycin expression plasmid ensuring all cells analysed overexpressed NHSL1. Mean track speed (dt = 3, TR = 4; see 'Methods' for calculation). Results are mean ± SEM (error bars), of four independent biological experiments. Each data point represents the mean speed of a cell from a total number of 106, 104, 108 cells for control, NHSL1 WT and NHSL1 SW Mut, respectively. Oneway ANOVA: P = 0.0007; F(2,395) = 7.385; and Dunnett's multiple comparisons tests: ***P = 0.0003; ns P = 0.0655; b, c speed and persistence of randomly migrating wild-type B16-F1 cells expressing Myc alone as control (black circles) or CRISPR 2 cells expressing either the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut, blue diamonds) or NHSL1 (NHSL1 WT, red crosses) or Myc alone as control (pink squares) plated on laminin after selection using a bicistronic blasticidin expression plasmid ensuring all cells analysed expressed NHSL1. Mean track speed (b) and persistence (c) (dt = 5, TR = 2; see 'Methods' for calculation). Each data point represents the mean speed of a cell. Results are mean ± SEM (error bars). b One-way ANOVA: P = 0.0077; F(3,302) = 4.044; and Dunnett's multiple comparisons test: **P = 0.0018; *P = 0.0364; ns P = 0.1215. c One-way ANOVA: P = 0.0034; F(3,302) = 4.648; and Dunnett's multiple comparisons tests: CRISPR 2 vs. WT control: *P = 0.0397; NHSL1 CRISPR2 vs. Rescue Myc-NHSL1 SW Mut: ns P = 0.9877; NHSL1 CRISPR2 vs. Rescue Myc-NHSL1 WT: ns P = 0.9442. d Mean Square Displacement analysis (log-log plot) of data shown in b, c. Results are mean values ± SEM (error bars). b-d n = 102 (wild-type cells Myc only), 55 (CRISPR 2 cells Myc only), 77 (CRISPR 2 Rescue Myc-NHSL1 SW Mut), 72 (CRISPR 2 Rescue Myc-NHSL1 WT) from five independent biological experiments. Source data are provided as a Source Data file.

NHSL1 negatively regulates cell migration via the Scar/WAVE complex. a Speed of randomly migrating B16-F1 cells overexpressing either wildtype NHSL1 (NHSL1 WT, black circles) or the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut, blue diamonds) or EGFP alone (red crosses) as control plated on fibronectin after selection using a bicistronic puromycin expression plasmid ensuring all cells analysed overexpressed NHSL1. Mean track speed (dt = 3, TR = 4; see 'Methods' for calculation). Results are mean ± SEM (error bars), of four independent biological experiments. Each data point represents the mean speed of a cell from a total number of 106, 104, 108 cells for control, NHSL1 WT and NHSL1 SW Mut, respectively. Oneway ANOVA: P = 0.0007; F(2,395) = 7.385; and Dunnett's multiple comparisons tests: ***P = 0.0003; ns P = 0.0655; b, c speed and persistence of randomly migrating wild-type B16-F1 cells expressing Myc alone as control (black circles) or CRISPR 2 cells expressing either the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut, blue diamonds) or NHSL1 (NHSL1 WT, red crosses) or Myc alone as control (pink squares) plated on laminin after selection using a bicistronic blasticidin expression plasmid ensuring all cells analysed expressed NHSL1. Mean track speed (b) and persistence (c) (dt = 5, TR = 2; see 'Methods' for calculation). Each data point represents the mean speed of a cell. Results are mean ± SEM (error bars). b One-way ANOVA: P = 0.0077; F(3,302) = 4.044; and Dunnett's multiple comparisons test: **P = 0.0018; *P = 0.0364; ns P = 0.1215. c One-way ANOVA: P = 0.0034; F(3,302) = 4.648; and Dunnett's multiple comparisons tests: CRISPR 2 vs. WT control: *P = 0.0397; NHSL1 CRISPR2 vs. Rescue Myc-NHSL1 SW Mut: ns P = 0.9877; NHSL1 CRISPR2 vs. Rescue Myc-NHSL1 WT: ns P = 0.9442. d Mean Square Displacement analysis (log-log plot) of data shown in b, c. Results are mean values ± SEM (error bars). b-d n = 102 (wild-type cells Myc only), 55 (CRISPR 2 cells Myc only), 77 (CRISPR 2 Rescue Myc-NHSL1 SW Mut), 72 (CRISPR 2 Rescue Myc-NHSL1 WT) from five independent biological experiments. 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...

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Context 1
... analysis revealed that NHSL1 is in complex with active Rac ( Fig. 3g and Supplementary Fig. 6a). This was verified in a reciprocal experiment in which EGFP-Rac1-Q61L or EGFP was pulled down with GFP-trap beads and the interaction with Myc-NHSL1 was evaluated by western blot against Myc ( Supplementary Fig. 6b). Again, we observed an interaction between dominant active Rac and NHSL1 suggesting that NHSL1 is a binding partner of active Rac. ...
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... data are provided as a Source Data file. Representative blots from three independent biological repeats (see Supplementary Fig. 6a for full western blots). h Western blot showing Myc-tagged DA Rac pulldowns of the four NHSL1 fragments (see panels a and b-e) from HEK cells expressing the EGFP-tagged fragments covering the entire length of NHSL1 or EGFP only as control co-expressed with Myc-tagged DA Rac. ...
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... the NHSL1 cDNA which cannot interact with the Abi SH3 domain and hence cannot interact with the Scar/WAVE complex ( Fig. 5g) (EGFP-NHSL1 SW Mut) in B16-F1 cells Supplementary Fig. 11a). We quantified random cell migration behaviour after plating the cells on fibronectin and observed a moderate but significant reduction in cell migration speed ( Fig. 6a) and a moderately reduced mean square displacement (Supplementary Fig. 11b) for cells overexpressing wild-type EGFP-NHSL1 compared to EGFP control. This is consistent with the result from the NHSL1 CRISPR cells, which displayed the opposite effect ( Fig. 2c-f). Cell migration persistence was increased upon overexpression of NHSL1 ( ...
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... NHSL1 Scar/WAVE binding mutant localises to the very edge of lamellipodia (Supplementary Fig. 11f and Supplementary Movie 7) like wild-type EGFP-NHSL1 (Fig. 1f, g and Supplementary Movie 1). In contrast, the NHSL1 Scar/WAVE binding mutant did not reduce migration speed (Fig. 6a) suggesting that NHSL1 negatively regulates cell migration speed via an interaction with the Scar/WAVE complex. To verify this and to test whether the observed phenotypes in the NHSL1 CRISPR knockout clones were not due to off-target effects, we re-expressed Myc-tagged wild-type NHSL1 (Myc-NHSL1 WT) or the NHSL1 Scar/WAVE complex ...
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... were not due to off-target effects, we re-expressed Myc-tagged wild-type NHSL1 (Myc-NHSL1 WT) or the NHSL1 Scar/WAVE complex binding mutant (Myc-NHSL1 SW Mut) in B16-F1 cells. After plating the cells on laminin we again observed an increase in random cell migration speed and persistence between wild-type control and NHSL1 CRISPR2 knockout cells (Fig. 6b, c) confirming our previous results ( Fig. 2c-e). Re-expression of neither wild-type Myc-NHSL1 nor Myc-NHSL1 SW Mut in CRISPR 2 cells rescued cell migration persistence (Fig. 6c, d and Supplementary Fig. 12a, b). This result and the increase in cell migration persistence upon NHSL1 overexpression ( Supplementary Fig. 11c-e) and NHSL1 ...
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... After plating the cells on laminin we again observed an increase in random cell migration speed and persistence between wild-type control and NHSL1 CRISPR2 knockout cells (Fig. 6b, c) confirming our previous results ( Fig. 2c-e). Re-expression of neither wild-type Myc-NHSL1 nor Myc-NHSL1 SW Mut in CRISPR 2 cells rescued cell migration persistence (Fig. 6c, d and Supplementary Fig. 12a, b). This result and the increase in cell migration persistence upon NHSL1 overexpression ( Supplementary Fig. 11c-e) and NHSL1 knockout ( Fig. 2e) together suggest that optimal expression levels of NHSL1 may be required for the lower cell migration persistence observed in the wild-type cells. However, ...
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... Fig. 11c-e) and NHSL1 knockout ( Fig. 2e) together suggest that optimal expression levels of NHSL1 may be required for the lower cell migration persistence observed in the wild-type cells. However, re-expression of wild-type Myc-NHSL1 but not Myc-NHSL1 SW Mut in CRISPR 2 cells resulted in a significant reduction in cell migration speed (Fig. 6b). This indicates that wild-type NHSL1 but not NHSL1 SW Mut (that cannot bind to the Scar/WAVE complex) can rescue the NHSL1 knockout phenotype and suggests that NHSL1 negatively regulates cell migration speed via an interaction with the Scar/WAVE ...

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
Full-text available
The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Using proteomics, we identified PPP2R1A among proteins differentially associated with the WAVE complex subunit ABI1 when RAC1 was activated and downstream generation of branched actin was blocked. PPP2R1A was found to associate at the lamellipodial edge with a novel form of WAVE complex, the WAVE Shell Complex (WSC), that contains NHSL1 instead of the Arp2/3 activating subunit WAVE as in the canonical WAVE Regulatory Complex (WRC). PPP2R1A was required for persistence in random and directed migration assays and for RAC1-dependent actin polymerization in cell extracts. PPP2R1A requirement was abolished by NHSL1 depletion. PPP2R1A mutations found in tumors impaired WSC binding and migration regulation, suggesting that this novel function of PPP2R1A is critical for its tumor suppressor activity.
... A large number of interacting ligands of the WRC have been identified, and the list is still rapidly growing . Like WASP/N-WASP, WRC directly interacts with small G proteins (Rho-family and Arf-family GTPases), inositol phospholipids (e.g., PIP 3 ), various kinases (Abl, Src, Cdk5, Cdk1, Erk, CK2, Pka, SepA), and many cytosolic or adaptor proteins, such as IRSp53, Nck, Lamellipodin, Ena/VASP, NHSL1, and WRP (Ardern et al., 2006;Dai and Pendergast, 1995;Danson et al., 2007;Kim et al., 2006;Kitamura et al., 1996;Kobayashi et al., 1998;Koronakis et al., 2011;Law et al., 2021Law et al., , 2013Leng et al., 2005;Mendoza, 2013;Miki et al., 2000;Miyamoto et al., 2008;Nakanishi et al., 2007;Oikawa et al., 2004;Pocha and Cory, 2009;Shi et al., 2021;V. Singh et al., 2020;S.P. Singh et al., 2020;Soderling et al., 2002;Stuart et al., 2006;Ura et al., 2012;Westphal et al., 2000;Xu and Quinn, 2012;Yamashita et al., 2011). ...
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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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