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

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 Syndrome-like 1 protein (NHSL1) as a direct binding partner of the Scar/WAVE complex, which co-localise at protruding lamellipodia. This interaction is mediated by the Abi SH3 domain and two binding sites in NHSL1. Furthermore, active Rac binds to NHSL1 at two regions that mediate leading edge targeting of NHSL1. Surprisingly, NHSL1 inhibits cell migration through its interaction with the Scar/WAVE complex. Mechanistically, NHSL1 may reduce cell migration efficiency by impeding Arp2/3 activity, as measured in cells using a Arp2/3 FRET-FLIM biosensor, resulting in reduced F-actin density of lamellipodia, and consequently impairing the stability of lamellipodia protrusions.

showing extent of reduction of NHSL1 expression in the clonal NHSL1 CRISPR B16-F1 cell lines 2 and 21 was probed with polyclonal NHSL1 antibodies and HSC70 antibodies as a loading control. Please note that lower molecular weight bands which may represent degradation products are also reduced in the CRISPR lines. Representative blot from three independent biological repeats. (b-d) Migration tracks of both NHSL1 CRISPR clones 1 and 21 in comparison to wild type B16-F1 cells. (e) Relative expression of NHSL1 in wild type B16-F1 (control) or NHSL1 CRISPR 2 and 21 cells by qPCR using isoform independent gene specific primer sets compared to expression of housekeeping gene GusB. Results are mean values +/-SEM (error bars) from n=3 independent biological experiments. One-way ANOVA: p=0.2066; F(2,6)=46.6; Dunnett's multiple comparisons test: *** p=0.0002; ns: not significant.

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Supplementary Figure 4. NHSL1 negatively regulates cell migration persistence. (a, b) Cell migration persistence was significantly increased for the NHSL1 CRISPR 2 or 21 cell line, respectively. (a) The directionality ratio is plotted to explore how it changes over the time of the movie. (b) Direction autocorrelation is shown, a measure of how the angle of displacement vectors correlate with themselves 18 which is independent of speed. (a-b) Results are mean values +/-SEM (error bars) from n= 177 (wild type), 140 (NHSL1 clones 2) and 156 (NHSL1 clones 21) cells from four independent biological repeats. Related to Figure 2 d-f. Source data are provided as a Source Data file. Figure 5. NHSL1 negatively regulates cell migration in MCF10A cells. (a) MCF10A normal breast epithelial cells were infected with lentiviruses harbouring control or NHSL1 specific shRNAs (shRNA A or B) and also conferring puromycin resistance and selected by puromycin. Stable MCF10A NHSL1 knockdown cell pools were grown until confluent before being scratched and imaged for 12 hours. Still images from live cell imaging showing one still image at the beginning (0 hours, left panel) and at the end of imaging (12 hours, right panel.) Scale bar: 300 µm. Representative images shown from four independent biological experiments. (b) The area of the scratch was measured at 0 and 12 h. Area closure is shown as percentage increase over control cells.

Supplementary
Results are mean ± SEM (error bars), from four independent biological repeats; full circles: control shRNA; empty circles: NHSL1 shRNA A; stars: NHSL1 shRNA B; One-   Fig. 3g: Western blot showing that dominant active (DA) Rac pulls down NHSL1 using Myc-trap beads from HEK cell lysates expressing Myctagged DA Rac1 co-expressed with EGFP-tagged NHSL1 or EGFP only as control. Representative blots from three independent biological repeats. (b) Western blot showing that dominant active (DA) Rac pulls down NHSL1 using GFP-trap beads from HEK cell lysates expressing Myc-tagged NHSL1 co-expressed with EGFP-tagged dominant active (DA) Rac1 or EGFP only as control. Representative blots shown from three independent biological experiments.

Supplementary Figure 7. Active Rac binds to two sites in NHSL. (a-b)
Nine overlapping EGFP-tagged subfragments covering fragments 2 and 3 of NHSL1 were generated (a) and expressed in HEK cells along with Myc-tagged DA Rac. After Myc-trap pulldown of Myc-DA-Rac, co-precipitation of EGFP-NHSL1 subfragments 1-9 was detected in a western blot with Myc antibody (b). Representative blots from three independent biological repeats. Source data are provided as a Source Data file.

Supplementary Figure 8. NHSL1 co-localises with Abi and the Scar/WAVE complex. (a-d)
Endogenous NHSL1 (NHSL1 pAb, magenta) co-localises with Abi1 (cyan) at the very edge of lamellipodia in B16-F1 mouse melanoma cells. (e-h) Endogenous NHSL1 (NHSL1 mAb, magenta) co-localises with Scar/WAVE2 (cyan) and at the very edge of lamellipodia in B16-F1 mouse melanoma cells. (a, e) Dual color merge of the insets shown in Fig. 4 (c,e): Scale bar in (a,e): 20 µm. (b-d;f-h) Three line scans (green, magenta, orange) were placed in arbitrary locations perpendicular to the leading edge and the intensity profile plotted; interrupted line: Abi1/WAVE2; continuous line: NHSL1; The Pearson's correlation coefficient (r 2 ) indicating degree of correlation of co-localisation was calculated and is displayed next to each pair of lines. (a,e) Representative images shown from three independent biological experiments. Fig. 5a: The Scar/WAVE complex coimmunoprecipitates with NHSL1. HEK cells were transfected with EGFP-NHSL1, and Myc-Pir121, -Nap-1, -WAVE2, -Abi2. NHSL-1 was immunoprecipitated (pAb 4457) from lysates and co-immunoprecipitation tested on a western blot with (a) Myc and reprobed with (b) EGFP antibodies. Representative blots from three independent experiments. (c) Negative control for experiment in Fig. 5d: GST-pull downs using purified Glutathionesepharose coupled GST-fusion proteins of Abi1 and c-Abl SH3 domains or GST alone from HEK cell lysates that were transfected with EGFP. Following GST-pulldown EGFP was detected in a western blot with anti-EGFP antibodies. Ponceau staining of membrane reveals GST or GST-tagged Abi-or Abl-SH3 domains used. Representative blots from three independent experiments. (d) HEK cells were transfected with EGFP-tagged Abi1 or Abi1-delta-SH3 or EGFP only as negative control and the remaining Myc-tagged Scar/WAVE components, PIR121, Nap1, Scar/WAVE2, HSPC300. After GFP-trap pulldown, co-precipitation was detected in a western blot with Myc antibody. Representative blots from three independent biological repeats. Source data are provided as a Source Data file. Figure 10. Abi SH3 domain binds to two fragments of NHSL1. (a) Far western overlay with purified MBP-tagged full-length Abi1 (MBP-Abi1 full length) or an MBP fusion protein with Abi1 in which the SH3 domain had been deleted (MBP-Abi1-delta-SH3) and MBP as control on a blot of different purified GST-NHSL1 fusion proteins covering the entire length of NHSL1. Representative blots from three independent experiments. Fragments 4 and 5 contain three putative SH3 binding sites. (b) Coomassie gel showing GST fragments covering the entire length of the NHSL1 amino acid sequence (see Fig. 1e for fragment sizes and location within NHSL1) and GST only as control which are used in the Far Western Blot in (a). Representative gel shown from three independent experiments.  ARPC3-mTurq2. (c-e) Quantification of FRET efficiency controls used to verify that any change in efficiency was a result of real changes in Arp2/3 activity and not skewed by, or an artefact of, the environment, vectors, or the FRET pair itself. The weighted mean average for each cell was calculated from the FRET efficiency histograms and were used instead of the normal mean in order to better represent the true FRET efficiency. Data is shown as weighted average mean ± SEM (error bars). Quantification from 4 independent biological repeats for B16-F1 cells expressing (c) control Myc plasmid and the donor ARPC3-mTurq2 (black circles; n=30 cells) or Myc DA Rac and the donor ARPC3-mTurq2 (brown upside-down triangles; n=30 cells); ns=0.9886; t=0.01436, df=58, Unpaired, two-tailed t-test. (d) control Myc plasmid and the donor ARPC3-mTurq2 (black circles; n=30 cells) or control Myc plasmid and the Arp2/3 biosensor (orange circles; n=31 cells), ns=0.6790, t=0.4158, df=59, Unpaired, two-tailed t-test and (e) Myc DA Rac and the donor ARPC3-mTurq2 (brown upside-down triangles; n=30 cells) or Myc DA Rac and the Arp2/3 biosensor (lilac upside-down triangles; n=26 cells), ****, p=0.000000001, t=7.379, df=54, Unpaired, two-tailed t-test. Source data are provided as a Source Data file.