Fig 2: Loss of Piezo1 leads to increased protrusion activity via Rac1 activation. (A,B) NC cells plated on a fibronectin-coated dish, expressing LifeAct-Ruby in control MO (A) and Piezo1 MO (B) at different time points (top). Note that cell protrusions are more abundant in Piezo1 MO compared with control MO. (C) Quantification of the protrusion duration in minutes from A,B. n=18 cells in each condition. (D) Time projection of NC cells from A,B at time 0 (green) and after 7 min of migration (red). Protrusion area is highlighted in red in the illustration. (E) Quantification of the area of cell protrusions from A,B. n=18 cells in each condition. (F) Immunostaining of Rac1-GTP (active Rac1) in control MO NC explants (top) and Piezo1 MO (bottom). (G) Quantification of fluorescence intensity of Rac1-GTP from F. r.u., relative units. n=70 cells in each condition. Each dot is the mean value of an independent experiment. All data are representative of at least three biological replicates. Error bars are ±s.e.m. **P=0.01, ****P=0.0001 (unpaired two-tailed Student's t-test). Scale bars: 10 µm.

Fig 3: Piezo1 prevents neural crest invasion via Rac1 inhibition. (A) NC graft of control (left), Piezo1 MO (middle) and Piezo1 MO plus dominant negative (dn) Rac1 (right). (B) Quantification of the percentage of embryos with normal NC migration from A. Normal NC migration of grafting experiments was determined by comparing the NC migratory streams to a whole-mount in situ hybridization. Note that expression of dnRac1 on top of Piezo1 MO partially rescues NC migration. n=5 embryos per condition. (C) Colour coded single cell tracks from boxed areas in A. Note that Piezo1 MO cells migrate laterally, whereas directional migration is restored in Piezo1 MO plus dnRac1. (D) Angles of cell migration from C. (E) Quantification of speed of cell migration from A. (F) Quantification of persistence of cell migration from A. Note that both speed and persistence of cell migration are rescued in Piezo1 MO plus dnRac1. n=20 in each condition (E,F). Error bars are ±s.e.m. Each dot is the mean value of an independent experiment. All data are representative of at least three biological replicates. **P=0.01, ***P=0.001, ****P=0.0001 (one-way ANOVA with a Dunnett's multiple comparisons post-test). n.s., non-significant. (G-I) Proposed model of the role of Piezo1 (P1) in NC migration. In control embryos (H) Piezo1 mechanical activation partially reduces Rac1 activity, which is further inhibited by chemical signals from Sema. This leads to a collapse of cell protrusions and inhibition of cell migration into the Sema region. In the absence of Piezo1 (I), there is no initial regulation of Rac1 levels: when NC cells reach the Sema region, Rac1 levels are only partially inhibited, and cells continue to invade the Sema region. Scale bar: 100 µm.

Fig 4: Schematic representation of RAC1 regulation by GTP.GAP GTPase-activating protein, GEF guanine nucleotide exchange factor, GDI GDP dissociation inhibitor.

Fig 5: Intracellular localization of IMPDH2 regulates GTP levels in cell protrusions, RAC1 activity, and cell invasion.a Cells transduced with the indicated constructs (Cl sh control shRNA, IMsh IMPDH2 shRNA, IM-LCK IMPDH2 with LCK membrane-localization domain, IM-GIA IMPDH2 with giantin Golgi localization domain) were separated into the plasma membrane and cytoplasm fractions as described in Methods, followed by immunoblotting with the indicated antibodies Shown are representative images of at least two independent experiments. b Immunofluorescence analysis for IMPDH2 (red) and actin (phalloidin, green), and nuclei (Hoechst, blue) in MDA-MB-231 cells transduced with the indicated constructs. Arrows denote the presence of IMPDH2 at the cell plasma membrane. Scale bar 20 µm. Shown are representative images of two independent experiments. c GTP levels were determined via mass spectroscopy as described in Methods. The data represents average ± SEM of two independent experiments performed in duplicates. Statistics performed by two-tailed unpaired Student’s t-test (****p < 0.0001), exact p value <0.00001 for both cases. d Quantification of GEVAL activity in cell bodies (CB) and cell protrusions (CP) of MDA-MB-231 cells transduced with the indicated constructs and with GEVAL30 or GEVALNull (30 CBs and 30 CPs per cell type). Horizontal bars represent average. Individual values are from two experiments (15 CB and 15CP per experiment). Statistics were performed by a two-tailed unpaired Student’s t-test. e Cells transduced with the indicated constructs were probed in RAC1 activity assay as described in Methods. Shown are representative images of three independent experiments. f Quantification of (e). The data represents the average ± SEM of three independent experiments. Statistics were performed by a two-tailed unpaired Student’s t-test. g Invasion assay of MDA-MB-231 cells transduced with the indicated constructs. The data represents the average ± SEM of three independent experiments performed in duplicates. Statistics were performed by two-tailed unpaired Student’s t-test.