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Fig 1. Overexpression of EC1-3-myc and treatment with recombinant EC1-3 increases Akt phosphorylation.
(A, upper) Injections of 200 pg mRNA for UGP alone or together with 800 pg of EC1-3-myc mRNA were performed on single blastomeres of 2-cell X.laevis embryos. CNC expressing UGP were dissected at neurula stages, lysed and analyzed for phospho-Akt (pAkt), phospho-MAPK (pMAPK) and GAPDH via western blot. (A, lower) Non-injected CNC were dissected and treated for 5 minutes with 10 ng/mL of recombinant EC1-3 (bacEC) before being processed as above. (B) As a control, bacEC was depleted from treatment solution using a specific bacEC antibody conjugated to agarose beads and then applied to CNC. Lysates were then analyzed as above. Immunodepletion using unconjugated beads was used as a control. (C) Significant increases in phospho-Akt were observed in CNC overexpressing EC1-3-myc (N = 5, p = 0.013) and those treated with bacEC (N = 5, p = 0.021). There were not significant (N.S.) increases in the phosphorylation of MAPK in CNC overexpressing EC1-3-myc (N = 5, p = 0.09), nor those treated with bacEC (N = 5, p = 0.178). GAPDH was used as a loading control for western blots and normalizer for calculation of phospho-Akt and phospho-MAPK. Error bars are one standard deviation to the mean. One-tailed, Student’s t-tests were performed to determine statistical significance. * p<0.05. N, number of experiments.
https://doi.org/10.1371/journal.pone.0188963.g001
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Fig 2. Inhibition of PI3K perturbs CNC migration.
(A,B) Lateral views of tailbud stage X.laevis embryos after in situ hybridization with Sox10 and Twist to visualize CNC positioning. Anterior is to the left, dorsal is up. Migration of CNC cells into the branchial arches is perturbed in embryos treated with 30 μM LY294002 (N = 4, n = 52). (C) Western blot of embryos treated with DMSO or 30 μM LY294002. Embryos treated from stage 17–18 to stage 25–26 with LY294002 had reduced levels of phosphorylated Akt (pAkt). One-embryo equivalents were loaded per well. (D,E) The length of CNC migration (shown in A) in each branchial arch was quantified and normalized to head size. LY294002 significantly reduced CNC migration into all branchial arches compared to DMSO controls (N = 4, n = 66). Treatment with 40 μM rapamycin (N = 4, n = 37) did not noticeably alter CNC positioning compared to DMSO controls (N = 4, n = 35, respectively). CNC cells migrated into the mandibular (M), hyoid (H), 3rd and 4th branchial arches. Ruler bars denote how CNC segments were measured. Error bars are one standard deviation to the mean. One-tailed, Student’s t-tests were performed to determine statistical significance. *** p<0.001. N, number of experiments; n, number of embryos.
https://doi.org/10.1371/journal.pone.0188963.g002
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Fig 3. EC1-3-myc and non-adhesive EC1-3-myc interact with growth factor receptors.
Western blots of EC1-3-myc (A) and non-adhesive EC1-3-myc (B; naEC1-3-myc) co-immunoprecipitations (coIPs). Receptors were immunoprecipitated using anti-HA or anti-flag antibodies. Both EC1-3 constructs co-IPed with all receptors: HA-tagged EGFR/ErbB1, HA-tagged ErbB3 and flag-tagged ErbB2, ErbB4, PDGFRα and FGFR1. (C) Western blot of EC1-3-myc coIP with cytosolic proteins ARID3a-flag and importinβ1-flag. To test if EC1-3 could interact non-specifically with proteins following extraction we tested Flag-tagged constructs overexpressed in Hek293T cells. EC1-3-myc did not associate with ARID3a-flag, nor importinβ1-flag. ErbB2-flag was used as a positive control. Results are representative of three independent experiments.
https://doi.org/10.1371/journal.pone.0188963.g003
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Fig 4. Recombinant EC1-3 (bacEC) increases Akt phosphorylation in ErbB2-transfected cells.
Hek293T cells were transfected with receptor tyrosine kinases, serum-starved and treated with bacEC for indicated lengths of time. Lysates were probed for phospho-Akt (pAkt) and GFP-myc. The latter was co-transfected with receptor constructs to account for variation in transfection efficiency that could result in changes to receptor protein levels. (A) Representative western blot of lysates from ErbB2-transfected cells (in triplicate). (B,C) Quantification of pAkt from cell lysates. GFP-myc was used for normalization. (B) ErbB2-expressing cells had significantly higher pAkt levels after exposure to bacEC (p = 0.018–0.036). The p values are p = 0.02, p = 0.018 and p = 0.036 for the 1, 5, and 60 minute time points. (C) Addition of 600 nM mubritinib to ErbB2-transfected cells abrogated the effect bacEC on Akt phosphorylation. Results are representative of three independent experiments. Means were calculated from triplicates and shown as bars. Data points are shown as stars. One-tailed, Student’s t-tests were performed to determine statistical significance. * p<0.05.
https://doi.org/10.1371/journal.pone.0188963.g004
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Fig 5. ErbB2-ErbB3 heterodimers bind PI3K p85 in the presence of recombinant EC1-3 (bacEC).
(A) Quantitative real-time PCR analysis of ErbB receptors from dissected CNC and stage 16 embryos. The relative gene expression in the CNC is compared to that of whole embryos at the same stage. ErbB2, 3, and 4 are enriched in the CNC compared to whole embryos. Slug was used as a positive control for CNC enrichment. GAPDH was used for normalization. (B) Western blot of co-immunoprecipitation experiments with ErbB3-HA and p85 in the presence or absence of bacEC and ErbB2-flag. Hek293T cells were transfected with ErbB3-HA alone or together with ErbB2-flag, serum starved, and treated with 10 ng/mL bacEC for 5 minutes before being lysed for co-immunoprecipitation experiments. ErbB3-HA modestly binds p85 when transfected alone. Co-transfection of ErbB3-HA with ErbB2-flag noticeably increases this interaction. Association of p85 with ErbB3-HA is further elevated in the presence of bacEC. (C) Co-precipitation of p85 with ErbB3-HA and ErbB2-flag significantly increases (p = 0.03) in the presence of bacEC. Immunoprecipitated ErbB3-HA was used for normalization. Results are representative of three independent experiments.
https://doi.org/10.1371/journal.pone.0188963.g005
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Fig 6. Knockdown of ErbB2 decreases phosphorylation of Akt and CNC migration.
(A) Western blot of non-injected (NI) and injected embryo extracts probed for flag. Single-cell embryos were injected with 800 pg DN-ErbB2-Flag mRNA with or without 25 ng of ErbB2 morpholino (MO ErbB2). Translation of DN-ErbB2-flag was hindered in embryos co-injected with MO ErbB2. GAPDH is shown as a loading control. One-embryo equivalents are loaded per lane. (B) Western blot of CNC extracts probed for phospho-Akt, total Akt and GAPDH. Eight-cell embryos were either injected with 200 pg RFP mRNA alone (N = 4, n = 32) or with 3.1 ng MO Erb2 (N = 3, n = 43) into CNC progenitor cells. CNC cells were then dissected from embryos once they reached stage 17–18. Injection of MO ErbB2 into CNC cells precursors dramatically reduced phosphorylation of Akt without altering the levels of control proteins. Ten CNC explants are loaded in each lane. (C-E) Early tailbud stage embryos labeled for Twist and Sox10 CNC markers using in situ hybridization. Embryos were injected as in part (B) along with a set injected with 300 pg rat ErbB2-flag and 1.6 ng MO ErbB2 (E; N = 2, n = 20). Embryos injected with RFP mRNA and MO ErbB2 showed defects in CNC migration (D) compared to RFP-injected controls (C). Migration was partially rescued by co-injecting the morpholino with rat ErbB2-flag mRNA (E). (F) CNC migration was measured in each branchial arch and normalized to head size from embryos in part (C-E). Co-injection of MO ErbB2 with RFP mRNA significantly reduced CNC migration in the mandibular (M), hyoid (H), 3rd and 4th branchial arches. Co-injection of MO ErbB2 with rat ErbB2 mRNA partially rescued migration. Error bars are one standard deviation to the mean. One-tailed, Student’s t-tests were performed to determine statistical significance. ** p<0.01, *** p<0.001. N, number of experiments; n, number of embryos.
https://doi.org/10.1371/journal.pone.0188963.g006
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Fig 7. Mubritinib and canertinib perturb CNC migration ex vivo.
(A-C) Lateral views of tailbud stage X.laevis embryos after in situ hybridization with Sox10 and Twist to visualize CNC positioning. Anterior is to the left, dorsal is up. Embryos treated with 40 μM of the ErbB2 inhibitor, mubritinib (N = 4, n = 72), or 25 μM of the pan-ErbB inhibitor, canertinib (N = 4, n = 68), show no difference in CNC migration compared to DMSO controls (N = 6, n = 100; N = 4, n = 69, respectively). (D-E) The distance of migration within each branchial arch was measured and normalized to the head size. CNC migration as observed in the mandibular (M), hyoid (H), 3rd and 4th branchial arches. (F-U) Time-lapse images of CNC explants migrating on fibronectin substrate and treated with 0.5% DMSO (N = 8, n = 24), 6 μM mubritinib (N = 4, n = 12), 10 μM canertinib (N = 4, n = 12), and 20 μM LY294002 (N = 3, n = 9) for indicated lengths of time. (V) Fold change in explant surface area over time. Areas were normalized to measurements calculated at initial time points (t = 0). All inhibitors significantly decreased CNC migration ex vivo. Error bars represent the 95% confidence interval. One-tailed, Student’s t-tests were performed to determine statistical significance. ** p<0.01, ***p<0.001. N, number of experiments; n, number of embryos or explants.
https://doi.org/10.1371/journal.pone.0188963.g007
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Fig 8. EC1-3 stimulates Akt and CNC migration.
During migration of cranial neural crest (CNC) cells, ADAM13 (blue) cleaves the extracellular domain of cadherin-11 (red) at the cell surface to release a soluble fragment (EC1-3) capable of promoting CNC migration. EC1-3 can induce cell migration by binding to ErbB receptors: EGFR/ErbB1 (teal), ErbB2 (yellow), ErbB3 (green) and ErbB4 (purple). In particular, binding of EC1-3 with ErbB2-ErbB3 dimers increases docking of the regulatory (p85) subunits of PI3K. Recruitment of PI3K to the cell membrane ultimately leads to the phosphorylation of Akt, which is important for CNC migration. Because inhibition of ErbB2 is less detrimental to cell migration than inhibition of all ErbB receptors, it is possible that EC1-3 utilizes Akt-independent pathways downstream of ErbB dimers that do not include ErbB2. Whether EC1-3 signaling first requires receptors to bind their cognate ligand still needs to be investigated.
https://doi.org/10.1371/journal.pone.0188963.g008
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Recombinant EC1-3 increases the persistence of migrating CNC cells.
(A) Persistence of cell migration was calculated by adding the distances cells traveled between time points (d) and dividing it by their net displacement (D). (B,C) Quantification of persistence and velocity for untreated and recombinant EC1-3 (bacEC) treated CNC cells. CNC explants were dissected from neurula stage embryos and incubated on fibronectin substrate for at least one hour before being treated with 10 ng/mL bacEC and monitored using time-lapse microscopy. Analysis of cell movement revealed that CNC cells treated with bacEC (N = 3, n = 6, c = 103) have a significantly higher persistence (p = 0.013) than untreated cells (N = 3, n = 6, c = 99). No statistical difference was observed in CNC cell velocity. Scatter plot shows median and interquartile range. One-tailed, Student’s t-tests were performed to determine statistical significance. * p<0.05. N, number of experiments; n, number of explants; c, number of cells.
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Co-expression of EC1-3 with ErbB2 increases Akt phosphorylation in vitro.
Hek293T cells were transfected with a nuclear-localized RFP (nucCherry), EC1-3-myc, ErbB2, or a combination of EC1-3-myc and ErbB2. Cells were then serum starved for at least 18 hours and lysed for western blotting. GAPDH was used as a loading control. Results are representative of three independent experiments.
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Mubritinib and canertinib do not inhibit Akt phosphorylation in embryos.
Western blot of embryos treated with 30 μM LY294002, 40 μM mubritinib, 25 μM canertinib or DMSO from stage 18 to stage 25–26. Of the inhibitors, only LY294002 was able to decrease phosphorylation of Akt (pAkt) compared to DMSO controls. GAPDH was used as a loading control. One-embryo equivalents were loaded per lane.
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Mubritinib inhibits Akt phosphorylation in Xenopus XTC cells.
Western blot of XTC cells transfected with X.laevis ErbB2 and GFP-myc, serum starved for 18 hours, and treated with either DMSO or 600 nM mubritinib for one hour. Mubritinib dramatically decreased phosphorylation in one of two Akt isoforms (pAkt) compared to DMSO-treated controls. GFP-myc was co-transfected with ErbB2 to account for variation in transfection efficiency that could result in changes to receptor protein levels.
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