Toro-Tapia G et al. (2018), The Ric-8A/Gα13/FAK signalling cascade controls...

XB-ART-55502

Development 2018 Nov 21;14522:. doi: 10.1242/dev.164269.

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The Ric-8A/Gα13/FAK signalling cascade controls focal adhesion formation during neural crest cell migration in Xenopus.

Toro-Tapia G , Villaseca S , Beyer A , Roycroft A , Marcellini S , Mayor R , Torrejón M .

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Ric-8A is a pleiotropic guanine nucleotide exchange factor involved in the activation of various heterotrimeric G-protein pathways during adulthood and early development. Here, we sought to determine the downstream effectors of Ric-8A during the migration of the vertebrate cranial neural crest (NC) cells. We show that the Gα13 knockdown phenocopies the Ric-8A morphant condition, causing actin cytoskeleton alteration, protrusion instability, and a strong reduction in the number and dynamics of focal adhesions. In addition, the overexpression of Gα13 is sufficient to rescue Ric-8A-depleted cells. Ric-8A and Gα13 physically interact and colocalize in protrusions of the cells leading edge. The focal adhesion kinase FAK colocalizes and interacts with the endogenous Gα13, and a constitutively active form of Src efficiently rescues the Gα13 morphant phenotype in NC cells. We propose that Ric-8A-mediated Gα13 signalling is required for proper cranial NC cell migration by regulating focal adhesion dynamics and protrusion formation.

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Species referenced: Xenopus
Genes referenced: cdh1 cdh2 fn1 gna13 itgb1 itk myc ptk2 pxn ric8a slc5a5 snai2 twist1
GO keywords: neural crest cell migration [+]
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	Fig. 1. GÎ±13 is required for cranial NC cell migration in vivo and acts downstream of Ric-8A. (A) X. tropicalis embryos were unilaterally injected with the indicated morpholinos, either alone or in combination with a Ric-8A or a GÎ±13 rescue mRNA, before being in situ hybridized for the NC marker snail2 at stages 23-24. For each specific experimental condition, the pictures show the non-injected side (Nis) and the injected side (Is) of the same embryo. White and black arrowheads show snail2-positive cells whose migration is severely inhibited and normal migration, respectively. n=40-100. (B) Quantification of the normal (white), mild (grey) and severe (black) phenotypes shown in A. The number of examined embryos is indicated at the top of each bar. (C) Lineage-tracing experiment showing that cranial NC explanted from a GÎ±13 morphant embryo to a normal embryo fails to migrate when transplanted into a wild-type host embryo at stage 23. GFP was used as a lineage tracer. Scale bars: 600 Î¼m.
	Fig. 2. Ric-8A and GÎ±13 downregulation inhibits cranial NC cell migration in vitro and GÎ±13 subunit rescues the Ric-8A morphant effect. (A) Cranial NC explants from injected X. laevis embryos were cultured on fibronectin-coated dishes, and each cellular condition was monitored over a 10â€…h period by time-lapse microscopy. (B) Delaunay triangulation was performed to quantify the cell dispersion. (C) Cell dispersion was quantified by the ratio between the final and the first frame and plotted at 10â€…h. Data are meanÂ±s.e.m., ***P<0.001; n=30. The images are representative of three independent experiments analysing 10 explants per condition. Scale bars: 100 Î¼m.
	Fig. 3. Ric-8A and GÎ±13 are localized mainly at the leading edge of cranial NC cells. Immunofluorescence assays were performed in X. tropicalis cranial NC explants using anti-GÎ±13 and anti-myc antibodies. GFP mRNA co-injection allowed membrane visualization. (A) Myc-Ric-8A (red) and endogenous GÎ±13 (pink) are localized in the lamellipodium (white arrowhead) and to a lesser extent in the cytoplasmic area. (B) The GÎ±13 localization is impaired in Ric-8AMO-treated cells (arrowheads). (C) The GÎ±13MO downregulation effect was demonstrated by immunofluorescence using an anti-GÎ±13 antibody (red). (D) GÎ±13 expression was evaluated in GÎ±13MO- or Ric-8AMO-treated embryos by western blot analysis, showing that GÎ±13 expression is similar in Ric-8AMO and control embryos. (E) Co-immunoprecipitation assays were performed using anti-GÎ±13 or anti-his (GÎ±13 tag) and anti-myc (Ric-8A tag) antibodies, demonstrating that both proteins interact in HEK293 (top) and Xenopus embryos (bottom). The images are representative of five independent experiments analysed. Scale bars: 10 Î¼m in A,B; 25 Î¼m in C.
	Fig. 4. Ric-8A and GÎ±13 cranial NC morphant cells present abnormal cortical actin cytoskeleton organization and instable cell protrusions. (A) Phalloidin staining was performed over different cellular conditions in X. tropicalis. GÎ±13MO- and Ric-8AMO-treated cells have an altered cortical actin cytoskeleton organization, and smaller and fewer lamellipodia than do control cells. Co-injection of Ric-8AMO and GÎ±13 mRNA rescued the abnormal phenotype. White arrowheads indicate normal F-actin. Red arrowheads indicate altered F-actin. (B) Time-lapse microscopy was performed every 15âs for 30âmin in X. laevis cranial NC explants from embryos treated with morpholino and lifeAct-Cherry. The panels show the protrusion stability in a representative assay. Green arrows indicate protrusions in growth. Red arrows indicate collapsed protrusions. The top graph in B shows the protrusion lifetime for each condition. The bottom graph in B shows de protrusion numbers for each condition, new protrusions formed and existing protrusions retracted per minute. GÎ±13 mRNA was able to rescue the Ric-8AMO phenotype. Data are meanÂ±s.d., *P<0.001, P<0.01, n=20. The images are representative of five independent experiments analysing four explants per each condition. Scale bars: 25 Î¼m in A; 10 Î¼m in B.
	Fig. 5. Ric-8A and GÎ±13 morphant cells have defects in focal adhesions. Embryos were injected with nuclear RFP and GÎ±13MO or Ric-8AMO or a Ric-8AMO and GÎ±13 mRNA mixture, and the cranial NC cells were extracted and cultured onto fibronectin. (A) Immunostaining against phospho-paxillin (green) and phalloidin (red) was performed in X. laevis cranial NC. Ric-8AMO and GÎ±13MO cells showed a decrease in the number and area of focal adhesions compared with control cells. GÎ±13 mRNA was able to rescue the Ric-8A morphant phenotype. (B) The graphs show the number of focal adhesions per condition normalized to the control, and the total area of focal adhesion per cell in each condition normalized to control. (C) Adhesion assay performed on GÎ±13 morphant cells to evaluate the attachment of the explants over fibronectin. GÎ±13 morphant explants do not attach properly to the extracellular matrix in comparison with the control explants, suggesting a severe defect in focal adhesions. MnCl2 rescues the GÎ±13MO phenotype, increasing the attachment in a dose-dependent fashion. Data are meanÂ±s.e.m., P<0.1, P<0.01,***P<0.001, n=40. The images are representative of four independent experiments analysing 10 explants per each condition. Scale bars: 25 Î¼m.
	Fig. 6. GÎ±13 morphant cells have defects in focal adhesion dynamics. (A) Control cells and GÎ±13MO-treated cells were analysed by time-lapse TIRF microscopy using FAK-GFP and lifeActCherry as a markers. Insets show the boxed areas in more detail (red, collapsed protrusions; green, growing protrusions). (B) Graphs showing that GÎ±13MO cells have decreased stability of focal adhesions, which are also shorter and smaller than controls. Data are meanÂ±s.d., ***P<0.001, n=24. (C) Co-immunoprecipitation was performed using FAK-GFP and myc-GÎ±13 in HEK293T cells, illustrating that FAK and GÎ±13 interact. (D) Immunostaining assays demonstrate that FAK-GFP (green) colocalizes partially with endogenous GÎ±13 (red) mainly at the leading edge of X. laevis cranial NC cells. The graph in D shows colocalization using the intensity from red and green channels. The images are representative of four independent experiments analysing seven explants per condition. Scale bars: 10 Î¼m in A; 50 Î¼m in D.
	Fig. 7. Constitutively active Src rescues the GÎ±13 morphant phenotype. (A) In situ hybridization was performed in stage 23-24 embryos that were previously injected with GÎ±13MO, GÎ±13MO+Src Y527F mRNA and Src Y527F mRNA alone to analyse cranial NC migration in vivo using snail2 and twist as NC markers. Quantification of each phenotype as a percentage of total embryos per condition was determined (graph). n=65-80. (B) Immunostaining against phospho-paxillin (green) was performed in explants with the conditions described above. Staining with phalloidin (red) was used. Arrowheads indicate focal adhesion at the protrusions. (C) The graphs show number of focal adhesions and total area of focal adhesion per cell in each condition normalized to control. Constitutively active Src Y527F mRNA rescues the GÎ±13 morphant phenotype, both in vivo and in vitro, suggesting that it participates in the same GÎ±13 signalling pathway during cranial NC migration. Data are meanÂ±s.e.m., P<0.1, **P<0.001, n=15. The images are representative of three independent experiments analysing five explants per condition. Scale bars: 600 Î¼m in A; 10 Î¼m in B.
	Fig. 8. Regulation of cranial NC cell migration through the regulation of adhesion properties by Ric-8A and GÎ±13 signalling. Ric-8A/GÎ±13 regulates focal adhesion proteins, stabilizing the protrusions at the leading edge during migration. The scheme shows a representation of the Ric-8A/GÎ±13 pathway during cranial NC migration. First, GÎ±13 is activated by a G-protein-coupled receptor bound to its ligand. Once the signalling is activated, the Ric-8A GEF activity maintains the monomeric GÎ± protein in an active state and dissociated from the GÎ²Î³ dimer, thereby amplifying the signal. In addition, Ric-8A coordinates GÎ±13 translocation at the leading edge of the cell, where GÎ±13 interacts with the focal adhesion complex, and regulates focal adhesion formation and/or turnover through FAK and Src as part of a protein complex with integrin (see Discussion).
	Figure S1: (a) In situ hybridization confirms the presence of GÎ±13 and Ric8A transcripts in migrating cranial NC cells at stage 26, as previously reported in Maldonado-Agurto et al. (2011) and Fuentealba et al. (2016). (b) GÎ±13 gain and loss of function controls. Left panel: Anti-Myc Western blot in embryos injected with a myc-GÎ±13 mRNA. Right panel: Anti-GÎ±13 Western blot showing that morphant embryos display diminished levels of endogenous GÎ±13 protein. GAPDH and Î±-tubulin were used as loading controls. (c) The rescue assay of the Ric-8A morpholino was performed using a myc-Ric-8A mRNA, as previously reported in Fuentealba et al.(2013). Snail2 was used as a migratory cranial NC marker at stage 23 (left panel). Snail2 expression is unaffected in stage 16 GÎ±13 morphant embryos (right panel). Legend: Is, injected side; Nis: non-injected side. All experiments shown here were performed in X. tropicalis.
	Figure S2: Cranial NC morphant cells for Ric-8A and GÎ±13 undergo EMT. (a) The panels show N-cadherin and E-cadherin expression in green and actin labeled with phalloidin in red at migratory stages of control, Ric-8A and GÎ±13 cranial NC morphant explants. E-cadherin expression at pre-migratory stages in control explant is also shown. (b) Intensity of N- and E- cadherin in green at cell-cell contact per each condition in migratory stages. Actin in red was labeled with phalloidin. A.u. arbitrary units
	Figure S3: (a) RT-PCR from Ric-8A morphant embryos shows no change at the GÎ±13 transcript. (b) Repetitions of co-immunoprecipitation assay from Figure 3e, was performed using anti-his (GÎ±13 tag) and anti-myc (Ric-8A tag) antibodies, confirming that both proteins interact. (c) Co- immunoprecipitation controls from Figure 3e showing that when each protein is expressed alone (without its partner) no pull-down occurs.
	Figure S4: Intensity of cortical actin labeled with phalloidin (red) at the cell edge in control, GÎ±13 and Ric-8A morphant and rescue (Ric-8AMO + GÎ±13 mRNA) conditions. A.u. : arbitrary units.
	Figure S5: (a) Visualization of b-integrin (immunofluorescence, green) and actin (fluorescent phalloidin, red) were performed in X. tropicalis cranial NC. Ric-8AMO and GÎ±13MO cells showed a decreased number of focal adhesions compared with control cells. Nuclear GFP was used as a lineage tracer. (b) 3D cell reconstruction was performed from an immunostaining of control and GÎ±13MO-treated cranial NC cells using phospho-paxillin (green) and phalloidin (red). Morphant cells exhibit an altered actin cytoskeleton and display abnormal focal adhesion. (c) Representative images from an adhesion assay performed on control and GÎ±13 morphant cells to evaluate the attachment of the explants over fibronectin. Phospho-paxillin (green) shows that treatment with MnCl2 rescues the GÎ±13MO phenotype, increasing the number of focal adhesion in a dose- dependent fashion.
	Figure S6: (a) Control cells and Ric-8AMO-treated cells were analyzed by time-lapse microscopy using FAK-GFP as a marker. The inset in each panel shows a zoom of the red dotted boxes, which indicate stable protrusion and focal adhesions in controls cells and collapsed protrusions and focal adhesions in Ric-8AMO cells (60Ã— magnification). (b) Graphs showing that Ric-8AMO cells exhibit a mildly decreased stability of focal adhesions, which are significantly shorter and smaller than controls. Error bar: Standard deviation, *** p<0.001. (c) Repetitions of co-immunoprecipitation from Figure 6c, was performed using FAK-GFP, Myc-Ric-8A and His-GÎ±13 in HEK293T cells, illustrating that FAK, GÎ±13 and Ric-8A interact. Hence Ric-8A, GÎ±13 and FAK belong to the same protein complex.
	ric8a (RIC8 guanine nucleotide exchange factor A) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.

References [+] :

Alfandari, Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin. 2003, Pubmed, Xenbase