Petridou NI , Stylianou P , Christodoulou N , Rhoads D , Guan JL , Skourides PA .

	Figure 1. FAK expression and phosphorylation during development. (A) Western Blots from extracts of equal numbers of embryos probed with a monoclonal antibody against the C-terminus of FAK or polyclonal antibodies against the phosphorylated tyrosine residues indicated. FAK is phosphorylated on all three residues both before and after gastrulation. The intensity values from the densitometry analysis of the western blots were normalized against total FAK amount. (B) Blastula (1st column), early gastrula (2nd column) and late gastrula embryos (3rd column) stained with P-Y397, P-Y576, P-Y861 and P-Y31paxillin antibodies as indicated. Phosphorylated FAK and paxillin can be detected on the plasma membrane from early blastula stages including the apical region of superficial blastomeres. During gastrulation elevated levels of phosphorylation are detected in the highly morphogenetic mesodermal tissues (white arrowheads). Scale bar: 400 �m. doi:10.1371/journal.pone.0042577.g001
	Figure 2. FAK is heavily phosphorylated in mesodermal tissues and integrin-free regions of cells. show more (A) Intensity color coded confocal section of the dorsal lip region from a whole mount immunostained gastrula stage embryo using a P-Y576 FAK antibody. Mesodermal cells (white arrow) display much higher levels of phospho-FAK than endodermal cells lining the forming archenteron (white arrowheads) and the endodermal cells of the blastopore (red arrow). (B) Same as A but showing the anterior mesendoderm and the animal cap from a whole mount immunostained gastrula stage embryo. The superficial cells of the animal cap (white arrowhead) show lower levels of phospho-FAK signal compared to deep cells (white arrow) and mesendodermal cells (red arrow). (C) High magnification color coded narrow optical section of superficial cells of the animal cap reveals that the apical surface of these cells display similar levels of phospho-FAK compared to the basolateral region while (D) the apical region of the deep cells of the animal cap facing the fibronectin ECM display significantly elevated levels of phospho-FAK compared to the basolateral region. In addition, in the deep cells of the ectoderm labeling of phospho-FAK in the basolateral region is relatively uniform but the apical region displays distinct foci of higher signal intensity (E�G) Confocal optical sections from whole mount immunostained embryos using integrin-β1 (green) and P-Y397 FAK antibodies (red). Integrin-β1 and P-Y397 FAK colocalize on the plasma membrane at cell�cell boundaries (white arrowhead). However phosphorylated FAK is also present on the apical region of the outermost cells of the embryo where integrin-β1 is absent (red arrow). (H�J) Same embryo as above but the cells facing the blastocoel cavity are imaged. Integrin-β1 and P-Y397 FAK colocalize on the apical surface of the cells facing the blastocoel (white arrows). Scale bars: (A) 100 �m, (B) 50 �m, (C, D) 10 �m, (E) 30 �m, (H) 20 �m. doi:10.1371/journal.pone.0042577.g002
	Figure 3. FRNK does not act as a dominant negative in early Xenopus embryos. show more (A�D) Optical sections of whole mount immunostained embryos injected with 1 ng GFP-FRNK at the two dorsal blastomeres at the four-cell stage. Embryos were stained with anti-GFP (A) and anti-P-Y397 (B). C is the merged image and D an intensity color coded image of the anti-P-Y397 signal. FRNK injected cells are indicated with red stars and control cells with white stars. FRNK expression fails to reduce the phosphorylation levels of endogenous FAK on tyrosine 397. (E�H) Same as A�D, but the embryos were stained with anti-GFP (E) and anti-P-Y576 (F). FRNK expressing cells display similar levels of phosphorylation on tyrosine 576 as neighboring control cells. (I�L) Confocal images of A6 Xenopus cells transfected with GFP-FRNK. Cells were stained with anti-GFP (I) and anti-P-Y397 (J). K is the merged image and L an intensity color coded image of the anti-P-Y397 signal. FRNK expression leads to reduction of the phosphorylation levels of FAK on tyrosine 397 at the focal adhesions. (M�P) Same as I�L but the cells were stained with anti-GFP (M) and anti-P-Y576 (N) antibodies. FRNK expression leads to downregulation of the endogenous phosphorylation levels of FAK on tyrosine 576 at the focal adhesions. (Q) Western blot analysis of control and injected gastrula stage embryos with 1 ng FRNK at the animal pole of both blastomeres of two cell stage embryos. FRNK expression fails to reduce endogenous FAK phosphorylation on tyrosine 397. FRNK expression was verified using a FAK antibody raised against the C-terminus of the protein. (R�S) Localization of P-Y397 FAK (R) and FRNK (S) in animal pole cells of stage 10 Xenopus embryos. P-Y397 FAK shows strong membrane localization while FRNK is primarily cytoplasmic in these cells. Scale bars: (A) 40 �m, (E) 30 �m, (I) 20 �m, (M) 20 �m, (R�S) 40 �m.
	Figure 4. The FERM domain is necessary and sufficient for membrane localization of FAK at integrin-free regions. show more Confocal images and intensity profiles of the indicated constructs after whole mount immunostaining. The first column are top views of superficial cells of the animal cap in intact embryos and the second column are views from sagittally sectioned embryos that reveal the localization of each construct on the apical surface of superficial cells. Apical region of superficial blastomeres is to the right. (A) The FERM domain shows strong plasma membrane localization in the top view and is strongly localized to the apical surface. (B) Endogenous phosphorylated FAK shows very strong plasma membrane localization in the top view and is localized on the basolateral and apical surface of the cell. (C) Full length FAK with the point mutation K38A exhibits strong membrane localization. (D) Deletion of the FERM domain (HA-Δ375 FAK construct) abolishes the plasma membrane localization of FAK. Scale bars: 25 �m. doi:10.1371/journal.pone.0042577.g004
	Figure 5. The FERM domain leads to activation of endogenous FAK in a tyrosine 397 dependent manner. show more HA-FERM and HA-FERM Y397F injected embryos in one blastomere at the animal pole of two cell stage embryos were processed for whole mount immunostaining using an HA antibody (green) to reveal expressing cells and the respective phospho-specific antibodies (red) as indicated. In each case individual signals for each secondary are shown in addition to a merged image and finally an intensity color coded image of the respective phospho-specific antibody signal. HA-FERM and HA-FERM Y397F injected cells are indicated with red stars and un-injected cells with white stars. (A�D) Levels of phosphorylated tyrosine 576 are elevated in HA-FERM overexpressing cells compared to controls. (E�H) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 576. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 576 with neighboring control cells. (I�L) Levels of phosphorylated tyrosine 861 are elevated in HA-FERM expressing cells compared to controls. (M�P) Overexpression of HA-FERM Y397F has no effect on the endogenous levels of phosphorylated tyrosine 861. HA-FERM Y397F expressing cells have the same levels of phosphorylated endogenous FAK on tyrosine 861 with neighboring control cells. (Q) Total lysates from HA-FERM injected gastrula stage embryos contain comparable levels of endogenous FAK as un-injected controls but elevated levels of phosphorylated FAK on tyrosines 397, 576 and 861. Blotting using the anti-P-Y397 antibody shows that the exogenously expressed FERM is heavily in trans phosphorylated on tyrosine 397 (2nd row). The intensity values from the densitometry analysis were normalized against total FAK and present the average increase in phosphorylation from three independent experiments (R�U) Confocal images of A6 Xenopus cells transfected with HA-FERM. Cells were stained with anti-HA (R) and anti-P-Y576 (S). T is the merged image and U an intensity color coded image of the anti-P-Y576 signal. Transfected cells are shown with red stars and controls with white stars. HA-FERM transfected cells show reduced levels of tyrosine 576 phosphorylation suggesting that FERM expression blocks FAK activation in these cells. Scale bars: (A) 40 �m, (E) 30 �m, (I) 20 �m, (M) 50 �m, (R) 20 �m. doi:10.1371/journal.pone.0042577.g005
	Figure 6. The FERM domain activates endogenous FAK leading to increased phosphorylation of FAK/Src targets. (A�D) Embryos injected with HA-FERM mRNA in two blastomeres, at the animal pole, at the four cell stage were processed for immunofluorescence using anti-HA (green) and anti-P-Y31 paxillin (red) antibodies. C is the merged image and D is an intensity color coded image. FERM expressing cells display elevated levels of phosphorylated paxillin (red stars) when compared with un-injected neighbouring cells (white stars). (E�H) Same as (A�D) but comparing phosphorylation levels of p130Cas on tyrosine 762 between FERM expressing and control cells. FERM expressing cells show elevated levels of phosphorylated p130Cas (red stars), when compared with un-injected cells (white stars). (I�L) Same as (A�D) but comparing levels of phosphorylated Akt on serine 473 between FERM expressing and control cells. Levels of phosphorylated Akt are comparable in FERM expressing cells to those of control neighboring cells. Scale bars: 20 �m. doi:10.1371/journal.pone.0042577.g006

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