Fig 2: Interactions between the members of the CMC guide mitotic spindle orientation.(a) Optical sections at the plane of each spindle pole and side views of FAK nulls, or FAK nulls transfected with the indicated constructs (signal shown in green) stained for β-tubulin and actin. (b) Scatter plot of the substrate to spindle angles of metaphases cells described in a. Mean±s.e.m.: FAK−/− 19.04±2.028°, n=50; FAK−/− +WT FAK 6.507±0.8685°, n=52; FAK−/− +FAK Y397F 12.14±1.263°, n=61; FAK−/− +FAK P712/715 A 18.63±2.501°, n=35; P values calculated by Mann–Whitney test; n, number of metaphase cells, two independent experiments (Kruskal–Wallis: FAK−/− P=0.5405, ns, +WT FAK P=0.2432, ns, +FAK Y397F P=0.1732, ns, +FAK P712/715 A P=0.0.8886, ns). (c) Optical sections at the plane of each spindle pole and side views of Cas nulls, Cas reconstituted cells or Cas nulls transfected with the indicated constructs (shown in green) stained for β-tubulin and actin. (d) Scatter plot of the substrate to spindle angles of metaphases cells described in c. Mean±s.e.m.: Cas−/− 18.97±2.102°, n=63; Cas+/+ 3.825±0.5526°, n=57; Cas−/−+WT Cas 5.805±1.062°, n=24; Cas−/−+Cas ΔSH3 18.24±3.173°, n=22; Cas−/−+Cas 15 F 20.09±3.660°, n=17; Cas−/−+Cas mPR 6.703±1.882°, n=23; P values calculated by Mann–Whitney test; n, number of metaphase cells, two independent experiments (Kruskal–Wallis: Cas−/− P=0.6765, ns, Cas+/+ P=0.1978, ns, +WT Cas P=3967, ns, +Cas ΔSH3 P=0.7745, ns, +Cas 15 F P=0.2913, ns, +Cas mPR P=0.1564, ns). Scale bar, 5 μm (a,c).

Fig 3: An integrin β1-based cortical mechanosensory complex is formed at the lateral cortex of mitotic cells.(a) Optical sections, colour intensity-coded images and side views of representative metaphase HeLa cells co-stained for active β1 and phosphorylated active forms of FAK, Cas or Src. The white arrowheads indicate the polarized cortical crescent of the phosphorylated forms of the above proteins. The white line represents the metaphase plate and the dashed line shows the basal surface. (b) Optical sections at the spindle plane of metaphase control HeLa cells or cells treated with the P4C10 antibody. All cells were imaged under the same conditions. Cells were co-stained for β-tubulin, active β1 (9EG7) and phosphorylated Cas or Src. The plots show the average cortical intensity of P-Cas and P-Src in control and P4C10-treated cells. mean±s.e.m.: P-Cas control 9.106±0.7618, n=33; P-Cas P4C10 3.691±0.3323, n=34; P-Src control 10.30±1.155, n=35; P-Src P4C10 3.916±0.3537, n=30. Analysed by Mann–Whitney test; n, number of metaphase cells, two independent experiments (Kruskal–Wallis: for P-Cas Control P=0.0715, ns, P4C10 P=0.2177, ns; for P-Src Control P=0.0657, ns, P4C10 P=0.2510, ns). Scale bar, 10 μm (a), 5 μm (b).

Fig 4: The FERM domain is necessary and sufficient for membrane localization of FAK at integrin-free regions.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 µm.

Fig 5: 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 µm.