DEV01462 Supplementary Material

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Files in this Data Supplement:

• Supplemental Tables (PDF)
• Supplemental Data (Adobe PDF) - Additional materials and methods
• Supplemental Figure 1 -

  Fig. S1. Alignment of the conserved Src binding tyrosine in FAK family proteins. Alignment of the amino acid sequences surrounding the conserved Src docking tyrosine in the N-terminal of FAK family proteins. Sequences shown are for HsFAK (Y³⁹⁷), DmFak56 (Y⁴³⁰) and HsPyk2 (Y⁴⁰²). The YAEI consensus sequence is shown in red, and identical residues are marked with an asterisk.

• Supplemental Figure 2 -

  Fig. S2. Ectopically expressed DFak56-GFP is efficiently phosphorylated in vivo. Overexpression of Fak56^GFP fusion protein using the Engrailed-GAL4 driver, results in a segmental expression pattern in the epidermis of the developing embryo. Overexpressed Fak56^GFP protein (A,B), is efficiently tyrosine phosphorylated as detected with anti-phospho-FAK^Y397 antibodies (A,C), and with anti-phosphotyrosine antibodies (A,D).

• Supplemental Figure 3 -

  Fig. S3. Fak56 protein is not expressed in Df(2R)ED3716^CG2 mutant animals. (A) Immunoblot analysis of (1) wild-type, (2) Fak56^CG1, (3) Fak56^CG1/Df(2R)ED3716^CG2, (4) Df(2R)ED3716^CG2/+ and (5) Fak56^CG1;FAK^{genomicrescue} Drosophila lysates using Fak56 specific antibodies. Fak56 protein is absent in Fak56^CG1 and Fak56^CG1/Df(2R)ED3716^CG2 mutant animals, but Fak56 protein expression is restored with the introduction of a Fak56 genomic rescue transgene. (B) The same blot was stripped and reprobed with anti-a-Tubulin antibodies to ensure equal loading.

• Supplemental Figure 4 -

  Fig. S4. Fak56 is not required for border cell migration during Drosophila oogenesis. Fak56 mutant oocytes were stained with phalloidin (A-D and A¢-D¢) in red, visualising actin filaments and DAPI in blue (A¢-D¢), labelling DNA in the nuclei. The migrating border cells are indicated by arrowheads, anterior is towards the left and dorsal is upwards.

• Supplemental Figure 5 -

  Fig. S5. Fak56 is not required for the migration of the Drosophila embryonic germ cells. Wild-type (A,C,E,G,I) and Fak56 mutant (B,D,F,H,J) embryos were stained with anti-Vasa antibodies (green) to visualise germ cells and with anti-ALK antibodies (red) for staging of embryos and visualisation of the visceral mesoderm. The primordial germ cells are formed at the posterior pole of the Drosophila embryo, underlying somatic cells that give rise to the posterior midgut (PMG) anlage (A,B). By stage 10, at gastrulation, germ cells adhering to the PMG anlage are carried inside the embryo whereupon they start to actively transmigrate through the midgut epithelium at stage 11 (C,D), moving from its apical to basal side. Once the germ cells have passed through the PMG, they continue to migrate along the midgut (E,F) to the mesoderm where they associate with three lateral clusters of gonadal mesoderm cells (somatic gonadal precursors) (G,H) and subsequently merge and coalesce into a gonad, by stage 14 (I,J). Neither the formation of Drosophila germ cells or their migration to the gonads appears to require Fak56 as no differences between wild-type and Fak56 mutant embryos can be detected.

• Supplemental Figure 6 -

  Fig. S6. Fak56 is not required for migration during the embryonic development of the Drosophila tracheal system. Wild-type embryos (A,C,E) and Fak56 mutant embryos (B,D,F) were stained with 2A12 antibodies, visualising the Drosophila tracheal system. The migration of tracheal cells does not appear to be dependent on Fak56 during Drosophila embryogenesis.

• Supplemental Figure 7 -

  Fig. S7. Overexpression of Fak56 perturbs the formation of focal-adhesion-like structures on the basal surface of the wing imaginal disc epithelium. (A,B) Expression of Fak56^GFP in one half of the wing imaginal disc (B, in the posterior compartment, with engrailed-GAL4) perturbed the shape of the wing compared with wild type (A). Green, Fak56^GFP fusion protein; red, integrin bPS subunit; blue, talin. In B, the boxes show the approximate regions of C and D (from separate samples). (C) An enlarged region of a wing disc, stained as in B, shows that overexpression of FAK caused the focal adhesions to become rounder and more diffuse, but that they still formed. (D) Further enlargement of a region of a disc with anti-phospho-FAK^Y397 in red, talin in blue and Fak56^GFP in green. As in C, overexpressed Fak56 changes the appearance of the focal adhesions, which contain phosphorylated Fak56. Scale bars: 10 mm.