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First published online 3 November 2004
doi: 10.1242/dev.01462

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Focal adhesion kinase is not required for integrin function or viability in Drosophila

Caroline Grabbe^1,*, Christos G. Zervas^2,*,, Tony Hunter³, Nicholas H. Brown² and Ruth H. Palmer^1,

¹ Umeå Center for Molecular Pathogenesis, Building 6L, Umeå University, Umeå, 901 87, Sweden
² The Wellcome Trust/Cancer Research UK, Gurdon Institute and Department of Anatomy, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
³ The Salk Institute, Molecular and Cell Biology Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037-1099, USA

View larger version (32K): [in a new window]   Fig. 1. Molecular organization of the Fak56 locus and characterization of Fak56CG1 mutant allele. (A) Schematic comparison of Fak56 with the mammalian FAK family kinases, hsFAK and hsPyk2. Conserved domains are schematized as follows; FERM domain (green), protein tyrosine kinase (PTK) domain (red) and FAT domain (blue). Percentage amino acid identity of FAK domains relative to the Drosophila Fak56 is indicated. (B) Genetic analysis of Fak56. Top panel: the Fak56 locus is depicted together with the surrounding genes (indicated as gray boxes). The arrows below indicate the orientation of Fak56 and the surrounding genes. The location of the P-element insertion used in the excision screen, P{SUPor-P}KG00304 is shown by an inverted triangle. Middle panel: the deduced structure of the approximately 7 kb Fak56 transcription unit is shown in expanded form. The 16 exons are represented by gray boxes. The first AUG initiation codon at the 5' end of the gene is indicated by a flag, and the TAA termination codon at the 3' end is marked by an asterisk. The horizontal gray bars represent the genomic probes employed to identify Fak56 mutants. The FAK I probe corresponds to bp 14-743 and the FAK II probe, bp 2300-3008 (where bp 1 corresponds to the A of the ATG start codon of Fak56). Lower panel: by mobilization of transposon P{SUPor-P}KG00304 373 bp upstream of the Fak56 ATG start codon, deletion Fak56CG1, which deletes the ATG start codon and eliminates 1263 bp of the Fak56 ORF, including exons 2-5 and a portion of exon 6, was recovered. (C) Mapping of the breakpoints in the Fak56CG1 chromosome. The sequence alteration in the Fak56CG1 deletion mutant is flanked by the bases indicated. (D) The 5' region of the Fak56 gene is absent in Fak56CG1. Genomic DNA from wild-type (1) and Fak56CG1 (2) flies were probed with FAKI and FAKII (covering 5' and 3' regions of the Fak56 gene respectively), as well as Spt5 (the 5' flanking gene) probes as indicated. Importantly, Southern analysis using the Spt5 probe indicates that this gene is intact in the Fak56CG1 mutant.

View larger version (48K): [in a new window]   Fig. 2. Fak56 protein is not expressed in Fak56CG1 mutant animals. (A) Immunoblot analysis of wild-type (1) and Fak56CG1 (2) embryonic lysates using Fak56-specific antibodies, indicates that Fak56 protein is absent in Fak56CG1 mutant animals. Protein extracts were immunoblotted with anti-Fak56 antibodies (upper panel). Subsequently, the same blot was stripped and reprobed with anti--Tubulin antibodies to ensure equal loading. (B) Wild-type and Fak56CG1 embryos were analyzed with anti-Fak56 antibodies [(i), (ii), (iv) and (v)], as well as anti-phospho-FAKY397 antibodies [(iii) and (vi)]. In wild-type embryos, Fak56 protein is ubiquitously expressed with particularly high levels in the embryonic CNS [(i) and (ii)]. In Fak56CG1 mutant embryos, Fak56 protein is absent [(iv) and (v)]. Similarly, in wild-type embryos, phosphorylated Fak56 was observed at muscle attachment sites (iii), and was strongly reduced in Fak56CG1 mutant embryos (vi). (C) Wild-type and Fak56CG1 embryos were analyzed with anti-myosin heavy chain (MHC) [(i) and (ii)]. No obvious muscle disruption phenotypes were observed in Fak56CG1 embryos when compared with wild-type. Embryo orientation is anterior to left and dorsal up.

View larger version (23K): [in a new window]   Fig. 3. Generation and characterization of the Df(2R)ED3716CG2 Fak56 deletion mutant. (A) Genetic analysis of Fak56. Upper panel: the Fak56 locus is depicted together with the surrounding genes (as before, Fig. 1). The two P-element insertions used to generate Df(2R)ED3716CG2, P(5-SZ-3124) and P(UM-8250-3) are shown by inverted triangles. Lower panel: FRT recombination between the 5-SZ-3124 and UM-8250-3 P elements resulted in a 12.8 kb deletion, named Df(2R)ED3716CG2, which uncovers both the entire Fak56 ORF as well as the neighboring Calpain A gene. The Fak56 genomic rescue fragment is also indicated (striped box). (B) Mapping of the breakpoints in the Df(2R)ED3716CG2 chromosome. The sequence flanking Df(2R)ED3716CG2 is shown. (C) Southern analysis of the Df(2R)ED3716CG2 mutant. The entire ORF of the Fak56 gene is absent in Df(2R)ED3716CG2. Genomic DNA from (i) Df(2R)ED3716CG2/Fak56 CG1, (ii) Df(2R)ED3716CG2/+, and (3) wild-type flies were probed with FAKI and FAKII (covering 5' and 3' regions of the Fak56 gene respectively) to show that the Fak56 ORF is completely absent in Df(2R)ED3716CG2 animals (* indicates the RFLP generated from the Fak56CG1 mutant).

View larger version (78K): [in a new window]   Fig. 4. Fak56 is not required for localization of components of adhesion complexes at muscle attachment sites. Wild-type and Fak56CG1 embryos were immunostained with antibodies recognizing proteins previously known to localize at muscle attachment sites. Three distinct protein components are shown, representing extracellular, transmembrane and intracellular components of the muscle attachment site. Wild-type and Fak56CG1 embryos were stained with anti-Tiggrin (A), anti-ßPS integrin (B), and anti-Talin (C) antibodies (green). Both genotypes showed wild-type localization at muscle attachment sites (arrowheads). All embryos were double-stained with Rhodamine-Phalloidin (red) to visualize actin filaments and muscle attachment sites. Low magnification is shown in (i) and (v). High magnifications of the muscle attachment sites are shown in (ii)-(iv) and (vi)-(viii). (D) Fak56 is not required for migration of the primordial midgut cells. The enhancer trap insertion line 258 was used to study the migration of the primordial midgut cells in Fak56 mutant embryos. The endodermal midgut arises from two primordia, the anterior midgut (AMG) and the posterior migut (PMG) primordium [(i) and (v), stage 10 embryos]. To form the midgut, these extend toward each other during stages 11 and 13 [(ii), (iii), (vi) and (vii)] and fuse laterally on both sides of the yolk, at stage 14 [(iv) and (viii)]. No obvious defects in the migration of primordial midgut cells could be observed in Fak56CG1 mutant embryos, when compared with wild-type controls.

View larger version (96K): [in a new window]   Fig. 5. Muscle attachments sites are unaffected in Df(2R)ED3716CG2 mutant embryos. Muscle attachment sites were analyzed in Df(2R)ED3716CG2 mutants. Df(2R)ED3716CG2 mutant (A-D), and control Df(2R)ED3716CG2/CyOwglacZ embryos (E-H) were stained with anti-ßPS integrin antibodies to visualize muscle attachment sites (A,C,E,G). Embryos were double-stained with anti-myosin heavy chain (MHC) to visualize gross muscle structure (B,D,F,H). Both genotypes showed wild-type localization of ßPS integrin at muscle attachment sites (arrowheads), as well as normal muscle morphology. Mutant embryos were identified by absence of lacZ balancer (red in F and H).

View larger version (47K): [in a new window]   Fig. 6. Overexpression of Fak56 results in embryonic muscle detachment. Wild-type embryos and embryos expressing Fak56 specifically in embryonic muscle, using the Mef2-GAL4 driver, were stained with anti-ßPS antibodies (green) to visualize integrins and Phalloidin (red) to visualize actin. Embryos overexpressing Fak56 specifically in embryonic muscle, show a potent muscle detachment phenotype (E,F, arrows), when compared with wild-type (B,C). Interestingly, in spite of obvious rounding up of somatic muscles, integrins, as monitored by anti-ßPS-integrin antibodies, were still localized at the ends of the muscles that remain attached in Fak56 overexpressing embryos (D, arrowhead), indistinguishable to wild-type embryos (A, arrowhead).

View larger version (89K): [in a new window]   Fig. 7. Overexpression of Fak56 downregulates integrin adhesion to the extracellular matrix without affecting linkage to the cytoskeleton. Muscles and their attachment sites in the epidermis are visualized in stage 16 embryos expressing either Fak56-GFP (A-F,M-P) or Fak56 (J-L) specifically in embryonic muscle, using the Mef2-GAL4 driver, or control embryos (G-I). (A-F) Embryos overexpressing Fak56-GFP specifically in muscle were stained with antibodies against PS2 integrin (red) (A,B), or Talin (red) (D,E), or antiphospho-FAKPY397 (blue) (B). Fak56-GFP is localized at the muscle ends (C,F, arrows), and its overexpression causes loss of muscle adhesion, without affecting the initial localization of PS2 integrin. However in muscles that detach, PS2 integrin remains at the ends of the detached muscles (A,B, arrowheads) indicating its dissociation from the extracellular matrix. A similar effect on Talin localization is observed (D,E, arrowheads). (G-L) Embryos expressing ILK-GFP are shown probed with anti-myosin heavy chain (MHC, red), and the extracellular matrix component Tiggrin (blue) (I,L). In embryos overexpressing Fak56 (J-L), ILK-GFP and Tiggrin are localized at the muscle ends similar to controls (G-I). Embryos expressing Fak56-GFP analyzed with anti-PS2 (red) and Tiggrin (blue) (M-P). In embryos overexpressing Fak56-GFP, Tiggrin can be seen at the attachment site (arrows), but is not particularly enriched at the ends of the detached muscles (arrowheads).

View larger version (32K): [in a new window]   Fig. 8. Fak56 and ßmys-integrin interact genetically in the Drosophila wing. Wing blister formation induced by expression of Fak56 in the developing wing disc is sensitized in an integrin mutant background. (A) Light micrographs of wings overexpressing Fak56. A(i) Engrailed-GAL4/+ control showing wild-type wing morphology. Wing veins L1-5, anterior cross vein (ACV) and posterior wing vein (PCV) are indicated. A(ii) Engrailed-GAL4/+;UAS-Fak56/+ male, A(iii) ßmysb43;Engrailed-GAL4/+;UAS-Fak56/+ male wings are shown. Arrows indicate wing defects or blisters. (B) Quantification of the wing blistering observed for the genotypes: Engrailed-GAL4/+;UAS-Fak56/+ female (n=241) and male (n=172), ßmysnj42;Engrailed-GAL4/+;UAS-Fak56/+ female (n=246) and male (n=117) and ßmysb43;Engrailed-GAL4/+;UAS-Fak56/+ female (n=160) and male (n=154) is shown. Numbers in brackets after the genotype indicate the number of flies scored.

View larger version (73K): [in a new window]   Fig. 9. Tyrosine phosphorylation of Fak56 at embryonic muscle attachment sites is integrin dependent. Wild-type and inflatedB4 mutant embryos were stained with anti-muscle myosin heavy chain (MHC) (green) and anti-phospho-FAKY397 antibodies (red). (A) In wild-type embryos phosphorylated Fak56 is localized at muscle attachment sites (arrowheads), whereas in inflatedB4 mutants no phosphorylated Fak56 can be observed (B).