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First published online 28 March 2006
doi: 10.1242/jcs.02859

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Multiple factors contribute to integrin-talin interactions in vivo

Guy Tanentzapf, Maria D. Martin-Bermudo^*, Marcus S. Hicks and Nicholas H. Brown

The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Anatomy, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK

View larger version (100K): [in a new window]   Fig. 1. Localisation of talin N- and C-terminal domains in the embryo. (A-G) Confocal images of muscles from two segments of a late-stage Drosophila embryo. A,B are anti-talin stained fixed embryos and C-G are images of GFP fluorescence in live embryos. (A) Talin is enriched at integrin adhesion sites at muscle ends (arrowheads). (B) Elevating talin expression with mef2::Gal4 and UAS::talin resulted in a modest increase in localized talin and a larger increase in cytoplasmic talin. The mef2::Gal4 driver was also used to express the following domain fusion proteins. TalinH-GFP (C) was localized to muscle ends, but expression in the cytoplasm and nuclei was also observed. GFP-talinC (D) localized to muscle ends and the developing Z-lines. (E,F) Reduction in endogenous talin caused an increase in talinH-GFP recruitment to muscle ends and a loss of GFP-talinC. (G) Removal of PS2ßPS integrin caused the loss of talinH-GFP from the muscle ends. (H) TalinH-GFP, detected with an anti-GFP antibody, remained intact despite the putative calpain cleavage site between talinH and GFP. Arrow indicates where cleaved GFP would migrate. (I) An antibody raised against the talin head domain stains muscle attachments in wild-type embryos. (J) embryos expressing talinH-GFP also showed nuclear and cytoplasmic staining, identical to the GFP fluorescence from this fusion protein (C). Bar, 20 µm.

View larger version (153K): [in a new window]   Fig. 2. Elevated levels of integrin increased talin head recruitment. (A-F) Developing muscles at different stages. (A) Wild type; (B-F) elevated integrin expression (UAS::ßPS/+; UAS::PS2/24BGal4). (A) In wild-type myoblasts, integrin levels are too low to recruit talinH-GFP, which was found in the nucleus. Overexpressed integrins (C) recruited talinH-GFP (B) to the cortex. In fully fused muscles overexpressed integrin recruited talinH-GFP (arrowheads) out of the nucleus and to the ends (D) (compare with Fig. 1C). Later on, the elevated integrin caused muscle detachment and talinH-GFP was recruited to the cortex (E), in contrast to GFP-talinC (F). Bars, 20 µm.

View larger version (64K): [in a new window]   Fig. 3. Chimeras that contain oligomerized, but not monomeric, cytoplasmic domain of the integrin ß subunit recruited talin head and FAK, but not other integrin-associated proteins. (A) A chimera containing the extracellular domain of monomeric CD2 fused to the cytoplasmic and transmembrane domains from ßPS (cßß), did not increase talinH-GFP recruitment to the muscle ends. (B) The `diß' chimera, containing extracellular and transmembrane domains of the oligomeric and/or dimeric protein Torso, recruited talinH-GFP out of the nucleus to the cortex. (C) Bar graphs show the average ratio of cortical to cytoplasmic GFP staining in living embryos for wild-type muscles and muscles overexpressing integrin and integrin cytoplasmic tail chimeras. Overexpression of integrins or expression of the diß chimera resulted in the recruitment of talinH-GFP to the cell cortex whereas expression of the cßß and ccß chimeras did not. (D) Examination of other proteins showed that the majority were not recruited by diß and the ratio of cortical to cytoplasmic stain was not changed between wild-type muscle and muscles that expressed diß. However, phosphorylated FAK was weakly recruited to the cortex by diß.

View larger version (133K): [in a new window]   Fig. 4. diß interferes with the endogenous integrin-cytoskeleton link and recruits talin. (A-C") The dominant negative phenotype in muscles expressing diß. (A) Integrin (anti-PS2, blue in A and white in A") remained with the tendon matrix (anti-tiggrin, red in A and white in A') rather than at the ends of the detached muscles (myristylated-GFP, (green in A and white in A"'). By contrast, talin was found at the ends of the detached muscles (red in B, arrowheads in B"), distinct from PS2 (green in B and B') and colocalized with diß (C). The later-stage muscles (C) contained intracellular accumulations of talin and diß that were not seen in earlier-stage muscles (B). Arrowhead in A', A"', C indicate ends of detached muscles. Bar, 20 µm.

View larger version (32K): [in a new window]   Fig. 5. Point mutations generated in the ßPS cytoplasmic tail within the dimerized chimera (diß).

View larger version (60K): [in a new window]   Fig. 6. Expression levels and localization of mutant forms of diß to muscle ends. (A) Western blot analysis shows relative levels of expression of diß and its mutated variants as expressed under the control of the mef2::Gal4 driver; actin is used as a loading control. The diß protein has 12 potential extracellular glycosylation sites and is similar to the wild-type Torso protein (Sprenger and Nusslein-Volhard, 1992), two bands are detected for diß. (B,C) Antibody staining reveals that the mutant forms of diß still localized to muscle ends (arrowheads) as illustrated for the N840A and D807V/E810A mutations. Bar, 20 µm.

View larger version (50K): [in a new window]   Fig. 7. Recruitment of talin head by diß mutants. (A) diß recruited talinH-GFP from the nucleus to the cell cortex of muscles of living late-stage embryos. (B) Mutating the proximal NPXY motif abolished this cortical recruitment in muscle. (C) A mutated ATST sequence in the integrin cytoplasmic tail still recruited talin head out of the nucleus and onto the cortex. (D) Quantification of the average ratio of cortical to cytoplasmic staining from multiple muscles showed that mutations in the ATST sequence of the integrin cytoplasmic tail or in the distal NPxY motif still recruited talin head to the muscle cell cortex, whereas mutations in the proximal region or the first NPxY did not. (E-E") In the absence of endogenous integrins (E", lacking of PS2) the diß mutant with a TST to AAA mutation in the integrin cytoplasmic tail (E') recruited endogenous talin to the muscle ends (E, arrow). Bar, 20 µm.

View larger version (58K): [in a new window]   Fig. 8. Complementation between different diß point mutations. Expression of diß in the wing caused a dominant negative wing-blister phenotype (A) that was lost in the mutant diß constructs (B), e.g. P841A. Dominant negative activity was restored by coexpression of two different diß mutants. The diß mutants P829A and P841A produced a weak wrinkled-wing phenotype (C), whereas N828A and P841A restored a wing-blister phenotype (D).

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