Tensin Stabilizes Integrin Adhesive Contacts in Drosophila Catherine N Torgler, Maithreyi Narasimha, Andrea L Knox, Christos G Zervas, Matthew C Vernon, Nicholas H Brown  Developmental Cell  Volume 6, Issue 3, Pages 357-369 (March 2004) DOI: 10.1016/S1534-5807(04)00055-3

Figure 1 The blistery Gene Encodes Tensin (A) The single exon blistery gene is diagrammed with the arrow indicating the direction of transcription; the coding region is in white and the untranslated regions are in black. The 3′ ends of the genes on either side of blistery are indicated. The P element insertion at the 5′ end of blistery is indicated by an inverted triangle. The deletion in the null mutation by33c is represented by a gap in the black line. DNA fragments used for the rescue construct and the probe for in situs in Figure 2 are indicated with black lines. (B) Tensin sequence conservation. Human, chicken, and fly tensin sequences are supported by cDNA data; the Anopheles sequence is a prediction from the genomic sequence. Tensin proteins are schematized as follows: the thick gray line represents the whole protein sequence, the black square is the phosphatase like domain (PTP), the light pentagon represents the SH2 domain, and the dark square the PTB domain. (C) SH2 domains of tensins are more similar to each other than to other SH2 domains. Phylogenetic tree of SH2 domains from human proteins and their Drosophila orthologs, assembled in MacVector with ClustalW. (D) The Drosophila tensin N-terminal domain binds actin. Shown are the results of an actin filament spin-down assay. Actin filaments were polymerized in the presence and absence of a glutathione-S-transferase (GST)-tensin fusion protein and centrifuged at 150,000 × g. Supernatent and pellet fractions are shown, detected with the anti-GST antibody (top) or by Coomassie blue staining (bottom). Developmental Cell 2004 6, 357-369DOI: (10.1016/S1534-5807(04)00055-3)

Figure 2 Tensin mRNA and Protein Expression (A–F) Distribution of tensin mRNA in developing embryos. Expression is first detected at stage 11 (C). Large arrows marked p in (E) and (F) indicate particularly strong staining found in the key hole region in the proventriculus. (F) By stage 16, staining is detected in the epidermal cells that make attachments to the muscles (small arrows labeled ma). (G–J) Localization of GFP-tagged tensin expressed from its own promoter (tensin-GFP is white in [G] and [I], green in [H] and [J], and actin is red). Surface view of stage 16 embryo (G and H) shows strong expression at muscle attachment sites, while optical sections of stage 17 embryos (I and J) show tensin-GFP in the developing gut, with strong expression in the proventriculus (p) and pharynx (ph), shown enlarged and labeled with actin in (J). (K and L) Tensin distribution at stage 16 muscle attachments, detected with an antibody against tensin N terminus. (M and N) Fluorescent confocal sections of pupal wings from wild-type flies (M) and flies containing the tensin-GFP genomic rescue construct (N). In between is a schematic showing the two layers of cells, joined by basal integrin-mediated junctions containing tensin. The apical surface of each layer of cells is autofluorescent and therefore visible in both samples. Scale bar 10 μm. Developmental Cell 2004 6, 357-369DOI: (10.1016/S1534-5807(04)00055-3)

Figure 3 Tensin Phenotype in Drosophila Wings Adult flies lacking tensin have fully penetrant wing blisters. (A) Wild-type and (B) blistery mutant (by33c in this and all subsequent figures). (C–F) Wing blisters appear later in the absence of tensin (D and F) compared to integrin mutants such as inflated (if3) (C and E); wings immediately after eclosion (C and D) or 5 min later (E and F). (G and H) Mechanical stress produces the blister in flies lacking tensin; gluing down the legs of a by mutant fly suppresses the wing blister (G) compared to an untreated by fly that can stroke its wings (H). (I and J) Overexpression of the tensin PTB domain (I) caused small blisters (arrow), while overexpression of the combined SH2 and PTB domains caused wing “wrinkling” (J). Developmental Cell 2004 6, 357-369DOI: (10.1016/S1534-5807(04)00055-3)

Figure 4 Tensin Recruitment Requires Integrin, Talin, and ILK Each pair of panels shows an optical section of embryonic muscle attachment sites with the epidermis at the top and the muscles below. The embryos all express tensin-GFP, shown on its own on the left, or in green on the right in combination with staining for either βPS integrin (A) or filamentous actin in red (B–F). One of the epidermal tendon cells (tc) is indicated in (A)–(E). (A) Tensin and βPS integrins colocalize. (B–F) Tensin localization in the absence of other components of the integrin adhesion complex. Tensin-GFP localization at muscle ends, as seen in the wild-type (B), is lost in the absence of βPS integrin (C), talin (D), and ILK (E), but remains localized in the absence of PINCH (F). Scale bar 20 μm. Developmental Cell 2004 6, 357-369DOI: (10.1016/S1534-5807(04)00055-3)

Figure 5 Structure-Function Analysis of Tensin The distribution of each of the GFP-tagged tensin constructs diagrammed on the left side of the figure is shown in embryonic muscles (left), 2nd instar larval muscles (center), and pupal wings (right). In the diagrams, the plain line represents the N terminus, the light pentagon represents the SH2 domain, and the dark square the PTB domain. The vertical line crossing the SH2 domain represents the SH2 point mutation (SH2*) that disrupts phosphotyrosine binding. The majority of constructs (A)–(H) were expressed with the GAL4 system, while the constructs in (I)–(K) were expressed using the endogenous tensin gene promoter (genomic rescue constructs) in a wild-type background. Dominant effects seen upon overexpression of the various constructs in the wing were scored on the basis of the penetrance of phenotypes (blistering or crumpling/crinkling of the wing blade) as follows: 0%–10% of flies have a defect = −; 11%–20% = +; 21%–60% = ++; 61%–80% = +++. Between 70 to 250 flies of the relevant genotype were scored for each construct. Scale bar 20 μm. Developmental Cell 2004 6, 357-369DOI: (10.1016/S1534-5807(04)00055-3)

Figure 6 Tensin and Tyrosine Phosphorylation (A) Tensin-GFP is colocalized with phosphotyrosine (pY; detected with the monoclonal antibody 4G10) at muscle ends. Phosphotyrosine labeling of epidermal adherens junctions is also observed. (B) Only a small fraction of tensin SH2 domain-GFP colocalizes with phosphotyrosine. (C) Loss of tensin (by) does not alter the overall levels of phosphorylated proteins at the muscle ends. (D and E) Loss of tensin (E) does not alter the levels of focal adhesion kinase (FAK) phosphorylation relative to wild-type (D). Scale bar 5 μm. (F) Model of tensin function. Developmental Cell 2004 6, 357-369DOI: (10.1016/S1534-5807(04)00055-3)