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. 2015 Mar 30;25(7):847-57.
doi: 10.1016/j.cub.2015.01.043. Epub 2015 Mar 5.

Alternative mechanisms for talin to mediate integrin function

Benjamin Klapholz  1 , Samantha L Herbert  1 , Jutta Wellmann  1 , Robert Johnson  1 , Maddy Parsons  2 , Nicholas H Brown  3
Affiliations

Alternative mechanisms for talin to mediate integrin function

Benjamin Klapholz et al. Curr Biol. .

Abstract

Cell-matrix adhesion is essential for building animals, promoting tissue cohesion, and enabling cells to migrate and resist mechanical force. Talin is an intracellular protein that is critical for linking integrin extracellular-matrix receptors to the actin cytoskeleton. A key question raised by structure-function studies is whether talin, which is critical for all integrin-mediated adhesion, acts in the same way in every context. We show that distinct combinations of talin domains are required for each of three different integrin functions during Drosophila development. The partial function of some mutant talins requires vinculin, indicating that recruitment of vinculin allows talin to duplicate its own activities. The different requirements are best explained by alternative mechanisms of talin function, with talin using one or both of its integrin-binding sites. We confirmed these alternatives by showing that the proximity between the second integrin-binding site and integrins differs, suggesting that talin adopts different orientations relative to integrins. Finally, we show that vinculin and actomyosin activity help change talin's orientation. These findings demonstrate that the mechanism of talin function differs in each developmental context examined. The different arrangements of the talin molecule relative to integrins suggest that talin is able to sense different force vectors, either parallel or perpendicular to the membrane. This provides a paradigm for proteins whose apparent uniform function is in fact achieved by a variety of distinct mechanisms involving different molecular architectures.

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Figures

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Graphical abstract
Figure 1
Figure 1
New Talin Mutants (A) The diagram shows the domains of talin: F0–F3 of the FERM domain (yellow), vinculin-binding sites (light gray), integrin-binding site 2 (dark blue), and actin-binding domain (red). IBS2 is also a VBS. Mutations are indicated above talin: 38 cause a truncation (black), and one causes a substitution (R367H; blue). The truncations are named according to their last in-frame talin residue (see Table S1). (B) GFP-talin and Δhead transgenes. The position of GFP (green) and deletion (dashed line) are indicated.
Figure 2
Figure 2
Talin Head, but Not IBS2, Is Essential for Integrin Function at Muscle Attachment Sites (A) Weak versus strong muscle attachment defects in talin mutant embryos. Muscle myosin heavy chain (Mhc) staining of embryonic muscles exhibiting no defect (wild-type; top), mild detachments (hypomorphic phenotype; center), or complete detachment (null phenotype; bottom). The scale bars represent 100 μm. (B) The average shortening of five dorsal muscles (pink arrowheads in A) was quantified per embryo homozygous for the indicated mutants and plotted as the reduction in muscle length relative to wild-type in the presence (top histogram) or absence (bottom histogram) of vinculin. Bar colors show three statistically distinct categories (∗p < 0.01; green bars are not significantly different from wild-type). At least five embryos were measured per genotype. Error bars are SD. Genotypes not analyzed do not have a bar.
Figure 3
Figure 3
In Germband Retraction, Talin Head and Vinculin Are Redundant, whereas ABD Is Essential (A) Embryos exhibiting no defect (wild-type; top) or a GBR defect (bottom), stained for Fasciclin 3 in lateral epidermal membranes. (B) The percentage of GBR defects was quantified in ≥50 embryos/mutant. Bar colors show two statistically distinct categories (∗p < 0.01; green bars are not significantly different from wild-type). Genotypes not analyzed do not have a bar.
Figure 4
Figure 4
Both Talin Head and IBS2 Are Used for Wing Adhesion (A) Representative pictures of newly hatched flies with normal wings (top) or one wing with a blister (bottom; pink arrow). (B) Percentage of blistered wings in flies with homozygous mutant clones for the mutants indicated (for all mutants, see Figure S3), from ≥100 flies/mutant. Bar colors show four statistically distinct categories (∗p < 0.01). Flat bars (horizontal lines) indicate no defect. Genotypes not analyzed do not have a bar. (C) Phenotypic differences between muscle and wing are not explained by differences in protein levels, determined by western blotting of talin646 and talin2509 (top histogram) and talin site-directed mutants (bottom histogram) in embryos (light gray) or pupal wings (dark gray) heterozygous for the talin mutation. The protein levels were normalized to wild-type talin in each sample. SD is shown from two independent experiments.
Figure 5
Figure 5
Talin IBS2 Is in Proximity to Integrins in Wing but Not in Muscle (A–D) FRET analysis to determine the proximity between βPS-GFP and talinIBS2-mCherry (A), vinculin-GFP and talinIBS2-mCherry (B), GFP-talin and vinculin-RFP (C), or GFP-talin and talinIBS2-mCherry (D) at integrin adhesion sites in muscle (left panels) and wing (right panels). Donor GFP (gray in top panels) and GFP lifetime heat maps (lower panels, with scale in ns) are shown. Increasing FRET shortens GFP lifetime; FRET efficiencies are indicated by the standard errors (n > 10) below the panels. (E) 3D-SIM shows that βPS-GFP (top) and GFP-talin (center) are separated from talinIBS2-mCherry at MASs, in contrast to vinculin-GFP and vinculin-RFP (control for chromatic aberration; bottom). (F) Diagram of integrin adhesions (orange) between the two epithelial cell layers of the wing. The x-y focal plane of the images is shown with the light blue horizontal plane. Some adhesions are tilted sufficiently to provide a transverse section (dashed black rectangle). Pictures: 3D-SIM x-y sections through wing adhesions show colocalization of βPS-GFP and talinIBS2-mCherry (top), GFP-talin and talinIBS2-mCherry (center), and vinculin-GFP and vinculin-RFP (bottom).
Figure 6
Figure 6
Muscle Myosin Heavy Chain and Vinculin Are Required to Separate IBS2 from Integrins at Muscle Attachments (A) FRET analysis at MASs shows that talinIBS2-mCherry and βPS-GFP are in closer proximity in the absence of muscle myosin (Mhc; middle panels) or vinculin (right panels) compared to wild-type (left panels). Donor GFP is shown in gray on top and GFP lifetime heat maps below. Increasing FRET shortens the lifetime; FRET efficiencies are indicated by the standard errors (n > 15) below the panels. The scale bars represent 20 μm. (B) 3D-SIM shows that βPS-GFP and talinIBS2-mCherry are separated at MASs in the absence of vinculin. (C) FRET analysis at wing adhesions shows that the proximity between talinIBS2-mCherry and βPS-GFP is not affected in the absence of vinculin. The scale bars represent 20 μm. (D) Pictures: muscle-specific overexpression of IBS2-GFP at MASs (pink arrowheads) in the presence (wild-type; top) or absence (ΔVinc; bottom) of vinculin. Histogram: quantitation of IBS2-GFP levels at MASs, normalized to wild-type levels. Error bars are SDs (n > 25).
Figure 7
Figure 7
Models for Three Mechanisms of Talin Action The top diagrams show models for the way talin functions in muscle and wing and during germband retraction. At muscle attachment sites (left), talin dimers are bound to integrins or membrane with their heads and to actin directly with the C-terminal ABD and indirectly with vinculin. Actomyosin activity and vinculin are important to separate IBS2 and integrins, likely by exerting force on the rod of talin that pulls it toward the inside of the cell (white arrows). In the wing (middle), talin is oriented parallel to the membrane, with each talin dimer using all IBSs. This talin dimer binds actin, directly with the C-terminal ABD and indirectly with vinculin. During germband retraction (right), talin dimers are bound to actin or membrane directly with the head and indirectly with vinculin (only membrane binding is shown), to integrins with IBS2s, and to actin with the C-terminal ABD. In wing and germband retraction (see below), we suggest IBS2 binds integrins but could interact with other functional binding partners. The logic that generated these models from the mutant phenotypes is demonstrated by depicting the phenotypic effect of the mutations in each model. In muscle, talin head is essential to bind integrins and membrane, as its absence (Δhead) resulted in a null phenotype (red background). The absence of ABD (talin2120) resulted in a hypomorphic phenotype (orange background), and this remaining function requires vinculin but it is not clear how vinculin is recruited. The additional loss of IBS2 (talin646) did not enhance the phenotype, but talin caused a null phenotype when all VBSs were deleted (talin511). In the wing, talin head is important but integrins can still be linked together through dimerized IBS2s. The absence of ABD resulted in a hypomorphic phenotype, and this remaining function requires vinculin. The additional loss of IBS2 resulted in a null phenotype, as integrins cannot be linked together by talin. During germband retraction, the head was not required (green background) but vinculin was essential in this context. It is not clear how vinculin is recruited to Δhead. All truncations deleting ABD activity resulted in a null phenotype.
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