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. 2016 May 9;213(3):371-83.
doi: 10.1083/jcb.201510012.

Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity

Abhishek Kumar  1 , Mingxing Ouyang  1 , Koen Van den Dries  1 , Ewan James McGhee  2 , Keiichiro Tanaka  1 , Marie D Anderson  3 , Alexander Groisman  4 , Benjamin T Goult  3 , Kurt I Anderson  2 , Martin A Schwartz  5
Affiliations

Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity

Abhishek Kumar et al. J Cell Biol. .

Erratum in

Abstract

Integrin-dependent adhesions are mechanosensitive structures in which talin mediates a linkage to actin filaments either directly or indirectly by recruiting vinculin. Here, we report the development and validation of a talin tension sensor. We find that talin in focal adhesions is under tension, which is higher in peripheral than central adhesions. Tension on talin is increased by vinculin and depends mainly on actin-binding site 2 (ABS2) within the middle of the rod domain, rather than ABS3 at the far C terminus. Unlike vinculin, talin is under lower tension on soft substrates. The difference between central and peripheral adhesions requires ABS3 but not vinculin or ABS2. However, differential stiffness sensing by talin requires ABS2 but not vinculin or ABS3. These results indicate that central versus peripheral adhesions must be organized and regulated differently, and that ABS2 and ABS3 have distinct functions in spatial variations and stiffness sensing. Overall, these results shed new light on talin function and constrain models for cellular mechanosensing.

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Figures

Figure 1.
Figure 1.
Construction and characterization of a talin-TS. (A) Schematic of the TS module in the relaxed (top) and tensed (bottom) states. (B) Schematic of talin-TS in the relaxed (top) and tensed (middle) state and the C-terminal, zero-tension control talin-CS (bottom). (C) Western blot for talin-TS and talin-CS in talin1−/− cells, probed for talin1 and vinculin, and stripped and reblotted with anti-GFP. (D) Localization of talin-TS in FAs marked by paxillin immunostaining. (E) FRAP of EGFP-talin (n = 38), talin-CS (n = 62), and talin-TS (n = 43) in FAs. Error bars are standard deviations. (F) Western blot of talin1−/− cells for talin2 after scrambled or talin2 siRNA transfection. (G) Spreading of talin1−/− cells with and without transfection of talin-TS and scrambled or talin2 siRNA. Cells were stained with Alexa Fluor 488–conjugated wheat germ agglutinin (red) to determine the cell area; talin-TS–positive cells are shown in green. (H) Normalized cell area for siScrambled (n = 99)-, siTalin2 (n = 107)-, and talin-TS–transfected cells (n = 50 for siScrambled+TS and n = 55 for siTalin2+TS). Bottom and top error bars represent 5th and 95th percentiles, respectively. Small rectangles in the box plots indicate means normalized to the control cells (scrambled siRNA, without talin-TS). Bars, 20 µm.
Figure 2.
Figure 2.
Tension on talin requires actomyosin contractility. (A) Pseudocolor map of FRET index for talin-CS and talin-TS within FAs of live cells on fibronectin. (B) Normalized FRET index for talin-CS (n = 30) and talin-TS (n = 32) within FAs. (C) FRET map of talin-CS and talin-TS in cells on 0.1% (wt/vol) poly-l-lysine–coated dishes. (D) Normalized FRET index of talin-CS (n = 20) and talin-TS (n = 15) from C. Error bars represent SEM. (E) Fluorimetric measurement of FRET for talin-CS and talin-TS in 293T cell lysates. n = 3. Error bars indicate standard deviation. (F) FRET map image time series of talin-TS after 5 µM blebbistatin treatment. (G) Plot of FA area and mean FRET index with time after blebbistatin treatment (n = 8). Error bars are standard deviations. (H) Histogram of time domain fluorescence lifetime measurement of EGFP in talin-TS, with mutated, nonfluorescent tagRFP and talin-CS as controls that indicate zero and maximal FRET (corresponding to maximal and minimal lifetimes), respectively. n > 60 each. The asterisk indicates a nonfluorescent mutant. (A, C, and F) Bars, 20 µm.
Figure 3.
Figure 3.
Spatial variation in talin tension within single cells. (A and E) Pseudocolor map of FRET index for talin-TS (A) and for talin-CS (E) within FAs of cells plated on fibronectin-coated dishes with cell boundaries drawn in white. (B and F) Normalized FRET index in central and peripheral FAs for talin-TS (n = 32; B) and for talin-CS (n = 30; F). Error bars represent SEM. (C and G) Vinculin/talin intensity ratio map for talin-TS (C)– and for talin-CS (G)–transfected talin1−/− cells fixed and stained for vinculin. (D and H) Histograms of pixel-wise vinculin/talin intensity ratio for talin-TS cells (n = 25; D) and for talin-CS (n = 30; H). Bars, 20 µm. Arb., arbitrary units.
Figure 4.
Figure 4.
Differential substrate stiffness sensing by talin and vinculin. (A) Representative FRET map and normalized FRET index of talin-TS in talin1−/− cells on stiff (∼30 kPa; n = 29) or soft (∼3 kPa; n = 30) substrates. (B) Talin-CS on stiff (n = 27) or soft (n = 26) substrates. (C) Vinculin-TS expressed in vinculin−/− cells on stiff (n = 50) or soft (n = 45) substrates. (D) Vinculin–tailless control (vinculin-TL; control for zero tension) on stiff (n = 25) or soft (n = 25) substrates. Bars, 20 µm. Error bars represent SEM.
Figure 5.
Figure 5.
Role of vinculin and ABS in regulating force on talin. (A) Immunostaining of vinculin (red) in talin1−/− cells transfected with scrambled or vinculin siRNA. The talin-TS is in green. (B) Western blot for vinculin after knockdown. IB, immunoblot. (C) Representative FRET map and normalized FRET index of talin-TS in scrambled (n = 30) and vinculin (n = 32) siRNA–treated cells. (D and E) Representative FRET map (D) and normalized FRET index (E) of talin-TS (n = 46) or ABS3 mutant talin-TS (n = 37) and ABS2 mutant talin-TS (n = 31) transfected in talin1−/− cells. (F) Frequency domain EGFP lifetime for talin-CS (n = 74), talin-TS (n = 67), ABS3 mutant talin-TS (n = 66), and ABS2 mutant talin-TS (n = 66). Bars, 20 µm. Error bars represent SEM.
Figure 6.
Figure 6.
Role of vinculin and ABSs in spatial variation and ECM stiffness sensing by talin. (A–C) Representative FRET map and normalized FRET index of talin-TS in vinculin–knocked down talin1−/− cells (n = 29; A), ABS3 mutant talin-TS in talin1−/− cells (n = 18; B), and ABS2 mutant talin-TS in talin1−/− cells (n = 28; C) for central and peripheral FAs. (D–F) Representative FRET map and normalized FRET index of talin-TS in vinculin−/− cells plated on stiff (n = 30) and soft (n = 25) substrates (D), ABS3 mutant talin-TS in talin1−/− cells plated on stiff (n = 25) and soft (n = 25) substrates (E), and ABS2 mutant talin-TS in talin1−/− cells plated on stiff (n = 25) and soft (n = 25) substrates (F). Bars, 20 µm. Error bars represent SEM.
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