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

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ABSTRACT

The cytoskeletal adapter protein talin plays a prominent role in adhesive structures connecting integrins to the actin cytoskeleton. In this work, Kumar et al. use a novel talin sensor to measure talin tension and provide insights into focal adhesion force transmission and mechanosensitivity.

No MeSH data available.


Related in: MedlinePlus

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.
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fig2: 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.

Mentions: To determine the tension on talin, we measured the FRET index in live cells as an approximation of FRET/molecule (Grashoff et al., 2010). This method measures FRET intensity, subtracts the background and the bleed through for the two fluorophores, and then normalizes to acceptor intensity. For these experiments, we analyzed FAs >0.25 µm2, as smaller adhesions are harder to identify and quantify. In cells plated on fibronectin, FRET for talin-TS within FAs was low compared with talin-CS (Fig. 2, A and B). In contrast, when cells were plated on poly-l-lysine, where talin does not localize to FAs, FRET was high for both talin-TS and CS (Fig. 2, C and D). To check whether the talin-TS is sensitive to conformational changes associated with talin activation, we took advantage of the fact that talin recruitment to membranes is dependent on its conformational opening (Lagarrigue et al., 2015). Thus, membrane-bound talin outside of adhesive areas should be activated but without tension. Cells plated on polylysine show a clear rim of membrane-bound talin against the diffuse cytoplasmic pool. Membrane-bound versus cytoplasmic talin in cells on polylysine, at a plane well above the coverslip, showed no difference in FRET index (Fig. S2, A and B). Additionally, when FRET efficiency was measured in cell lysates by fluorimetry (Grashoff et al., 2010), talin-TS and CS were equivalent (Fig. 2 E). FRET index for talin-TS was similar in 3T3 cells that expressed endogenous talin (Fig. S2, C and D), indicating that the method is applicable to other cell types. Together, these data show that talin in FAs is under tension.


Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity
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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig2: 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.
Mentions: To determine the tension on talin, we measured the FRET index in live cells as an approximation of FRET/molecule (Grashoff et al., 2010). This method measures FRET intensity, subtracts the background and the bleed through for the two fluorophores, and then normalizes to acceptor intensity. For these experiments, we analyzed FAs >0.25 µm2, as smaller adhesions are harder to identify and quantify. In cells plated on fibronectin, FRET for talin-TS within FAs was low compared with talin-CS (Fig. 2, A and B). In contrast, when cells were plated on poly-l-lysine, where talin does not localize to FAs, FRET was high for both talin-TS and CS (Fig. 2, C and D). To check whether the talin-TS is sensitive to conformational changes associated with talin activation, we took advantage of the fact that talin recruitment to membranes is dependent on its conformational opening (Lagarrigue et al., 2015). Thus, membrane-bound talin outside of adhesive areas should be activated but without tension. Cells plated on polylysine show a clear rim of membrane-bound talin against the diffuse cytoplasmic pool. Membrane-bound versus cytoplasmic talin in cells on polylysine, at a plane well above the coverslip, showed no difference in FRET index (Fig. S2, A and B). Additionally, when FRET efficiency was measured in cell lysates by fluorimetry (Grashoff et al., 2010), talin-TS and CS were equivalent (Fig. 2 E). FRET index for talin-TS was similar in 3T3 cells that expressed endogenous talin (Fig. S2, C and D), indicating that the method is applicable to other cell types. Together, these data show that talin in FAs is under tension.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

The cytoskeletal adapter protein talin plays a prominent role in adhesive structures connecting integrins to the actin cytoskeleton. In this work, Kumar et al. use a novel talin sensor to measure talin tension and provide insights into focal adhesion force transmission and mechanosensitivity.

No MeSH data available.


Related in: MedlinePlus