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Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism.

Riveline D, Zamir E, Balaban NQ, Schwarz US, Ishizaki T, Narumiya S, Kam Z, Geiger B, Bershadsky AD - J. Cell Biol. (2001)

Bottom Line: Narumiya. 1999.Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts.Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Physical Spectrometry (CNRS), UMR 5588, Joseph Fourier University, French National Center for Scientific Research, BP87, 38402 Saint-Martin d'Hères Cedex, France.

ABSTRACT
The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136-143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

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Reversible suppression of cell contractility by BDM and Y-27632. SV-80 cells growing on the silicon rubber substrate and producing wrinkles (A and F) were treated with either 30 mM actomyosin ATPase inhibitor BDM (top) or 10 μM ROCK inhibitor Y-27632 (bottom). Images of BDM-treated cells were taken at 2.5 (B), 5 (C), and 9.5 min (D) after drug addition; the drug was washed off after 30 min incubation, and the last image (E) was taken at 45 min after washing. Images of the cell treated with Y-27632 were captured at 2.5 (G), 6 (H), and 9.5 min (I) after drug addition; the drug was washed off after 30 min incubation, and the image (J) was taken 50 min later. Bar, 20 μm.
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Figure 7: Reversible suppression of cell contractility by BDM and Y-27632. SV-80 cells growing on the silicon rubber substrate and producing wrinkles (A and F) were treated with either 30 mM actomyosin ATPase inhibitor BDM (top) or 10 μM ROCK inhibitor Y-27632 (bottom). Images of BDM-treated cells were taken at 2.5 (B), 5 (C), and 9.5 min (D) after drug addition; the drug was washed off after 30 min incubation, and the last image (E) was taken at 45 min after washing. Images of the cell treated with Y-27632 were captured at 2.5 (G), 6 (H), and 9.5 min (I) after drug addition; the drug was washed off after 30 min incubation, and the image (J) was taken 50 min later. Bar, 20 μm.

Mentions: In contrast, force-induced formation of focal contacts did not appear to require actin–myosin-mediated cell contraction. Indeed, the application of force led to focal contact formation even in the presence of BDM, a potent inhibitor of myosin ATPase (Higuchi and Takemori 1989; Cramer and Mitchison 1995) (Fig. 6, C–D′). Treatment of SV-80 cells with the same concentration of BDM rapidly but reversibly blocked their contractility, as revealed by observations of wrinkling of flexible substrate (Fig. 7, A–E). Another method for blocking actin–myosin contractility was transfection of cells with a construct encoding the regulatory protein caldesmon, a physiological inhibitor of actin-activated myosin ATPase (Helfman et al. 1999). The inhibitory effect of caldesmon transfection on focal contact formation and contractility of SV-80 cells was extensively documented in our previous studies (Helfman et al. 1999). After caldesmon overexpression, application of a local force still induced the formation of elongated focal contacts despite a severely altered cell morphology (Fig. 6, E–F′). Thus, the application of an external force can replace actomyosin contractility during the process of focal contact formation, as long as actin polymerization and actin filament integrity are not disturbed.


Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism.

Riveline D, Zamir E, Balaban NQ, Schwarz US, Ishizaki T, Narumiya S, Kam Z, Geiger B, Bershadsky AD - J. Cell Biol. (2001)

Reversible suppression of cell contractility by BDM and Y-27632. SV-80 cells growing on the silicon rubber substrate and producing wrinkles (A and F) were treated with either 30 mM actomyosin ATPase inhibitor BDM (top) or 10 μM ROCK inhibitor Y-27632 (bottom). Images of BDM-treated cells were taken at 2.5 (B), 5 (C), and 9.5 min (D) after drug addition; the drug was washed off after 30 min incubation, and the last image (E) was taken at 45 min after washing. Images of the cell treated with Y-27632 were captured at 2.5 (G), 6 (H), and 9.5 min (I) after drug addition; the drug was washed off after 30 min incubation, and the image (J) was taken 50 min later. Bar, 20 μm.
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getmorefigures.php?uid=PMC2192034&req=5

Figure 7: Reversible suppression of cell contractility by BDM and Y-27632. SV-80 cells growing on the silicon rubber substrate and producing wrinkles (A and F) were treated with either 30 mM actomyosin ATPase inhibitor BDM (top) or 10 μM ROCK inhibitor Y-27632 (bottom). Images of BDM-treated cells were taken at 2.5 (B), 5 (C), and 9.5 min (D) after drug addition; the drug was washed off after 30 min incubation, and the last image (E) was taken at 45 min after washing. Images of the cell treated with Y-27632 were captured at 2.5 (G), 6 (H), and 9.5 min (I) after drug addition; the drug was washed off after 30 min incubation, and the image (J) was taken 50 min later. Bar, 20 μm.
Mentions: In contrast, force-induced formation of focal contacts did not appear to require actin–myosin-mediated cell contraction. Indeed, the application of force led to focal contact formation even in the presence of BDM, a potent inhibitor of myosin ATPase (Higuchi and Takemori 1989; Cramer and Mitchison 1995) (Fig. 6, C–D′). Treatment of SV-80 cells with the same concentration of BDM rapidly but reversibly blocked their contractility, as revealed by observations of wrinkling of flexible substrate (Fig. 7, A–E). Another method for blocking actin–myosin contractility was transfection of cells with a construct encoding the regulatory protein caldesmon, a physiological inhibitor of actin-activated myosin ATPase (Helfman et al. 1999). The inhibitory effect of caldesmon transfection on focal contact formation and contractility of SV-80 cells was extensively documented in our previous studies (Helfman et al. 1999). After caldesmon overexpression, application of a local force still induced the formation of elongated focal contacts despite a severely altered cell morphology (Fig. 6, E–F′). Thus, the application of an external force can replace actomyosin contractility during the process of focal contact formation, as long as actin polymerization and actin filament integrity are not disturbed.

Bottom Line: Narumiya. 1999.Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts.Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Physical Spectrometry (CNRS), UMR 5588, Joseph Fourier University, French National Center for Scientific Research, BP87, 38402 Saint-Martin d'Hères Cedex, France.

ABSTRACT
The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136-143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

Show MeSH
Related in: MedlinePlus