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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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Covering of the substrate with fibronectin is essential for force-induced growth of focal contacts. GFP–vinculin-transfected SV-80 cells plated on poly-l-lysine in serum-free medium do not respond to pulling with a fibronectin-coated pipette by formation of focal contacts (D–F), whereas cells plated on substrate precoated with poly-l-lysine and then coated with fibronectin (fibronectina) produce normal focal contacts (A–C). Under standard conditions (fibronectinb), cells were plated in serum-containing medium and then serum starved. Pulling of these cells with a poly-l-lysine–coated pipette still produces growth of focal contacts (G–I). The positions of pipette immediately after shift are indicated in B, E, and H. Images labeled before and after were taken 1–10 min before and 3–7 min after pipette shift. Bars: (B, E, and H) 10 μm; (C, F, and I) 5 μm. In each before–after pair, the pictures were taken with the same magnification.
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Figure 5: Covering of the substrate with fibronectin is essential for force-induced growth of focal contacts. GFP–vinculin-transfected SV-80 cells plated on poly-l-lysine in serum-free medium do not respond to pulling with a fibronectin-coated pipette by formation of focal contacts (D–F), whereas cells plated on substrate precoated with poly-l-lysine and then coated with fibronectin (fibronectina) produce normal focal contacts (A–C). Under standard conditions (fibronectinb), cells were plated in serum-containing medium and then serum starved. Pulling of these cells with a poly-l-lysine–coated pipette still produces growth of focal contacts (G–I). The positions of pipette immediately after shift are indicated in B, E, and H. Images labeled before and after were taken 1–10 min before and 3–7 min after pipette shift. Bars: (B, E, and H) 10 μm; (C, F, and I) 5 μm. In each before–after pair, the pictures were taken with the same magnification.

Mentions: To examine the ECM dependence of force-induced focal contact formation, we compared responses to pipette pulling of cells plated on fibronectin versus poly-l-lysine–coated substrates in serum-free medium. The assay was performed 1–3 h after seeding, before substantial deposition of ECM proteins by the cells plated on poly-l-lysine occurred. Cells plated on the fibronectin-coated coverslips (Bershadsky et al. 1996) responded to the local force by developing focal adhesions as described above (Fig. 5, A–C and G–I). In contrast, cells plated in serum-free medium on coverslips coated with poly-l-lysine did not form focal contacts after the application of force (Fig. 5, D–F). Thus, engagement of integrin receptors with the corresponding ECM proteins on the substrate is an essential requirement for the induction of focal contact formation by external force. In contrast, the nature of the adhesive material used to coat the pipette tip did not affect its capacity to stimulate focal adhesion formation. Indeed, force applied with a poly-l-lysine–coated pipette was as efficient as a fibronectin coated pipette in the induction of focal adhesions (Fig. 5, compare I with C).


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)

Covering of the substrate with fibronectin is essential for force-induced growth of focal contacts. GFP–vinculin-transfected SV-80 cells plated on poly-l-lysine in serum-free medium do not respond to pulling with a fibronectin-coated pipette by formation of focal contacts (D–F), whereas cells plated on substrate precoated with poly-l-lysine and then coated with fibronectin (fibronectina) produce normal focal contacts (A–C). Under standard conditions (fibronectinb), cells were plated in serum-containing medium and then serum starved. Pulling of these cells with a poly-l-lysine–coated pipette still produces growth of focal contacts (G–I). The positions of pipette immediately after shift are indicated in B, E, and H. Images labeled before and after were taken 1–10 min before and 3–7 min after pipette shift. Bars: (B, E, and H) 10 μm; (C, F, and I) 5 μm. In each before–after pair, the pictures were taken with the same magnification.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192034&req=5

Figure 5: Covering of the substrate with fibronectin is essential for force-induced growth of focal contacts. GFP–vinculin-transfected SV-80 cells plated on poly-l-lysine in serum-free medium do not respond to pulling with a fibronectin-coated pipette by formation of focal contacts (D–F), whereas cells plated on substrate precoated with poly-l-lysine and then coated with fibronectin (fibronectina) produce normal focal contacts (A–C). Under standard conditions (fibronectinb), cells were plated in serum-containing medium and then serum starved. Pulling of these cells with a poly-l-lysine–coated pipette still produces growth of focal contacts (G–I). The positions of pipette immediately after shift are indicated in B, E, and H. Images labeled before and after were taken 1–10 min before and 3–7 min after pipette shift. Bars: (B, E, and H) 10 μm; (C, F, and I) 5 μm. In each before–after pair, the pictures were taken with the same magnification.
Mentions: To examine the ECM dependence of force-induced focal contact formation, we compared responses to pipette pulling of cells plated on fibronectin versus poly-l-lysine–coated substrates in serum-free medium. The assay was performed 1–3 h after seeding, before substantial deposition of ECM proteins by the cells plated on poly-l-lysine occurred. Cells plated on the fibronectin-coated coverslips (Bershadsky et al. 1996) responded to the local force by developing focal adhesions as described above (Fig. 5, A–C and G–I). In contrast, cells plated in serum-free medium on coverslips coated with poly-l-lysine did not form focal contacts after the application of force (Fig. 5, D–F). Thus, engagement of integrin receptors with the corresponding ECM proteins on the substrate is an essential requirement for the induction of focal contact formation by external force. In contrast, the nature of the adhesive material used to coat the pipette tip did not affect its capacity to stimulate focal adhesion formation. Indeed, force applied with a poly-l-lysine–coated pipette was as efficient as a fibronectin coated pipette in the induction of focal adhesions (Fig. 5, compare I with C).

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