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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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Force-induced recruitment of GFP–vinculin is accompanied by growth of focal contacts as revealed by IRM observations. GFP–vinculin (A and D) and IRM (B and E) images essentially overlap each other both 7 min before (A and B) and 9 min after (D and E) pulling. Phase image after pulling is shown in the middle (C). Bars: (C) 20 μm; (A, B, D, and E; shown in E) 5 μm.
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Figure 4: Force-induced recruitment of GFP–vinculin is accompanied by growth of focal contacts as revealed by IRM observations. GFP–vinculin (A and D) and IRM (B and E) images essentially overlap each other both 7 min before (A and B) and 9 min after (D and E) pulling. Phase image after pulling is shown in the middle (C). Bars: (C) 20 μm; (A, B, D, and E; shown in E) 5 μm.

Mentions: To demonstrate that the recruitment of GFP–vinculin corresponded to the real growth of focal adhesions, we visualized the process with IRM. The dark areas in the IRM images, indicating regions of closest apposition between the lower cell surface and the substrate (Abercrombie and Dunn 1975; Izzard and Lochner 1976), overlapped with the bright areas in the GFP–vinculin fluorescence, both in the case of initial dot-like focal complexes (Fig. 4A and Fig. B) and in the case of elongated focal contacts (Fig. 4D and Fig. E) formed after pipette pulling (Fig. 4 C). Experiments with cells transfected with GFP–actin revealed GFP fluorescence overlapping with the dark areas in the IRM images of focal contacts formed in response to applied stress (not shown). Thus, force-induced formation of focal contacts is accompanied by the recruitment of vinculin, paxillin, and actin, and the elongation of the corresponding dark areas in the IRM images. These results indicate that focal contacts induced by external force have the same structural characteristics as those produced by cells during spreading and locomotion.


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)

Force-induced recruitment of GFP–vinculin is accompanied by growth of focal contacts as revealed by IRM observations. GFP–vinculin (A and D) and IRM (B and E) images essentially overlap each other both 7 min before (A and B) and 9 min after (D and E) pulling. Phase image after pulling is shown in the middle (C). Bars: (C) 20 μm; (A, B, D, and E; shown in E) 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Force-induced recruitment of GFP–vinculin is accompanied by growth of focal contacts as revealed by IRM observations. GFP–vinculin (A and D) and IRM (B and E) images essentially overlap each other both 7 min before (A and B) and 9 min after (D and E) pulling. Phase image after pulling is shown in the middle (C). Bars: (C) 20 μm; (A, B, D, and E; shown in E) 5 μm.
Mentions: To demonstrate that the recruitment of GFP–vinculin corresponded to the real growth of focal adhesions, we visualized the process with IRM. The dark areas in the IRM images, indicating regions of closest apposition between the lower cell surface and the substrate (Abercrombie and Dunn 1975; Izzard and Lochner 1976), overlapped with the bright areas in the GFP–vinculin fluorescence, both in the case of initial dot-like focal complexes (Fig. 4A and Fig. B) and in the case of elongated focal contacts (Fig. 4D and Fig. E) formed after pipette pulling (Fig. 4 C). Experiments with cells transfected with GFP–actin revealed GFP fluorescence overlapping with the dark areas in the IRM images of focal contacts formed in response to applied stress (not shown). Thus, force-induced formation of focal contacts is accompanied by the recruitment of vinculin, paxillin, and actin, and the elongation of the corresponding dark areas in the IRM images. These results indicate that focal contacts induced by external force have the same structural characteristics as those produced by cells during spreading and locomotion.

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