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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 focal contact formation as revealed by GFP–paxillin fluorescence. (A and C) GFP–paxillin distribution at the leading edge of serum-starved SV-80 cell 2 min before (A) and 1.5 min after (C) development of the pulling force. (B) Phase image showing the pipette location after the shift. Bar, 10 μm.
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Figure 3: Force-induced focal contact formation as revealed by GFP–paxillin fluorescence. (A and C) GFP–paxillin distribution at the leading edge of serum-starved SV-80 cell 2 min before (A) and 1.5 min after (C) development of the pulling force. (B) Phase image showing the pipette location after the shift. Bar, 10 μm.

Mentions: Force-induced changes of adhesion sites visualized in live cells by expression of GFP–paxillin were similar to those visualized by GFP–vinculin fluorescence. Incubation of cells in serum-free medium greatly reduced the size and intensity of GFP–paxillin spots at the cell edge (Fig. 3 A), whereas the pipette shift (Fig. 3 B) induced formation of typical focal contacts elongated in the direction of pulling (Fig. 3 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)

Force-induced focal contact formation as revealed by GFP–paxillin fluorescence. (A and C) GFP–paxillin distribution at the leading edge of serum-starved SV-80 cell 2 min before (A) and 1.5 min after (C) development of the pulling force. (B) Phase image showing the pipette location after the shift. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Force-induced focal contact formation as revealed by GFP–paxillin fluorescence. (A and C) GFP–paxillin distribution at the leading edge of serum-starved SV-80 cell 2 min before (A) and 1.5 min after (C) development of the pulling force. (B) Phase image showing the pipette location after the shift. Bar, 10 μm.
Mentions: Force-induced changes of adhesion sites visualized in live cells by expression of GFP–paxillin were similar to those visualized by GFP–vinculin fluorescence. Incubation of cells in serum-free medium greatly reduced the size and intensity of GFP–paxillin spots at the cell edge (Fig. 3 A), whereas the pipette shift (Fig. 3 B) induced formation of typical focal contacts elongated in the direction of pulling (Fig. 3 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