The fully linked HTML version of this article has now been published.
JCS ePress
online publication date 23 Jan 2003
doi: 10.1242/jcs.00284
Research Article
Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption
Jochen Kirchner,
Zvi Kam,
Gila Tzur,
Alexander D. Bershadsky,
and
Benjamin Geiger*
* Author for correspondence (e-mail: benny.geiger{at}weizmann.ac.il)
Tyrosine phosphorylation of focal adhesion components is involved in the regulation of focal adhesion formation and turnover, yet the underlying molecular mechanisms are still poorly defined. In the present study, we have used quantitative fluorescence microscopy to investigate the dynamic relationships between the incorporation of new components into growing focal adhesions and tyrosine phosphorylation of these sites. For this purpose, a new approach for monitoring phosphotyrosine levels in live cells was developed, based on a 'phosphotyrosine reporter' consisting of yellow fluorescent protein fused to two consecutive phosphotyrosine-binding Src-homology 2 (SH2)-domains derived from pp60c-Src. This YFP-dSH2 localized to cell-matrix adhesions and its intensity was linearly correlated with that of an anti-phosphotyrosine antibody labeling. The differential increase in vinculin and phosphotyrosine levels was examined in live cells by two-color time-lapse movies of CFP-vinculin and YFP-dSH2. In this study, focal adhesion growth was triggered by microtubule disruption, which was previously shown to stimulate focal adhesion development by inducing cellular contraction. We show here that, 2 minutes after addition of the microtubule-disrupting drug nocodazole, the local densities of the focal adhesion-associated proteins vinculin, paxillin and focal adhesion kinase (FAK) are significantly elevated and the focal adhesion area is increased, whereas elevation in tyrosine phosphorylation inside the growing adhesions occurs only a few minutes later. Phosphotyrosine and FAK density reach their maximum levels after 10 minutes of treatment, whereas vinculin and paxillin levels as well as focal adhesion size continue to grow, reaching a plateau at about 30 minutes. Our findings suggest that protein recruitment and growth of focal adhesions are an immediate and direct result of increased contractility induced by microtubule disruption, whereas tyrosine phosphorylation is activated later.
This article has been cited by other articles:
|
|
|
|
 
Y. Lim, S.-T. Lim, A. Tomar, M. Gardel, J. A. Bernard-Trifilo, X. L. Chen, S. A. Uryu, R. Canete-Soler, J. Zhai, H. Lin, et al.
PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility
J. Cell Biol.,
January 10, 2008;
180(1):
187 - 203.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. Humphries, P. Wang, C. Streuli, B. Geiger, M. J. Humphries, and C. Ballestrem
Vinculin controls focal adhesion formation by direct interactions with talin and actin
J. Cell Biol.,
December 3, 2007;
179(5):
1043 - 1057.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W.-h. Guo and Y.-l. Wang
Retrograde Fluxes of Focal Adhesion Proteins in Response to Cell Migration and Mechanical Signals
Mol. Biol. Cell,
November 1, 2007;
18(11):
4519 - 4527.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Schmitter, S. Pils, S. Weibel, F. Agerer, L. Peterson, A. Buntru, K. Kopp, and C. R. Hauck
Opa Proteins of Pathogenic Neisseriae Initiate Src Kinase-Dependent or Lipid Raft-Mediated Uptake via Distinct Human Carcinoembryonic Antigen-Related Cell Adhesion Molecule Isoforms
Infect. Immun.,
August 1, 2007;
75(8):
4116 - 4126.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. D.-A. Tran, T. P. Marmo, A. A. Salam, S. Che, E. Finkelstein, R. Kabarriti, H. S. Xenias, R. Mazitschek, C. Hubbert, Y. Kawaguchi, et al.
HDAC6 deacetylation of tubulin modulates dynamics of cellular adhesions
J. Cell Sci.,
April 15, 2007;
120(8):
1469 - 1479.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. G. Deschesnes, A. Patenaude, J. L. C. Rousseau, J. S. Fortin, C. Ricard, M.-F. Cote, J. Huot, R. C.-Gaudreault, and E. Petitclerc
Microtubule-Destabilizing Agents Induce Focal Adhesion Structure Disorganization and Anoikis in Cancer Cells
J. Pharmacol. Exp. Ther.,
February 1, 2007;
320(2):
853 - 864.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Ballestrem, N. Erez, J. Kirchner, Z. Kam, A. Bershadsky, and B. Geiger
Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer
J. Cell Sci.,
March 1, 2006;
119(5):
866 - 875.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Zlatanov, T. Groth, A. Lendlein, and G. Altankov
Dynamics of {beta}1-Integrins in Living Fibroblasts--Effect of Substratum Wettability
Biophys. J.,
November 1, 2005;
89(5):
3555 - 3562.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Robles, S. Woo, and T. M. Gomez
Src-Dependent Tyrosine Phosphorylation at the Tips of Growth Cone Filopodia Promotes Extension
J. Neurosci.,
August 17, 2005;
25(33):
7669 - 7681.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Kodama, T. Lechler, and E. Fuchs
Coordinating cytoskeletal tracks to polarize cellular movements
J. Cell Biol.,
October 25, 2004;
167(2):
203 - 207.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Watt
Best Paper Award 2003
J. Cell Sci.,
April 15, 2004;
117(10):
1871 - 1871.
[Full Text]
[PDF]
|
|
|
© The Company of Biologists Ltd 2003
