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First published online 21 September 2004
doi: 10.1242/jcs.01385
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Research Article |
1 CIHR Group in Matrix Dynamics, University of Toronto, Fitzgerald Building, 150 College Street, Toronto, Ontario, M5S 3E2, Canada
2 Department of Oral Biological and Medical Sciences Faculty of Dentistry, J. B. Macdonald Building, The University of British Columbia, 2199 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada
3 Department of Surgery, University of Toronto, Banting Institute, 100 College Street, Toronto, ON M5G 1L5, Canada
* Author for correspondence (e-mail: t.elsayegh{at}utoronto.ca)
Accepted 24 June 2004
The regulation of N-cadherin-mediated intercellular adhesion strength in fibroblasts is poorly characterized; this is due, in part, to a lack of available quantitative models. We used a recombinant N-cadherin chimeric protein and a Rat 2 fibroblast, donor-acceptor cell model, to study the importance of cortical actin filaments and cortactin in the strengthening of N-cadherin adhesions. In wash-off assays, cytochalasin D (1 µM) reduced intercellular adhesion by threefold, confirming the importance of cortical actin filaments in strengthening of N-cadherin-mediated adhesions. Cortactin, an actin filament binding protein, spatially colocalized to, and directly associated with, nascent N-cadherin adhesion complexes. Transfection of Rat-2 cells with cortactin-specific, RNAi oligonucleotides reduced cortactin protein by 85% and intercellular adhesion by twofold compared with controls (P<0.005) using the donor-acceptor model. Cells with reduced cortactin exhibited threefold less N-cadherin-mediated intercellular adhesion strength compared with controls in wash-off assays using N-cadherin-coated beads. Immunoprecipitation and immunoblotting showed that N-cadherin-associated cortactin was phosphorylated on tyrosine residue 421 after intercellular adhesion. While tyrosine phosphorylation of cortactin was not required for recruitment to N-cadherin adhesions it was necessary for cadherin-mediated intercellular adhesion strength. Thus cortactin, and phosphorylation of its tyrosine residues, are important for N-cadherin-mediated intercellular adhesion strength.
Key words: Cortactin, Cadherin, Actin, Adhesion strength
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