Cortactin associates with N-cadherin adhesions and mediates intercellular adhesion strengthening in fibroblasts.
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 microM) 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.
El Sayegh
Tarek Y
TY
CIHR Group in Matrix Dynamics, University of Toronto, Fitzgerald Building, 150 College Street, Ontario, M5S 3E2, Canada. t.elsayegh@utoronto.ca
Arora
Pamela D
PD
Laschinger
Carol A
CA
Lee
Wilson
W
Morrison
Charlotte
C
Overall
Christopher M
CM
Kapus
Andras
A
McCulloch
Christopher A G
CA
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D015820:Cadherins / Q000378:metabolism*; D002448:Cell Adhesion; D002460:Cell Line; D002470:Cell Survival; D051356:Cortactin; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008954:Models, Biological; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
D000199:Actins; D015820:Cadherins; D051356:Cortactin; C495870:Cttn protein, rat; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D009841:Oligonucleotides; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine