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Mechanobiology of Disease
Poster Abstracts
46
7-POS
Board 7
The p130Cas Focal Adhesion Targeting (FAT) Domain Confers Mechanosensing Function
Peta Bradbury
1,2
, Kylie Turner
1
, Camilla Mitchell
1
, Kaitlyn Griffin
1
, Loretta Lau
1
, Rebecca
Dagg
1
, Elena Taran
3
, Justin Cooper-White
3
, Ben Fabry
4
, Geraldine M. O'Neill
1,2
.
2
University of Sydney, Sydney, Australia,
1
Kids Research Institute at The Children's Hospital at
Westmead, Sydney, Australia,
3
University of Queensland, Brisbane, Australia,
4
University of
Erlangen-Nuremberg, Erlangen, Germany.
The Cas family of focal adhesion docking proteins contain a C-terminal focal adhesion targeting
(FAT) domain that is highly conserved both at the primary sequence level and structurally. The
FAT domain regulates the molecular exchange of NEDD9 and p130Cas members of the Cas
protein family at focal adhesions. However, while p130Cas increases focal adhesion turnover,
NEDD9 conversely stabilizes focal adhesions. In order to determine the role of the FAT domain
in this differential behaviour we have compared the function of wildtype exogenous NEDD9
with an expression construct in which the NEDD9 FAT domain is exchanged for the p130Cas
FAT domain. Strikingly, substitution of the FAT domain amplifies the effects of wildtype
NEDD9. Thus, FRAP analysis reveals significantly slowed exchange of the fusion protein at
focal adhesions and this correlated with significantly slower 2D migration. Atomic Force
Microscopy (AFM) analysis of cell membrane compliance suggests that changes in migration
speed are not due to altered cell membrane stiffness. Further, analysis of cell adhesion force
using a magnetic tweezer device revealed no difference in adhesion force indicating that slowed
migration in cells expressing the p130Cas FAT domain exchanged construct were not due to
changes in adhesion. Instead, we find that the p130Cas FAT domain significantly increases the
tyrosine phosphorylation of the NEDD9 substrate domain. Phosphorylation of this domain has
been previously established to be required for NEDD9 modulation of cell speed. Analysis of cell
migration on surfaces of increasing rigidity revealed a striking reduction in cell motility in cells
expressing the FAT domain exchanged protein. Collectively our data therefore suggest that the
p130Cas FAT domain confers mechanosensing function.