Biophysical Society Thematic Meeting | Singapore

Mechanobiology of Disease

Poster Abstracts

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.

46