Investigating the interactions between paxillin, vinculin and talin in regulating the dynamics of cell-extracellular matrix adhesions

Focal adhesions provide a bi-directional, highly dynamic interface between the extracellular and intracellular environments. The abundance and diversity of proteins recruited to the adhesion sites enable focal adhesions to act as highly efficient mechanosensitive signalling hubs that regulate multiple aspects of cell behaviour including cell movement. Paxillin is one of the proteins identified to localise to focal adhesions. This study explored the interaction of paxillin with the focal dhesionassociated proteins talin, vinculin and focal adhesion kinase, and how such interactions are regulated. Biochemical characterisation using a suite of assays, including fluorescence polarisation, microscale thermophoresis, nuclear magnetic resonance, size exclusion chromatography and pulldowns, identified two novel interactions: 1) the paxillin LIM domains bind talin R9-R12, and 2) multiple paxillin LD motifs cooperatively bind to the head and talin domains of vinculin. We propose a model whereby talin facilitates the localisation of paxillin in proximity of vinculin at adhesions. Once recruited, paxillin functions to control the activation state of vinculin and thus regulate adhesion dynamics, influencing a whole host of cellular activities including cell migration and apopotosis.