A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions

TLDR

Integrin-mediated adhesions include focal complexes, focal adhesions, and fibrillar adhesions, which drive cell spreading, robust ECM attachment, and matrix remodeling, yet the mechanisms controlling their spatial and temporal distribution remain unknown. The authors set out to determine whether paxillin tyrosine phosphorylation controls the assembly and turnover of these adhesion sites. They employed live‑cell imaging, phosphospecific‑antibody labeling, and overexpression of a paxillin tyrosine phosphomimetic mutant to probe this regulation. They found that phosphorylated paxillin promotes lamellipodial protrusion, while non‑phosphorylated paxillin is required for fibrillar adhesion and fibronectin fibrillogenesis; focal adhesion kinase preferentially binds the phosphomimetic form, driving high turnover of focal complexes and focal adhesion translocation, and a mathematical model confirmed that paxillin phosphorylation acts as a major switch governing cellular adhesive phenotype.

Abstract

Diverse cellular processes are carried out by distinct integrin-mediated adhesions. Cell spreading and migration are driven by focal complexes; robust adhesion to the extracellular matrix by focal adhesions; and matrix remodeling by fibrillar adhesions. The mechanism(s) regulating the spatio-temporal distribution and dynamics of the three types of adhesion are unknown. Here, we combine live-cell imaging, labeling with phosphospecific-antibodies and overexpression of a novel tyrosine phosphomimetic mutant of paxillin, to demonstrate that the modulation of tyrosine phosphorylation of paxillin regulates both the assembly and turnover of adhesion sites. Moreover, phosphorylated paxillin enhanced lamellipodial protrusions, whereas non-phosphorylated paxillin was essential for fibrillar adhesion formation and for fibronectin fibrillogenesis. We further show that focal adhesion kinase preferentially interacted with the tyrosine phosphomimetic paxillin and its recruitment is implicated in high turnover of focal complexes and translocation of focal adhesions. We created a mathematical model that recapitulates the salient features of the measured dynamics, and conclude that tyrosine phosphorylation of the adaptor protein paxillin functions as a major switch, regulating the adhesive phenotype of cells.