To determine how cells use integrins to sense and respond to their environment, we investigate the relationship between integrin clustering and the intracellular protein focal adhesion kinase (FAK) experimentally. FAK senses integrin clustering by phosphorylating itself at locations where integrins cluster , and it regulates integrin cluster size by associating with a host of proteins that affect integrin binding and clustering . Because FAK acts as both a sensor and a regulator of integrin clustering, it has been identified as a key protein that facilitates integrin function in both their adhesive role and their signaling role. In this work, we use an in vitro experimental system to investigate how cells use integrin clustering and FAK phosphorylation to sense and respond to ECM protein concentration.
Cells stably expressing the IIb3 integrin were allowed to spread on varying concentrations of the ECM molecule fibrinogen (Fg), a specific ligand for IIb3. By measuring integrin cluster sizes on different concentrations of Fg, we show that lower concentrations of Fg result in larger integrin clusters. To investigate how cells sense and respond to changes in Fg concentration, we examined the role of FAK in integrin cluster regulation by measuring FAK phosphorylation as a function of Fg concentration. Differences in the magnitude and dynamics of FAK phosphorylation resulting from cell adhesion to different ECM ligand densities suggest bi-directional relationships between FAK activation and integrin clustering. The quantitative relationships between integrin clustering and FAK phosphorylation that we elucidate provide new insight into how integrin clustering and intracellular signaling are coupled to relay important information about the ECM into the cell and simultaneously move the cell across the ECM.
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