Polarized focal adhesion kinase activity within a focal adhesion during cell migration.

Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK-separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK-SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.

The (13)C amide I band is still sensitive to conformation change when the regular amide I band cannot be distinguished at the typical position in H2O.

The attenuated total reflection technique was utilized to obtain FTIR spectra of (13)C-labeled peptides with a sequence of (AAAAK)4AAAAY in H2O. The regular amide I band was not at the typical position as reported in globular proteins, whereas the (13)C amide I band was still sensitive to conformation change.