Elucidating how protein turnover in cell-ECM adhesion stabilizes tissue structure during development

Morphogenesis is the process by which cells rearrange to form complex three dimensional structures. Cell to extracellular matrix (ECM) adhesion, primarily mediated by Integrins, is essential for the formation and maintenance of tissue architecture. A critical way to regulate cell-ECM adhesion is by modulating the turnover of Integrins and their adhesion complex, and thereby modulating the stability of Integrin-based adhesions. We previously showed that mechanical force stabilizes Integrin-based adhesions during development by modulating Integrin turnover. Here, we extend our studies to understand how mechanical stress impacts the dynamics of the cytoplasmic adaptor protein Talin, a critical regulator of Integrin function. Using Fluorescence Recovery After Photobleaching (FRAP) analysis in combination with a newly developed mathematical model that encompasses the complexities of Talin turnover, we determined that mechanical force stabilizes cell-ECM adhesion by increasing the rate of assembly of Talin-mediated adhesion complexes. To dissect the mechanisms that regulate Talin turnover downstream of mechanical force, we used point mutations of Talin which abrogate specific functions of the Integrin adhesion complex and measured turnover kinetics. We found that the activation of Integrins, resulting in increased affinity for ECM ligands, is a crucial process to regulate adhesion complex turnover. To further investigate the role of Integrin activation in regulating adhesion stability, we introduced small molecules known to induce “outside-in activation” of Integrins in vitro into live, intact embryos. This approach revealed that outside-in activation stabilizes cell-ECM adhesion by decreasing Integrin endocytosis; similarly to what we have previously seen when mechanical force is increased. Based on this finding, we propose that mechanical force may induce changes in Integrin activation in order to stabilize cell-ECM adhesions. Overall, we show that Integrin activation is a key mechanism that regulates cell-ECM adhesion stabilization during embryogenesis.Medicine, Faculty ofGraduat