Project Summary The over-arching goal of this proposal is to establish the predictive multi-scale mathematical model to decipher the mechanism of durotaxis. Durotaxis is the preference of cells migrating toward a stiffer extracellular matrix (ECM) and has important roles in many biological processes, ranging from embryo development to tumor metastasis. Focal adhesion (FA) is the functional unit of durotaxis; it an integrin-based multi-protein transmembrane linkage, through which cell exerts actin cytoskeleton-based traction force to tug the ECM and sense the stiffness. Despite the high relevance to biomedical applications, it is not well understood how FA mediates mechanosensing of ECM stiffness and drives durotaxis, largely because predictive mathematical models lag behind the descriptive experimental finding in the field. At single-FA level, while previous models explain molecular-clutch behaviors in FA mechanosensing, they cannot explain how and why FA- localized protein activities adapt to environments by distinctive spatial-temporal patterns (akin to footprints) that are demonstrated to be essential for durotaxis. The full underlying mechanisms of the FA-localized “footprint” and its exact roles in durotaxis are thus unknown. Further, durotaxis must coordinate movements of cell body and protrusion/retraction of cell edge. While the FA-mediated tractions drive the cell body, how the FA-localized mechanosensing events coordinate with the cell edge dynamics is unknown. Last, at a single-cell level, there exist many FAs at different developmental stages at any time. It is not understood how the cell integrates the mechanosensing activities of individual FAs to drive durotaxis. A predictive model that meaningfully engages with experiments is desirable and likely holds the key to decipher durotaxis. Toward this goal, we have been and will uniquely integrate mathematical modeling in iterative dialogues with experimental testing. The central hypothesis is: FA-localized spatial-temporal dynamics of the traction force generation and transmission defines FA-mediated mechanosensing and durotaxis. The basis of this proposal is our previous findings. We built the first mathematical model that captures the essence of entire FA maturation process. That is, FA evolves from a nascent complex, the centripetally growing FA that couples the retrograde flux of branching actin network, to the mature FA that transmits the stress fiber (SF)-mediated contractions onto ECM. This model uniquely links the FA-localized fine features of protein activities – emerging from FA maturation process – to FA mechanosensing events. The model predicted and was experimentally confirmed that a negative feedback between the elongation and contractility of the FA-engaging SF underlies the FA-localized traction oscillation and mechanosensing of ECM stiffness. Ushered by these findings, our specific aims are to determine: 1) how FA force-transmission and SF elongation cross-talk in FA ...