Abstract
Focal adhesions (FAs) are force-bearing multiprotein complexes, whose nanoscale organization and signaling are essential for cell growth and differentiation. However, the specific organization of FA components to exert spatiotemporal activation of FA proteins for force sensing and transduction remains unclear. In this study, we unveil the intricacies of FA protein nanoarchitecture and that its dynamics are coordinated by a molecular scaffold protein, BNIP-2, to initiate downstream signal transduction for cardiomyoblast differentiation. Within the FAs, BNIP-2 regulates the nano-organization of focal adhesion kinase (FAK), and the dynamics of FAK, paxillin, and vinculin. Depletion of BNIP-2 resulted in altered focal adhesion numbers and sizes per cell, reduced traction force, and decreased FA sensitivity for mechanosensing. At the molecular level, the loss of BNIP-2 disrupted the FAK-paxillin signaling axis, where FAK inhibition reproduces the effects of BNIP-2 loss by impairing the phosphorylation of both FAK and paxillin. Mechanistically, BNIP-2 preferentially binds to constitutively active FAK and acts as a molecular scaffold to mediate interactions between FAK and paxillin and between paxillin and vinculin. We have validated BNIP-2's role in the FAK-paxillin signaling axis in human embryonic stem cells (hESC). Furthermore, we showed that depletion of BNIP-2 resulted in changes in signature gene targets at the cardiac progenitor stage of differentiation. In summary, we showed that the intricate interplay of FA nanoarchitecture and dynamics, governed by BNIP-2, is crucial for force transduction and biochemical signaling in driving cardiomyoblast differentiation.