Probing the conformations of fibronectin in cell culture

Summary form only given. Discovering how mechanical force can regulate the function of proteins and subsequently cell signaling is needed to reveal the molecular basis of several diseases where mechanical forces play a critical role in their onset or progression, including cardiovascular diseases and osteoporosis. We have developed nanoanalytical tools to investigate whether cells can mechanically unfold proteins, and how mechanical stretching changes their function. Fibronectin is an ideal model system to study the effect of force on function, since it mechanically couples the extracellular matrix of cells, via the transmembrane integrins, to the cytoskeleton. Fibronectin is composed of repeating modules that regulates many cellular functions, including cell adhesion, cell migration and proliferation. Fluorescence resonance energy transfer (FRET) between multiple donor and acceptor fluorophores attached to single fibronectin molecules was utilized as a tool to distinguish a range of protein conformations in cell culture, from compact to extended, to hyperextended with several functional modules being unfolded by cells stretching fibronectin fibrils. Structural predictions how mechanical forces may change the functional states of fibronectin where obtained using steered molecular dynamic simulations. Learning how cells can alter the structure and function of ECM proteins by mechanical stretching has profound implications for the design of biomaterials and in tissue engineering.