Analysis of chemomechanical behavior of stress fibers by continuum mechanics-based FRAP.

ABSTRACT

Fluorescence recovery after photobleaching (FRAP) is a common technique to analyze the turnover of molecules in living cells. Numerous physicochemical models have been developed to quantitatively evaluate the rate of turnover driven by chemical reaction and diffusion that occurs in a few seconds to minutes. On the other hand, they have limitations in interpreting long-term FRAP responses where intracellular active movement inevitably provides target molecular architectures with additional effects other than chemical reaction and diffusion, namely directed transport and structural deformation. To overcome the limitations, we develop a continuum mechanics-based model that allows for decoupling FRAP response into the intrinsic turnover rate and subcellular mechanical characteristics such as displacement vector and strain tensor. Our approach was validated using fluorescently labeled ß-actin in an actomyosin-mediated contractile apparatus called stress fibers, revealing spatially distinct patterns of the multi-physicochemical events, in which the turnover rate, which represents effective off-rate of ß-actin, was significantly higher at the center of the cell. We also found that the turnover rate is negatively correlated with the rate of displacement or velocity along stress fibers but, interestingly, not with the absolute magnitude of strain. Moreover, stress fibers are subjected to centripetal flow that is facilitated by the circulation of actin molecules. Taken together, this novel framework for long-term FRAP analysis allows for unveiling the contribution of overlooked microscopic mechanics to molecular turnover in living cells.