Analysis of chemical and mechanical behaviors in living cells 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 beta-actin in an actomyosin-mediated contractile apparatus called stress fibers, revealing spatially distinct patterns of the multi-physicochemical events, in which the turnover rate of beta-actin was significantly higher at the center of the cell. We also found that the turnover rate is negatively correlated with the strain rate along stress fibers but, interestingly, not with the absolute strain magnitude. Moreover, stress fibers are subjected to centripetal flow as well as both contractile and tensile strains along them. 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.

Macromolecular crowding affects protein diffusion in stress fibers as revealed by fluorescence correlation spectroscopy

“…Given the delayed diffusion of proteins inside stress fibers found in the present study, the birdcage effect may be necessary to expose the inside to the surrounding environment to compensate for the limitation in diffusion because a prompt disassembly of stress fibers is key for cellular adaptation to changes in the surrounding environment [Saito et al, 2022a, Saito et al, 2022b. Thus, our findings seem consistent with the adaptive natures of stress fibers with the highly packed structures.…”