Advancing FRAP for cell studies: Where there is a new method, there is a new field

抄録

Intracellular proteins are continuously replaced over time by chemical reaction called molecular turnover. Fluorescence recovery after photobleaching (FRAP) is a powerful technique to evaluate the turnover in living cells. In short-term FRAP measurements, individual proteins involved in the turnover are transported by the Brownian motion-based diffusion. In long-term measurements, by contrast, intracellular flow can no longer be ignored, which transports proteins in specific directions within cells and accordingly shifts the spatial distribution of the local chemical equilibrium state. In addition to that, regions initially marked by photobleaching are subject to not only the spatial movement but also microscopic deformations in the presence of contractility produced by active dynamics of motor proteins. Evaluating the complex molecular turnover composed of these multiple physicochemical factors remains an open challenge. Motivated by this situation, FRAP-based novel approaches have been extensively developed to unveil unknown quantities associated with turnover. In other words, advance in FRAP method can potentially open up new ways in cell biology and related physics, in which turnover is critically involved. In this paper aiming at reviewing recent advances in FRAP analysis, we categorize the turnover-associated timescale into (i) the short-term case (~sec) composed of molecular diffusion and the long-term one (~sec–min) driven by (ii) flow-like movement or by (iii) structural deformation. In the case of (i), FRAP combined with a reaction-diffusion model and genetic engineering allows us to distinguish between the pure diffusion-related quantities and the domain-level equilibrium constant. In the case of (ii) and (iii), continuum mechanics-based FRAP (CM-FRAP) model allows for simultaneously quantifying chemical and mechanical behaviors such as the off-rate of fluorescently labeled proteins, the spatially directed movements, and the microscopic deformation. Thus, we describe these recent advances in FRAP analysis as well as conventional techniques, which have greatly contributed to deciphering the complicated intracellular turnover.