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.

Mechanosensing-based cell orientation and migration in microenvironments, such as microgrooved surfaces, are essential in biological tissue growth and repair. These responses might be cell type-dependent as they are deeply involved in cellular functions. Despite their orientation and migration responses are depended on cell-substrate adhesion, which is deeply involved with focal adhesions (FAs) of cells, we have limited information on the cell behavior on FA-sized microgrooved surfaces. Here, we systematically investigated the cell orientation and migration behavior of primary porcine aortic smooth muscle cells (PASMs), embryonic rat aortic smooth muscle cells with fibroblast-like phenotype (A7r5), and human cervical cancer cells (HeLa) on a substrate comprised of superficial grooves with three widths (1 μm, 5 μm, and 10 μm) and 150-nm depth, which are the same order of magnitude of the three-dimensional size of FAs. PASM and A7r5 cells including thick bundles of actin fibers with elongated FAs showed pronounced cell polarization and directional migration on the 1 and 5 μm wide grooves. PASMs were more sensitive to the superficial grooves than A7r5 cells. HeLa cells adhering to the grooves had non-oriented actin fibers with smaller FAs, and they did not show specific orientation nor directional migration in any groove type. Atomic force microscopy elucidated that the mechanical tension of the actin fibers in live cells had a significant positive correlation with the length/width ratio of FAs, reflecting the cell alignment and directional migration on the grooves. The increase or decrease in the mechanical tension of the actin fibers improved or diminished the mechanosensing for the grooves, respectively. The results strongly suggested that the differences in cell type-specific alignment and migration become much more pronounced on the microgrooves which are similar in three-dimensional size of FA, and that is useful for clarifying slight differences in force-dependent cellular mechanosensing.