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.

The Young’s modulus of normal lung tissues ranges from 1 to 5 kPa, whereas fibrotic or lung tumor tissues can be ~30 times stiffer. However, the lung parenchyma microscopically consists of various tissues, and cancer cells in tumors contact stiff collagen-containing fibers during invasion. In this study, we investigated the effect of stiff substrates on A549 cell migration. We fabricated stiff polydimethylsiloxane (PDMS) substrates with different stiffness levels (1.4, 3.4, and 18.3 MPa) by adjusting the cross-linking agent ratio. We examined the distance and the trajectory orientation of A549 cell migration on PDMS substrates and much stiffer 7.7 GPa glass with scratch-wound assays. A549 cells exhibited a collective sheet-like migration pattern toward the wounded area on stiff substrates. The migration range at 18.3 MPa was significantly higher than at 1.4 and 3.4 MPa, and that on glass was significantly higher than that at 18.3 MPa. The cell trajectory orientation on stiff PDMS substrates gradually increased and became constant, whereas that on glass was constant from the initial migration. Our results revealed that stiff substrates affect A549 cells migration in this range.

Despite considerable advancements in biological measurement technologies, capturing the simultaneous temporal changes in various biomolecular concentrations remains a challenge. Overcoming this technical difficulty via data preprocessing could not only clarify the principles of biological functions but also reduce the costs associated with advancing measurement technologies. This review introduces a novel approach to harmonizing heterogeneous time-series data related to molecular signaling and cellular movement. In response to this challenge, we developed and employed a motion-trigger average (MTA) algorithm. The MTA comprehensively screens and averages intracellular molecular activities that coincide with targeted velocity patterns of the moving cell edge. Given that the MTA filters out cell individuality-dependent noise from the data, a straightforward regression equation can correlate edge moving velocities with the molecular activities of various species within the cell. This methodology not only integrates fragmented datasets but also enables the reuse of past data for new analyses. The crux of our discovery is the elucidation of the role that Rho GTPases play in regulating cellular edge dynamics, a finding made possible by adopting the MTA algorithm. Our study suggests that the MTA could become an indispensable tool in data-driven biology, potentially paving the way for considerable insights into dynamic cellular behaviors and the underlying biological principles.

Atomic force microscopy (AFM) has been extensively used to measure the mechanical properties of single cells and tissues with a high force sensitivity. AFM has been established to quantify mechanical differences between cells, e.g., between normal and disease cells, and between untreated (controlled) and treated cells. However, since these biological samples are intrinsically heterogeneous and hierarchical materials, AFM often suffers from the quantification of cell and tissue mechanics due to the high spatial resolution of AFM from the nanoscale to the microscale, comparable to the spatial variation and fluctuation of living systems. Thus, it is still challenging to elucidate universal nano- and micro-mechanical features of living systems using AFM data. This review addresses how AFM can quantify the heterogeneities and hierarchies of cell systems. For single-cell mechanical analysis, AFM has been combined with micropatterned substrate to control cell shape and precisely define the AFM measurement within cells, allowing us to analyze the cell-to-cell mechanical variation. For tissue mechanical analysis, we introduce AFM with a wide-scan range to map multicellular samples from a few hundred to millimeter scales, depending on the type of scanner, allowing us to quantify the spatial mechanical variation in multicellular systems. The reliability and the possibility of AFM to apply mechanics studies on cells and tissues with a range of Pascal (Pa) to MPa are addressed.

Cell adhesion to the extracellular matrix critically influences essential cellular functions such as proliferation, motility, and differentiation, all of which are crucial for maintaining tissue homeostasis. Achieving precise control over cell adhesion to artificial substrates is a pivotal challenge in biomaterials engineering. This minireview aims to elucidate the multifaceted considerations for substrate surface design, grounded in the detailed molecular mechanisms of the cell adhesion complex. We systematically outline key design variables for controlling cell adhesion, such as the spatial arrangement of adhesion ligands, matrix stiffness, and surface lateral deformation. The review also delves into the emerging role of mechanobiology of membrane glycocalyx, with a particular focus on the impact on the formation of focal adhesion complexes. Collectively, these considerations offer a methodology for fine-tuned control of cell adhesion and its subsequent cellular functions on engineered biomaterials.

Atrophy is characterized by muscle weakness and reduced mobility and results from inactivity, aging, cancer cachexia, and excessive glucocorticoid use. Skeletal muscle is essential for locomotion and energy production and accounts for a significant portion of body weight. Loss of muscle function adversely affects athletic performance and metabolism, affecting quality of life. Since there is no definitive cure, preventive measures such as diet and exercise are taken. The development of direct muscle atrophy therapeutics is urgently needed. Historically, animal studies have been used to predict pharmaceutical and food-derived functional ingredients efficacy in humans and to assess pharmacokinetics. However, in recent years there has been a global shift towards non-animal methods (alternatives to animal testing) due to animal welfare concerns. This study introduces a versatile assay for the development of Pharmaceutical and food-derived functional ingredients for the treatment of muscle atrophy using an artificial skeletal muscle contractility-based drug quantification platform. The effectiveness of this platform was demonstrated with quercetin, revealing its direct role in enhancing muscle contractility and promoting myotube formation via upregulation of Myh3 and cell migration by Cxcl5. This method provides a bridge between cellular assays and human studies and will advance research into the treatment of myopathies.

Cell migration plays a crucial role in the physiological and pathological phenomena in our body such as morphogenesis, wound healing, immune response, and cancer metastasis. Cell migration can be regulated by chemical cues, although negative side effects on cell behavior may potentially be induced due to chemical treatments. Mechanical cues are also known to regulate cell migration behavior, however, how cell migration responds to mechanical cues is still under investigation. Therefore, in this study, we developed a confined PDMS-based microchannel with three different widths of 25 μm, 50 μm and 75 μm to study the effect of the width of the microchannel device on cell migration behavior. After the fabrication of the microchannel device, MDCK cells are introduced in the microchannel device for cell culture experiments. When the cells reached the confluent state, the cell migration behavior is observed using time lapse microcopy for 12 h. As a result, the velocity of cells increased with increasing the width of the microchannel from 9.7 μm/h with 25 μm in width to 26.6 μm/h with 75 μm in width. The morphology of the cells and nuclei were not elongated in the middle regions of the microchannels and the cells were oriented parallel to the long axes of the microchannels near the walls. The information on the cell response inside a confined microchannel device can be useful in developing strategies for tissue engineering and cancer metastasis therapies.