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Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) comprises the heterogeneous environment outside of cells in a biological system. The ECM is dynamically organized and regulated, and many biomolecules secreted from cells diffuse throughout the ECM, regulating a variety of cellular processes. Therefore, investigation of the diffusive behaviors of biomolecules in the extracellular environment is critical. In this study, we investigated the diffusion coefficients of biomolecules of various sizes, measuring from 1 to 10 nm in radius, by fluorescence correlation spectroscopy in contracted collagen gel caused by fibroblasts, a traditional culture model of dynamic rearrangement of collagen fibers. The diffusion coefficients of the biomolecules in control collagen gel without cells decreased slightly as compared to those in solution, while the diffusion coefficients of biomolecules in the contracted gel at the cell vicinity decreased dramatically. Additionally, the diffusion coefficients of biomolecules were inversely correlated with molecular radius. In collagen gels populated with fibroblasts, the diffusion coefficient at the cell vicinity clearly decreased in the first 24 h of culture. Furthermore, molecular diffusion was greatly restricted, with a central focus on the populated cells. By using the obtained diffusion coefficients of biomolecules, we calculated the collagen fiber condensation ratio by fibroblasts in the cell vicinity at 3 days of culture to represent a 52-fold concentration. Thus, biomolecular diffusion is restricted in the vicinity of the cells where collagen fibers are highly condensed. [ABSTRACT FROM AUTHOR]

The mechanical properties of cells are unique indicators of their states and functions. Though, it is difficult to recognize the degrees of mechanical properties, due to small size of the cell and broad distribution of the mechanical properties. Here, we developed a simple virtual reality system for presenting the mechanical properties of cells and their dispersion using a haptic device and a PC. This system simulates atomic force microscopy (AFM) nanoindentation experiments for floating cells in virtual environments. An operator can virtually position the AFM spherical probe over a round cell with the haptic handle on the PC monitor and feel the force interaction. The Young's modulus of mesenchymal stem cells and HEK293 cells in the floating state was measured by AFM. The distribution of the Young's modulus of these cells was broad, and the distribution complied with a log-normal pattern. To represent the mechanical properties together with the cell variance, we used lognormal distribution-dependent random number determined by the mode and variance values of the Young's modulus of these cells. The represented Young's modulus was determined for each touching event of the probe surface and the cell object, and the haptic device-generating force was calculated using a Hertz model corresponding to the indentation depth and the fixed Young's modulus value. Using this system, we can feel the mechanical properties and their dispersion in each cell type in real time. This system will help us not only recognize the degrees of mechanical properties of diverse cells but also share them with others. [ABSTRACT FROM AUTHOR]