Mechanical stiffness softening and cell adhesion are coordinately regulated by ERM dephosphorylation in KG-1 cells.

Mechanical stiffness is closely related to cell adhesion and rounding in some cells. In leukocytes, dephosphorylation of ezrin/radixin/moesin (ERM) proteins is linked to cell adhesion events. To elucidate the relationship between surface stiffness, cell adhesion, and ERM dephosphorylation in leukocytes, we examined the relationship in the myelogenous leukemia line, KG-1, by treatment with modulation drugs. KG-1 cells have ring-shaped cortical actin with microvilli as the only F-actin cytoskeleton, and the actin structure constructs the mechanical stiffness of the cells. Phorbol 12-myristate 13-acetate and staurosporine, which induced cell adhesion to fibronectin surface and ERM dephosphorylation, caused a decrease in surface stiffness in KG-1 cells. Calyculin A, which inhibited ERM dephosphorylation and had no effect on cell adhesion, did not affect surface stiffness. To clarify whether decreasing cell surface stiffness and inducing cell adhesion are equivalent, we examined KG-1 cell adhesion by treatment with actin-attenuated cell softening reagents. Cytochalasin D clearly diminished cell adhesion, and high concentrations of Y27632 slightly induced cell adhesion. Only Y27632 slightly decreased ERM phosphorylation in KG-1 cells. Thus, decreasing cell surface stiffness and inducing cell adhesion are not equivalent, but these phenomena are coordinately regulated by ERM dephosphorylation in KG-1 cells.

Alpha-mangostin reduces mechanical stiffness of various cells.

Alpha-mangostin (α-mangostin) has been identified as a naturally occurring compound with potential anticancer properties. It can induce apoptosis and inhibit the growth and metastasis of cancer cells. Moreover, α-mangostin reduces the mechanical stiffness of lung cancer cells. The objective of this study was to determine the effect of α-mangostin on the mechanical stiffness of various cells, as well as cell viability. The following cell types were examined: human fibroblast TIG-1 cells, human cancerous HeLa cells, human embryonic kidney HEK293 cells, mouse macrophage RAW 264.7 cells, and human myeloblasts KG-1 cells. Cells were treated with α-mangostin, and then examined for cell viability, actin cytoskeletal structures, and surface mechanical stiffness using atomic force microscopy. α-Mangostin demonstrated cytotoxicity against TIG-1, HeLa, HEK293, and KG-1 cells, but not against RAW 264.7 cells. The cytotoxic effect of α-mangostin varies according to cell type. On the other hand, α-mangostin reduced the mechanical stiffness of all cell types, including RAW 264.7 cells. Upon treatment with α-mangostin, F-actin was slightly reduced but the actin cytoskeletal structures were little altered in these cells. Thus, reducing mechanical stiffness of animal cells is an inherent effect of α-mangostin. Our results show that α-mangostin is a naturally occurring compound with potential to change the actin cytoskeletal micro-structures and reduce the surface stiffness of various cells.

Osteogenic cells form mineralized particles, a few µm in size, in a 3D collagen gel culture.

Osteogenic cells form mineralized matrices in vitro, as well as in vivo. The formation and shape of the mineralized matrices are highly regulated by the cells. In vitro formation of mineralized matrices by osteogenic cells can be a model for in vivo osteogenesis. In this study, using a three-dimensional (3D) collagen gel culture system, we developed a new in vitro model for the formation of mineralized particles, a few µm in size, by the osteogenic cells. Human osteosarcoma (HOS) cells formed spherical mineralized matrices (about 12 µm) at approximately 7 days when cultured with ß-glycerophosphate (ß-GP)-containing culture media on 2D tissue culture plates. Alternately, when they were cultured in a 3D collagen gel containing ß-GP, they formed mineralized particles with about 1.7 µm in the gel at approximately 3 days. Calcium precipitation in the gel was evaluated by measuring the gel turbidity. This type of mineralization of HOS cells, which formed mineralized particles inside the gel, was also observed in a peptide-based hydrogel culture. The mineralized particles were completely diminished by inhibiting the activity of Pit-1, phosphate cotransporter, of the HOS cells. When mouse osteoblast-like MC3T3-E1 cells, which form large and flat mineralized matrices in 2D osteogenic conditions at approximately 3 weeks of culture, were cultured in a 3D collagen gel, they also formed mineralized particles in the gel, similar to those in HOS cells, at approximately 18 days. Thus, osteogenic cells cultured in the 3D collagen gel form mineralized particles over a shorter period, and the mineralization could be easily determined by gel turbidity. This 3D gel culture system of osteogenic cells acts as a useful model for cells forming particle-type mineralized matrices, and we assume that the mineralized particles in the 3D hydrogel are calcospherulites, which are derived from matrix vesicles secreted by osteogenic cells.

Alpha-mangostin inhibits the migration and invasion of A549 lung cancer cells.

Several studies have indicated that α-mangostin exerts anti-metastasis and anti-subsistence effects on several types of cancer cells. Especially, the anti-metastatic effect of α-mangostin on cancer cells is a prospective function in cancer treatment. However, the metastasis process is complicated, and includes migration, invasion, intravasation, and extravasation; thus, the main target of anti-metastatic effect of α-mangostin is not known. In this study, we investigated the effects of α-mangostin on the invasion, subsistence, and migration of lung cancer cells under co-culture conditions with normal cells and regular mono-culture conditions. We found that α-mangostin killed the lung cancer and normal cells in a dose-dependent manner. Furthermore, the alteration in the surface mechanical properties of cells was examined by using atomic force microscopy. Although the α-mangostin concentrations of 5 and 10 µM did not affect the short-term cell viability, they considerably decreased the Young's modulus of lung cancer cells implying a decline in cell surface actin cytoskeletal properties. Additionally, these concentrations of α-mangostin inhibited the migration of lung cancer cells. In co-culture conditions (cancer cells with normal cells), the invasive activities of cancer cells on normal cells were discernibly observed, and was inhibited after treatment with 5 and 10 µM of α-mangostin. Taken together, α-mangostin suppressed the subsistence of lung cancer cells and displayed anti-metastatic activities by inhibiting the migration and invasion, and reducing the actin cytoskeleton of cancer cells. Our findings suggest that α-mangostin could be a potential therapeutic agent for cancer treatment.

Cysteine-rich protein 2 accelerates actin filament cluster formation.

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.

In silico characterization of cell-cell interactions using a cellular automata model of cell culture.

BACKGROUND: Cell proliferation is a key characteristic of eukaryotic cells. During cell proliferation, cells interact with each other. In this study, we developed a cellular automata model to estimate cell-cell interactions using experimentally obtained images of cultured cells. RESULTS: We used four types of cells; HeLa cells, human osteosarcoma (HOS) cells, rat mesenchymal stem cells (MSCs), and rat smooth muscle A7r5 cells. These cells were cultured and stained daily. The obtained cell images were binarized and clipped into squares containing about 104 cells. These cells showed characteristic cell proliferation patterns. The growth curves of these cells were generated from the cell proliferation images and we determined the doubling time of these cells from the growth curves. We developed a simple cellular automata system with an easily accessible graphical user interface. This system has five variable parameters, namely, initial cell number, doubling time, motility, cell-cell adhesion, and cell-cell contact inhibition (of proliferation). Within these parameters, we obtained initial cell numbers and doubling times experimentally. We set the motility at a constant value because the effect of the parameter for our simulation was restricted. Therefore, we simulated cell proliferation behavior with cell-cell adhesion and cell-cell contact inhibition as variables. By comparing growth curves and proliferation cell images, we succeeded in determining the cell-cell interaction properties of each cell. Simulated HeLa and HOS cells exhibited low cell-cell adhesion and weak cell-cell contact inhibition. Simulated MSCs exhibited high cell-cell adhesion and positive cell-cell contact inhibition. Simulated A7r5 cells exhibited low cell-cell adhesion and strong cell-cell contact inhibition. These simulated results correlated with the experimental growth curves and proliferation images. CONCLUSIONS: Our simulation approach is an easy method for evaluating the cell-cell interaction properties of cells.

Distinct mechanical behavior of HEK293 cells in adherent and suspended states.

The mechanical features of individual animal cells have been regarded as indicators of cell type and state. Previously, we investigated the surface mechanics of cancer and normal stromal cells in adherent and suspended states using atomic force microscopy. Cancer cells possessed specific mechanical and actin cytoskeleton features that were distinct from normal stromal cells in adherent and suspended states. In this paper, we report the unique mechanical and actin cytoskeletal features of human embryonic kidney HEK293 cells. Unlike normal stromal and cancer cells, the surface stiffness of adherent HEK293 cells was very low, but increased after cell detachment from the culture surface. Induced actin filament depolymerization revealed that the actin cytoskeleton was the underlying source of the stiffness in suspended HEK293 cells. The exclusive mechanical response of HEK293 cells to perturbation of the actin cytoskeleton resembled that of adherent cancer cells and suspended normal stromal cells. Thus, with respect to their special cell-surface mechanical features, HEK293 cells could be categorized into a new class distinct from normal stromal and cancer cells.

Transfer printing of transfected cell microarrays from poly(ethylene glycol)-oleyl surfaces onto biological hydrogels.

We have developed a novel technique for constructing microarrays of transfected mammalian cells on or in extracellular matrix (ECM) hydrogels by transfer printing from patterned poly(ethylene glycol) (PEG)-oleyl surfaces. A mixed solution of small interfering RNA (siRNA) and a transfection reagent was spotted on PEG-oleyl-coated glass slides using an ink-jet printer, and the cells were then transiently immobilized on the patterned transfection mixtures. After overlaying an ECM hydrogel sheet onto the immobilized cells, the cells sandwiched between the glass slide and the hydrogel sheet were incubated at 37°C for simultaneous transfection of siRNA into cells and adhesion of cells to the hydrogel sheet. Transfer of the adhered, transfected cells was completed by peeling off the hydrogel sheet. The knockdown of a model gene in the transferred cell microarray by the transfected siRNA was successfully confirmed. Transfected cell microarrays were also embedded within three-dimensional ECM hydrogels. In the three-dimensional hydrogel, the inhibition effect of siRNA on cancer cell invasion was evaluated by quantifying the size of cell clusters on the microarrays. These results indicate that transfection of cell microarrays on or in a biological matrix is a promising technique for high-throughput screening of disease-related genes by direct observation of cellular phenomena in a physiologically relevant environment.

Actin-based biomechanical features of suspended normal and cancer cells.

The mechanical features of individual cells have been regarded as unique indicators of their states, which could constantly change in accordance with cellular events and diseases. Particularly, cancer progression was characterized by the disruption and/or reorganization of actin filaments causing mechanical changes. Thus, mechanical characterization of cells could become an effective cytotechnological approach for early detection of cancer. To develop mechanical cytotechnology, it would be necessary to clarify the mechanical properties in various cell adhesion states. In this study, we investigated the surface mechanical behavior of cancer and normal cells in the adherent and suspended states using atomic force microscopy. Adherent normal stromal cells showed high surface stiffness due to developed actin cap structures on their apical surface, whereas cancer cells did not have developed filamentous actin structures, and their surface stiffness was low. Upon cell detachment from the substrate, filamentous actin structures of adherent normal stromal cells reorganized to the cortical region and their surface stiffness decreased consequently however, the stiffness of suspended normal cells remained higher than that of cancer cells. These suspended state actin structures were similar, regardless of the cell type. Furthermore, the mechanical responses of the cancer and normal stromal cells to perturbation of the actin cytoskeleton were different, suggesting distinct regulatory mechanisms for actin cytoskeleton in cancer and normal cells in both adherent and suspended states. Therefore, cancer cells possess specific mechanical and actin cytoskeleton features different from normal stromal cells.

Rapidly serum-degradable hydrogel templating fabrication of spherical tissues and curved tubular structures.

Development of the techniques for fabricating three-dimensional tissues still poses significant challenges for tissue engineering. We used hydrogels obtained from phenol-substituted amylopectin (AP-Ph) as templates for preparing multicellular spherical tissues (MSTs) and endothelialized curved tubular structures in type I collagen gel. AP-Ph hydrogel microparticles of diameter 200 µm and fibers of diameter 500 µm disappeared within hours of soaking in a serum-containing medium. HeLa cells and human endothelial cells were enclosed in the microparticles and hydrogel fibers, respectively, and then embedded in Ca-alginate microcapsules or the collagen gel. The enclosed cells were released in cavities formed by hydrogel degradation in the serum-containing medium. The released HeLa cells in the spherical cavities grew and formed MSTs, eventually filling the cavities. The spherical tissues were easily harvested by liquefying the Ca-alginate hydrogel microcapsule membrane by chelation using sodium citrate. The released endothelial cells grew on the tubular cavity surfaces and formed tubular structures. An endothelial cell network was formed by cell migration into the collagen gel. These results demonstrate the potential of serum-degradable AP-Ph hydrogels in constructing three-dimensional tissues.

Cortical rigidity of round cells in mitotic phase and suspended state.

This paper describes the results of the analysis of cortical rigidity in two round cell states: mitotic round cells and detached round cells after trypsinization using atomic force microscopy (AFM). These two states are primary cell events with dynamic morphological alterations in vitro. The trypsinized detached cells were fixed on the substrate of membrane anchoring oleyl surface. Fluorescent images taken by confocal laser scanning microscopy revealed diverse cell surface protrusions and cortical actin development in the round cells under different conditions. Although the cortical actin of these cells seemed to develop similarly, cortical rigidity of the trypsinized round cells showed greater stiffness than that of mitotic round cells. The elasticity measurements by AFM may detect invisible information about the maturation or strength of F-actin structures and such measurements may indicate that the strength of the actomyosin cortex would be higher in trypsinized round cells compared to mitotic cells. The mechanical properties can help provide better insights into the characteristics of the actin cytoskeleton network in vicinity of cell surface during dynamic morphological alterations.

Simple display system of mechanical properties of cells and their dispersion.

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 log-normal 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.

Mechanical force-based probing of intracellular proteins from living cells using antibody-immobilized nanoneedles.

We developed a method combining atomic force microscopy (AFM) and antibody-immobilized nanoneedles to discriminate living cells by probing intracellular cytoskeletal proteins without the need for cell labeling. The nanoneedles are ultra-thin AFM probes sharpened to 200 nm in diameter. While retracting a nanoneedle inserted into a cell, we measured the mechanical force needed to unbind the antibody-target protein complex. Using this method, the intermediate filament protein, nestin and neurofilament were successfully detected in mouse embryonic carcinoma P19 cells and rat primary hippocampal cells within a minute for a single cell and cell differentiation states could be determined. Additionally, the measured magnitude of the force detecting nestin was indicative of the malignancy of breast cancer cells. This method was shown to affect neither the doubling time of cells nor does it leave extrinsic antibodies within the examined cells, allowing to be used in subsequent analyses in their native state.

Regulation of cysteine-rich protein 2 localization by the development of actin fibers during smooth muscle cell differentiation.

Cysteine-rich protein 2 (CRP2) is a cofactor for smooth muscle cell (SMC) differentiation. Here, we examined the mechanism of CRP2 distribution dynamics during SMC differentiation. CRP2 protein directly associated with F-actin through its N-terminal LIM domain and Gly-rich region, as determined by ELISA. In undifferentiated cells that contain few actin stress fibers, CRP2 was broadly distributed throughout the whole cell, including the nucleus. After induction of SMC differentiation, CRP2 localized to actin stress fibers as they formed. The stress fiber-localized CRP2 entered the nucleus because of induced actin depolymerization. These CRP2 dynamics were reproduced by in silico simulation. CRP2 localization dynamics, which affect CRP2 function, are regulated by the formation of actin stress fibers in conjunction with SMC differentiation.

Physical properties of mesenchymal stem cells are coordinated by the perinuclear actin cap.

Mesenchymal stem cells (MSCs) have been extensively investigated for their applications in regenerative medicine. Successful use of MSCs in cell-based therapies will rely on the ability to effectively identify their properties and functions with a relatively non-destructive methodology. In this study, we measured the surface stiffness and thickness of rat MSCs with atomic force microscopy and clarified their relation at a single-cell level. The role of the perinuclear actin cap in regulating the thickness, stiffness, and proliferative activity of these cells was also determined by using several actin cytoskeleton-modifying reagents. This study has helped elucidate a possible link between the physical properties and the physiological function of the MSCs, and the corresponding regulatory role of the actin cytoskeleton.

Exogenous type I collagen facilitates osteogenic differentiation and acts as a substrate for mineralization of rat marrow mesenchymal stem cells in vitro.

We cultured rat mesenchymal stem cells (MSCs) in a medium containing beta-glycerophosphate, ascorbic acid, and dexamethasone to show in vitro osteogenic differentiation of the MSCs. The differentiation was enhanced by adding solubilized type I collagen to the medium as evidenced by higher alkaline phosphatase activity as well as more calcium deposition than that without collagen. The exogenous collagen integrated well with the mineralized bone matrix and maintained the native triple helical structure. These findings indicate that exogenously supplemented type I collagen acts as a component of the extracellular matrix of MSCs, and deposited type I collagen facilitates osteogenic differentiation followed by maturation of mineralized bone matrix.

Reconstituted type V collagen fibrils as cementing materials in the formation of cell clumps in culture.

Previous studies have reported that type V collagen is an anti-adhesive substrate for cultured cells in that the cells detach from culture dishes coated with type V collagen molecules or polypeptides derived from them. We have noticed that human fetal lung fibroblasts (TIG-1) initially show no reduction in adherence to and spreading on a dish coated with reconstituted type V collagen fibrils but eventually detach from the dish and form cell clumps. To determine the way in which reconstituted type V collagen fibrils are involved in cell clump formation, we have followed the fate of the fluorescence of type V collagen fibrils pre-labeled with fluorescein isothiocyanate. Essentially, all the fluorescence disappeared from the dish surface as the cells detached and was condensed in the cell clumps. The cells that were recovered from clumps and dissociated into separate cells by trypsin treatment proliferated normally after they were seeded on a bare culture dish. This result and those from gel electrophoresis, fluorescence microscopy, and a cell proliferation assay indicate that the cell detachment from the dish is not caused by cell necrosis or apoptosis but by cellular motility together with the unique features of type V collagen fibrils. Not only the adherence of type V collagen fibrils to TIG-1 cells is much stronger than that to the culture dish, but the fibrils are retained on the cellular surface. The strong adherence of type V collagen fibrils to cells plays a role in cementing TIG-1 cells together.

Three-dimensional visualization analysis of in vitro cultured bone fabricated by rat marrow mesenchymal stem cells.

Marrow mesenchymal stem cells are well known for their differentiation into bone-forming osteoblasts and in vitro mineralized tissue formation. However, process details, including tissue structure and cellular environments, remain unclear. The present study demonstrates three-dimensional visualization of tissue fabricated by culturing MSCs in the presence of calcein, a fluorescent marker for bone mineralization. The 3D visualization was performed by computer-assisted confocal laser scanning microscopy and revealed that the in vitro tissue consisted of layers of a mineralized matrix with round cells in the matrix lacunae, an unmineralized matrix (osteoid), and osteoblastic cells on the osteoid surface. The findings show that the mineralization by cultured MSCs is an in vitro counterpart of in vivo bone formation and indicate that the novel technique of visualization without tissue fixation could be useful for continuous monitoring of tissue organization in an ongoing culture.