Mutations in the SARS-CoV-2 spike proteins affected the ACE2-binding affinity during the development of Omicron pandemic variants.

Mutations in SARS-CoV-2 caused multiple waves of pandemics. To identify the function of such mutations, we investigated the binding affinity of the S protein with its receptor, ACE2. Omicron BA.1 showed significantly lower binding affinity with human ACE2 than prototype SARS-CoV-2 and Alpha strain, indicating that pre-Omicron to Omicron transition was not mediated by increasing the ACE2-binding affinity. Meanwhile, the later Omicron variants, BA.5 and XBB.1.5, showed significantly higher ACE2-binding affinity, suggesting that the increased ACE2-binding could be involved in the variant transition within Omicron strains. Furthermore, Alpha and Omicron variants, but not prototype SARS-CoV-2, bound mouse ACE2, which lead to a hypothesis that early Omicron strains evolved from Alpha strain by acquiring multiple mutations in mice.

Alpha-mangostin dephosphorylates ERM to induce adhesion and decrease surface stiffness in KG-1 cells.

Surface stiffness is a unique indicator of various cellular states and events and needs to be tightly controlled. α-Mangostin, a natural compound with numerous bioactivities, reduces the mechanical stiffness of various cells; however, the mechanism by which it affects the actin cytoskeleton remains unclear. We aimed to elucidate the mechanism underlying α-mangostin activity on the surface stiffness of leukocytes. We treated spherical non-adherent myelomonocytic KG-1 cells with α-mangostin; it clearly reduced their surface stiffness and disrupted their microvilli. The α-mangostin-induced reduction in surface stiffness was inhibited by calyculin A, a protein phosphatase inhibitor. α-Mangostin also induced KG-1 cell adhesion to a fibronectin-coated surface. In KG-1 cells, a decrease in surface stiffness and the induction of cell adhesion are largely attributed to the dephosphorylation of ezrin/radixin/moesin proteins (ERMs); α-mangostin reduced the levels of phosphorylated ERMs. It further increased protein kinase C (PKC) activity. α-Mangostin-induced KG-1 cell adhesion and cell surface softness were inhibited by the PKC inhibitor GF109203X. The results of the present study suggest that α-mangostin decreases stiffness and induces adhesion of KG-1 cells via PKC activation and ERM dephosphorylation.

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.

Activation of PKC induces leukocyte adhesion by the dephosphorylation of ERM.

Although circulating leukocytes are non-adherent cells, they also undergo adhesion in response to external stimuli. To elucidate this switch mechanism, we investigated PMA-induced cell adhesion in myelomonocytic KG-1 cells. PMA induced microvillius collapse, decrease of cell surface rigidity and exclusion of sialomucin from adhesion sites. All these adhesion-contributing events are linked to dephosphorylation of Ezrin/Radixin/Moesin (ERM) proteins. Indeed, PMA-treatment induced quick decrease of phosphorylated ERM proteins, while expression of Moesin-T558D, a phospho-mimetic mutant, inhibited PMA-induced cell adhesion. PMA-induced cell adhesion and ERM-dephophorylation were inhibited by PKC inhibitors or by a phosphatase inhibitor, indicating the involvement of PKC and protein phophatase in these processes. In peripheral T lymphocytes, ERM-dephosphorylation by adhesion-inducing stimuli was inhibited by a PKC inhibitor. Combined, these findings strongly suggest that external stimuli induce ERM-dephosphorylation via the activation of PKC in leukocytes and that ERM-dephosphorylation leads to leukocytes' adhesion.

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.

Probing actin filament and binding protein interaction using an atomic force microscopy.

Actin filaments play essential roles in many kinds of cellular functions by interacting with hundreds of actin binding proteins. Here we probe the interaction between actin filament and a binding protein, α-actinin, using an atomic force microscopy (AFM) and dynamic force spectroscopy (DFS). The distribution of rupturing events including specific and non-specific interactions of actin filament/α- actinin and BSA/α-actinin were analyzed. The rupture force of the actin filament/α-actinin binding was significantly larger than that of the BSA/α-actinin non-specific interaction, and the peaks represent typical multiple parallel bonds. In addition, based on the rupture forces in different loading rate DFS experiments, the dissociation constant of actin filament/α-actinin binding was estimated. The value is in good agreement with a previously reported value obtained by optical tweezer measurement. We expect that the present method will be useful for interaction measurement of actin filaments and many kinds of binding protein.

Measurement of biomolecular diffusion in extracellular matrix condensed by fibroblasts using fluorescence correlation spectroscopy.

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.

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.

Fabrication of in vitro three-dimensional multilayered blood vessel model using human endothelial and smooth muscle cells and high-strength PEG hydrogel.

We fabricated a three-dimensional multilayered blood vessel model using human cells and high-strength PEG hydrogel. The hydrogel tube was physically suitable for perfusion culture, and cells were cultured on the hydrogel surface by binding with fibronectin. Using the layer-by-layer cell multilayered technique, we successfully constructed an artificial blood vessel.

Evaluation of the actin cytoskeleton state using an antibody-functionalized nanoneedle and an AFM.

A cell diagnosis technique was developed, which uses an Atomic Force Microscope (AFM) and an ultra-thin AFM probe sharpened to a diameter of 200 nm (nanoneedle). Due to the high aspect ratio of the nanoneedle, it was successfully inserted into a living cell without affecting its viability. Furthermore, by functionalizing the nanoneedle with specific antibodies and measuring the unbinding forces ('fishing forces') during evacuation of the nanoneedle from the cell, it was possible to measure specific mechanical interactions between the antibody-functionalized nanoneedle and the intracellular contents of the cell. In this study, an anti-actin-antibody-functionalized nanoneedle was used to evaluate the actin cytoskeleton state in living cells. To examine the effect of cytoskeleton condition on the measured fishing forces, the cytoskeleton-disrupting drugs cytochalasin D (cytD) and Y-27632 were used, showing a marked decrease in the measured fishing forces following incubation with either of the drugs. Furthermore, the technique was used to measure the time course changes in a single cell during incubation with cytD, showing a gradual time-dependent decrease in fishing forces. Even minute doses of the drugs, the effects of which were hardly evident by optical and fluorescence methods, could be clearly detected by the measurement of nanoneedle-protein fishing forces, pointing to the high sensitivity of this detection method. This technique may prove beneficial for the evaluation of cytoskeleton conditions in health and disease, and for the selection of specific cells according to their intracellular protein contents, without the need for introduction of marker proteins into the cell.

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.

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.

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.

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.