ABSTRACT
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.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Mutation , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Humans , Animals , Mice , COVID-19/virology , COVID-19/metabolism , PandemicsABSTRACT
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.
Subject(s)
Cell Adhesion/drug effects , Cytoskeletal Proteins/metabolism , Leukocytes/cytology , Leukocytes/drug effects , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Protein Kinase C/metabolism , Cell Line , Cytoskeletal Proteins/chemistry , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Humans , Leukocytes/metabolism , Membrane Proteins/chemistry , Microfilament Proteins/chemistry , Phorbol Esters/pharmacology , Phosphorylation/drug effects , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Actin Cytoskeleton/chemistry , Microscopy, Atomic Force/methods , Actin Cytoskeleton/metabolism , Molecular Probes , Protein BindingABSTRACT
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.
Subject(s)
Cell Culture Techniques/methods , Microarray Analysis/methods , Animals , Drug Evaluation, Preclinical/methods , Ethylene Glycol , High-Throughput Screening Assays/methods , Humans , Hydrogels , MammalsABSTRACT
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.
Subject(s)
Hydrogel, Polyethylene Glycol Dimethacrylate/metabolism , Serum/metabolism , Tissue Engineering/methods , Absorbable Implants , Alginates/metabolism , Amylopectin/metabolism , Cells, Cultured , Citrates/metabolism , Collagen/metabolism , Endothelial Cells , Epithelial Cells , Glucuronic Acid/metabolism , Hexuronic Acids/metabolism , Humans , Sodium CitrateABSTRACT
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.
Subject(s)
Cell Adhesion/drug effects , Cytoskeletal Proteins/metabolism , Elasticity/drug effects , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Xanthones/pharmacology , Cells, Cultured , Enzyme Inhibitors/pharmacology , Humans , Indoles/pharmacology , Maleimides/pharmacology , Phosphorylation/drug effects , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolismABSTRACT
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.
Subject(s)
Actins/ultrastructure , Cell Proliferation , Mechanical Phenomena , Mesenchymal Stem Cells/physiology , Mesenchymal Stem Cells/ultrastructure , Animals , Cells, Cultured , Cytoskeleton/ultrastructure , Male , Microscopy, Atomic Force , Rats , Rats, Inbred F344 , Stress Fibers/ultrastructureABSTRACT
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.
Subject(s)
Actins/metabolism , Carrier Proteins/metabolism , Cell Differentiation , Myocytes, Smooth Muscle/cytology , Stress Fibers/metabolism , Animals , Carrier Proteins/genetics , Cell Line , LIM Domain Proteins , Mice , Myocytes, Smooth Muscle/metabolism , PhotobleachingABSTRACT
In this study, we have developed a method of mechanical force detection for ligands bound to receptors on a cell surface, both of which are involved in a signal transduction pathway. This pathway is an autocrine pathway, involving the production of insulin-like growth factor-II (IGF-II) and activation of the IGF-I receptor, involved in myoblast differentiation induced by MyoD in C3H10T1/2 mouse mesenchymal stem cells. Differentiation of C3H10T1/2 was induced with the DNA demethylation agent 5-azacytidine (5-aza). The etched AFM tip used in the force detection had a flat surface of which about 10 µm(2) was in contact with a cell surface. The forces required to rupture the interactions of IGF-IIs on a cell and anti mouse IGF-II polyclonal antibody immobilized on an etched AFM tip were measured within 5 days of induction of differentiation. The mean unbinding force for a single paired antibody-ligand on a cell was about 81 pN, which was measured at a force loading rate of about 440 nN/s. The percentage of unbinding forces over 100 pN increased to 32% after 2 days from the addition of 5-aza to the medium. This method could be used in non-invasive and successive evaluation of a living cell's behavior.
Subject(s)
Insulin-Like Growth Factor II/metabolism , Microscopy, Atomic Force , Receptors, Cell Surface/metabolism , Animals , Antibodies, Monoclonal/metabolism , Azacitidine/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Line , Cytological Techniques , Mice , Receptor, IGF Type 1/metabolism , Recombinant Proteins/metabolismABSTRACT
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.
Subject(s)
Cell Adhesion/drug effects , Cell Adhesion/physiology , Cytoskeletal Proteins/metabolism , Elasticity/physiology , Leukemia, Myeloid/pathology , Leukocytes/metabolism , Leukocytes/physiology , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Actins/metabolism , Amides/pharmacology , Cell Adhesion/genetics , Cell Line, Tumor , Cytochalasin D/pharmacology , Elasticity/drug effects , Fibronectins/metabolism , Humans , Leukemia, Myeloid/metabolism , Microvilli/drug effects , Microvilli/metabolism , Phorbol Esters/pharmacology , Phosphorylation/drug effects , Pyridines/pharmacology , Staurosporine/pharmacologyABSTRACT
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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Biomechanical Phenomena/drug effects , Neoplasms/pathology , Xanthones/pharmacology , Animals , HeLa Cells , Humans , Mice , RAW 264.7 CellsABSTRACT
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.
ABSTRACT
We reported previously that human fibroblasts form clumps when cultured on a dish coated with reconstituted type V collagen fibrils. Essentially all the type V collagen fibrils, initially coated on the dish, were recovered in the cell clumps that had eventually formed during the culture. We interpreted that type V collagen fibrils adhere to cells more strongly than to the dish and are detached by cell movements. In this study, type V collagen was suspended with fibroblasts to examine the fate of the type V collagen fibrils and to determine whether the fibrils affect the behaviour of the cells directly adherent to the dish. The added type V collagen accumulated in the intercellular space concomitantly with the local aggregation of fibroblasts. scanning electron microscope examination indicated that type V collagen fibrils were found in the vicinity of cells in cultures without ascorbic acid where essentially no collagen secretion takes place. These results indicate that type V collagen forms fibrils and the fibrils are accumulated in the intercellular spaces. The accumulated type V collagen fibrils work as a cementing material for cell clump formation. This phenomenon is discussed in relation to the possible involvement of type V collagen fibrils in tissue organization.
Subject(s)
Cell Adhesion/physiology , Collagen Type V , Fibroblasts/metabolism , Animals , Cattle , Cell Proliferation , Cells, Cultured , Collagen Type V/chemistry , Collagen Type V/metabolism , Fibroblasts/cytology , Humans , MiceABSTRACT
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.
ABSTRACT
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.
Subject(s)
Cell Communication , Computer Simulation , Models, Biological , Animals , Cell Adhesion , Cell Count , Cell Culture Techniques , Cell Proliferation , HeLa Cells , Humans , Male , Rats, Inbred F344 , User-Computer InterfaceABSTRACT
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.
Subject(s)
Actin Cytoskeleton/metabolism , Carrier Proteins/metabolism , LIM Domain Proteins/metabolism , Protein Multimerization , Actin Cytoskeleton/chemistry , Animals , Carrier Proteins/chemistry , LIM Domain Proteins/chemistry , Mice , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant ProteinsABSTRACT
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.
ABSTRACT
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.
Subject(s)
Extracellular Matrix/metabolism , Spectrometry, Fluorescence/methods , Cell Line , Collagen/metabolism , Diffusion , HumansABSTRACT
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.
Subject(s)
Blood Vessels/cytology , Endothelium, Vascular/cytology , Hydrogels/chemistry , Muscle, Smooth, Vascular/cytology , Polyethylene Glycols/chemistry , Cells, Cultured , Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/cytology , Humans , Models, Anatomic , Myocytes, Smooth Muscle/cytology , Tissue Engineering/methodsABSTRACT
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.