Fascin in lamellipodia contributes to cell elasticity by controlling the orientation of filamentous actin.

Fascin, an actin-bundling protein, is present in the filopodia and lamellipodia of growth cones. However, few studies have examined lamellipodial fascin because it is difficult to observe. In this study, we evaluated lamellipodial fascin. We visualized the actin meshwork of lamellipodia in live growth cones by super-resolution microscopy. Fascin was colocalized with the actin meshwork in lamellipodia. Ser39 of fascin is a well-known phosphorylation site that controls the binding of fascin to actin filaments. Fluorescence recovery after photobleaching experiments with confocal microscopy showed that binding of fascin was controlled by phosphorylation of Ser39 in lamellipodia. Moreover, TPA, an agonist of protein kinase C, induced phosphorylation of fascin and dissociation from actin filaments in lamellipodia. Time series images showed that dissociation of fascin from the actin meshwork was induced by TPA. As fascin dissociated from actin filaments, the orientation of the actin filaments became parallel to the leading edge. The angle of actin filaments against the leading edge was changed from 73° to 15°. This decreased the elasticity of the lamellipodia by 40%, as measured by atomic force microscopy. These data suggest that actin bundles made by fascin contribute to elasticity of the growth cone.

Measuring viscoelasticity of soft biological samples using atomic force microscopy.

Mechanical properties play important roles at different scales in biology. At the level of a single cell, the mechanical properties mediate mechanosensing and mechanotransduction, while at the tissue and organ levels, changes in mechanical properties are closely connected to disease and physiological processes. Over the past three decades, atomic force microscopy (AFM) has become one of the most widely used tools in the mechanical characterization of soft samples, ranging from molecules, cell organoids and cells to whole tissue. AFM methods can be used to quantify both elastic and viscoelastic properties, and significant recent developments in the latter have been enabled by the introduction of new techniques and models for data analysis. Here, we review AFM techniques developed in recent years for examining the viscoelastic properties of cells and soft gels, describe the main steps in typical data acquisition and analysis protocols, and discuss relevant viscoelastic models and how these have been used to characterize the specific features of cellular and other biological samples. We also discuss recent trends and potential directions for this field.

Spontaneous Spatial Correlation of Elastic Modulus in Jammed Epithelial Monolayers Observed by AFM.

For isolated single cells on a substrate, the intracellular stiffness, which is often measured as the Young's modulus, E, by atomic force microscopy (AFM), depends on the substrate rigidity. However, little is known about how the E of cells is influenced by the surrounding cells in a cell population system in which cells physically and tightly contact adjacent cells. In this study, we investigated the spatial heterogeneities of E in a jammed epithelial monolayer in which cell migration was highly inhibited, allowing us to precisely measure the spatial distribution of E in large-scale regions by AFM. The AFM measurements showed that E can be characterized using two spatial correlation lengths: the shorter correlation length, lS, is within the single cell size, whereas the longer correlation length, lL, is longer than the distance between adjacent cells and corresponds to the intercellular correlation of E. We found that lL decreased significantly when the actin filaments were disrupted or calcium ions were chelated using chemical treatments, and the decreased lL recovered to the value in the control condition after the treatments were washed out. Moreover, we found that lL decreased significantly when E-cadherin was knocked down. These results indicate that the observed long-range correlation of E is not fixed within the jammed state but inherently arises from the formation of a large-scale actin filament structure via E-cadherin-dependent cell-cell junctions.

Cricket tympanal organ revisited: morphology, development and possible functions of the adult-specific chitin core beneath the anterior tympanal membrane.

Vertebrates and insects are phylogenetically separated by millions of years but have commonly developed tympanal membranes for efficiently converting airborne sound to mechanical oscillation in hearing. The tympanal organ of the field cricket Gryllus bimaculatus, spanning 200 µm, is one of the smallest auditory organs among animals. It indirectly links to two tympana in the prothoracic tibia via tracheal vesicles. The anterior tympanal membrane is smaller and thicker than the posterior tympanal membrane and it is thought to have minor function as a sound receiver. Using differential labeling of sensory neurons/surrounding structures and three-dimensional reconstructions, we revealed that a shell-shaped chitin mass and associated tissues are hidden behind the anterior tympanal membrane. The mass, termed the epithelial core, is progressively enlarged by discharge of cylindrical chitin from epithelial cells that start to aggregate immediately after the final molt and it reaches a plateau in size after 6 days. The core, bridging between the anterior tracheal vesicle and the fluid-filled chamber containing sensory neurons, is supported by a taut membrane, suggesting the possibility that anterior displacements of the anterior tracheal vesicle are converted into fluid motion via a lever action of the core. The epithelial core did not exist in tympanal organ homologs of meso- and metathoracic legs or of nymphal legs. Taken together, the findings suggest that the epithelial core, a potential functional homolog to mammalian ossicles, underlies fine sound frequency discrimination required for adult-specific sound communications.

Temporal Variation in Single-Cell Power-Law Rheology Spans the Ensemble Variation of Cell Population.

Changes in the cytoskeletal organization within cells can be characterized by large spatial and temporal variations in rheological properties of the cell (e.g., the complex shear modulus G∗). Although the ensemble variation in G∗ of single cells has been elucidated, the detailed temporal variation of G∗ remains unknown. In this study, we investigated how the rheological properties of individual fibroblast cells change under a spatially confined environment in which the cell translational motion is highly restricted and the whole cell shape remains unchanged. The temporal evolution of single-cell rheology was probed at the same measurement location within the cell, using atomic force microscopy-based oscillatory deformation. The measurements reveal that the temporal variation in the power-law rheology of cells is quantitatively consistent with the ensemble variation, indicating that the cell system satisfies an ergodic hypothesis in which the temporal statistics are identical to the ensemble statistics. The autocorrelation of G∗ implies that the cell mechanical state evolves in the ensemble of possible states with a characteristic timescale.

Time-lapse imaging of morphological changes in a single neuron during the early stages of apoptosis using scanning ion conductance microscopy.

Apoptosis plays an important role in many physiologic and pathologic conditions. The biochemical and morphological characteristics of apoptosis including cellular volume decrease, cell membrane blebbing, and phosphatidylserine translocation from the inner to the outer leaflet of the cell membrane are considered important events for phagocyte detection. Despite its importance, the relationship between the biological and morphological changes in a living cell has remained controversial. Scanning ion conductance microscopy is a suitable technique for investigating a series of these changes, because it allows us to observe the morphology of living cells without any mechanical interactions between the probe and the sample surface with a high resolution. Here, we investigated the biochemical and morphological changes in single neurons during the early stages of apoptosis, including apoptotic volume decrease, membrane blebbing and phosphatidylserine translocation, by using scanning ion conductance microscopy. Time-course imaging of apoptotic neurons showed there was a reduction in apoptotic volume after exposure to staurosporine and subsequent membrane bleb formation, which has a similar onset time to phosphatidylserine translocation. Our results show that a reduction in cellular volume is one of the earliest morphological changes in apoptosis, and membrane blebbing and phosphatidylserine translocation occur as subsequent biological and morphological changes. This is the first report to describe this series of morphological and biochemical changes ranging from an apoptotic volume decrease to membrane blebbing and PS translocation by scanning ion conductance microscopy (SICM). This new and direct imaging technique will provide new insight into the relationship between biochemical events inside a cell and cellular morphological changes.

Enhancement of neural crest formation by mechanical force in Xenopus development.

In vertebrate development, ectoderm is specified into neural plate (NP), neural plate border (NPB), and epidermis. Although such patterning is thought to be achieved by molecular concentration gradients, it has been revealed, mainly by in vitro analysis, that mechanical force can regulate cell specification. During in vivo patterning, cells deform and migrate, and this applies force to surrounding tissues, shaping the embryo. However, the role of mechanical force for cell specification in vivo is largely unknown. In this study, with an aspiration assay and atomic force microscopy, we have demonstrated that tension on ectodermal cells decreases laterally from the midline in Xenopus early neurula. Ectopically applied force laterally expanded the neural crest (NC) region, a derivative of the NPB, whereas force relaxation suppressed it. Furthermore, force application activated both the FGF and Wnt pathways, which are required for NC formation during neuroectodermal patterning. Taken together, mechanical force is necessary for NC formation in order to regulate signaling pathways. Furthermore, molecular signals specify the NP and generate force on neighboring tissue, the NPB, with its closure. This force activates signals, possibly determining the appropriate width of a narrow tissue, the NC.

Quantifying cell-to-cell variation in power-law rheology.

Among individual cells of the same source and type, the complex shear modulus G(∗) exhibits a large log-normal distribution that is the result of spatial, temporal, and intrinsic variations. Such large distributions complicate the statistical evaluation of pharmacological treatments and the comparison of different cell states. However, little is known about the characteristic features of cell-to-cell variation. In this study, we investigated how this variation depends on the spatial location within the cell and on the actin filament cytoskeleton, the organization of which strongly influences cell mechanics. By mechanically probing fibroblasts arranged on a microarray, via atomic force microscopy, we observed that the standard deviation σ of G(∗) was significantly reduced among cells in which actin filaments were depolymerized. The parameter σ also exhibited a subcellular spatial dependence. Based on our findings regarding the frequency dependence of σ of the storage modulus G('), we proposed two types of cell-to-cell variation in G(') that arise from the purely elastic and the frequency-dependent components in terms of the soft glassy rheology model of cell deformability. We concluded that the latter inherent cell-to-cell variation can be reduced greatly by disrupting actin networks, by probing at locations within the cell nucleus boundaries distant from the cell center, and by measuring at high loading frequencies.

Mechanical properties of epithelial cells in domes investigated using atomic force microscopy.

As epithelial cells in vitro reach a highly confluent state, the cells often form a microscale dome-like architecture that encloses a fluid-filled lumen. The domes are stabilized by mechanical stress and luminal pressure. However, the mechanical properties of cells that form epithelial domes remain poorly characterized at the single-cell level. In this study, we used atomic force microscopy (AFM) to measure the mechanical properties of cells forming epithelial domes. AFM showed that the apparent Young's modulus of cells in domes was significantly higher when compared with that in the surrounding monolayer. AFM also showed that the stiffness and tension of cells in domes were positively correlated with the apical cell area, depending on the degree of cell stretching. This correlation disappeared when actin filaments were depolymerized or when the ATPase activity of myosin II was inhibited, which often led to a large fluctuation in dome formation. The results indicated that heterogeneous actomyosin structures organized by stretching single cells played a crucial role in stabilizing dome formation. Our findings provide new insights into the mechanical properties of three-dimensional deformable tissue explored using AFM at the single-cell level.

Pathogenic Roles of Cardiac Fibroblasts in Pediatric Dilated Cardiomyopathy.

Background Dilated cardiomyopathy (DCM) is a major cause of heart failure in children. Despite intensive genetic analyses, pathogenic gene variants have not been identified in most patients with DCM, which suggests that cardiomyocytes are not solely responsible for DCM. Cardiac fibroblasts (CFs) are the most abundant cell type in the heart. They have several roles in maintaining cardiac function; however, the pathological role of CFs in DCM remains unknown. Methods and Results Four primary cultured CF cell lines were established from pediatric patients with DCM and compared with 3 CF lines from healthy controls. There were no significant differences in cellular proliferation, adhesion, migration, apoptosis, or myofibroblast activation between DCM CFs compared with healthy CFs. Atomic force microscopy revealed that cellular stiffness, fluidity, and viscosity were not significantly changed in DCM CFs. However, when DCM CFs were cocultured with healthy cardiomyocytes, they deteriorated the contractile and diastolic functions of cardiomyocytes. RNA sequencing revealed markedly different comprehensive gene expression profiles in DCM CFs compared with healthy CFs. Several humoral factors and the extracellular matrix were significantly upregulated or downregulated in DCM CFs. The pathway analysis revealed that extracellular matrix receptor interactions, focal adhesion signaling, Hippo signaling, and transforming growth factor-ß signaling pathways were significantly affected in DCM CFs. In contrast, single-cell RNA sequencing revealed that there was no specific subpopulation in the DCM CFs that contributed to the alterations in gene expression. Conclusions Although cellular physiological behavior was not altered in DCM CFs, they deteriorated the contractile and diastolic functions of healthy cardiomyocytes through humoral factors and direct cell-cell contact.

Atomic force microscopy identifies the alteration of rheological properties of the cardiac fibroblasts in idiopathic restrictive cardiomyopathy.

Restrictive cardiomyopathy (RCM) is a rare disease characterized by increased ventricular stiffness and preserved ventricular contraction. Various sarcomere gene variants are known to cause RCM; however, more than a half of patients do not harbor such pathogenic variants. We recently demonstrated that cardiac fibroblasts (CFs) play important roles in inhibiting the diastolic function of cardiomyocytes via humoral factors and direct cell-cell contact regardless of sarcomere gene mutations. However, the mechanical properties of CFs that are crucial for intercellular communication and the cardiomyocyte microenvironment remain less understood. In this study, we evaluated the rheological properties of CFs derived from pediatric patients with RCM and healthy control CFs via atomic force microscopy. Then, we estimated the cellular modulus scale factor related to the cell stiffness, fluidity, and Newtonian viscosity of single cells based on the single power-law rheology model and analyzed the comprehensive gene expression profiles via RNA-sequencing. RCM-derived CFs showed significantly higher stiffness and viscosity and lower fluidity compared to healthy control CFs. Furthermore, RNA-sequencing revealed that the signaling pathways associated with cytoskeleton elements were affected in RCM CFs; specifically, cytoskeletal actin-associated genes (ACTN1, ACTA2, and PALLD) were highly expressed in RCM CFs, whereas several tubulin genes (TUBB3, TUBB, TUBA1C, and TUBA1B) were down-regulated. These results implies that the signaling pathways associated with cytoskeletal elements alter the rheological properties of RCM CFs, particularly those related to CF-cardiomyocyte interactions, thereby leading to diastolic cardiac dysfunction in RCM.

Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia.

Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. Ischemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2h after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes.

Artificial polymeric receptors on the cell surface promote the efficient cellular uptake of quantum dots.

In our previous paper, secondary-amine appended cationic polymer 1 was used as a scaffold to display artificial receptors on a cell surface (R. Kamitani et al., ChemBioChem, 2009, 10, 230). This polymer can be retained on the cell surface for more than 30 min before being slowly internalized into the cells. In this study, our aim is to achieve the efficient internalization of quantum dots (QDs) into target cells via artificial receptors on the polymer. As a receptor molecule, N-acetylglucosamine (GlcNAc) moieties were introduced into the polymer, and GlcNAc binding protein-displaying QDs were used as a ligand. We found that ligand-presenting QDs could be internalized effectively into cells via polymer-mediated endocytosis, whereas QDs were not internalized into untreated cells. These data suggest that our method based on cell-surface engineering using polymers affords a new approach to the delivery of various poorly permeable nanoparticles into cells.

Spatiotemporal dynamics of single cell stiffness in the early developing ascidian chordate embryo.

During the developmental processes of embryos, cells undergo massive deformation and division that are regulated by mechanical cues. However, little is known about how embryonic cells change their mechanical properties during different cleavage stages. Here, using atomic force microscopy, we investigated the stiffness of cells in ascidian embryos from the fertilised egg to the stage before gastrulation. In both animal and vegetal hemispheres, we observed a Rho kinase (ROCK)-independent cell stiffening that the cell stiffness exhibited a remarkable increase at the timing of cell division where cortical actin filaments were organized. Furthermore, in the vegetal hemisphere, we observed another mechanical behaviour, i.e., a ROCK-associated cell stiffening, which was retained even after cell division or occurred without division and propagated sequentially toward adjacent cells, displaying a characteristic cell-to-cell mechanical variation. The results indicate that the mechanical properties of embryonic cells are regulated at the single cell level in different germ layers.

Pharmacological Alteration of Cellular Mechanical Properties in Pulmonary Arterial Smooth Muscle Cells of Idiopathic Pulmonary Arterial Hypertension.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is a progressive disease caused by vascular remodeling of the pulmonary arteries with elevated pulmonary vascular resistance. Recently, various pulmonary vasodilator drugs have become available in the clinical field, and have dramatically ameliorated the prognosis of IPAH. However, little is known about how the mechanical properties of pulmonary arterial smooth muscle cells (PASMCs) are altered under drug supplementation. METHODS: Atomic force microscopy (AFM) was used to investigate the mechanical properties of PASMCs derived from a patient with IPAH (PAH-PASMCs) and a healthy control (N-PASMCs) which received the supplementation of clinically used drugs for IPAH: sildenafil, macitentan, and riociguat. RESULTS: PASMCs derived from PAH-PASMCs were stiffer than those derived from N-PASMCs. With sildenafil treatment, the apparent Young's modulus (E 0) of cells significantly decreased in PAH-PASMCs but remained unchanged in N-PASMCs. The decrease in E 0 of PAH-PASMCs was also observed in macitentan and riociguat treatment. The stress relaxation AFM revealed that the decrease in E 0 of PAH-PASMCs resulted from a decrease in the cell elastic modulus and/or increase in cell fluidity. The combination treatment of macitentan and riociguat showed an additive effect on cell mechanical properties, implying that this clinically accepted combination therapy for IPAH influences the intracellular mechanical components. CONCLUSIONS: Pulmonary vasodilator drugs affect the mechanical properties of PAH-PASMCs, and there exists a mechanical effect of combination treatment on PAH-PASMCs.

Influence of hydrophobic structures on the plasma membrane permeability of lipidlike molecules.

A series of FITC-labeled hydrophobic molecules (1-8) were prepared, and their cellular uptakes have been investigated using cell-cycle-synchronized HeLa cells. The cellular membrane permeability of compounds strongly depended on both the chemical structure and the cell-cycle phase. In the G1/S phase, branched hydrocarbon-containing 3 and cis-olefin-containing 2 and 8 were efficiently internalized into cells by passive diffusion. In contrast, linear alkyl chain-containing 1 and 7 were retained on the membrane without rapid internalization. In the M phase, rapid permeation was suppressed for all molecules.

Observation of the three-dimensional structure of actin bundles formed with polycations.

Three-dimensional structures of actin bundles formed with polycations were observed by using transmission electron microtomography and atomic force microscopy. We found, for the first time, that the cross-sectional morphology of actin bundles depends on the polycation species and ionic strength, while it is insensitive to the degree of polymerization and concentration of polycation. Actin bundles formed with poly-N-[3-(dimethylamino)propyl] acrylamide methyl chloride quaternary show a ribbon-like cross-sectional morphology in low salt concentrations that changes to cylindrical cross-sectional morphology with hexagonal packing of the actin filaments in high salt concentrations. Contrastingly, actin bundles formed with poly-L-lysine show triangular cross-sectional morphology with hexagonal packing of the actin filaments. These variations in cross-sectional morphology are discussed in terms of anisotropy in the electrostatic energy barrier.

Origami-based self-folding of co-cultured NIH/3T3 and HepG2 cells into 3D microstructures.

This paper describes an origami-inspired self-folding method to form three-dimensional (3D) microstructures of co-cultured cells. After a confluent monolayer of fibroblasts (NIH/3T3 cells) with loaded hepatocytes (HepG2 cells) was cultured onto two-dimensional (2D) microplates, degradation of the alginate sacrificial layer in the system by addition of alginate lyase triggered NIH/3T3 cells to self-fold the microplates around HepG2 cells, and then 3D cell co-culture microstructures were spontaneously formed. Using this method, we can create a large number of 3D cell co-culture microstructures swiftly with ease in the same time. We find that HepG2 cells confined in the 3D cell co-culture microstructures have an ability to enhance the secreted albumin compared to 2D system in a long culture period. The result indicates that the origami-based cell self-folding technique presented here is useful in regenerative medicine and the preclinical stage of drug development.

Regulation of neuritogenesis in hippocampal neurons using stiffness of extracellular microenvironment.

The mechanosensitivity of neurons in the central nervous system (CNS) is an interesting issue as regards understanding neuronal development and designing compliant materials as neural interfaces between neurons and external devices for treating CNS injuries and disorders. Although neurite initiation from a cell body is known to be the first step towards forming a functional nervous network during development or regeneration, less is known about how the mechanical properties of the extracellular microenvironment affect neuritogenesis. Here, we investigated the filamentous actin (F-actin) cytoskeletal structures of neurons, which are a key factor in neuritogenesis, on gel substrates with a stiffness-controlled substrate, to reveal the relationship between substrate stiffness and neuritogenesis. We found that neuritogenesis was significantly suppressed on a gel substrate with an elastic modulus higher than the stiffness of in vivo brain. Fluorescent images of the F-actin cytoskeletal structures showed that the F-actin organization depended on the substrate stiffness. Circumferential actin meshworks and arcs were formed at the edge of the cell body on the stiff gel substrates unlike with soft substrates. The suppression of F-actin cytoskeleton formation improved neuritogenesis. The results indicate that the organization of neuronal F-actin cytoskeletons is strongly regulated by the mechanical properties of the surrounding environment, and the mechanically-induced F-actin cytoskeletons regulate neuritogenesis.

Visualising the dynamics of live pancreatic microtumours self-organised through cell-in-cell invasion.

Pancreatic ductal adenocarcinoma (PDAC) reportedly progresses very rapidly through the initial carcinogenesis stages including DNA damage and disordered cell death. However, such oncogenic mechanisms are largely studied through observational diagnostic methods, partly because of a lack of live in vitro tumour imaging techniques. Here we demonstrate a simple live-tumour in vitro imaging technique using micro-patterned plates (micro/nanoplates) that allows dynamic visualisation of PDAC microtumours. When PDAC cells were cultured on a micro/nanoplate overnight, the cells self-organised into non-spheroidal microtumours that were anchored to the micro/nanoplate through cell-in-cell invasion. This self-organisation was only efficiently induced in small-diameter rough microislands. Using a time-lapse imaging system, we found that PDAC microtumours actively stretched to catch dead cell debris via filo/lamellipoedia and suction, suggesting that they have a sophisticated survival strategy (analogous to that of starving animals), which implies a context for the development of possible therapies for PDACs. The simple tumour imaging system visualises a potential of PDAC cells, in which the aggressive tumour dynamics reminds us of the need to review traditional PDAC pathogenesis.