Glyoxal-methyl-ethylene sulfonic acid fixative enhances the fixation of cytoskeletal structures for Förster resonance energy transfer measurements.

Förster resonance energy transfer (FRET) serves as a tool for measuring protein-protein interactions using various sensor molecules. The tension sensor module relies on FRET technology. In our study, this module was inserted within the actinin molecule to measure the surface tension of the cells. Given that the decay curve of FRET efficiency correlates with surface tension increase, precise and accurate efficiency measurement becomes crucial. Among the methods of FRET measurements, FRET efficiency remains the most accurate if sample fixation is successful. However, when cells were fixed with 4% paraformaldehyde (PFA), the actinin-FRET sensor diffused across the cytoplasm; this prompted us to explore fixation method enhancements. Glyoxal fixative has been reported to improve cytoskeletal morphologies compared to PFA. However, it was not known whether glyoxal fits FRET measurements. Glyoxal necessitates an acetic acid solution for fixation; however, acidic conditions could compromise fluorescence stability. We observed that the pH working range of glyoxal fixative aligns closely with MES (methyl-ethylene sulfonic acid) Good's buffer. Initially, we switched the acidic solution for MES buffer and optimized the fixation procedure for in vitro and in vivo FRET imaging. By comparing FRET measurements on hydrogels with known stiffness to tumor nodules in mouse lung, we estimated in vivo stiffness. The estimated stiffness of cancerous tissue was harder than the reported stiffness of smooth muscle. This discovery shed lights on how cancer cells perceive environmental stiffness during metastasis.

Hierarchical Development of Motile Polarity in Durotactic Cells Just Crossing an Elasticity Boundary.

Cellular durotaxis has been extensively studied in the field of mechanobiology. In principle, asymmetric mechanical field of a stiffness gradient generates motile polarity in a cell, which is a driving factor of durotaxis. However, the actual process by which the motile polarity in durotaxis develops is still unclear. In this study, to clarify the details of the kinetics of the development of durotactic polarity, we investigated the dynamics of both cell-shaping and the microscopic turnover of focal adhesions (FAs) for Venus-paxillin-expressing fibroblasts just crossing an elasticity boundary prepared on microelastically patterned gels. The Fourier mode analysis of cell-shaping based on a persistent random deformation model revealed that motile polarity at a cell-body scale was established within the first few hours after the leading edges of a moving cell passed through the boundary from the soft to the stiff regions. A fluorescence recovery after photobleaching (FRAP) analysis showed that the mobile fractions of paxillin at FAs in the anterior part of the cells exhibited an asymmetric increase within several tens of minutes after cells entered the stiff region. The results demonstrated that motile polarity in durotactic cells is established through the hierarchical step-wise development of different types of asymmetricity in the kinetics of FAs activity and cell-shaping with a several-hour time lag.Key words: Microelasticity patterned gel, durotaxis, cell polarity, focal adhesions, paxillin.

Cellular Durotaxis Revisited: Initial-Position-Dependent Determination of the Threshold Stiffness Gradient to Induce Durotaxis.

Directional cell movement from a softer to a stiffer region on a culture substrate with a stiffness gradient, so-called durotaxis, has attracted considerable interest in the field of mechanobiology. Although the strength of a stiffness gradient has been known to influence durotaxis, the precise manipulation of durotactic cells has not been established due to the limited knowledge available on how the threshold stiffness gradient (TG) for durotaxis is determined. In the present study, to clarify the principles for the manipulation of durotaxis, we focused on the absolute stiffness of the soft region and evaluated its effect on the determination of TG required to induce durotaxis. Microelastically patterned gels that differed with respect to both the absolute stiffness of the soft region and the strength of the stiffness gradient were photolithographically prepared using photo-cross-linkable gelatins, and the TG for mesenchymal stem cells (MSCs) was examined systematically for each stiffness value of the soft region. As a result, the TG values for soft regions with stiffnesses of 2.5, 5, and 10 kPa were 0.14, 1.0, and 1.4 kPa/µm, respectively, i.e., TG markedly increased with an increase in the absolute stiffness of the soft region. An analysis of the area and long-axis length for focal adhesions revealed that the adhesivity of MSCs was more stable on a stiffer soft region. These results suggested that the initial location of cells starting durotaxis plays an essential role in determining the TG values and furthermore that the relationship between the position-dependent TG and intrinsic stiffness gradient (IG) of the culture substrate should be carefully reconsidered for inducing durotaxis; IG must be higher than TG (IG ≥ TG). This principle provides a fundamental guide for designing biomaterials to manipulate cellular durotaxis.

[Design of mechanobio-materials for cell manipulation and its application for stem cell manipulation.]

Recently, control of mechanobiologic response of cells has been a strong attractive issue for biomaterials sciences in relation to the requirements for optimization of cell-materials interactions. In this mini-review, we survey the typical parameters for designing the biomaterials to manipulate cell mechanobiology, i.e., mechanobio-materials. In addition, from the view of regenerative biomedical engineering, we introduce our recent approaches on the development of mechanobio-materials for stem cell manipulation that ensures the high-qualified stemness.

Time-dependent migratory behaviors in the long-term studies of fibroblast durotaxis on a hydrogel substrate fabricated with a soft band.

Durotaxis, biased cell movement up a stiffness gradient on culture substrates, is one of the useful taxis behaviors for manipulating cell migration on engineered biomaterial surfaces. In this study, long-term durotaxis was investigated on gelatinous substrates containing a soft band of 20, 50, and 150 µm in width fabricated using photolithographic elasticity patterning; sharp elasticity boundaries with a gradient strength of 300 kPa/50 µm were achieved. Time-dependent migratory behaviors of 3T3 fibroblast cells were observed during a time period of 3 days. During the first day, most of the cells were strongly repelled by the soft band independent of bandwidth, exhibiting the typical durotaxis behavior. However, the repellency by the soft band diminished, and more cells crossed the soft band or exhibited other mixed migratory behaviors during the course of the observation. It was found that durotaxis strength is weakened on the substrate with the narrowest soft band and that adherent affinity-induced entrapment becomes apparent on the widest soft band with time. Factors, such as changes in surface topography, elasticity, and/or chemistry, likely contributing to the apparent diminishing durotaxis during the extended culture were examined. Immunofluorescence analysis indicated preferential collagen deposition onto the soft band, which is derived from secretion by fibroblast cells, resulting in the increasing contribution of haptotaxis toward the soft band over time. The deposited collagen did not affect surface topography or surface elasticity but did change surface chemistry, especially on the soft band. The observed time-dependent durotaxis behaviors are the result of the mixed mechanical and chemical cues. In the studies and applications of cell migratory behavior under a controlled stimulus, it is important to thoroughly examine other (hidden) compounding stimuli in order to be able to accurately interpret data and to design suitable biomaterials to manipulate cell migration.

A plasmonic metasurface reveals differential motility of breast cancer cell lines at initial phase of adhesion.

A plasmonic metasurface composed of a self-assembled monolayer of gold nanoparticles allows for fluorescence imaging with high spatial resolution, owing to the collective excitation of localized surface plasmon resonance. Taking advantage of fluorescence imaging confined to the nano-interface, we examined actin organization in breast cancer cell lines with different metastatic potentials during cell adhesion. Live-cell fluorescence imaging confined within tens of nanometers from the substrate shows a high actin density spanning < 1 µm from the cell edge. Live-cell imaging revealed that the breast cancer cell lines exhibited different actin patterns during the initial phase of cell adhesion (∼ 1 h). Non-tumorous MCF10A cells exhibited symmetric actin localization at the cell edge, whereas highly metastatic MDA-MB-231 cells showed asymmetric actin localization, demonstrating rapid polarization of MDA-MB-231 cells upon adhesion. The rapid actin organization observed by our plasmonic metasurface-based fluorescence imaging provides information on how quickly cancer cells sense the underlying substrate.

Detection of prion protein oligomers by single molecule fluorescence imaging.

The degree of polymerization of PrP has a close relationship with the pathological mechanisms of prion diseases. We examined, at the molecular level, the polymerization state of PrP in lysates of prion-infected cells using total internal reflection fluorescence microscopy (TIRFM). The crude lysates were fractionated by gel-filtration spin columns according to their molecular size. Both the oligomer-rich and the monomer-rich fractions were probed with fluorescein-labeled anti-PrP antibodies (mAb SAF70 and mAb 8G8). Fluorescent spots of varying intensity were detected, with the ratio of intense fluorescent spots being greater in the oligomer fraction samples with mAb SAF70 than those with 8G8, the specific epitope of which is thought to be buried in abnormal PrP molecules. The results indicated that PrP oligomers could be specifically detected and conformational changes of abnormal PrP molecules observed. Imaging by TIRFM may aid in determining the polymerization state and properties of PrP oligomers in pathological processes.

Local pH mapping in the cell adhesion nano-interfaces on a pH-responsive fluorescence-dye-immobilized substrate.

Cell-substrate adhesion nano-interfaces can, in principle, exhibit a spatial distribution of local pH values under the influence of the weakly acidic microenvironment of glycocalyx grafted on lipid bilayer cell membrane which is compressed and closely attached to culture substrate in the vicinity of integrin-adhesion complexes. However, a simple local pH distribution imaging methodology has not been developed. In this study, to visualize the local pH distribution at the cell adhesion interface, we prepared glass substrates chemically modified with a pH-responsive fluorescent dye fluorescein isothiocyanate (FITC), observed the distribution of FITC fluorescence intensity at the adhesion interface of fibroblast (NIH/3T3) and cancer cells (HeLa), and compared the FITC images with the observed distribution of focal adhesions. FITC images were converted to pH mapping based on the pH-fluorescence calibration data of surface-immobilized FITC pre-measured in different pH media, which showed significantly larger regions with lowered pH level (6.8-7.0) from outside the cell (pH 7.4) were observed at the thick inner periphery of HeLa cells while 3T3 cells exhibited smaller lowered pH regions at the thin periphery. The lowered pH regions overlapped with many focal adhesions, and image analysis showed that larger focal adhesions tend to possess more lowered pH sites inside, reflecting enhanced glycocalyx compression due to accumulated integrin-adhesion ligand binding. This tendency was stronger for HeLa than for 3T3 cells. The role of glycocalyx compression and the pH reduction at the cell adhesive interface is discussed.

Guideline for design of substrate stiffness for mesenchymal stem cell culture based on heterogeneity of YAP and RUNX2 responses.

Mesenchymal stem cells (MSCs) have the potential for self-renewal and multipotency to differentiate into various lineages. Thus, they are of great interest in regenerative medicine as a cell source for tissue engineering. Substrate stiffness is one of the most extensively studied exogenous physical factors; however, consistent results have not always been reported for controlling MSCs. Conventionally used stiff culture substrates, such as tissue-culture polystyrene and glass, enhance nuclear localization of a mechanotransducer YAP and a pre-osteogenic transcription factor RUNX2, and bias MSCs towards the osteogenic lineage, even without osteogenic-inducing soluble factors. The mechanosensitive nature and intrinsic heterogeneity present challenges for obtaining reproducible results. This review summarizes the heterogeneity in human MSC response, specifically, nuclear/cytoplasmic localization changes in the mechanotransducer yes-associated protein (YAP) and the osteogenic transcription factor RUNX2, in response to substrate stiffness. In addition, a perspective on the intracellular factors attributed to response heterogeneity is discussed. The optimal range of stiffness parameters, Young's modulus, for MSC expansion culture to suppress osteogenic differentiation bias through the suppression of YAP and RUNX2 nuclear localization, and cell cycle progression is likely to be surprisingly narrow for a cell population from an identical donor and vary among cell populations from different donors. We believe that characterization of the heterogeneity of MSCs and understanding their biological meaning is an exciting research direction to establish guidelines for the design of culture substrates for the sophisticated control of MSC properties.

Actin crosslinking by α-actinin averts viscous dissipation of myosin force transmission in stress fibers.

Contractile force generated in actomyosin stress fibers (SFs) is transmitted along SFs to the extracellular matrix (ECM), which contributes to cell migration and sensing of ECM rigidity. In this study, we show that efficient force transmission along SFs relies on actin crosslinking by α-actinin. Upon reduction of α-actinin-mediated crosslinks, the myosin II activity induced flows of actin filaments and myosin II along SFs, leading to a decrease in traction force exertion to ECM. The fluidized SFs maintained their cable integrity probably through enhanced actin polymerization throughout SFs. A computational modeling analysis suggested that lowering the density of actin crosslinks caused viscous slippage of actin filaments in SFs and, thereby, dissipated myosin-generated force transmitting along SFs. As a cellular scale outcome, α-actinin depletion attenuated the ECM-rigidity-dependent difference in cell migration speed, which suggested that α-actinin-modulated SF mechanics is involved in the cellular response to ECM rigidity.

Rigidity matching between cells and the extracellular matrix leads to the stabilization of cardiac conduction.

Biomechanical dynamic interactions between cells and the extracellular environment dynamically regulate physiological tissue behavior in living organisms, such as that seen in tissue maintenance and remodeling. In this study, the substrate-induced modulation of synchronized beating in cultured cardiomyocyte tissue was systematically characterized on elasticity-tunable substrates to elucidate the effect of biomechanical coupling. We found that myocardial conduction is significantly promoted when the rigidity of the cell culture environment matches that of the cardiac cells (4 kiloPascals). The stability of spontaneous target wave activity and calcium transient alternans in high frequency-paced tissue were both enhanced when the cell substrate and cell tissue showed the same rigidity. By adapting a simple theoretical model, we reproduced the experimental trend on the rigidity matching for the synchronized excitation. We conclude that rigidity matching in cell-to-substrate interactions critically improves cardiomyocyte-tissue synchronization, suggesting that mechanical coupling plays an essential role in the dynamic activity of the beating heart.

2D-DIGE proteomic analysis of mesenchymal stem cell cultured on the elasticity-tunable hydrogels.

The present study focuses on mechanotransduction in mesenchymal stem cells (MSCs) in response to matrix elasticity. By using photocurable gelatinous gels with tunable stiffness, proteomic profiles of MSCs cultured on tissue culture plastic, soft (3 kPa) and stiff (52 kPa) matrices were deciphered using 2-dimensional differential in-gel analysis (2D-DIGE). The DIGE data, tied to immunofluorescence, indicated abundance and organization changes in the cytoskeletonal proteins as well as differential regulation of important signaling-related proteins, stress-responsing proteins and also proteins involved in collagen synthesis. The major CSK proteins including actin, tubulin and vimentin of the cells cultured on the gels were remarkably changed their expressions. Significant down-regulation of α-tubulin and ß-actin can be observed on gel samples in comparison to the rigid tissue culture plates. The expression abundance of vimentin appeared to be highest in the MSCs cultured on hard gels. These results suggested that the substrate stiffness significantly affects expression balances in cytoskeletal proteins of MSCs with some implications to cellular tensegrity.

Interplay among cell migration, shaping, and traction force on a matrix with cell-scale stiffness heterogeneity.

In living tissues where cells migrate, the spatial distribution of mechanical properties, especially matrix stiffness, is generally heterogeneous, with cell scales ranging from 10 to 1000 µm. Since cell migration in the body plays a critical role in morphogenesis, wound healing, and cancer metastasis, it is essential to understand the migratory dynamics on the matrix with cell-scale stiffness heterogeneity. In general, cell migration is driven by the extension and contraction of the cell body owing to the force from actin polymerization and myosin motors in the actomyosin cytoskeleton. When a cell is placed on a matrix with a simple stiffness gradient, directional migration called durotaxis emerges because of the asymmetric extension and contraction of the pseudopodia, which is accompanied by the asymmetric distribution of focal adhesions. Similarly, to determine cell migration on a matrix with cell-scale stiffness heterogeneity, the interaction between cell-scale stiffness heterogeneity and cellular responses, such as the dynamics of the cell-matrix adhesion site, intracellular prestress, and cell shape, should play a key role. In this review, we summarize systematic studies on the dynamics of cell migration, shaping, and traction force on a matrix with cell-scale stiffness heterogeneity using micro-elastically patterned hydrogels. We also outline the cell migration model based on cell-shaping dynamics that explains the general durotaxis induced by cell-scale stiffness heterogeneity. This review article is an extended version of the Japanese article, Dynamics of Cell Shaping and Migration on the Matrix with Cell-scale Stiffness-heterogeneity, published in SEIBUTSU BUTSURI Vol. 61, p. 152-156 (2021).

Avoiding tensional equilibrium in cells migrating on a matrix with cell-scale stiffness-heterogeneity.

Intracellular stresses affect various cell functions, including proliferation, differentiation and movement, which are dynamically modulated in migrating cells through continuous cell-shaping and remodeling of the cytoskeletal architecture induced by spatiotemporal interactions with extracellular matrix stiffness. When cells migrate on a matrix with cell-scale stiffness-heterogeneity, which is a common situation in living tissues, what intracellular stress dynamics (ISD) emerge? In this study, to explore this issue, finite element method-based traction force microscopy was applied to cells migrating on microelastically patterned gels. Two model systems of microelastically patterned gels (stiff/soft stripe and stiff triangular patterns) were designed to characterize the effects of a spatial constraint on cell-shaping and of the presence of different types of cues to induce competing cellular taxis (usual and reverse durotaxis) on the ISD, respectively. As the main result, the prolonged fluctuation of traction stress on a whole-cell scale was markedly enhanced on single cell-size triangular stiff patterns compared with homogeneous gels. Such ISD enhancement was found to be derived from the interplay between the nomadic migration of cells to regions with different degrees of stiffness and domain shape-dependent traction force dynamics, which should be an essential factor for keeping cells far from tensional equilibrium.

Designing Elastic Modulus of Cell Culture Substrate to Regulate YAP and RUNX2 Localization for Controlling Differentiation of Human Mesenchymal Stem Cells.

To establish a guideline for the design of cell culture substrates to control human mesenchymal stem cell (MSC) differentiation, we quantitatively characterized the heterogeneity in the responsiveness of MSCs to the elastic modulus of culture substrates. We analyzed the elastic modulus-dependent dynamics of a mechanotransducer, YAP, and an osteogenic differentiation factor, RUNX2, in three different MSC lots using a styrenated gelatin gel with controllable elastic modulus. The percentage of cells with YAP in the nucleus increased linearly with increases in the elastic modulus, reaching a plateau at 10 kPa for all the lots analyzed. The increase in the percentage with the substrate elastic modulus was described by the same linear function. The percentage of cells with RUNX2 nuclear localization also increased linearly with increases in the substrate elastic modulus, plateauing at 5 kPa, although the regression lines to the linearly increasing regions varied between lots. These similarities and differences in YAP and RUNX2 dynamics among cell populations are basis to design the substrate elastic modulus to manipulate YAP and RUNX2 localizations.

Thermo-switching of the conformation of genomic DNA in solutions of poly(N-isopropylacrylamide).

We used poly(N-isopropylacrylamide) (PNIPAM) to control the conformation of genomic DNA by changing the temperature of a reaction solution and studied the DNA transition at the level of single DNA molecules. With this method, the conformation of long genomic DNA can be readily and reversibly switched between a very compact condensate and an unfolded macromolecule.

General cellular durotaxis induced with cell-scale heterogeneity of matrix-elasticity.

Stiffness-gradient-induced cellular taxis, so-called durotaxis, has been extensively studied on a substrate with a single broad or steep stiffness gradient. However, in actual living tissues, cells should sense cell-scaled heterogeneous elasticity distribution in the extracellular matrix. In this study, to clarify the effect of the cell-scale heterogeneity of matrix-elasticity on durotaxis, we examined the motility of different types of cells on microelastically-striped patterned gels with different cell-sized widths. We found that cells accumulated in stiff regions with specific width on cell-type-dependency, even when a stiffness gradient is too small to induce usual durotaxis with a monotonic stiffness gradient. Fibroblast cells accumulated in a wide stiff region of multicellular size, while mesenchymal stem cells localized in a narrow stiff region of single-cell size. It was revealed that durotactic activity is critically affected not only with the cell type but also with the cell-scale heterogeneity of matrix-elasticity. Based on the shape-fluctuation-based analysis of cell migration, the dynamics of the pseudopodia were found to play a key role in determining the behaviors of general durotaxis. Our results suggest that design of cell-scale heterogeneity of matrix-elasticity is pivotal in controlling directional cell migration, the spontaneous cell-patterning, and development of the tissue on the biomaterials surfaces.

Mechanisms of endothelial cell coverage by pericytes: computational modelling of cell wrapping and in vitro experiments.

Pericytes (PCs) wrap around endothelial cells (ECs) and perform diverse functions in physiological and pathological processes. Although molecular interactions between ECs and PCs have been extensively studied, the morphological processes at the cellular level and their underlying mechanisms have remained elusive. In this study, using a simple cellular Potts model, we explored the mechanisms for EC wrapping by PCs. Based on the observed in vitro cell wrapping in three-dimensional PC-EC coculture, the model identified four putative contributing factors: preferential adhesion of PCs to the extracellular matrix (ECM), strong cell-cell adhesion, PC surface softness and larger PC size. While cell-cell adhesion can contribute to the prevention of cell segregation and the degree of cell wrapping, it cannot determine the orientation of cell wrapping alone. While atomic force microscopy revealed that PCs have a larger Young's modulus than ECs, the experimental analyses supported preferential ECM adhesion and size asymmetry. We also formulated the corresponding energy minimization problem and numerically solved this problem for specific cases. These results give biological insights into the role of PC-ECM adhesion in PC coverage. The modelling framework presented here should also be applicable to other cell wrapping phenomena observed in vivo.

Frustrated differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are one of the most useful cell resources for clinical application in regenerative medicine. However, standardization and quality assurance of MSCs are still essential problems because the stemness of MSCs depends on such factors as the collection method, individual differences associated with the source, and cell culture history. As such, the establishment of culture techniques which assure the stemness of MSCs is of vital importance. One important factor affecting MSCs during culture is the effect of the mechanobiological memory of cultured MSCs built up by their encounter with particular mechanical properties of the extracellular mechanical milieu. How can we guarantee that MSCs will remain in an undifferentiated state? Procedures capable of eliminating effects related to the history of the mechanical dose for cultured MSCs are required. For this problem, we have tried to establish the design of microelastically patterned cell-culture matrix which can effectively induce mechanical oscillations during the period of nomadic migration of cells among different regions of the matrix. We have previously observed before that the MSCs exposed to such a growth regimen during nomadic culture keep their undifferentiated state-with this maintenance of stemness believed due to lack of a particular regular mechanical dosage that is likely to determine a specific lineage. We have termed this situation as "frustrated differentiation". In this minireview, I introduce the concept of frustrated differentiation of MSCs and show possibility of purposeful regulation of this phenomenon.

Brain-stiffness-mimicking tilapia collagen gel promotes the induction of dorsal cortical neurons from human pluripotent stem cells.

The mechanical properties of the extracellular microenvironment, including its stiffness, play a crucial role in stem cell fate determination. Although previous studies have demonstrated that the developing brain exhibits spatiotemporal diversity in stiffness, it remains unclear how stiffness regulates stem cell fate towards specific neural lineages. Here, we established a culture substrate that reproduces the stiffness of brain tissue using tilapia collagen for in vitro reconstitution assays. By adding crosslinkers, we obtained gels that are similar in stiffness to living brain tissue (150-1500 Pa). We further examined the capability of the gels serving as a substrate for stem cell culture and the effect of stiffness on neural lineage differentiation using human iPS cells. Surprisingly, exposure to gels with a stiffness of approximately 1500 Pa during the early period of neural induction promoted the production of dorsal cortical neurons. These findings suggest that brain-stiffness-mimicking gel has the potential to determine the terminal neural subtype. Taken together, the crosslinked tilapia collagen gel is expected to be useful in various reconstitution assays that can be used to explore the role of stiffness in neurogenesis and neural functions. The enhanced production of dorsal cortical neurons may also provide considerable advantages for neural regenerative applications.