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.

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.

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.

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).

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.

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.

High-resolution imaging of a cell-attached nanointerface using a gold-nanoparticle two-dimensional sheet.

This paper proposes a simple, effective, non-scanning method for the visualization of a cell-attached nanointerface. The method uses localized surface plasmon resonance (LSPR) excited homogeneously on a two-dimensional (2D) self-assembled gold-nanoparticle sheet. The LSPR of the gold-nanoparticle sheet provides high-contrast interfacial images due to the confined light within a region a few tens of nanometers from the particles and the enhancement of fluorescence. Test experiments on rat basophilic leukemia (RBL-2H3) cells with fluorescence-labeled actin filaments revealed high axial and lateral resolution even under a regular epifluorescence microscope, which produced higher quality images than those captured under a total internal reflection fluorescence (TIRF) microscope. This non-scanning-type, high-resolution imaging method will be an effective tool for monitoring interfacial phenomena that exhibit relatively rapid reaction kinetics in various cellular and molecular dynamics.

Reversible Monolayer/Spheroid Cell Culture Switching by UCST-Type Thermoresponsive Ureido Polymers.

Multicellular spheroids have been studied in the fields of oncology, stem cell biology, and tissue engineering. In this study, we found a new polymer material for thermo-controlled spheroid/monolayer cell culture switching. The polymers that have pendant ureido groups (ureido polymers) exhibited upper critical solution temperature-type phase separation behavior. Cells in monolayer culture were converted to spheroids by the addition of ureido polymers below phase separation temperature (Tp). Time-lapse observations indicated that cells began to migrate and aggregate to form the spheroids to avoid contact with phase-separated polymer (coacervates) on the surface of the culture dish. We supposed that the coacervates seemingly suppressed interaction between cell and the dish surface or extracellular matrices. By increasing culture temperature above Tp, the spheroids began to collapse into a monolayer of cells due to dissolution of the coacervates. These results indicated that cell morphology could be repeatedly switched by changing the culture temperature in the presence of ureido polymers.

Fabrication of Elasticity-Tunable Gelatinous Gel for Mesenchymal Stem Cell Culture.

Surface elasticity or stiffness of an underlying substrate may regulate cellular functions such as adhesion, proliferation, signaling, differentiation, and migration. Recent studies have reported on the development of biomaterials to control stem cell fate determination via the stiffness of the culture substrates. In this chapter, we provide a detailed protocol for fabricating elasticity-tunable gelatinous hydrogels for stem cell culture with photo-induced or thermo-induced crosslinking of well-developed styrenated gelatin (StG). We also include the detailed application of gelatinous gel for mesenchymal stem cell (MSC) culture and sample collection for transcriptional and proteomic analysis.

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.

Time-programmed dual release formulation by multilayered drug-loaded nanofiber meshes.

To develop a drug carrier that enables time-programmed dual release in a single formulation, multilayered drug-loaded biodegradable nanofiber meshes were designed using sequential electrospinning with the following construction: (i) first drug-loaded mesh (top), (ii) barrier mesh, (iii) second drug-loaded mesh, and (iv) basement mesh (bottom). The drug release speed and duration were controlled by designing morphological features of the electrospun meshes such as the fiber diameter and mesh thickness. Control of the timed release of the second drug-the retardation period-was accomplished by appropriate design of the barrier mesh thickness. An in vitro release experiment demonstrated that the tetra-layered construction described above with appropriate morphological features of each component mesh can provide timed dual release of the respective drugs. The time-programmed dual release system using the multilayered electrospun nanofiber meshes was demonstrated as a useful formulation for advanced multidrug combination therapy requiring regiospecific administration of different drugs at different times. The potential use of the present multilayered formulation is discussed for application to biochemical modulation as one administrative strategy for use in sequential chemotherapy employing multiple anti-tumor drugs.

Nanoscale elongating control of the self-assembled protein filament with the cysteine-introduced building blocks.

Self-assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self-assembling proteins, named "Nanolego." Nanolego consists of "structural elements" of a structurally stable symmetrical homo-oligomeric protein and "binding elements," which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self-assembly process. The stabilization method is mediated by disulfide bonds between Cysteine-residues incorporated into the binding elements, and the termination method uses "capping Nanolegos," in which some of the binding elements in the Nanolego are absent for the self-assembled ends. With these technologies, we successfully constructed timing-controlled and size-regulated filament-shape complexes via Nanolego self-assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.

Dynamic force spectroscopy of the specific interaction between the PDZ domain and its recognition peptides.

To characterize the molecular basis of specific interactions of PDZ proteins, dynamic force spectroscopy (DFS) for the PDZ protein Tax-interacting protein-1 (TIP-1) and its recognition peptide (PDZ-pep) derived from beta-catenin was performed using an atomic force microscope (AFM), together with measurement of thermodynamic and kinetic parameters using surface plasmon resonance (SPR). The unbinding force of this pair was measured under different conditions of AFM tip-retraction velocity. The relationship between the unbinding force and the logarithmic force-loading rate, that is, the dynamic force spectrum, exhibited two different rate regimes, for each of which the forces increased linearly with the force-loading rate. On the basis of the theoretical treatment of the Bell-Evans model, the positions of two different activation barriers in the reaction coordinate and dissociation rate constants in each barrier were evaluated from slopes and x-intercepts of the two linear regimes (first barrier: 0.04 nm and 1.10 x 10 s(-1); second barrier: 0.21 nm and 2.77 x 10(-2) s(-1), respectively). Although two-step unbinding kinetics between TIP-1 and PDZ-pep was suggested from the DFS analysis, SPR results showed single-step dissociation kinetics with a rate constant of 2.89 x 10(-1) s(-1). Different shapes of the free energy profile of the unbinding process were deduced from each result of DFS and SPR. The reason for such topographic differences in the energy landscape is discussed in relation to the differences in the pathways of forced unbinding and spontaneous dissociation.

Force measurement for antigen-antibody interaction by atomic force microscopy using a photograft-polymer spacer.

To determine the intermolecular force on protein-protein interaction (PPI) by atomic force microscopy (AFM), a photograft-polymer spacer for protein molecules on both surfaces of the substrate and AFM probe tip was developed, and its effectiveness was assessed in a PPI model of a pair of human serum albumin (HSA) and its monoclonal antibody (anti-HSA). A carboxylated photoiniferter, N-(dithiocarboxy)sarcosine, was derivatized on both surfaces of the glass substrate and AFM probe tip, and subsequently water-soluble nonionic vinyl monomers, N,N-dimethylacrylamide (DMAAm), were graft-polymerized on them upon ultraviolet light irradiation. DMAAm-photograft-polymerized spacers with carboxyl groups at the growing chain end but with different chain lengths on both surfaces were prepared. The proteins were covalently bound to the carboxyl terminus of the photograft-polymer chain using a water-soluble condensation agent. The effects of the graft-spacer length on the profile of the force-distance curves and on the unbinding characteristics (unbinding force and unbinding distance) were examined in comparison with those in the case of the commercially available poly(ethylene glycol) (PEG) spacer. The frequency of the nonspecific adhesion force profile was markedly decreased with the use of the photograft spacers. Among the force curves detected, a high frequency of single-peak curves indicating the unbinding process of a single pair of proteins and a very low frequency of multiple-peak profiles were observed for the photograft spacers, regardless of the graft chain length, whereas a high frequency of no-force peaks was noted. These observations were in marked contrast with those for the PEG spacer. The force peak values determined ranged from 88 to 94 pN, irrespective of the type of spacer, while the standard deviation of force distribution observed for the photograft spacer was lower than that for the PEG spacer, indicating that the photograft spacers provide a higher accuracy of force determination.