Recent progress in mechanobiology sheds light on the regulation of cellular phenotypes by dissipative property of matrices, i.e., viscosity, fluidity, and stress relaxation, in addition to extensively studied elasticity. However, most researches have focused on bulk mechanics, despite cells in 2D culture can only interact with matrix interface directly. Here, we studied the impact of interfacial viscosity as well as elasticity of substrates on the early stage of adhesion behaviors of epithelial cells through new material design and mechanical characterization. The materials are copolymers of ε-caprolactone and d,l-lactide photocrosslinked by benzophenone. The substrate viscoelasticity changes depending on the polymer molecular weight and irradiation time. The interfacial elasticity and relaxation were determined by atomic force microscopy with modes of nanoindentation and tip-dwelling, respectively. MDCK cells changed morphologically, ranging from loose beaded assembly to more compact spheroids and eventual spread monolayer clusters, in response to the interfacial viscoelasticity change. Such morphological changes were mainly determined by substrate interfacial relaxation, rather than interfacial elasticity. Single-cell tracking identified biphasic motility with the minimum speed at intermediate relaxation time (~350 ms), where cells showed transitional morphologies between epithelial and mesenchymal traits. In that relaxation level, partially deformed cells moved around to coalesce with surrounding cells, eventually assembling into compact cellular aggregates. These results highlight, unlike the conventional hanging-drop technique, an appropriate level of interfacial relaxation is critical for efficient cell aggregate maturation on adhesive viscoelastic matrices. This work not only elucidates that the interfacial relaxation as the essential mechanical parameter for epithelial cell adhesion and migration, but also gives useful tips for creating physiologically relevant drug screening platform.

The original activity of epidermal growth factor (EGF) is to promote cell growth or block their apoptosis. However, its activity changes to proapoptotic, completely opposite to the original one, upon conjugation to nanoparticles. We have recently identified that this unique activity conversion was mediated by the confinement of EGF receptor (EGFR) within membrane rafts and signal condensation therein. In this study, we investigated the effect of interfacial parameters between the EGF molecule immobilized at the nanoparticle surface and the cell-surface membrane receptors and analyzed how their interactions were transduced to downstream signaling leading to apoptotic responses. We also studied the cell-type selective apoptotic responses and compared them with EGFR expression level to demonstrate the potential of the nanoparticle conjugate as a new type of anti-cancer drug activating EGFR rather than conventional blocking approaches.

Nanaomycin K, derived from Streptomyces rosa subsp. notoensis OS-3966T, has been discovered to have inhibitory bioactivity on epithelial-mesenchymal transition (EMT), an important mechanism of cancer cell invasion and migration. In this study, we examined the anti-EMT and anti-tumor effect of nanaomycin K in bladder cancer, where EMT has important roles in progression. We treated two bladder cancer lines, non-muscle-invasive KK47 and muscle-invasive T24, with nanaomycin K to determine the effects on cell proliferation, apoptosis and expression of EMT markers in vitro. Wound-healing assays were performed to assess cell invasion and migration. We conducted an in vivo xenograft study in which mice were inoculated with bladder cancer cells and treated with intratumoral administration of nanaomycin K to investigate its anti-tumor and EMT inhibition effects. As the results, nanaomycin K (50 µg/mL) significantly inhibited cell proliferation in KK47 (p < 0.01) and T24 (p < 0.01) in the presence of TGF-β, which is an EMT-inducer. Nanaomycin K (50 µg/mL) also significantly inhibited cell migration in KK47 (p < 0.01) and T24 (p < 0.01), and induced apoptosis in both cell lines in the presence of TGF-β (p < 0.01). Nanaomycin K increased the expression of E-cadherin and inhibited the expression of N-cadherin and vimentin in both cell lines. Nanaomycin K also decreased expression of Snail, Slug, phospho-p38 and phospho-SAPK/JNK especially in T24. Intratumoral administration of nanaomycin K significantly inhibited tumor growth in both KK47 and T24 cells at high dose (1.0 mg/body) (p = 0.009 and p = 0.003, respectively) with no obvious adverse events. In addition, nanaomycin K reversed EMT and significantly inhibited the expression of Ki-67 especially in T24. In conclusion, we demonstrated that nanaomycin K had significant anti-EMT and anti-tumor effects in bladder cancer cells, suggesting that nanaomycin K may be a therapeutic candidate for bladder cancer treatment.

Mechanics of the extracellular matrix (ECM) exhibit changes during many biological events. During disease progression, such as cancer, matrix stiffening or softening occurs due to crosslinking of the collagen matrix or matrix degradation through cell-secreted enzymes. Engineered hydrogels have emerged as a prime in vitro model to mimic such dynamic mechanics during disease progression. Although there have been a variety of engineered hydrogels, few can offer both stiffening and softening properties under the same working principle. In addition, to model individual disease progression, it is desirable to control the kinetics of mechanical changes. To this end, we describe a photoresponsive hydrogel that undergoes stiffness changes by the photo-induced phase transition. The hydrogel was composed of a copolymer of azobenzene acrylate monomer (AzoAA) and N,N-dimethyl acrylamide (DMA). By tuning the amount of azobenzene, the phase transition behavior of this polymer occurs solely by light irradiation, because of the photoisomerization of azobenzene. This phase behavior was confirmed at 37 °C by turbidity measurements. In addition, the crosslinked poly(AzoAA-r-DMA) gel undergoes reversible swelling-deswelling upon photoisomerization by ultraviolet or visible light. Furthermore, the poly(AzoAA-r-DMA) sheet gels exhibited modulus changes at different isomerization states of azobenzene. When MCF-7 cells were cultured on the gels, stiffening at different timepoints induced varied responses in the gene expression levels of E-cadherin. Not only did this suggest an adaptive behavior of the cells against changes in mechanics during disease progression, this also demonstrated our material's potential towards in vitro disease modeling. STATEMENT OF SIGNIFICANCE: During disease progression such as cancer, cellular microenvironment called extracellular matrix (ECM) undergoes stiffness changes. Hydrogels, which are swollen network of crosslinked polymers, have been used to model such dynamic mechanical environment of the ECM. However, few could offer both stiffening and softening properties under the same working principle. Herein, we fabricated a novel photoresponsive hydrogel with switchable mechanics, activated by photo-induced structural change of the polymer chains within the hydrogel. When breast cancer cells were cultured on our dynamic hydrogels, gene expression and morphological observation suggested that cells react to changes in stiffness by a transient response, as opposed to a sustained one. The photoresponsive hydrogel offers possibility for use as a patient-specific model of diseases.

The bio-industrial application of microalgae has gained much attention in recent years. One of the challenges in this field is to lower the cultivation and harvest costs and to achieve the steady productivity. To address this, new systems have been proposed, in which the products are extracted without killing the algal cells. These non-destructive extraction systems are called "milking." Some of the milking systems reported so far are continuous processes where culturing and milking occur simultaneously, and the others are a periodic process where cells are cyclically cultured and milked. These systems are based on the organic solvent extraction of non-polar products, such as lipids, terpenes, and carotenoids. However, a special facility required for handling organic solvents increases the costs and solvents lost during milking need to be recouped. In this study, we examined a solvent-free method, based on mechanical milking using a shearing disperser (glass homogenizer). We cultured the N-2-fixing filamentous cyanobacteria, Tolypothrbc sp. PCC7601 in non-sterile agricultural water and performed long-term (87 days) milking cycles to harvest the extracellular carbohydrates and phycobiliproteins. As a result, the productivity of extracellular carbohydrates and the cell densities remained constant throughout the milking cycle, yielding 90-140 mg/L of extracellular carbohydrates every 3 weeks. Our results demonstrated that mechanical milking is a practical and effective method that can be used to harvest products from algae steadily.

A new nanaomycin analog, nanaomycin K, was isolated from a cultured broth of actinomycete strain "Streptomyces rosa subsp. notoensis" OS-3966. Nuclear magnetic resonance (NMR) analyses revealed that the planar structure of nanaomycin K had an ergothioneine moiety. To determine the absolute configuration, nanaomycin K was semisynthesized using standards of nanaomycin E and l-ergothioneine. The natural and semisynthetic nanaomycin K were identified as the same compounds based on retention time, mass spectrometry, 1H NMR, and optical rotation data. Nanaomycin K showed cytotoxicity against Madin-Darby canine kidney (MDCK) cells undergoing transforming growth factor (TGF) β1-induced epithelial-mesenchymal transition.

There is a growing interest in the development of dynamic adaptive biomaterials for regulation of cellular functions. However, existing materials are limited to two-state switching of the presentation and removal of cell-adhesive bioactive motifs that cannot emulate the native extracellular matrix (ECM) in vivo with continuously adjustable characteristics. Here, tunable adaptive materials composed of a protein monolayer assembled at a liquid-liquid interface are demonstrated, which adapt dynamically to cell traction forces. An ultrastructure transition from protein monolayer to hierarchical fiber occurs through interfacial jamming. Elongated fibronectin fibers promote formation of elongated focal adhesion structures, increase focal adhesion kinase activation, and enhance neuronal differentiation of stem cells. Cell traction force results in spatial rearrangement of ECM proteins, which feeds back to alter stem cell fate. The reported biomimetic adaptive liquid interface enables dynamic control of stem cell behavior and has potential translational applications.

Epithelial-mesenchymal transition (EMT), a qualitative change in cell migration behavior during cancer invasion and metastasis, is becoming a new target for anticancer drugs. Therefore, it is crucial to develop in vitro assays for the evaluation of the abilities of drug candidates to control EMT progression. We herein report on a method for the quantification of the EMT based on particle image velocimetry and correlation functions. The exponential fitting of the correlation curve gives an index (lambda), which represents transforming growth factor (TGF)-beta 1-induced EMT progression and its suppression by inhibitors. Moreover, real-time monitoring of the lambda value illustrates a time-dependent EMT progressing process, which occurs earlier than the bio-chemical changes in an EMT marker protein expression. The results demonstrate the usefulness of the present method for kinetic studies of EMT progression as well as EMT inhibitor screening.

Two new nanaomycin analogs, nanaomycin I and J, were isolated from a cultured broth of an actinomycete strain, "Streptomyces rosa subsp. notoensis" OS-3966. In our previous study, we have confirmed the occurrence of nanaomycin I (m/z = 482 [M + H]+) that lacks a pseudo-disaccharide from the mycothiol of nanaomycin H under same culture condition. In this study, to confirm the structure of nanaomycin I, the strain "S. rosa subsp. notoensis" OS-3966 was re-cultured and the target compound with m/z = 482 [M + H]+ was isolated. Furthermore, we discovered another new analog, designated as nanaomycin J in isolating nanaomycin I. The NMR analyses revealed that the structures of nanaomycin I and J are N-acetylcysteine S-conjugates without a pseudo-disaccharide and N-acetylcysteine S-conjugates without a myo-inositol of nanaomycin H, respectively. The relative configurations of the tetrahydropyrane moiety of nanaomycin I and J were determined by rotating-frame overhauser effect spectroscopy (ROESY) analysis. Absolute configurations of the N-acetylcysteine moiety of nanaomycin I and J were determined by advanced Marfey's analyses for acid hydrolysis of de-sulfurized nanaomycin I and J with Raney nickel. Nanaomycin I and J showed moderate cytotoxicity against several human tumor cell lines.

The proliferation epidermal growth factor (EGF) is known to acquire contradictory apoptotic activities upon conjugation with gold nanoparticles (GNPs) through hitherto unknown mechanisms. Here, we identified an essential role of membrane rafts in the drastic activity switching of EGF-GNPs through the following intracellular signaling. (1) In contrast to the rapid diffusion of activated EGF receptor after the soluble EGF stimulation, the receptor is confined within membrane rafts upon binding to the EGF-GNPs. (2) This initial receptor confinements switch its endocytosis process from normal clathrin-mediated endocytosis to caveolin-mediated one, changing the phosphorylation dynamics of essential downstream kinases, i.e., extracellular signal-regulated kinase and AKT. Importantly, the destruction of membrane rafts by β-cyclodextrin reversed this trafficking and signaling, restoring EGF-GNPs to lost anti-apoptotic property. These results reveal the importance of GNP-mediated signal condensation at membrane rafts in conferring the unique apoptotic activity on EGF-nanoparticle conjugates. STATEMENT OF SIGNIFICANCE: Epidermal growth factor (EGF) is a small secretory protein that induces cell proliferation upon binding to its receptor existed on cellular plasma membranes. One interesting feature of the protein in the nanobiology field is, its acquisition of apoptosis-inducing (cellular suicide) activity rather than proliferative one upon conjugation to gold nanoparticles through hitherto unknown mechanisms. Here, we identified the involvement of membrane rafts, plasma membrane nanodomains enriched with cholesterol, in the apoptosis processes by changing the receptor trafficking and downstream signal transduction pathways. Moreover, the destruction of lipid rafts restored the EGF-nanoparticle conjugates with lost anti-apoptotic activity. These finding highlight potential applications of EGF-nanoparticle conjugates to cancer therapy, as the EGF receptor are highly expressed in cancer cells.

Epithelial-mesenchymal transition (EMT), phenotypic changes in cell adhesion and migration, is involved in cancer invasion and metastasis, hence becoming a target for anti-cancer drugs. In this study, we report a method for the evaluation of EMT inhibitors by using a photoactivatable gold substrate, which changes from non-cell-adhesive to cell-adhesive in response to light. The method is based on the geometrical confinement of cell clusters and the subsequent migration induction by controlled photoirradiation of the substrate. As a proof-of-concept experiment, a known EMT inhibitor was successfully evaluated in terms of the changes in cluster area or leader cell appearance, in response to biochemically and mechanically induced EMT. Furthermore, an application of the present method for microbial secondary metabolites identified nanaomycin H as an EMT inhibitor, potentially killing EMTed cells in disseminated conditions. These results demonstrate the potential of the present method for screening new EMT inhibitors.

Epithelial cells migrate as multicellular units. The directionality and speed of these units are determined by actively moving leader cells. It is important to understand how external cues affect the appearance of these leader cells in physiological and pathological processes. However, the impact of extracellular matrices (ECMs) is still controversial, because physically-adsorbed ECM proteins are amenable to protein remodeling, and uncontrolled cluster geometry can vary migration phenotypes. Here, we demonstrate a photoactivatable substrate, which we used to study the impact of a cyclic Arg-Gly-Asp (cRGD) ligand on leader cell formation in MDCK cells. This robust platform allowed us to investigate the effect of cRGD density on leader cell formation, in any given cluster geometry, with minimized ECM remodeling. Our results show a biphasic response of leader cell appearance upon reducing the surface cRGD density. The increase, in leader cell appearance, within the higher density range, is not only associated with the weakening of circumferential actomyosin belts, but also reduction of cellular mechanical tension and intercellular junctional E-cadherin. These results indicate that cRGD-mediated cell-ECM interactions positively regulate mechanical and biochemical coupling within cell clusters
both are critical for the coordination of cell collectives and eventual reduction in the appearance of leader cells.

There is growing evidence to suggest that the prevailing physical microenvironment and mechanical stress I I regulate cellular functions, including adhesion, proliferation, and differentiation. Moreover, the physical microenvironment determines the stem-cell lineage depending on stiffness of the substrate relative to biological tissues as well as the stress relaxation properties of the viscoelastic substrates used for cell culture. However, there is little known regarding the biological effects of a fluid substrate, where viscoelastic stress is essentially absent. Here, we demonstrate the regulation of myogenic differentiation on fluid substrates by using a liquid liquid interface as a scaffold. C2C12 myoblast cells were cultured using water-perfluorocarbon (PFC) interfaces as the fluid microenvironment. We found that, for controlled in vitro culture at water PFC interfaces, expression of myogenin, myogenic regulatory factors (MRF) family gene, is remarkably attenuated even when myogenic differentiation was induced by reducing levels of growth factors, although MyoD was expressed at the usual level (MyoD up-regulates myogenin under an elastic and/or viscoelastic environment). These results strongly suggest that this unique regulation of myogenic differentiation can be attributed to the fluid microenvironment of the interfacial culture medium. This interfacial culture system represents a powerful tool for investigation of the mechanisms by which physical properties regulate cellular adhesion and proliferation as well as their differentiation. Furthermore, we successfully transferred the cells cultured at such interfaces using Langmuir-Blodgett (LB) techniques. The combination of the interfacial culture system with the LB approach enables investigation of the effects of mechanical compression on cell functions.

Photoactivatable substrates, which show changes in surface cell adhesiveness in response to photoirradiation, are promising platforms for cell manipulation with high spatiotemporal resolution. In addition to having applications in cell and tissue engineering, these materials are unique tools for basic biological sciences research, and they complement conventional genetic engineering technologies. One of the most useful applications is in the study of cell migration, which occurs in various physiological and pathological processes. In this personal account, I provide a brief overview of the development of photoactivatable substrates and their applications, highlighting in particular the contributions of our research group to collective cell migration studies. This material-based approach is useful for dissecting the molecular biological and mechanobiological aspects of the regulatory mechanisms in the cellular social activities.

Physicochemical screening identified a new nanaomycin analog, nanaomycin H, which was isolated from a culture broth of Streptomyces rosa subsp. notoensis OS-3966. This microorganism is already known to produce seven nanaomycin compounds, (nanaomycin A to G). Structural elucidation of nanaomycin H showed it to be a pyranonaphthoquinone with a mycothiol moiety. A N-acetylcysteine S-conjugate of nanaomycin H, without oc-glucosamine linked to myo-inositol moiety, mercapturic acid derivative, was also detected in the same culture broth. Mercapturic acid derivatives of secondary metabolites are known to be produced for xenobiotic metabolism outside microbial cells. Mycothiol acts as a detoxifier to help prevent cell damage from factors such as oxidative stress. The production of O-2(-). generated by reduction of nanaomycin A is correlated with antibacterial activity. Mycothiol-containing nanaomycin H proved to be markedly decreased in O-2(-). and did not express any notable antimicrobial activity. It is suggested that nanaomycin H is produced in the detoxification process. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

A synergistic effect of biochemical and mechanical cues emanating from the extracellular matrix (ECM) on inducing malignant transformation of epithelial cells has been observed recently. However, the effect of quantitative changes in biochemical stimuli on cell phenotype, without changes in ECM component and rigidity, remains unknown. To determine this effect, we grew Madin-Darby canine kidney epithelial cells (MDCK) on gold surfaces immobilized with varying densities of cyclic arginine-glycine-aspartate (cRGD) peptide and analyzed cell morphology, cell migration, cytoskeletal organization, and protein expression. Cells grown on a surface presenting a higher density of cRGD displayed an epithelial morphology and grew in clusters, while those grown on a diluted cRGD surface transformed into an elongated, fibroblast-like form with extensive scattering. Time-lapse imaging of cell clusters grown on the concentrated cRGD surface revealed collective migration with intact cell-cell contacts accompanied by the development of cortical actin. In contrast, cells migrated individually and formed stress fibers on the substrate with sparse cRGD. These data point towards transdifferentiation of epithelial cells to mesenchymal-like cells when plated on a diluted cRGD surface. Supporting this hypothesis, immunofluorescence microscopy and western blot analysis revealed increased membrane localization and total expression of N-cadherin and vimentin in cells undergoing mesenchymal-like transition. Taken together, these results suggest a possible role of decreased biochemical stimuli from the ECM in regulating epithelial phenotype switching.
Statement of Significance
Epithelial-mesenchymal transition (EMT) is a process where adherent epithelial cells convert into individual migratory mesenchymal phenotype. It plays an important role both in physiological and pathological processes. Recent studies demonstrate that the program is not only governed by soluble factors and gene expressions, but also modulated by biochemical and mechanical cues in ECMs. In this study, we developed chemically defined surfaces presenting controlled ECM ligand densities and studied their impact on the EMT progression. Morphological and biochemical analyses of epithelial cells cultured on the surfaces indicate the cells undergo an EMT-like transition on the diluted cRGD surface while retaining epithelial characteristics on the cRGD-rich substrate, suggesting an important role of the ECM ligand density in epithelial phenotype switching. (C) 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Most of us may mistakenly believe that sciences within the nano regime are a simple extension of what is observed in micrometer regions. We may be misled to think that nanotechnology is merely a far advanced version of microtechnology. These thoughts are basically wrong. For true developments in nanosciences and related engineering outputs, a simple transformation of technology concepts from micro to nano may not be perfect. A novel concept, nanoarchitectonics, has emerged in conjunction with well-known nanotechnology. In the first part of this review, the concept and examples of nanoarchitectonics will be introduced. In the concept of nanoarchitectonics, materials are architected through controlled harmonized interactions to create unexpected functions. The second emerging concept is to control nano-functions by easy macroscopic mechanical actions. To utilize sophisticated forefront science in daily life, high-tech-driven strategies must be replaced by low-tech-driven strategies. As a required novel concept, hand-operation nanotechnology can control nano and molecular systems through incredibly easy action. Hand-motion-like macroscopic mechanical motions will be described in this review as the second emerging concept. These concepts are related bio-processes that create the third emerging concept, mechanobiology and related mechano-control of bio-functions. According to this story flow, we provide some incredible recent examples such as atom-level switches, operation of molecular machines by hand-like easy motions, and mechanical control of cell fate. To promote and activate science and technology based on these emerging concepts in nanotechnology, the contribution and participation of polymer scientists are crucial. We hope that some readers would have interests within what we describe.

Activities of cells are dependent on extracellular niches, which change quite dynamically. Dynamic substrates whose chemical and/or physical properties can be controlled by an external stimulus are useful to resolve how cells respond to such dynamic environmental changes. Moreover, they are useful for cell manipulations, such as heterotypic cell coculturing, cell migration induction, and cell sheet harvesting. We have developed for the first time a dynamic substrate based on the photocleavage reaction of the 2-nitrobenzyl group. One of the biggest advantages of the photochemical approach is its high spatiotemporal resolution. However, there is a lot of room for innovation to improve the switching efficiency and to expand the time window of its usage. This chapter discusses the developmental history of our photoactivatable substrates. Readers will notice how it is technically challenging to meet these two requirements on a single substrate. We also briefly refer to their applications to cell migration studies.

A method was developed for photocontrolling cell adhesion on a gel substrate with defined mechanical properties. Precise patterning of geometrically controlled cell clusters and their migration induction became possible by spatiotemporally controlled photo-irradiation of the substrate. The clusters exhibited unique collective motion that depended on substrate stiffness and cluster geometry.

Besides its well-known hormonal effects initiated in the nucleus, estradiol (E2) also activates non-nuclear pathways through interactions with receptors located on the cell plasma membrane. Micropatterned substrates consisting of gold dots bearing tethered E2 distributed on a cell-adhesive substrate were prepared and shown to trigger specifically E2 non-genomic effects in cells grown on the substrates.

Cell migration is an essential cellular activity in various physiological and pathological processes, such as wound healing and cancer metastasis. Therefore, in vitro cell migration assays are important not only for fundamental biological studies but also for evaluating potential drugs that control cell migration activity in medical applications. In this regard, robust control over cell migrating microenvironments is critical for reliable and quantitative analysis as cell migration is highly dependent upon the microenvironments. Here, we developed a facile method for making a commercial glass-bottom 96-well plate photoactivatable for cell adhesion, aiming to develop a versatile and multiplex cell migration assay platform. Cationic poly-D-lysine was adsorbed to the anionic glass surface via electrostatic interactions and, subsequently, functionalized with poly(ethylene glycol) (PEG) bearing a photocleavable reactive group. The initial PEGylated surface is non-cell-adhesive. However, upon near-ultraviolet (UV) irradiation, the photorelease of PEG switches the surface from non-biofouling to cell-adhesive. With this platform, we assayed cell migration in the following procedure: (1) create cell-attaching regions of precise geometries by controlled photo-irradiation, (2) seed cells to allow them to attach selectively to the irradiated regions, (3) expose UV light to the remaining PEGylated regions to extend the cell-adhesive area, (4) analyse cell migration using microscopy. Surface modification of the glass surface was characterized by zeta-potential and contact angle measurements. The PEGylated surface showed cell-resistivity and became cell-adhesive upon releasing PEG by near-UV irradiation. The method was applied for parallelly evaluating the effect of model drugs on the migration of epithelial MDCK cells in the multiplexed platform. The dose-response relationship for cytochalasin D treatment on cell migration behavior was successfully evaluated with high reproducibility. Interestingly, the impact of blebbistatin on cell migration was dependent upon the widths of the migrating regions, resulting in both cases of migration acceleration and deceleration. These results clearly demonstrate that the cellular response to certain drugs is highly affected by their migrating geometries. Therefore, the obtained novel photoactivatable 96-well plate serves as a useful high-throughput platform for the identification of drug candidates that have an effect on cell migration behavior.

Stem cell responsiveness to extracellular matrix (ECM) composition and mechanical cues has been the subject of a number of investigations so far, yet the molecular mechanisms underlying stem cell mechano-biology still need full clarification. Here we demonstrate that the paralog proteins YAP and TAZ exert a crucial role in adult cardiac progenitor cell mechano-sensing and fate decision. Cardiac progenitors respond to dynamic modifications in substrate rigidity and nanopattern by promptly changing YAP/TAZ intracellular localization. We identify a novel activity of YAP and TAZ in the regulation of tubulogenesis in 3D environments and highlight a role for YAP/TAZ in cardiac progenitor proliferation and differentiation. Furthermore, we show that YAP/TAZ expression is triggered in the heart cells located at the infarct border zone. Our results suggest a fundamental role for the YAP/TAZ axis in the response of resident progenitor cells to the modifications in microenvironment nanostructure and mechanics, thereby contributing to the maintenance of myocardial homeostasis in the adult heart. These proteins are indicated as potential targets to control cardiac progenitor cell fate by materials design.

Collective cell migration is involved in many biological and pathological processes. Various factors have been shown to regulate the decision to migrate collectively or individually, but the impact of cell-extracellular matrix (ECM) interactions is still debated. Here, we developed a method for analyzing collective cell migration by precisely tuning the interactions between cells and ECM ligands. Gold nanoparticles are arrayed on a glass substrate with a defined nanometer spacing by block copolymer micellar nanolithography (BCML), and photocleavable poly(ethylene glycol) (Mw = 12 kDa, PEG12K) and a cyclic RGD peptide, as an ECM ligand, are immobilized on this substrate. The remaining glass regions are passivated with PEG2K-silane to make cells interact with the surface via the nanoperiodically presented cyclic RGD ligands upon the photocleavage of PEG12K. On this nanostructured substrate, HeLa cells are first patterned in photo-illuminated regions, and cell migration is induced by a second photocleavage of the surrounding PEG12K. The HeLa cells gradually lose their cell-cell contacts and become disconnected on the nanopatterned substrate with 10-nm particles and 57-nm spacing, in contrast to their behavior on the homogenous substrate. Interestingly, the relationship between the observed migration collectivity and the cell-ECM ligand interactions is the opposite of that expected based on conventional soft matter models. It is likely that the reduced phosphorylation at tyrosine-861 of focal adhesion kinase (FAK) on the nanopatterned surface is responsible for this unique migration behavior. These results demonstrate the usefulness of the presented method in understanding the process of determining collective and non-collective migration features in defined micro-and nano-environments and resolving the crosstalk between cell-cell and cell-ECM adhesions.

Cellular activity is highly dependent on the extracellular environment, which is composed of surrounding cells and extracellular matrices. This focus review summarizes recent advances in chemically and physically engineered switchable substrates designed to control such cellular microenvironments by application of an external stimulus. Special attention is given to their molecular design, switching strategies, and representative examples for bioanalytical and biomedical applications.

This protocol describes a method for dynamic patterning cells on a glass coverslip. The glass substrate is first functionalized with photocleavable silane bearing 2-nitrobenzyl group, thereafter a cell-repellent polymer, poly(ethylene glycol) (PEG), is conjugated. Upon absorption of near-UV light, the PEG is cleaved from he surface, changing the surface from non-cell-adhesive to cell-adhesive. The method allows not only for spatially controlling cell attachment on the substrate (conventional patterning), but also inducing cell migration or coculturing heterotypic cells (dynamic patterning). Furthermore, it should be emphasized that the surface is compatible with fluorescence imaging in a high-resolution inverted objective setup as it is composed of a normal glass coverslip functionalized with the thin layers. In this chapter, I describe the procedure for the synthesis of the silane molecule, the preparation of the photoactivatable surface, and its application for dynamic cell patterning.

Dynamic substrates whose cell adhesiveness changes in response to an external stimulus are useful not only for patterning cells in various geometries but also for inducing cell migration or arraying heterotypic cells. The requirements for such applications are high switching efficiency in cell adhesiveness and long-term persistence of the created cellular patterns. In this study, we prepared a dynamic substrate bearing photocleavable poly(ethylene glycol) (PEG) and examined the effect of the surface PEG density and the charge of cationic base materials on the above-mentioned key requirements. An amino-terminated substrate with a certain amino group density and charge was functionalized with photocleavable PEG5K, with and without subsequent backfilling of photocleavable PEG2K. The PEG chains made the surface non-cell-adhesive, but subsequent near-UV irradiation of the substrate induced photocleavage of the PEG, eventually making the surface cell-adhesive. The substrates were analyzed by atomic force microscopy, contact angle measurements, ellipsometry, and zeta potential measurements, complemented with protein adsorption observations. Although the density of amino group in the base material affected both the grafting efficiency of the backfilling PEG and the electrokinetic potential mainly in the positive range, the latter mainly determined the protein- and cell-repelling abilities of the substrates. Furthermore, varying the surface compositions had almost no effect on the switching efficiency in the early stage of the culture, but it became more significant after culturing cells for a longer time; the cells fouled the nonirradiated PEGylated regions earlier on the surfaces with higher positive zeta potentials. These results indicate that the zeta potential is an essential factor in the long-term persistence of cellular patterns on photoactivatable substrates. This study not only provides a recipe for the development of a dynamic substrate with an adequate time frame but also clarifies how the interfacial nanoarchitectures, composed of the nanometer-scale PEG brushes and charged base materials, affect the biocompatibility.

We have developed a colorimetric measurement chip that measures triglycerides, total cholesterol, and high-density lipoprotein in 6 mL of whole blood collected with a painless needle. The chip can be used by patients to self-monitor certain health conditions at home. This chip contains a sharp 150 mm diameter stainless steel (SUS) needle that collects blood painlessly. The chip consists of three layers of injection-molded poly(methyl methacrylate) bonded together with two double-sided tapes. Two commercial reagents are used, and the volume ratio of plasma to reagent is doubled from the reagent specification to reduce the optical absorption length (and chip mass) by half. Centrifugal force separates the plasma from the blood, and then weighs out and mixes the plasma and reagents. A zigzag channel allows mixing of the plasma with the reagents mainly by vortex motion due to the centrifugal force generated at the corners of the channel. The measured values correlated well with conventionally tested values.

In addition to its role in the regulation of sex-related processes, 17 beta-estradiol (E2) participates in the prevention and treatment of cardiovascular diseases via nongenomic pathways mediated by estrogen receptors (ER-alpha) located in the cell membrane. To achieve specific nongenomic activity of E2, we linked E2 (4.4 mol %) to chitosan-phosphorylcholine (CH-PC) (20 mol % PC). Injections of ER-alpha solutions (S to 100 nmol L-1) over rehydrated CH-PC-E2 thin films led to permanent adsorption of ER-alpha to the film surface, as detected by quartz crystal microbalance with dissipation (QCM-D). However, ER-alpha did not bind onto CH-PC-E2 films formed in situ and never dried. X-ray photoelectron spectroscopy (XPS) analysis of spin-cast CH-PC-E2 films revealed significant E2 enrichment of the topmost section of the film, attributed to the preferential migration of E2 toward the film/air interface upon drying. Mechanical analysis of CH-PC-E2 films in the frequency domain probed by QCM-D indicated that rehydrated films behave as an entangled network with junction points formed by self-assembly of hydrophobic E2 moieties and by ion pairing among PC groups, whereas films formed in situ are entangled polymer solutions with temporary junctions. The structural analysis presented offers useful guidelines for the study of amphiphilic biomacromolecules designed for therapeutic use as thin films.

A great number of the neurites interconnect neuronal cells in a brain to form the complicate neural circuits, whose structures are dynamically changed with changing the numbers and destinations of the neurites. Fabricating a model of neural network in vitro is one of the promising methods to precisely assay the signal transmission and processing within the circuit as well as to examine behaviors of individual cells. In this study, aiming to fabricate the dynamically alterable neural network in vitro, the chemically modified surface with the photo-reactive self-assembled monolayer was applied to navigate the neurite outgrowth activities of differentiated PC12 cell in the spatially and temporally controlled manner. Numbers of the cell soma were effectively adhered and simultaneously arrayed according to the 25 mu m square patterns, which were easily fabricated with a single shot of the 365-nm ultraviolet (UV) irradiation and pre-coated with the extracellular matrix (ECM) protein. Narrow neurites were successively guided along the 5 mu m line patterns drawn on the surface by stepwise irradiation of the UV light in the intended designs and at appropriate timing. Sprouting number, elongating direction, bending, branching, and formation of autapse-like structure were controllable. The rate of neurite elongation was dependent on the ECM species, that were pre-coated beneath the cell soma, suggesting the ECM stimulated the basal side of the cell soma and affected the outgrowth process of the neurite. Navigation of the neurite elongation along the microline pattern for a primary rat brain cortex neuron was also achieved. (C) 2011 Elsevier B.V. All rights reserved.

Collective cell migration plays a major role in cancer metastasis and wound healing, therefore, several in vitro assays for studying such behavior have been developed. Using photoswitchable surfaces, we studied collective cell expansion behavior from initially precisely controlled adhesive patterns. A non-adhesive poly(ethylene glycol) (PEG) layer is conjugated to a glass coverslip via 2-nitrobenzyl groups, which cleave upon exposure to UV light, changing the surface from non-cell-adhesive to cell-adhesive without mechanical interference. Initial cell attaching areas are generated in arbitrary shapes via projection exposure through a photomask. After a growth phase, epithelial cell sheets are released from their confinement by a second illumination allowing for collective cell expansion. Our experiments with epithelial cells show that cluster size and boundary curvature modulate the expansion of the cell sheet and the formation of leader cells. At a certain cluster size, characteristics of the expansion behavior change and cells in the core are hardly affected by the boundary release. With donut-like ring structures, we demonstrate a break in symmetry between the behavior of cells along the outer convex boundary and along the inner concave boundary. Additionally, we observe that collective migration characteristics are modulated by the initial incubation time of the cell sheet. (C) 2011 Elsevier Ltd. All rights reserved.

The display of PHB depolymerase (PhaZRpiT1) from R. pickettii T1 on the surface of E. coli JM109 cells is realized using OprI of P. aeruginosa as the anchoring motif. The fusion protein is stably expressed and its surface localization is verified by immunofluorescence microscopy. The displayed PhaZRpiT1 retains its cleaving ability for soluble substrates as well as its ability to adsorb to the PHB surface, and also remains catalycically active in the degradation of insoluble polyester materials, in spite of the possible suppression of the enzyme movement on the polymer surface. The results demonstrate that PhaZRpiT1-displaying E. coli shows potential for use as a whole-cell biocatalyst for the production of (R)-3-hydroxybutyrate monomers from insoluble PHB materials.

The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG(7)). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7-10) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

It is well known that cell migrations play a key role in the living systems. From the basic technical point of view, cell migration control is one of the important and useful ways to clarify the mechanism of the cell migration qualitatively and quantitatively. For the purpose of high-resolution control and observation of the cell migrations, which is the goal of this research, in this study, an attempt was made to establish the fabrication process of micro-patterns on caged cell-culturing substrates using inverted microscope with high magnification objective lens. With the combination of fluorescence antibody technique, the adequate ultraviolet (UV) lay exposure time was obtained for the formation of micro-patterns of fibronectin on the caged cell-culturing substrates. We also confirmed that it was possible to control the position of micro-patterns by the primary and secondary UV exposure. In addition, Swiss 3T3 cell culture experiment demonstrated that the initial shape of the cell could be restricted by the primary UV exposure and that the secondary UV exposure was remarkably useful to control the cell migration. © 2011 The Institute of Electrical Engineers of Japan.

Dynamic control of cell adhesion on substrates is a useful technology in tissue engineering and basic biology. This paper describes a method for the control of cell adhesion on amino-bearing surfaces by reversible conjugation of an anti-fouling polymer, poly(ethylene glycol) (PEG), via a newly developed photocleavable linker, 1-(5-methoxy-2-nitro-4-prop-2-ynyloxyphenyl)ethyl N-succinimidyl carbonate (1). This molecule has alkyne and succinimidyl carbonate at each end, which are connected by photocleavable 2-nitrobenzyl ester. Under this molecular design, the molecule crosslinked azides and amines, whose linkage cleaved upon application of near-UV light. By using aminosilanised glass and silicon as model substrates, we studied their reversible surface modification with PEG-azide (M-w = 5000) based on contact angle measurements, ellipsometry, and AFM morphological observations. Protein adsorption and cell adhesion dramatically changed by PEGylation and the following irradiation, which can be used for cellular patterning. Also, the capability of the substrate to change cell adhesiveness by photoirradiation during cell cultivation was demonstrated by inducing cell migration. We believe this method will be useful for dynamic patterning of cells on protein-based scaffolds.

Dynamic control of cell-substrate interactions is useful for exploring signaling processes involved in cell adhesion and migration as well as patterning heterotypic cells to mimic living tissues. We have developed photoactivatable substrates whose surface was modified with photoreleasable cell-repellent blocking agents. The surfaces are initially non-cell adhesive because of the presence of the blocking agents, but become cell adhesive upon near-UV irradiation of the substrates via the photo-induced desorption of the blocking agents. Here, I overview the developmental process of our photoactivatable substrates and demonstrate their potential uses for bioanalysis.

Patterned immobilization of synthetic and biological ligands on material surfaces with controlled surface densities is important for various bioanalytical and cell biological purposes. This paper describes the synthesis, characterization, and application of a novel silane coupling agent bearing a photoremovable succinimidyl carbonate, which enables the photopatterning of various primary amines on glass and silicon surfaces. The silane coupling agent is 1-[5-methoxy-2-nitro-4-(3-trimethoxysilylpropyloxy)phenyl]ethyl N-succinimidyl carbonate. The distinct feature of this molecule is that it has a photocleavable 2-nitrobenzyl switch between a trimethoxysilyl group and a succinimidyl Carbonate, each reactive to the hydroxy groups of inorganic oxides and primary amines. Based on this molecular design, the compound allows for the one-step introduction Of succinimidyl carbonates onto the surface of glass and silicon, immobilization of primary amines, and region-selective and dose-dependent release of the amines by near-UV irradiation. Therefore, we were able to pattern amine ligands on the substrates in given Surface densities and arbitrary geometries by controlling the closes and regions of photoirradiation. These features were verified by UV-vis spectroscopy, contact angle measurements, infrared (IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force Microscopy (AFM). The compound was applied to form a chemical density gradient of amino-biotin on a Silicon Substrate in a range of 0.87-0.12 chains/nm(2) by controlling photoirradiation under a standard fluorescence microscope. Furthermore, we also succeeded in forming a chemical density gradient at a lower surface density range (0.15-0.011 chains/nm(2)) on the Substrate by diluting the feed amino-biotin with an inert control amine. (C) 2009 Elsevier B.V. All rights reserved.

The shape of cells is a key determinant of cellular fates and activities. In this study, we demonstrate a method for controlling the cellular shape on a chemically modified glass coverslip with micropatterned cell adhesiveness. The glass surface was chemically modified with an alkylsiloxane monolayer having a caged carboxyl group, where single-cell-sized hydrophilic islands with hydrophobic background were created by irradiating the substrate in contact with a photomask to produce the carboxyl group. Thus, the created surface hydrophilicity pattern was converted to a negative pattern of a protein-repellent amphiphilic polymer, Pluronic F108, according to its preferential adsorption to the hydrophobic surfaces. The following addition of a cell-adhesive protein, fibronectin, resulted in its selective adsorption to the irradiated regions. In this way, cell-adhesive islands were produced reproductively, and the cells formed a given shape on the islands. As examples of the cell-shape control, we seeded HeLa cells and NIH3T3 cells to an array of triangular spots, and fluorescently imaged the dynamic motions of cell protrusions extended from the periphery of the cells. The present method will not only be useful for studying the molecular mechanism of cell polarity formation, but also for studying other shape-related cellular events such as apoptosis, differentiation and migration.

Activities of the cells are regulated by extracellular cues, including soluble factors, surrounding cells and extracellular matrices. This paper describes photoresponsive substrates and nanoparticles that enable us to control the extracellular cues both in time and space. Arraying heterotypic cells and their induction of migration and proliferation have been accomplished by physical or chemical adsorption of blocking agents to the photoresponsive substrates. Photo-induced production of biological active substances has become possible by tethering them to photoresponsive nanoparticles. These materials provide new methodology for engineering and exploring cellular functions.

In this study, we examined the construction of biointerfaces on a commodity plastic surface using the non-equilibrium atmospheric pressure low frequency plasma (LF plasma) and poly(ethylene glycol) (PEG) macromonomers with different terminal functional groups. The surface of polypropylene (PP) was spin coated with poly(4-chloromethylstyrene) (PCMS) followed by PEG macromonomers and irradiated with LF plasma. The chemical immobilization of PEG proceeded rapidly and mostly completed within 30 s, which was confirmed from the contact angle measurements. It is interesting to note that the surface properties were remarkably different by the end group of PEG macromonomers used in this study. Monoacrylated PEG macromonomer-treated substrate exhibited high anti-biofouling property whereas dimethacrylated end PEG did not show anti-biofouling property. This method is simple and applicable for constructing PEG-based biointerface on a variety of commodity plastics.

A photoresponsive nanocarrier for amines based on gold nanoparticles (GNPs) having a photocleavable succinimidyl ester has been developed. It offers a useful platform for the synthesis of caged compounds. Using the GNPs, we have developed caged histamine for the first time and applied it to evoke intracellular signaling by controlled near-UV irradiation. The present work will allow for new possibilities in studies of inter- and intracellular signaling networks.

This article describes a photochemical method for the site-selective assembly of heterotypic cells on a glass substrate modified with a silane coupling agent having a caged functional group. Silane coupling agents having a carboxyl (COOH), amino (NH2), hydroxyl (OH), or thiol (SH) group protected by a photocleavable 2-nitrobenzyl group were synthesized to modify the surfaces of glass coverslips. The caged substrates were first coated by the adsorption of a blocking agent, bovine serum albumin (BSA), to make the entire surface non-cell-adhesive and then irradiated at 365 nm under a standard fluorescence microscope. The photocleavage reaction on the surface was followed by contact angle measurements and X-ray photoelectron spectroscopy. When COS7, NIH3T3, and HEK293 cells were seeded onto these substrates in a serum-free medium, the cells adhered selectively and efficiently to the irradiated regions on the caged NH2 Substrate, whereas the other caged COOH, SH, and OH substrates were nonphotoactivatable for cell adhesion. Qualitative and quantitative analysis of BSA adsorbed to the uncaged substrates revealed that this highly efficient photoactivation on the caged NH2 substrate arose because of the following reasons: (i) upon photoactivation, BSA adsorbed in advance on the 2-nitrobenzyl groups was readsorbed onto the uncaged functional groups and (ii) BSA readsorbed onto the NH2 groups became unable to passivate the surface against cell adhesion whereas BSA on the other groups still had normal passivating activity. It was also demonstrated that heterotypic single COS7, NIH3T3, and HEK293 cells were positioned at any desired arrangement on the caged NH2 substrate by repeating the UV irradiation at optimized array spot sizes and cell seeding in optimized cell concentrations. The present method will be particularly useful in studying the dynamic processes of cell-cell interactions at a single-cell level.

A novel method to produce a multifunctional microarray in which different types of self-assembled monolayers (SAMs) are positioned on predefined surface sites on an oxide-covered silicon substrate is described. To achieve this, a liquid-transportation system called "liquid manipulation lithography" (LML) is developed. This system allows the delivery of different varieties of molecular inks, trialkoxysilanes, onto each predefined surface position of the given substrate even under ambient conditions. Under optimum conditions, the transferred trialkoxysilane inks first form one-molecule-thick microstructures at each surface position through the hydrolysis of the reactive silanes with surface water adsorbed on the substrate, followed by a condensation reaction. Three types of trialkoxysilanes with long alkyl-chains, specifically triethoxysilylundecanal (TESUD), N-(6-amino-hexyl)-3-aminopropyltrimethoxysilane (AHAPS), and octadecyltrimethoxysilane (OTS), are used as model molecular inks due to their high-end group-functionalities in biological and electronic applications. The precise positioning of the ink with sub-micrometer edge resolution is performed by carefully controlling a femtoliter-scale liquid-injection micromanipulator under a microscope. To ensure that the prepared SAM microarray is available for parallel analysis of biomolecular interactions, the area-selective immobilization of a protein molecule is explored. Successful observation of the area-selective biomolecular attachment confirmed a high industrial potential for the method as a lithography-free process for the miniaturization of a multifunctional SAM array on an oxide substrate.

This paper describes a glass substrate having a photocleavable poly(ethylene glycol) (PEG) designed for light-induced cell micropatterning. The Substrate changed from non-cell-adhesive to cell-adhesive by the photocleavage of PEG. Cellular patterns maintained for more than 17 days, and they were able to be changed to control cell migration and proliferation at multi- and single-cell levels by irradiating their adjacent regions during cell cultivation.

Cell micropatterning is an important technique for a wide range of applications, such as tissue engineering, cell-based drug screening, and fundamental cell biology studies. This paper overviews cell patterning techniques based on chemically modified substrates with different degrees of cell adhesiveness. In particular, the focus is on dynamic substrates that change their cell adhesiveness in response to external stimuli, such as heat, voltage, and light. Such substrates allow researchers to achieve an in situ alteration of patterns of cell adhesiveness, which is useful for co-culturing multiple cell types and analyzing dynamic cellular activities. As an example of dynamic substrates, we introduce a dynamic substrate based on a caged compound, where we accomplished a light-driven alteration of cell adhesiveness and the analysis of a single cell's motility.

Spatiotemporal control of cell migration was achieved on a photoactivatable cell-culturing substrate. Single cells were micropatterned on the substrate and were induced to extend protrusions led by lamellipodia or filopodia alternatively by the subsequent formation of wide or narrow paths in their surroundings, respectively. By tracking the migration of single cells in a microarray format, we performed quantitative analysis of the migration rates of single cells.

Irradiation of a patterned benzophenone-terminated boron-doped diamond (BDD) surface with UV light (lambda = 350 nm) in the presence of a 15(mer) oligonucleotide resulted in the covalent linking of the DNA strand to the BDD interface.

Control of cell adhesion is a key technology for cell-based drug screening and for analyses of cellular processes. We developed a method to spatiotemporally control cell adhesion using a photochemical reaction. We prepared a cell-culturing substrate by modifying the surface of a glass coverslip, with a self-assembled monolayer of an alkylsiloxane having a photocleavable 2-nitrobenzyl group. Bovine serum albumin (BSA) was adsorbed onto the substrate to make the surface inert to cell adhesion. When exposed to UV light, the alkylsiloxane underwent a photocleavage reaction, leading to the release of BSA from the surface. Fibronectin, a protein promoting cell adhesion, was added to cover the irradiated regions and made them cell-adhesive. Seeding of cells on this substrate resulted in their selective adhesion to the illuminated regions. By controlling the sizes of the illuminated regions, we formed cell-adhesive spots smaller than single cells and located focal adhesions of the cells. Moreover, by subsequently illuminating the region alongside the cells patterned on the substrate in advance, we released their geometrical confinements and induced migration and proliferation. These manipulations were conducted under a conventional fluorescence microscope without any additional instruments. The present method of cell manipulation will be useful for cell biological studies as well as for the formation of cell arrays. (c) 2006 Elsevier B.V. All rights reserved.

The beta(2) adrenergic receptor (beta(2)AR) is a G protein-coupled receptor that is selective to epinephrine. We demonstrate herein monitoring of an agonist-induced conformational change Of PAR in living cells. The monitoring method is based on fluorescence resonance energy transfer from a cyan fluorescent protein (CFP) to a biarsenical fluorophore, FlAsH, attached to the C-terminus, and the third intracellular loop (ICL3), respectively. Recombinant beta(2)ARs exhibited agonist-induced increases in the FlAsH/CFP emission ratio, indicating that the ICL3 approached the C-terminus upon activation. Since the emission ratio changes were on a time scale of seconds, the conformational change of beta(2)AR in living cells was more rapid than that of purified beta(2)AR measured in vitro. Interestingly, the direction of the emission ratio change Of PAR was opposite to that of the norepinephrine-responsive alpha(2A) adrenergic receptor reported recently. It was suggested that this discrepancy corresponds directly to the diametric biological functions, i.e., the activation or inactivation of adenylyl cyclase. (c) 2006 Elsevier Inc. All rights reserved.

We demonstrate herein a new protein conformation indicator based on biarsenical fluorescein with an extended benzoic acid moiety. The present indicator is reactive to a genetically introduced tetracysteine motif (Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is a noncysteine amino acid) of proteins. Compared to the original biarsenical fluorescein (FlAsH) and the biarsenical Nile red analogue (BArNile), the present indicator exhibited larger fluorescence intensity changes in response to Ca2+-induced conformational rearrangements of calmodulin. A calculation of the highest occupied molecular orbital (HOMO) level of the benzoic acid moiety of the indicator molecule supports possible involvement of a photoinduced electron transfer (PET) process. These results indicate that the present indicator is useful for sensitive detection of protein conformational changes.

Potentiometric selectivity coefficients, K-A,B(pot), have been collected for ionophore-based ion-selective electrodes (ISEs) for inorganic anions reported during 1988-1998. In addition to the actual numerical values of K-A,B(pot) together with the methods and conditions for their determination, response slopes, linear concentration (activity) ranges, chemical compositions, and ionophore structures for the ISE membranes are tabulated.

We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + i, i + 4, and i + 5 positions of an alpha -helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity, The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.

A new method for evaluating chemical selectivity of agonists for the NMDA subtype of glutamate receptor (GluR) channels is described. The method is based on the magnitude of Ca2+ release from GluR-incorporated liposomes, which is measured by a Ca2+ ion-selective electrode with a thin-layer mode. The partially purified GluRs from rat whole brain were reconstituted into Ca2+-loaded liposomes. Small aliquots (each 50 mu l) of the proteoliposomes, in the presence of an antagonist DNQX for blocking non-NMDA subtype, were subjected to potentiometric measurements of Ca2+ release under stimulation by three kinds of agonists, i.e. NMDA, L-glutamate and L-CCG-IV. The amount of the Ca2+-ion flux through the GluR channel induced by the agonists was found to increase in the order of NMDA &lt; L-glutamate &lt; L-CCG-IV, which was consistent with that of binding affinity of the agonists toward the NMDA subtype. However, the range of selectivity of the relevant agonists was much smaller compared with results based on binding affinities. The present method provides physiologically more relevant values for the agonist selectivity of GluRs as compared to that of the conventional binding assay in the sense that the selectivity is based on the very magnitude of Ca2+ flux through the NMDA receptor, i.e. the extent of signal transduction by a given agonist. The evaluation of agonist selectivity based on Na+ release was also investigated by using a Na+ ion-selective electrode, but agonist-induced Na+ release was not detected, because of low permeability of Na+ through the NMDA subtype. (C) 1999 Elsevier Science B.V. All rights reserved.

A new method for evaluating chemical selectivity of agonists towards receptor ion channel proteins is proposed by using glutamate receptor (GluR) ion channel proteins and their agonists N-methyl-D-aspartic acid (NMDA), L-glutamate, and (2S, 3R, 4S) isomer of 2-(carboxycyclopropyl)glycine (L-CCGIV). Integrated multi-channel currents, corresponding to the sum of total amount of ions passed through the multiple open channels, were used as a measure of agonists' selectivity to recognize ion channel proteins and induce channel currents. GluRs isolated from rat synaptic plasma membranes were incorporated into planar bilayer lipid membranes (BLMs) formed by the folding method. The empirical factors that affect the selectivity were demonstrated: (i) the number of GluRs incorporated into BLMs varied from one membrane to another; (ii) each BLM contained different subtypes of GluRs (NMDA and/or non-NMDA subtypes); and (iii) the magnitude of multi-channel responses induced by L-glutamate at negative applied potentials was larger than at positive potentials, while those by NMDA and L-CCG-IV were linearly related to applied potentials. The chemical selectivity among NMDA, L-glutamate and L-CCG-IV for NMDA subtype of GluRs was determined with each single BLM in which only NMDA subtype of GluRs was designed to be active by inhibiting the non-NMDA subtypes using a specific antagonist DNQX. The order of selectivity among the relevant agonists for the NMDA receptor subtype was found to be L-CCG-IV&gt;L-glutamate&gt;NMDA, which is consistent with the order of binding affinity of these agonists towards the same NMDA subtypes. The potential use of this approach for evaluating chemical selectivity towards non-NMDA receptor subtypes of GluRs and other receptor ion channel proteins is discussed. (C) 1997 Elsevier Science Limited.