Language：English   Publishing type：Research paper (scientific journal)  

Stem cell-derived sheet engineering has been developed as the next-generation treatment for myocardial infarction (MI) and offers attractive advantages in comparison with direct stem cell transplantation and scaffold tissue engineering. Furthermore, induced pluripotent stem cell-derived cell sheets have been indicated to possess higher potential for MI therapy than other stem cell-derived sheets because of their capacity to form vascularized networks for fabricating thickened human cardiac tissue and their long-term therapeutic effects after transplantation in MI. To date, stem cell sheet transplantation has exhibited a dramatic role in attenuating cardiac dysfunction and improving clinical manifestations of heart failure in MI. In this review, we retrospectively summarized the current applications and strategy of stem cell-derived cell sheet technology for heart tissue repair in MI.

DOI： 10.1186/s13287-019-1536-y

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The application of mechanical stimuli to cells often induce increases in intracellular calcium, affecting the regulation of a variety of cell functions. Although the mechanism of mechanotransduction-induced calcium increases has not been fully resolved, the involvement of mechanosensitive ion channels in the plasma membrane and the endoplasmic reticulum has been reported. Here, we demonstrate that voltage-gated L-type calcium channels play a critical role in the mechanosensitive calcium response in H9c2 rat cardiomyocytes. The intracellular calcium level in H9c2 cells increased in a reproducible dose-dependent manner in response to uniaxial stretching. The stretch-activated calcium response (SICR) completely disappeared in calcium-free medium, whereas thapsigargin and cyclopiazonic acid, inhibitors of sarcoendoplasmic reticulum calcium ATPase, partially reduced the SICR. These findings suggest that both calcium influx across the cell membrane and calcium release from the sarcoendoplasmic reticulum are involved in the SICR. Nifedipine, diltiazem, and verapamil, inhibitors of L-type calcium channels, reduced the SICR in a dose-dependent manner. Furthermore, small interfering RNA against the L-type calcium channel α1c subunit diminished the SICR dramatically. Nifedipine also diminished the mechanosensitivity of Langendorff-perfused rat heart. These results suggest that the SICR in H9c2 cardiomyocytes involves the activation of L-type calcium channels and subsequent calcium release from the sarcoendoplasmic reticulum.

DOI： 10.1016/j.ceca.2019.02.008

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Language：Japanese   Publisher：(NPO)日本歯周病学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

DOI： 10.1016/j.bpj.2016.11.1471

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Language：Japanese   Publisher：(一社)日本生理学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMP-response element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons. To visualize CREB at the single-molecule level, fluorescent-tagged CREB in living dissociated cortical neurons was observed by highly inclined and laminated optical sheet microscopy. We found that a significant fraction of CREB spots resided in the restricted locations in the nucleus for several seconds (dissociation rate constant: 0.42 s(-1)). In contrast, two mutant CREBs, which cannot bind to the cAMP-response element, scarcely exhibited long-term residence. To test the possibility that CREB dynamics depends on neuronal activity, pharmacological treatments and an optogenetic method involving channelrhodopsin-2 were applied to cultured cortical neurons. Increased neuronal activity did not appear to influence the residence time of CREB spots, but markedly increased the number of restricted locations (hot spots) where CREB spots frequently resided with long residence times (>1s). These results suggest that neuronal activity promotes CREB-dependent transcription by increasing the frequency of CREB binding to highly localized genome locations.

DOI： 10.1523/JNEUROSCI.0943-16.2017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Integrins mediate cell adhesion to the extracellular matrix and enable the construction of complex, multicellular organisms, yet fundamental aspects of integrin-based adhesion remain poorly understood. Notably, the magnitude of the mechanical load experienced by individual integrins within living cells is unclear, due principally to limitations inherent to existing techniques. Here we use Forster resonance energy transfer-based molecular tension sensors to directly measure the distribution of loads experienced by individual integrins in living cells. We find that a large fraction of integrins bear modest loads of 1-3 pN, while subpopulations bearing higher loads are enriched within adhesions. Further, our data indicate that integrin engagement with the fibronectin synergy site, a secondary binding site specifically for alpha(5)beta(1) integrin, leads to increased levels of alpha(5)beta(1) integrin recruitment to adhesions but not to an increase in overall cellular traction generation. The presence of the synergy site does, however, increase cells' resistance to detachment by externally applied loads. We suggest that a substantial population of integrins experiencing loads well below their peak capacities can provide cells and tissues with mechanical integrity in the presence of widely varying mechanical loads.

DOI： 10.1021/acsnano.6b03314

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Proper spatiotemporal gene expression is achieved by selective DNA binding of transcription factors in the genome. The most intriguing question is how dynamic interactions between transcription factors and their target sites contribute to gene regulation by recruiting the basal transcriptional machinery. Here we demonstrate individual binding and dissociation events of the transcription factor cAMP response element-binding protein (CREB), both in vitro and in living cells, using single-molecule imaging. Fluorescent-tagged CREB bound to its target sequence cAMP-response element (CRE) for a remarkably longer period (dissociation rate constant: 0.21 s(-1)) than to an unrelated sequence (2.74 s(-1)). Moreover, CREB resided at restricted positions in the living cell nucleus for a comparable period. These results suggest that CREB stimulates transcription by binding transiently to CRE in the time range of several seconds.

DOI： 10.1038/srep10662

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.

DOI： 10.1371/journal.pone.0126262

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Focal adhesions (FAs) are micron-sized protein assemblies that coordinate cell adhesion, migration, and mechanotransduction. How the many proteins within FAs are organized into force sensing and transmitting structures is poorly understood. We combined fluorescent molecular tension sensors with super-resolution light microscopy to visualize traction forces within FAs with <100 nm spatial resolution. We find that alpha(v)beta(3) integrin selectively localizes to high force regions. Paxillin, which is not generally considered to play a direct role in force transmission, shows a higher degree of spatial correlation with force than vinculin, talin, or alpha-actinin, proteins with hypothesized roles as force transducers. These observations suggest that alpha(v)beta(3) integrin and paxillin may play important roles in mechanotransduction.

DOI： 10.1021/nl5047335

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Living cells are exquisitely responsive to mechanical cues, yet how cells produce and detect mechanical force remains poorly understood due to a lack of methods that visualize cell-generated forces at the molecular scale. Here we describe Forster resonance energy transfer (FRET)-based molecular tension sensors that allow us to directly visualize cell-generated forces with single-molecule sensitivity. We apply these sensors to determine the distribution of forces generated by individual integrins, a class of cell adhesion molecules with prominent roles throughout cell and developmental biology. We observe strikingly complex distributions of tensions within individual focal adhesions. FRET values measured for single probe molecules suggest that relatively modest tensions at the molecular level are sufficient to drive robust cellular adhesion.

DOI： 10.1021/nl4005145

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Transformations between G-(monomeric) and F-actin (polymeric) are important in cellular behaviors such as migration, cytokinesis, and morphing. In order to understand these transitions, we combined single-molecule Forster resonance energy transfer with total internal reflection fluorescence microscopy to examine conformational changes of individual actin protomers. We found that the protomers can take different conformational states and that the transition interval is in the range of hundreds of seconds. The distribution of these states was dependent on the environment, suggesting that actin undergoes spontaneous structural changes that accommodate itself to polymerization.

DOI： 10.1371/journal.pone.0045864

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DOI： 10.1016/j.bpj.2010.12.1832

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DOI： 10.1016/j.bpj.2010.12.385

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DOI： 10.1016/j.bpj.2009.12.827

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Language：English  

DOI： 10.1016/j.bpj.2009.12.848

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S54_5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN ACAD  

Until recently, actin was thought to act merely as a passive track for its motility partner, myosin, during actomyosin interactions. Yet a recent report; having observed dynamical conformational changes in labeled skeletal muscle alpha-actin suggests that actin has a more active role. Because the labeling technique was still immature, however. conclusions regarding the significance of the different conformations are difficult to make. Here, we describe the preparation of fully active alpha-actin obtained from a, baculovirus expression system. We developed alpha-actin recombinants, of which subdomains 1 and 2 have specific sites for fluorescent probes. This specific labeling technique offers to significantly expand the information acquired from actin studies.

DOI： 10.2183/pjab.85.491

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S172_2

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S101_1

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.46.S221_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：VSP BV  

A simple microfabrication method for a controlled-re lease drug-delivery system has been designed using biodegradable polymeric microchips. Microholes were made in a poly(L-lactic acid) plate and dyes were cast in each well. After drying, the wells were sealed with polymers having different biodegradation rates using a mold that had hollows corresponding to the wells. The polymers were prepared by mixing polylactides with the co-polymers. The sealing was confirmed by ultrasonication. The plate was incubated in phosphate-buffered saline and the dye released from the plate as the degradation proceeded was detected spectrophotometrically. The higher the degradation rate of the polymer sealing, the faster the sealed dye was released. This biodegradable biochip is useful for the design of controlled-re lease drug-delivery systems.

DOI： 10.1163/1568562054414621

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S248_1

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：CELL PRESS  

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

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