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Arai S.,National University of Singapore | Arai S.,Waseda University | Lee S.-C.,Agency for Science, Technology and Research Singapore | Zhai D.,National University of Singapore | And 4 more authors.
Scientific Reports | Year: 2014

The dynamics of cellular heat production and propagation remains elusive at a subcellular level. Here we report the first small molecule fluorescent thermometer selectively targeting the endoplasmic reticulum (ER thermo yellow), with the highest sensitivity reported so far (3.9%/°C). Unlike nanoparticle thermometers, ER thermo yellow stains the target organelle evenly without the commonly encountered problem of aggregation, and successfully demonstrates the ability to monitor intracellular temperature gradients generated by external heat sources in various cell types. We further confirm the ability of ER thermo yellow to monitor heat production by intracellular Ca2+ changes in HeLa cells. Our thermometer anchored at nearly-zero distance from the ER, i.e. the heat source, allowed the detection of the heat as it readily dissipated, and revealed the dynamics of heat production in real time at a subcellular level.

Chiba M.,Waseda University | Miyazaki M.,Waseda University | Ishiwata S.,Waseda University | Ishiwata S.,Waseda Bioscience Research Institute in Singapore WABIOS
Biophysical Journal | Year: 2014

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models. © 2014 Biophysical Society.

Sato K.,RIKEN | Kuramoto Y.,International Institute for Advanced Studies | Ohtaki M.,Waseda University | Shimamoto Y.,Rockefeller University | And 2 more authors.
Physical Review Letters | Year: 2013

At an intermediate activation level, striated muscle exhibits autonomous oscillations called SPOC, in which the basic contractile units, sarcomeres, oscillate in length, and various oscillatory patterns such as traveling waves and their disrupted forms appear in a myofibril. Here we show that these patterns are reproduced by mechanically connecting in series the unit model that explains characteristics of SPOC at the single-sarcomere level. We further reduce the connected model to phase equations, revealing that the combination of local and global couplings is crucial to the emergence of these patterns. © 2013 American Physical Society.

Oguchi Y.,Waseda University | Uchimura S.,RIKEN | Ohki T.,Waseda University | Mikhailenko S.V.,Waseda University | And 2 more authors.
Nature Cell Biology | Year: 2011

During cell division the replicated chromosomes are segregated precisely towards the spindle poles. Although many cellular processes involving motility require ATP-fuelled force generation by motor proteins, most models of the chromosome movement invoke the release of energy stored at strained (owing to GTP hydrolysis) plus ends of microtubules. This energy is converted into chromosome movement through passive couplers, whereas the role of molecular motors is limited to the regulation of microtubule dynamics. Here we report, that the microtubule-depolymerizing activity of MCAK (mitotic centromere-associated kinesin), the founding member of the kinesin-13 family, is accompanied by the generation of significant tensiongremarkably, at both microtubule ends. An MCAK-decorated bead strongly attaches to the microtubule side, but readily slides along it in either direction under weak external loads and tightly captures and disassembles both microtubule ends. We show that the depolymerization force increases with the number of interacting MCAK molecules and is ∼1-pN per motor. These results provide a simple model for the generation of driving force and the regulation of chromosome segregation by the activity of MCAK at both kinetochores and spindle poles through aside-sliding, end-catchinmechanism. © 2011 Macmillan Publishers Limited. All rights reserved.

Sato K.,Tohoku University | Ohtaki M.,Waseda University | Shimamoto Y.,Waseda University | Shimamoto Y.,Rockefeller University | And 2 more authors.
Progress in Biophysics and Molecular Biology | Year: 2011

It is widely accepted that muscle cells take either force-generating or relaxing state in an all-or-none fashion through the so-called excitation-contraction coupling. On the other hand, the membrane-less contractile apparatus takes the third state, i.e., the auto-oscillation (SPOC) state, at the activation level that is intermediate between full activation and relaxation. Here, to explain the dynamics of all three states of muscle, we construct a novel theoretical model based on the balance of forces not only parallel but also perpendicular to the long axis of myofibrils, taking into account the experimental fact that the spacing of myofilament lattice changes with sarcomere length and upon contraction. This theory presents a phase diagram composed of several states of the contractile apparatus and explains the dynamic behavior of SPOC, e.g., periodical changes in sarcomere length with the saw-tooth waveform. The appropriate selection of the constant of the molecular friction due to the cross-bridge formation can explain the difference in the SPOC periods observed under various activating conditions and in different muscle types, i.e., skeletal and cardiac. The theory also predicts the existence of a weak oscillation state at the boundary between SPOC and relaxation regions in the phase diagram. Thus, the present theory comprehensively explains the characteristics of auto-oscillation and contraction in the contractile system of striated muscle. © 2010 Elsevier Ltd.

Ishiwata S.,Waseda University | Ishiwata S.,Waseda Bioscience Research Institute in Singapore WABIOS | Shimamoto Y.,Rockefeller University | Fukuda N.,Jikei University School of Medicine
Progress in Biophysics and Molecular Biology | Year: 2011

It is widely known that the contractile system of muscle takes on either the state of contraction (force-generating) or the state of relaxation (non-force-generating), which is known as the " all-or-nothing" principle. However, it is important to note that under intermediate activation conditions there exists a third state, which demonstrates auto-oscillatory properties and is termed SPOC (SPontaneous Oscillatory Contraction) state. We present a phase diagram, in which the states of the contractile system of muscle are divided into three regions consisting of contraction, relaxation and SPOC states. In the present review, experimental data related to the characteristics of SPOC are summarized and the mechanism of SPOC is described. We propose that the bio-motile system itself is an auto-oscillator, even in a membrane-less supra-molecular structure composed of an assembly of molecular motors and cytoskeletons (actin filaments and microtubules). Finally, the physiological significance of SPOC is discussed. © 2010 Elsevier Ltd.

Sakai H.,Waseda Bioscience Research Institute in Singapore WABIOS
Artificial cells, nanomedicine, and biotechnology (Print) | Year: 2013

Hemoglobin-based oxygen carriers (HBOCs) have been developed as a transfusion alternative and oxygen therapy. The Hb source is usually outdated donated human blood or cow blood obtained from cattle industries because of its abundance. This study examined the feasibility of using swine Hb ((S)Hb) for preparation of cellular-type HBOCs, hemoglobin-vesicles (HbV). Purification of (S)Hb from fresh swine whole blood was conducted with processes including carbonylation ((S)HbO(2) --> (S)HbCO), pasteurization (60 °C, 15 hours) and tangential flow ultrafiltration, with yield of 90%. Actually, differential scanning calorimetric analysis showed a denaturation temperature of (S)HbCO at 83 °C and assures its stability during pasteurization. Concentrated (S)HbCO together with pyridoxal 5'-phosphate (PLP) as an allosteric effector was encapsulated in phospholipid vesicles to prepare (S)HbV. After decarbonylation ((S)HbCO --> (S)HbO(2)), the oxygen affinity (P(50)) of (S)Hb changes mainly by PLP, and the influence of Cl(-) was small, in a manner similar to that of human Hb ((H)Hb). However, after encapsulation, vesicles of (S)HbV showed much lower oxygen affinity (higher P(50)) than (H)HbV did. Autoxidation of (S)HbV was slightly faster than (H)HbV. Although some differences are apparent in oxygen affinity and autoxidation rates, results clarified that (S)Hb is useful as a starting material for HbV production.

Miyazaki M.,Waseda University | Chiba M.,Waseda University | Eguchi H.,Waseda University | Ohki T.,Waseda University | And 2 more authors.
Nature Cell Biology | Year: 2015

During cell division, many animal cells transform into a spherical shape and assemble a contractile ring composed of actin filaments and myosin motors at the equator to separate the cell body into two. Although actomyosin regulatory proteins are spatio-temporally controlled during cytokinesis, the direct contribution of cell shape and actomyosin activity to the contractile ring assembly remains unclear. Here, we demonstrated in vitro that actin polymerization inside cell-sized spherical droplets induced the spontaneous formation of single ring-shaped actin bundles in the presence of bundling factors. Despite a lack of spatial regulatory signals, the rings always assembled at the equator to minimize the elastic energy of the bundles. Myosin promoted ring formation by the dynamic remodelling of actin networks, and an increase in the effective concentration of myosin triggered ring contraction. These results will help us understand how animal cells coordinate cell shape and actomyosin activities to direct cytokinesis. © 2015 Macmillan Publishers Limited.

Takagi J.,Waseda University | Itabashi T.,Waseda University | Suzuki K.,Waseda University | Kapoor T.M.,Rockefeller University | And 3 more authors.
Cell Reports | Year: 2013

The polymerization/depolymerization dynamics of microtubules (MTs) have been reported to contribute to control of the size and shape of spindles, but quantitative analysis of how the size and shape correlate with the amount and density of MTs in the spindle remains incomplete. Here, we measured these parameters using 3D microscopy of meiotic spindles thatself-organized in Xenopus egg extracts and presented a simple equation describing the relationship among these parameters. To examine the validity of the equation, we cut the spindle into two fragments along the pole-to-pole axis by micromanipulation techniques that rapidly decrease the amount of MTs. The spheroidal shape spontaneously recovered within 5min, but the size of each fragment remained small. The equation we obtained quantitatively describes how the spindle size correlates with the amount of MTs while maintaining the shape and the MT density

Mikhailenko S.V.,Waseda University | Oguchi Y.,Waseda University | Ishiwata S.,Waseda University | Ishiwata S.,Waseda Bioscience Research Institute in Singapore WABIOS
Journal of the Royal Society Interface | Year: 2010

In cells, ATP (adenosine triphosphate)-driven motor proteins, both cytoskeletal and nucleic acid-based, operate on their corresponding 'tracks', that is, actin, microtubules or nucleic acids, by converting the chemical energy of ATP hydrolysis into mechanical work. During each mechanochemical cycle, a motor proceeds via several nucleotide states, characterized by different affinities for the 'track' filament and different nucleotide (ATP or ADP) binding kinetics, which is crucial for a motor to efficiently perform its cellular functions. The measurements of the rupture force between the motor and the track by applying external loads to the individual motor-substrate bonds in various nucleotide states have proved to be an important tool to obtain valuable insights into the mechanism of the motors' performance.We review the application of this technique to various linear molecular motors, both processive and nonprocessive, giving special attention to the importance of the experimental geometry. © 2010 The Royal Society.

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