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ABSTRACT: Baculovirus infection of Sf9 cells at high densities, such as during mid- and late exponential phase, often results in a significant reduction of protein yield per cell, compared to the early exponential phase. Nutrient depletion has been considered as a major cause for the decreased protein yield. In this study, we report that the addition of nutrients (glucose, yeastolate ultrafiltrate, and lactalbumin hydrolysate) and small fraction of fresh medium at time of infection restores the expression level of actin and myosin V-HMM at late exponential phase (11.3 × 10(6) cells/ml) to that at early exponential phase (1.0 × 10(6) cells/ml). The relative yields of actin and myosin V-HMM were approximately equal at both phases (typically 200 mg of actin and 5 mg of myosin V-HMM per 10(10) cells), i.e., the volumetric yield of proteins from the cell culture at late exponential phase was approximately tenfold higher than at early exponential phase. The functionality of the recombinant actin and myosin V-HMM was confirmed by measuring the rate of actin polymerization, actin-activated ATPase, and the gliding velocity of actin filaments in an in vitro motility assay.
Journal of Muscle Research and Cell Motility 09/2012; 33(5):351-8. · 1.98 Impact Factor
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ABSTRACT: This paper describes a convenient screening method using ion trap electrospray ionization mass spectrometry to classify ligands to a target molecule in terms of kinetic parameters. We demonstrate this method in the screening of ligands to a hexahistidine tag from a pooled library synthesized by click chemistry. The ion trap mass spectrometry analysis revealed that higher stabilities of ligand-target complexes in the gas phase were related to lower dissociation rate constants, i.e., off-rates in solution. Finally, we prepared a fluorescent probe utilizing the ligand with lowest off-rate and succeeded in performing single molecule observations of hexahistidine-tagged myosin V walking on actin filaments.
ACS combinatorial science. 07/2012; 14(8):451-5.
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ABSTRACT: Chromosome segregation machinery is controlled by mechanochemical regulation. Tension in a mitotic spindle, which is balanced by molecular motors and polymerization-depolymerization dynamics of microtubules, is thought to be essential for determining the timing of chromosome segregation after the establishment of the kinetochore-microtubule attachments. It is not known, however, whether and how applied mechanical forces modulate the tension balance and chemically affect the molecular processes involved in chromosome segregation. Here we found that a mechanical impulse externally applied to mitotic HeLa cells alters the balance of forces within the mitotic spindle. We identified two distinct mitotic responses to the applied mechanical force that either facilitate or delay anaphase onset, depending on the direction of force and the extent of cell compression. An external mechanical impulse that physically increases tension within the mitotic spindle accelerates anaphase onset, and this is attributed to the facilitation of physical cleavage of sister chromatid cohesion. On the other hand, a decrease in tension activates the spindle assembly checkpoint, which impedes the degradation of mitotic proteins and delays the timing of chromosome segregation. Thus, the external mechanical force acts as a crucial regulator for metaphase progression, modulating the internal force balance and thereby triggering specific mechanochemical cellular reactions.
Proceedings of the National Academy of Sciences 04/2012; 109(19):7320-5. · 9.68 Impact Factor
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ABSTRACT: We fabricated fluorescent nanoparticles which monitor temperature changes without sensitivity to pH (4-10) and ionic strength (0-500 mM). The nanothermometers spontaneously enter living HeLa cells via endocytosis, enclosed in acidic organelles, i.e., endosome/lysosome, and then transported along microtubules in a temperature-dependent manner, working as "walking nanothermometers".
Lab on a Chip 03/2012; 12(9):1591-3. · 5.67 Impact Factor
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ABSTRACT: We here review the use of quantum dots (QDs) for the imaging of sarcomeric movements in cardiac muscle. QDs are fluorescence substances (CdSe) that absorb photons and reemit photons at a different wavelength (depending on the size of the particle); they are efficient in generating long-lasting, narrow symmetric emission profiles, and hence useful in various types of imaging studies. Recently, we developed a novel system in which the length of a particular, single sarcomere in cardiomyocytes can be measured at ~30 nm precision. Moreover, our system enables accurate measurement of sarcomere length in the isolated heart. We propose that QDs are the ideal tool for the study of sarcomere dynamics during excitation-contraction coupling in healthy and diseased cardiac muscle.
Journal of Biomedicine and Biotechnology 01/2012; 2012:313814. · 2.44 Impact Factor
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ABSTRACT: To establish α-tropomyosin (Tm)'s structure-function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, and wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm-actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasi-equivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca(2+). Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm-actin interaction cooperatively affects actin to result in generation of greater force and velocity.
Journal of Molecular Biology 12/2011; 414(5):667-80. · 4.00 Impact Factor
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ABSTRACT: It was recently demonstrated that laser irradiation can control the beating of cardiomyocytes and hearts, however, the precise mechanism remains to be clarified. Among the effects induced by laser irradiation on biological tissues, temperature change is one possible effect which can alter physiological functions. Therefore, we investigated the mechanism by which heat pulses, produced by infra-red laser light under an optical microscope, induce contractions of cardiomyocytes. Here we show that microscopic heat pulses induce contraction of rat adult cardiomyocytes. The temperature increase, ΔT, required for inducing contraction of cardiomyocytes was dependent upon the ambient temperature; that is, ΔT at physiological temperature was lower than that at room temperature. Ca(2+) transients, which are usually coupled to contraction, were not detected. We confirmed that the contractions of skinned cardiomyocytes were induced by the heat pulses even in free Ca(2+) solution. This heat pulse-induced Ca(2+)-decoupled contraction technique has the potential to stimulate heart and skeletal muscles in a manner different from the conventional electrical stimulations.
Biochemical and Biophysical Research Communications 12/2011; 417(1):607-12. · 2.48 Impact Factor
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ABSTRACT: Electron microscopy has shown that cross-bridges (CBs) are formed at the target zone that is periodically distributed on the thin filament in striated muscle. Here, by manipulating a single bead-tailed actin filament with optical tweezers, we measured the unbinding events of rigor CBs one by one on the surface of the A-band in rabbit skeletal myofibrils. We found that the spacings between adjacent CBs were not always the same, and instead were 36, 72, or 108 nm. Tropomyosin and troponin did not affect the CB spacing except for a relative increase in the appearance of longer spacing in the presence of Ca(2+). In addition, in an in vitro assay where myosin molecules were randomly distributed, were obtained the same spacing, i.e., a multiple of 36 nm. These results indicate that the one-dimensional distribution of CBs matches with the 36-nm half pitch of a long helical structure of actin filaments. A stereospecific model composed of three actin protomers per target zone was shown to explain the experimental results. Additionally, the unbinding force (i.e., the binding affinity) of CBs for the reconstituted thin filaments was found to be larger and smaller relative to that for actin filaments with and without Ca(2+), respectively.
Biophysical Journal 12/2011; 101(11):2740-8. · 3.65 Impact Factor
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ABSTRACT: In skeletal muscle, active force production varies as a function of sarcomere length (SL). It has been considered that this SL dependence results simply from a change in the overlap length between the thick and thin filaments. The purpose of this study was to provide a systematic understanding of the SL-dependent increase in Ca(2+) sensitivity in skeletal muscle, by investigating how thin filament "on-off" switching and passive force are involved in the regulation. Rabbit psoas muscles were skinned, and active force measurements were taken at various Ca(2+) concentrations with single fibers, in the short (2.0 and 2.4 μm) and long (2.4 and 2.8 μm) SL ranges. Despite the same magnitude of SL elongation, the SL-dependent increase in Ca(2+) sensitivity was more pronounced in the long SL range. MgADP (3 mM) increased the rate of rise of active force and attenuated SL-dependent Ca(2+) activation in both SL ranges. Conversely, inorganic phosphate (Pi, 20 mM) decreased the rate of rise of active force and enhanced SL-dependent Ca(2+) activation in both SL ranges. Our analyses revealed that, in the absence and presence of MgADP or Pi, the magnitude of SL-dependent Ca(2+) activation was (1) inversely correlated with the rate of rise of active force, and (2) in proportion to passive force. These findings suggest that the SL dependence of active force in skeletal muscle is regulated via thin filament "on-off" switching and titin (connectin)-based interfilament lattice spacing modulation in a coordinated fashion, in addition to the regulation via the filament overlap.
The Journal of Physiological Sciences 09/2011; 61(6):515-23. · 1.61 Impact Factor
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ABSTRACT: As the dynamic properties of cardiac sarcomeres are markedly changed in response to a length change of even ∼0.1 μm, it is imperative to quantitatively measure sarcomere length (SL). Here we show a novel system using quantum dots (QDs) that enables a real-time measurement of the length of a single sarcomere in cardiomyocytes. First, QDs were conjugated with anti-α-actinin antibody and applied to the sarcomeric Z disks in isolated skinned cardiomyocytes of the rat. At partial activation, spontaneous sarcomeric oscillations (SPOC) occurred, and QDs provided a quantitative measurement of the length of a single sarcomere over the broad range (i.e., from ∼1.7 to ∼2.3 μm). It was found that the SPOC amplitude was inversely related to SL, but the period showed no correlation with SL. We then treated intact cardiomyocytes with the mixture of the antibody-QDs and FuGENE HD, and visualized the movement of the Z lines/T tubules. At a low frequency of 1 Hz, the cycle of the motion of a single sarcomere consisted of fast shortening followed by slow relengthening. However, an increase in stimulation frequency to 3-5 Hz caused a phase shift of shortening and relengthening due to acceleration of relengthening, and the waveform became similar to that observed during SPOC. Finally, the anti-α-actinin antibody-QDs were transfected from the surface of the beating heart in vivo. The striated patterns with ∼1.96-μm intervals were observed after perfusion under fluorescence microscopy, and an electron microscopic observation confirmed the presence of QDs in and around the T tubules and Z disks, but primarily in the T tubules, within the first layer of cardiomyocytes of the left ventricular wall. Therefore, QDs are a useful tool to quantitatively analyze the movement of single sarcomeres in cardiomyocytes, under various experimental settings.
AJP Cell Physiology 08/2011; 301(5):C1116-27. · 3.54 Impact Factor
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ABSTRACT: Long-term disuse results in atrophy in skeletal muscle, which is characterized by reduced functional capability, impaired locomotor condition, and reduced resistance to fatigue. Here we show how long-term disuse affects contractility and fatigue resistance in single fibers of soleus muscle taken from the hindlimb immobilization model of the rat. We found that long-term disuse results in depression of caffeine-induced transient contractions in saponin-treated single fibers. However, when normalized to maximal Ca(2+)-activated force, the magnitude of the transient contractions became similar to that in control fibers. Control experiments indicated that the active force depression in disused muscle is not coupled with isoform switching of myosin heavy chain or troponin, or with disruptions of sarcomere structure or excessive internal sarcomere shortening during contraction. In contrast, our electronmicroscopic observation supported our earlier observation that interfilament lattice spacing is expanded after disuse. Then, to investigate the molecular mechanism of the reduced fatigue resistance in disused muscle, we compared the inhibitory effects of inorganic phosphate (Pi) on maximal Ca(2+)-activated force in control vs. disused fibers. The effect of Pi was more pronounced in disused fibers, and it approached that observed in control fibers after osmotic compression. These results suggest that contractile depression in disuse results from the lowering of myofibrillar force-generating capacity, rather than from defective Ca(2+) mobilization, and the reduced resistance to fatigue is from an enhanced inhibitory effect of Pi coupled with a decrease in the number of attached cross bridges, presumably due to lattice spacing expansion.
Journal of Applied Physiology 06/2011; 111(4):1080-7. · 3.75 Impact Factor
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ABSTRACT: The microtubule-based metaphase spindle is subjected to forces that act in diverse orientations and over a wide range of timescales. Currently, we cannot explain how this dynamic structure generates and responds to forces while maintaining overall stability, as we have a poor understanding of its micromechanical properties. Here, we combine the use of force-calibrated needles, high-resolution microscopy, and biochemical perturbations to analyze the vertebrate metaphase spindle's timescale- and orientation-dependent viscoelastic properties. We find that spindle viscosity depends on microtubule crosslinking and density. Spindle elasticity can be linked to kinetochore and nonkinetochore microtubule rigidity, and also to spindle pole organization by kinesin-5 and dynein. These data suggest a quantitative model for the micromechanics of this cytoskeletal architecture and provide insight into how structural and functional stability is maintained in the face of forces, such as those that control spindle size and position, and can result from deformations associated with chromosome movement.
Cell 06/2011; 145(7):1062-74. · 32.40 Impact Factor
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ABSTRACT: 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 tension-remarkably, 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 a 'side-sliding, end-catching' mechanism.
Nature Cell Biology 05/2011; 13(7):846-52. · 19.49 Impact Factor
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ABSTRACT: 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.
Progress in Biophysics and Molecular Biology 12/2010; 105(3):199-207. · 3.20 Impact Factor
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ABSTRACT: 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.
Progress in Biophysics and Molecular Biology 12/2010; 105(3):187-98. · 3.20 Impact Factor
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ABSTRACT: The regulation of actin filament networks by various proteins has essential roles in the growth cone dynamics. In this study we focused on the actin-myosin interaction which has been suggested to be an important player in the neurite extension. We examined in vitro how the decoration of actin filaments with a side-binding protein, drebrin-E, affects the motile properties of an intracellular transporter myosin V. Single myosin V molecules landed on the drebrin-E-decorated actin filaments with a lower frequency and ran over shorter distances; however, their velocities were normal. Furthermore, the analysis of the movement of myosin V molecules in the optical trap revealed that the decoration of actin filaments with drebrin-E markedly increased the load-sensitivity of the myosin V stepping. These results are attributable to the delay in the attachment of the motor's leading head (ADP·P(i) state) to actin, induced by the competitive binding of drebrin-E to actin, whereas the rate of ADP release from the trailing head (the rate-limiting step in the ATPase cycle of myosin V) is unaffected. Our study indicates that, in addition to the regulation of binding affinity of myosin V, drebrin-E also modulates the chemo-mechanical coupling in the motile myosin V molecules, presumably affecting the movement of the growth cone.
Biochemical and Biophysical Research Communications 10/2010; 400(4):643-8. · 2.48 Impact Factor
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ABSTRACT: Cardiac sarcomeres produce greater active force in response to stretch, forming the basis of the Frank-Starling mechanism of the heart. The purpose of this study was to provide the systematic understanding of length-dependent activation by investigating experimentally and mathematically how the thin filament "on-off" switching mechanism is involved in its regulation. Porcine left ventricular muscles were skinned, and force measurements were performed at short (1.9 µm) and long (2.3 µm) sarcomere lengths. We found that 3 mM MgADP increased Ca(2+) sensitivity of force and the rate of rise of active force, consistent with the increase in thin filament cooperative activation. MgADP attenuated length-dependent activation with and without thin filament reconstitution with the fast skeletal troponin complex (sTn). Conversely, 20 mM of inorganic phosphate (Pi) decreased Ca(2+) sensitivity of force and the rate of rise of active force, consistent with the decrease in thin filament cooperative activation. Pi enhanced length-dependent activation with and without sTn reconstitution. Linear regression analysis revealed that the magnitude of length-dependent activation was inversely correlated with the rate of rise of active force. These results were quantitatively simulated by a model that incorporates the Ca(2+)-dependent on-off switching of the thin filament state and interfilament lattice spacing modulation. Our model analysis revealed that the cooperativity of the thin filament on-off switching, but not the Ca(2+)-binding ability, determines the magnitude of the Frank-Starling effect. These findings demonstrate that the Frank-Starling relation is strongly influenced by thin filament cooperative activation.
The Journal of General Physiology 10/2010; 136(4):469-82. · 3.84 Impact Factor
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ABSTRACT: The organization of biomotile systems possesses structural and functional hierarchy, building up from single molecules via protein assemblies and cells further up to an organ. A typical example is the hierarchy of cardiac muscle, on the top of which is the heart. The heartbeat is supported by the rhythmic contraction of the muscle cells that is controlled by the Ca(2+) oscillation triggered by periodic electrical excitation of pacemaker cells. Thus, it is usually believed that the heartbeat is governed by the control system based on a sequential one-way chain with the electrical∕chemical information transfer from the upper to the lower level of hierarchy. On the other hand, it has been known for many years that the contractile system of muscle, i.e., skinned muscle fibers and myofibrils, itself possesses the auto-oscillatory properties even in the constant chemical environment. A recent paper [Plaçais, et al. (2009), Phys. Rev. Lett. 103, 158102] demonstrated the auto-oscillatory movement∕tension development in an in vitro motility assay composed of a single actin filament and randomly distributed myosin II molecules, suggesting that the auto-oscillatory properties are inherent to the contractile proteins. Here we discuss how the molecular motors may acquire the higher-ordered auto-oscillatory properties while stepping up the staircase of hierarchy.
HFSP journal. 06/2010; 4(3-4):100-4.
