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To bridge the gap between the contractile system in muscle and in vitro motility assay, we have devised an A-band motility assay system. A glycerinated skeletal myofibril was treated with gelsolin to selectively remove the thin filaments and expose a single A-band. A single bead-tailed actin filament trapped by optical tweezers was made to interact with the inside or the outer surface of the A-band, and the displacement of the bead-tailed filament was measured in a physiological ionic condition by phase-contrast and fluorescence microscopy. We observed large back-and-forth displacement of the filament accompanied by a large change in developed force. Despite this large tension fluctuation, we found that the average force was proportional to the overlap inside and outside the A-band up to approximately 150 nm and 300 nm from the end of the A-band, respectively. Consistent with the difference in the density of myosin molecules, the average force per unit length of the overlap inside the A-band (the time-averaged force/myosin head was approximately 1 pN) was approximately twice as large as that outside. Thus, we conclude that the A-band motility assay system described here is suitable for studying force generation on a single actin filament, and its sliding movement within a regular three-dimensional thick filament lattice.

Src family kinases are major non-receptor tyrosine kinase in cells and Src-mediated signal transduction involves various cellular functions. In activation process, Src molecules translocate to cell membrane and the subdomains in Src, SH2 and SH3 domains, are exposed. For Src to serve as a kinase, the interaction with its substrates via SH2 and/or SH3 domains is required. Although the activation mechanism of Src has been well-studied, the dynamics of Src at the cell membrane is still unclear. In this study, we examined the role of Src subdomains, especially SH2 and SH3 domains, on the dynamics of Src at the cell membrane. To achieve this, we constructed PAmCherry-tagged wild-type Src (SrcWT), SrcW121A and SrcR178A mutants that decrease the binding of Src to its substrate(s) via SH3 and SH2 domains, respectively, and traced individual Src molecules in the cell membrane with gentle activation of PAmCherry. SrcWT dynamically moved on the cell membrane in the range of 0.27±0.01 μm2/s within a few seconds. The dynamics of SrcR178A mutant was comparable with that of SrcWT, whereas SrcW121A mutant exhibited less mobility (0.16±0.01 μm2/s) at the cell membrane compared with SrcWT. Since both SrcW121A and SrcR178A mutants showed higher phosphorylation level than SrcWT, the result indicates that the less mobility of SrcW121A in the cell membrane seems not to depend upon Src activation status. We further demonstrate that SrcW121A mutant showed ∼30% increase in the Src molecules residence time at focal adhesion compared with SrcWT, which is mediated by slower dissociation from adhesion site. Taken together with enhanced localization of SrcW121A at focal adhesion, our findings show that the SH3 domain of Src molecules governs dynamics of Src at the cell membrane, which may be involved in the rapid signal transduction in cells.

Embryonic stem cell (ESC) is pluripotent cell, which is capable of self-renewal and can differentiate to all types of cells. The ESCs collectively differentiate to the right cell in the right place in a group during tissue development. Because the mouse ESC (mESC) can be considered as a simple spontaneously fluctuating individual that does not have any oscillators or circadian clock, it is thought that the collective differentiation in stem cell conceals the mechanism to generate orderliness from assemble of randomness. In order to find the basic role to generate the collective differentiation, we here investigate relationships of heterogeneity of cells and of colonies during the early differentiation stage in mESC by using single cell microscopy. We collected and analyzed total of 100 images of mESC colony containing approximately 30 cells every day for 8 days after differentiation induction. The cellular state could be defined by the fluorescent intensities of the reporters, Venus and mKate2, respectively reporting expressions of Nanog and Oct4, which are core transcription factors for pluripotency maintenance. The heterogeneity in cellular state could be exhibited not only in individual cell but also in colony. We found that the cell tended to resemble its state to the neighboring cells with some kinds of cooperativity, causing the colonial heterogeneity with restricting the cellular fluctuation in a colony. The quantitative analysis of the experimental data lead us the hypothesis that the imbalance of spontaneous fluctuation in a cell and cooperativity between cells realizes a flashing Brownian ratchet, causing the avalanche-like state transition of differentiation state. The present hypothesis was further reinforced by the numerical toy-model with simple rules. The fluctuation and cooperativity play a role for preserving the fractal heterogeneity in multilayer, and the flashing Brownian ratchet is possibly an intrinsic common feature in collective decision making in fluctuating individuals.

Skinned skeletal and cardiac muscle fibers exhibit spontaneous oscillatory contraction (SPOC) in the presence of MgATP, MgADP, and inorganic phosphate (Pi), but the molecular mechanism underlying this phenomenon is not yet clear. We have investigated the role of regulatory proteins in SPOC using cardiac muscle fibers of which the actin filaments had been reconstituted without tropomyosin and troponin, according to a previously reported method (Fujita et al., 1996. Biophys. J. 71:2307–2318). That is, thin filaments in glycerinated cardiac muscle fibers were selectively removed by treatment with gelsolin. Then, by adding exogenous actin to these thin filament-free cardiac muscle fibers under polymerizing conditions, actin filaments were reconstituted. The actin filament-reconstituted cardiac muscle fibers generated active tension in a Ca2+-insensitive manner because of the lack of regulatory proteins. Herein we have developed a new solvent condition under which SPOC occurs, even in actin filament-reconstituted fibers: the coexistence of 2,3-butanedione 2-monoxime (BDM), a reversible inhibitor of actomyosin interactions, with MgATP, MgADP and Pi. The role of BDM in the mechanism of SPOC in the actin filament-reconstituted fibers was analogous to that of the inhibitory function of the tropomyosin-troponin complex (-Ca2+) in the control fibers. The present results suggest that SPOC is a phenomenon that is intrinsic to the actomyosin motor itself.

The cell environment is very crowded, containing various molecules, proteins and nucleotides. This crowded condition is an indispensable factor for cellular functions of proteins. In the past, the diffusion coefficient of a chemical probe has been used as an evaluation index of the intracellular crowded condition. However, crowding depends not only on the mobility, but also the density of the crowding agents. We have succeeded in making a yellow fluorescent protein (YFP) that senses crowding density via hydrophobicity by inserting into the YFP a glycine and conjugating it to cyan fluorescent protein (CFP), which is insensitive to Forster resonance energy transfer (FRET) probe. This probe has been named GimRET (Glycine inserted mutant FRET probe). GimRET enabled us to visualize the dynamic changes of the intracellular crowding density during cell division. Because GimRET can distinguish the crowding density from the viscosity of the solution, crowding can be evaluated by the GimRET fluorescence intensity ratio and diffusion coefficient, which respectively reflects the density and mobility of the crowding agents. While the diffusion coefficient of GimRET linearly relates to the fluorescence intensity ratio, the slope also depends on the location of the cell, i.e., nucleus or cytoplasm, indicating that the diffusion coefficient alone is insufficient for defining crowding. Here, we propose the simultaneous observation of the GimRET intensity ratio and the diffusion coefficient as a way to evaluate intracellular crowding.

In the past couple of decades, single molecule measurement techniques have tremendously unveiled dynamic behavior and function of individual motor protein molecules. In many living systems, however, multiple molecules work with sort of cooperativity to increase the energy efficiency and the robustness. To understand the physicochemical mechanism of the motor protein systems, the attention is now shifting to upper hierarchy; from individual elements to cooperative motion of multiple protein molecules. We have proposed a novel technique for simultaneous nanometric tracking of dynamic behavior of multiple motor proteins along a one-dimensional rail protein filament. By simple combination of a total internal reflection fluorescence microscope with an imaging spectrometer, multiple motor proteins labeled with quantum dots of different colors can be simultaneously observed with the millisecond time resolution. Even two or more adjacent motor proteins working within nanometric scale, which is much smaller than spatial resolution of optical microscopy, can be separately tracked. Our microscopy system has successfully tracked one-dimensional processive movement of multiple myosins (myosin VI) with a few nanometers precision, and realized observation of how they were chasing each other back and forth on an actin filament. We also applied the microscopy to dynamic observation of two heads of a single myosin (myosin V), and showed the hand-over-hand walking manner, which is consistent with previous works. Furthermore, we extended the technique to two dimensional tracking and applied it to tracking of multiple myosin V on two-dimensional plane. We believe that this would be a useful tool to investigate cooperative movement of motor protein molecules in nanoscale.

Single particle tracking is widely used to study protein movement with high spatiotemporal resolution both in vitro and in cells. Quantum dots, which are semiconductor nanoparticles, have recently been employed in single particle tracking because of their intense and stable fluorescence. Although single particles inside cells have been tracked in three spatial dimensions (X, Y, Z), measurement of the angular orientation of a molecule being tracked would significantly enhance our understanding of the molecule’s function. In this study, we synthesized highly polarized, rod-shaped quantum dots (Qrods) and developed a coating method that optimizes the Qrods for biological imaging. We describe a Qrod-based single particle tracking technique that blends optical nanometry with nanomaterial science to simultaneously measure the three-dimensional and angular movements of molecules. Using Qrods, we spatially tracked a membrane receptor in living cells in four dimensions with precision close to the single-digit range in nanometers and degrees.

The muscle contractile apparatus has a highly ordered liquid crystalline structure. The molecular mechanism underlying the formation of this apparatus remains, however, to be elucidated. Selective removal and reconstitution of the components are useful means of examining this mechanism. In addition, this approach is a powerful technique for examining the structure and function of a specific component of the contractile system. In this study we have achieved the structural and functional reconstitution of thin filaments in the cardiac contractile apparatus. First, all thin filaments other than short fragments at the Z line were removed by treatment with gelsolin. Under these conditions no active tension could be generated. By incorporating exogenous actin into these thin filament-free fibers, actin filaments were reconstituted, and active tension, which was insensitive to Ca2+, was restored. The active tension after the reconstitution of thin filaments reached 135 +/- 64% of the original level. The augmentation of tension was attributable to the elongation of reconstituted filaments. As another possibility for augmented tension generation, we suggest the presence of an inhibitory system that was not reconstituted. In any case, the thin filaments of the cardiac contractile apparatus are considered to be assembled so as not to develop the highest degree of tension. Incorporation of the tropomyosin-troponin complex fully restored Ca2+ sensitivity without affecting maximum tension. The present results indicate that a muscle contractile apparatus with a higher order structure and function can be constructed by the self-assembly of constituent proteins.

We investigated the effect of pH on isometric tension in actin filament-reconstituted and thin filament-reconstituted bovine cardiac muscle fibers in the pH range of 6.0-7.4. Thin filament was reconstituted from purified G-actin with either bovine cardiac tropomyosin (Tm) or rabbit skeletal Tm in conjunction with cardiac or skeletal troponin (Tn). Results showed that isometric tension decreased linearly with a decrease in pH. The slope of the pH-tension relation, DeltaF/DeltapH (Deltarelative tension/Deltaunit pH), was 0.28 and 0.44 in control cardiac fibers and skeletal fibers, respectively. In actin filament-reconstituted fibers without regulatory proteins, DeltaF/DeltapH was 0.62, namely larger than that in cardiac or skeletal fibers. When reconstituted with cardiac Tm-Tn complex (nTm), DeltaF/DeltapH recovered to 0.32, close to the value obtained in control cardiac fibers. When reconstituted with skeletal nTm, DeltaF/DeltapH recovered to 0.48, close to the value for control skeletal fibers. To determine whether Tm or Tn is responsible for the inhibitory effects of nTm on the tension decrease caused by reduced pH, thin filament was reconstituted with cardiac Tm and skeletal Tn, or with skeletal Tm and cardiac Tn. When cardiac Tm was used, pH dependence of isometric tension coincided with that of control cardiac fibers. When skeletal Tm was used, the pH dependence coincided with that of control skeletal fibers. Furthermore, closely similar results were obtained in fibers reconstituted with actin and either cardiac or skeletal Tm without Tn. These results demonstrate that Tm but not Tn modulates the pH dependence of active tension.