Madoka Suzuki

A nanoparticle-based ratiometric and self-calibrated fluorescent thermometer for single living cells

The homeostasis of body temperature and energy balance is one of the major principles in biology. Nanoscale thermometry of aqueous solutions is a challenging but crucial technique to understand the molecular basis of this essential process. Here, we developed a ratiometric nanothermometer (RNT) for intracellular temperature measurement in real time. Both the thermosensitive fluorophore, β-diketonate chelate europium(III) thenoyltrifluoroacetonate, and the thermoinsensitive fluorophore, rhodamine 101, which was used as a self-reference, are embedded in a polymeric particle that protects the fluorophores from intracellular conditions. The ratiometric measurement of single RNT spots is independent of the displacement of the RNT along the z-axis. The temperature is therefore determined at the location of each RNT under an optical microscope regardless of the dynamic movement of living cells. As a demonstration of the spot-by-spot intracellular thermometry, we successfully followed the temperature change in individual RNT spots in a single cell together with the Ca(2+) burst induced by the Ca(2+) ionophore ionomycin. The temperature increases differently among different spots, implying heterogeneous heat production in the cell. We then show that, in some spots, the temperature gradually decreases, while in others it remains high. The average temperature elevation within a cell is positively correlated to the increase in Ca(2+), suggesting that the activity and/or number of heat sources are dependent on the Ca(2+) concentration.

A Molecular Fluorescent Probe for Targeted Visualization of Temperature at the Endoplasmic Reticulum

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.

Inter-sarcomere coordination in muscle revealed through individual sarcomere response to quick stretch.

The force generation and motion of muscle are produced by the collective work of thousands of sarcomeres, the basic structural units of striated muscle. Based on their series connection to form a myofibril, it is expected that sarcomeres are mechanically and/or structurally coupled to each other. However, the behavior of individual sarcomeres and the coupling dynamics between sarcomeres remain elusive, because muscle mechanics has so far been investigated mainly by analyzing the averaged behavior of thousands of sarcomeres in muscle fibers. In this study, we directly measured the length-responses of individual sarcomeres to quick stretch at partial activation, using micromanipulation of skeletal myofibrils under a phase-contrast microscope. The experiments were performed at ADP-activation (1 mM MgATP and 2 mM MgADP in the absence of Ca2+) and also at Ca2+-activation (1 mM MgATP at pCa 6.3) conditions. We show that under these activation conditions, sarcomeres exhibit 2 distinct types of responses, either “resisting” or “yielding,” which are clearly distinguished by the lengthening distance of single sarcomeres in response to stretch. These 2 types of sarcomeres tended to coexist within the myofibril, and the sarcomere “yielding” occurred in clusters composed of several adjacent sarcomeres. The labeling of Z-line with anti-α-actinin antibody significantly suppressed the clustered sarcomere “yielding.” These results strongly suggest that the contractile system of muscle possesses the mechanism of structure-based inter-sarcomere coordination.

Highly thermosensitive Ca2+ dynamics in a HeLa cell through IP3 receptors

Intracellular Ca2+ distribution and its dynamics are essential for various cellular functions. We show with single HeLa cells that a microscopic heat pulse induces Ca2+ uptake into intracellular stores during heating and Ca2+ release from them at the onset of recooling, and the overshoot of Ca2+ release occurs above the critical value of a temperature change, which decreases from 1.5 to 0.2 °Con increasing the experimental temperature from 22 to 37 °C. This highly thermosensitive Ca2+ dynamics is probably attributable to the altered balance between Ca2+ uptake by endoplasmic reticulum Ca2+‐ATPases and Ca2+ release via inositol 1,4,5‐trisphosphate receptors. These results suggest that Ca2+ signaling is extremely sensitive to temperature changes, especially around body temperature, in cells expressing inositol 1,4,5‐trisphosphate receptors.

A novel method of thermal activation and temperature measurement in the microscopic region around single living cells.

We present a simple approach to bring fast and reversible temperature steps of a wide range of amplitudes from the temperature of the experimental chamber up to the boiling point of water in a desired position, with rise and fall times of around 10 ms in a microvolume of microm in size, such as in a single cell. For this purpose, we applied a technique for illuminating a metal aggregate (1-2 microm in diameter) placed at the tip of a glass micropipette with a focused infrared (1064 nm) laser beam under an optical microscope. Stable temperature gradients were created around the metal aggregate using an appropriate neutral density filter set for the laser output. To monitor the local temperature, we devised a new microthermometer composed of the tip of a micropipette filled with thermosensitive fluorescent dye Europium-TTA possessing steep temperature-dependent phosphorescence upon 365 nm excitation. The microm size of the tip of this pipette was able to measure the local temperature with 0.1 degrees C precision and microm spatial resolution. This new approach is compatible with standard electrophysiological and imaging techniques.

Mitochondria-targeted fluorescent thermometer monitors intracellular temperature gradient

Intracellular thermometry at the microscopic level is currently a hot topic. Herein we describe a small molecule fluorescent thermometer targeting mitochondria (Mito thermo yellow). Mito thermo yellow successfully demonstrates the ability to monitor the intracellular temperature gradient, generated by exogenous heating, in various cells.

Nonlinear Force-Length Relationship in the ADP-Induced Contraction of Skeletal Myofibrils

The regulatory mechanism of sarcomeric activity has not been fully clarified yet because of its complex and cooperative nature, which involves both Ca2+ and cross-bridge binding to the thin filament. To reveal the mechanism of regulation mediated by the cross-bridges, separately from the effect of Ca2+, we investigated the force-sarcomere length (SL) relationship in rabbit skeletal myofibrils (a single myofibril or a thin bundle) at SL > 2.2 μm in the absence of Ca2+ at various levels of activation by exogenous MgADP (4–20 mM) in the presence of 1 mM MgATP. The individual SLs were measured by phase-contrast microscopy to confirm the homogeneity of the striation pattern of sarcomeres during activation. We found that at partial activation with 4–8 mM MgADP, the developed force nonlinearly depended on the length of overlap between the thick and the thin filaments; that is, contrary to the maximal activation, the maximal active force was generated at shorter overlap. Besides, the active force became larger, whereas this nonlinearity tended to weaken, with either an increase in [MgADP] or the lateral osmotic compression of the myofilament lattice induced by the addition of a macromolecular compound, dextran T-500. The model analysis, which takes into account the [MgADP]- and the lattice-spacing-dependent probability of cross-bridge formation, was successfully applied to account for the force-SL relationship observed at partial activation. These results strongly suggest that the cross-bridge works as a cooperative activator, the function of which is highly sensitive to as little as ≤1 nm changes in the lattice spacing.

The effect of tropomyosin on force and elementary steps of the cross-bridge cycle in reconstituted bovine myocardium

The role of tropomyosin (Tm) in the elementary steps of the cross‐bridge cycle in bovine myocardium was investigated. The thin filament was selectively removed using gelsolin (thin filament severing protein), and the actin filament was reconstituted from G‐actin. Tm was further reconstituted without troponin (Tn), and the kinetic constants of the elementary steps of the cross‐bridge cycle were deduced using sinusoidal analysis at pCa ≤ 4.66, pH 7.00, and 25°C. The association constant of MgATP to cross‐bridges (K1) after reconstitution of Tm was 20.7 ± 2.3 mm−1, which was about 2 × the control (untreated) myocardium (9.1 ± 1.3 mm−1). Following reconstitution of Tm, the equilibrium constant of the cross‐bridge detachment step (K2), the phosphate (Pi) association constant (K5) and the equilibrium constant of the force‐generation step (K4), which significantly changed in the actin filament‐reconstituted myocardium, recovered to those of the control myocardium. Active tension after reconstitution of Tm was 0.69 × the control myocardium, a value between the control (1.00 ×) and the actin filament‐reconstituted myocardium (0.59 ×). Tm‐reconstituted myocardium was further reconstituted with Tn, and the effect of MgATP on the rate constants (K1, K2) was studied. Following reconstitution with Tn, the myocardium regained the Ca2+‐sensitivity and the active tension became 0.83 × the control myocardium. In addition, K1 recovered to the value of the control myocardium with Tn reconstitution. These results indicate that both Tm and Tn enhance the force generated by each cross‐bridge, and that Tm is primarily responsible for the change in the kinetic constants of the elementary steps of the cross‐bridge cycle.

Microscopic heat pulses induce contraction of cardiomyocytes without calcium transients

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.

Gold Nanoshell-Mediated Remote Myotube Activation

Mild heat stimulation of muscle cells within the physiological range represents an intriguing approach for the modulation of their functions. In this work, photothermal conversion was exploited to remotely stimulate striated muscle cells by using gold nanoshells (NSs) in combination with near-infrared (NIR) radiation. Temperature increments of approximately 5 °C were recorded by using an intracellular fluorescent molecular thermometer and were demonstrated to efficiently induce myotube contraction. The mechanism at the base of this phenomenon was thoroughly investigated and was observed to be a Ca2+-independent event directly involving actin-myosin interactions. Finally, chronic remote photothermal stimulations significantly increased the mRNA transcription of genes encoding heat shock proteins and sirtuin 1, a protein which in turn can induce mitochondrial biogenesis. Overall, we provide evidence that remote NIR + NS muscle excitation represents an effective wireless stimulation technique with great potential in the fields of muscle tissue engineering, regenerative medicine, and bionics.