Published papers

During the developmental processes of embryos, cells undergo massive deformation and division that are regulated by mechanical cues. However, little is known about how embryonic cells change their mechanical properties during different cleavage stages. Here, using atomic force microscopy, we investigated the stiffness of cells in ascidian embryos from the fertilised egg to the stage before gastrulation. In both animal and vegetal hemispheres, we observed a Rho kinase (ROCK)-independent cell stiffening that the cell stiffness exhibited a remarkable increase at the timing of cell division where cortical actin filaments were organized. Furthermore, in the vegetal hemisphere, we observed another mechanical behaviour, i.e., a ROCK-associated cell stiffening, which was retained even after cell division or occurred without division and propagated sequentially toward adjacent cells, displaying a characteristic cell-to-cell mechanical variation. The results indicate that the mechanical properties of embryonic cells are regulated at the single cell level in different germ layers.

Marine invertebrate larvae are known to begin metamorphosis in response to environmentally derived cues. However, little is known about the relationships between the perception of such cues and internal signalling for metamorphosis. To elucidate the mechanism underlying the initiation of metamorphosis in the ascidian, Ciona intestinalis type A (Ciona robusta), we artificially induced ascidian metamorphosis and investigated Ca 2+ dynamics from pre- to post-metamorphosis. Ca 2+ transients were observed and consisted of two temporally distinct phases with different durations before tail regression which is the early event of metamorphosis. In the first phase, Phase I, the Ca 2+ transient in the papillae (adhesive organ of the anterior trunk) was coupled with the Ca 2+ transient in dorsally localized cells and endoderm cells just after mechanical stimulation. The Ca 2+ transients in Phase I were also observed when applying only short stimulation. In the second phase, Phase II, the Ca 2+ transient in papillae was observed again and lasted for approximately 5-11 min just after the Ca 2+ transient in Phase I continued for a few minutes. The impaired papillae by Foxg-knockdown failed to induce the second Ca 2+ transient in Phase II and tail regression. In Phase II, a wave-like Ca 2+ propagation was also observed across the entire epidermis. Our results indicate that the papillae sense a mechanical cue and two-round Ca 2+ transients in papillae transmits the internal metamorphic signals to different tissues, which subsequently induces tail regression. Our study will help elucidate the internal mechanism of metamorphosis in marine invertebrate larvae in response to environmental cues.

The Ciona notochord has emerged as a simple and tractable in vivo model for tubulogenesis. Here, using a chemical genetics approach, we identified UTKO1 as a selective small molecule inhibitor of notochord tubulogenesis. We identified 14-3-3ϵa protein as a direct binding partner of UTKO1 and showed that 14-3-3ϵa knockdown leads to failure of notochord tubulogenesis. We found that UTKO1 prevents 14-3-3ϵa from interacting with ezrin/radixin/moesin (ERM), which is required for notochord tubulogenesis, suggesting that interactions between 14-3-3ϵa and ERM play a key role in regulating the early steps of tubulogenesis. Using live imaging, we found that, as lumens begin to open between neighboring cells, 14-3-3ϵa and ERM are highly colocalized at the basal cortex where they undergo cycles of accumulation and disappearance. Interestingly, the disappearance of 14-3-3ϵa and ERM during each cycle is tightly correlated with a transient flow of 14-3-3ϵa, ERM, myosin II, and other cytoplasmic elements from the basal surface toward the lumen-facing apical domain, which is often accompanied by visible changes in lumen architecture. Both pulsatile flow and lumen formation are abolished in larvae treated with UTKO1, in larvae depleted of either 14-3-3ϵa or ERM, or in larvae expressing a truncated form of 14-3-3ϵa that lacks the ability to interact with ERM. These results suggest that 14-3-3ϵa and ERM interact at the basal cortex to direct pulsatile basal accumulation and basal-apical transport of factors that are essential for lumen formation. We propose that similar mechanisms may underlie or may contribute to lumen formation in tubulogenesis in other systems.

Temperature governs states and dynamics of all biological molecules, and several cellular processes are often heat sources and/or sinks. Technical achievement of intracellular thermometry enables us to measure intracellular temperature, and it can offer novel perspectives in biology and medicine. However, little is known that changes of intracellular temperature throughout the cell-cycle and the manner of which cells regulates their thermogenesis in response to fluctuation of the environmental temperature. Here, cell-cycle-dependent changes of intracellular temperature were reconstructed from the snapshots of cell population at single-cell resolution using ergodic analysis for asynchronously cultured HeLa cells expressing a genetically encoded thermometry. Intracellular temperature is highest at G1 phase, and it gradually decreases along cell-cycle progression and increases abruptly during mitosis. Cells easily heated up are harder to cool down and vice versa, especially at G1/S phases. Together, intracellular thermogenesis depends on cell-cycle phases and it maintains intracellular temperature through compensating environmental temperature fluctuations.

For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1–5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6–17], free divalent cations such as Mg2+ and Ca2+, which condense chromatin or chromosomes in vitro [18–28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like “beads on a string” by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg2+, have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg2+ indicator that monitors free Mg2+ dynamics throughout the cell cycle. By combining this indicator with Ca2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg2+, but not Ca2+, increase during mitosis. The Mg2+ increase is coupled with a decrease in ATP, which is normally bound to Mg2+ in the cell [33]. ATP inhibited Mg2+-dependent chromatin condensation in vitro. Chelating Mg2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg2+-ATP balance. How the negatively charged long genomic DNA is organized into mitotic chromosome remains unclear. Using a newly developed Mg2+ indicator, Maeshima et al. demonstrate a transient rise in free Mg2+ released from ATP-Mg during mitosis and suggest that the rise contributes to mitotic chromosome condensation by charge neutralization.

In some types of meditation, such as mindfulness and Zen, breathing is the focus of attention, whereas during an excessive, short-period of anaerobic exercise, the muscles become the focus of attention. Thus, during both efforts, one's attention is focused on a certain feature of the body. Both meditation and exercise generally provide mental refreshment to humans. We hypothesized that the same brain regions are activated by both efforts in humans. To examine this hypothesis, we engaged participants in 3 tasks: meditation, exercise, and a control task. After each task, the participants underwent a 2-back test to concentrate their thoughts, while changes in their blood hemoglobin levels were simultaneously monitored using near-infrared spectroscopy (NIRS). Seventeen participants (20-24 years of age; 11 men, 6 women) were enrolled. We applied a fast-Fourier transform (FFT) analysis to the NIRS wave data and calculated the correlation coefficients of the FFT data between (1) meditation and control, (2) exercise and control, and (3) meditation and exercise, at the orbitofrontal cortex (OFC) and dorsolateral prefrontal cortex (DLPFC), brain areas that are generally involved in mental refreshment. A significant difference in the correlation coefficients between the OFC and DLPFC was detected in the meditation and exercise analysis, and signal source analysis confirmed that the NIRS waves spread from the right and left OFC edges (i.e., right and left temples) toward the center. Our results suggest that both meditation and exercise activate the OFC, which is involved in emotional reactions and motivation behavior, resulting in mental refreshment.