Shunsuke Yuba

Molecular analysis of zebrafish photolyase/cryptochrome family: two types of cryptochromes present in zebrafish

Cryptochromes (CRY), members of the DNA photolyase/cryptochrome protein family, regulate the circadian clock in animals and plants. Two types of animal CRYs are known, mammalian CRY and Drosophila CRY. Both CRYs participate in the regulation of circadian rhythm, but they have different light dependencies for their reactions and have different effects on the negative feedback loop which generates a circadian oscillation of gene expression. Mammalian CRYs act as a potent inhibitor of transcriptional activator whose reactions do not depend on light, but Drosophila CRY functions as a light‐dependent suppressor of transcriptional inhibitor.

Infrared laser-mediated gene induction in targeted single cells in vivo.

We developed infrared laser–evoked gene operator (IR-LEGO), a microscope system optimized for heating cells without photochemical damage. Infrared irradiation causes reproducible temperature shifts of the in vitro microenvironment in a power-dependent manner. When applied to living Caenorhabditis elegans, IR-LEGO induced heat shock–mediated expression of transgenes in targeted single cells in a more efficient and less deleterious manner than a 440-nm dye laser and elicited physiologically relevant phenotypic responses.

Induction of Pluripotent Stem Cells from Human Third Molar Mesenchymal Stromal Cells

The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30–100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.

Three Distinct Classes of the α-Subunit of the Nuclear Pore-targeting Complex (Importin-α) Are Differentially Expressed in Adult Mouse Tissues

The process of active nuclear protein transport is mediated by the nuclear localization signal (NLS). An NLS-containing karyophile forms a stable complex, termed the nuclear pore-targeting complex, to target nuclear pores. The α-subunit of the complex (importin-α) binds to the NLS and the β-subunit (importin-β-) carries the α-subunit, bound to the NLS substrate, into the nucleus. To date, five mouse α-subunits have been identified and classified into three subfamilies (α-P, α-Q, and α-S). The expression of these α-subunits and the β-subunit in various adult mouse tissues was examined by immunoblotting and immunohistochemistry using antibodies specific for each subfamily of the α-subunit or the β-subunit. The β-subunit was found to be ubiquitously expressed, whereas each subfamily of the α-subunit showed a unique expression pattern in various tissues, especially in brain and testis. In brain, the expression of α-P was not observed, whereas α-S was significantly expressed in Purkinje cells, and pyramidal cells of the hippocampus and cerebral cortex. In testis, α-P was expressed predominantly in primary spermatocytes, whereas α-Q was found mainly in Leydig cells. Expression of α-S was detected in almost all cells in convoluted seminiferous tubules and Leydig cells to a similar extent. These results suggest that nuclear protein import may be controlled in a tissue-specific manner by α-subunit family proteins.

Real-time and Single Fibril Observation of the Formation of Amyloid β Spherulitic Structures

In Alzheimer disease, amyloid β, a 39-43-residue peptide produced by cleavage from a large amyloid precursor protein, undergoes conformational change to form amyloid fibrils and deposits as senile amyloid plaques in the extracellular cerebral cortices of the brain. However, the mechanism of how the intrinsically linear amyloid fibrils form spherical senile plaques is unknown. With total internal reflection fluorescence microscopy combined with the use of thioflavin T, an amyloid-specific fluorescence dye, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 μm on the chemically modified quartz surface. Real-time observation at a single fibrillar level revealed that, in the absence of tight contact with the surface, the cooperative and radial growth of amyloid fibrils from the core leads to a huge spherulitic structure. The results suggest the underlying physicochemical mechanism of senile plaque formation, essential for obtaining insight into prevention of Alzheimer disease.

A Neural Mechanism Underlying Mating Preferences for Familiar Individuals in Medaka Fish

Familiarity Does Not Breed Contempt Female mating preference is influenced by social familiarity in various species from fish to primates. Okuyama et al. (p. 91) showed in Japanese rice fish that females prefer to mate with visually familiarized males over unfamiliar males and that this preference is mediated by specific neuromodulatory neurons in the female brain. A particular class of neurons regulates female fish mating preference based on social familiarity. Social familiarity affects mating preference among various vertebrates. Here, we show that visual contact of a potential mating partner before mating (visual familiarization) enhances female preference for the familiarized male, but not for an unfamiliarized male, in medaka fish. Terminal-nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons, an extrahypothalamic neuromodulatory system, function as a gate for activating mating preferences based on familiarity. Basal levels of TN-GnRH3 neuronal activity suppress female receptivity for any male (default mode). Visual familiarization facilitates TN-GnRH3 neuron activity (preference mode), which correlates with female preference for the familiarized male. GnRH3 peptides, which are synthesized specifically in TN-GnRH3 neurons, are required for the mode-switching via self-facilitation. Our study demonstrates the central neural mechanisms underlying the regulation of medaka female mating preference based on visual social familiarity.

Infrared laser-mediated local gene induction in medaka, zebrafish and Arabidopsis thaliana

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single‐cell gene induction using an infrared laser‐evoked gene operator (IR‐LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR‐LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR‐LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.

Non-neuronal acetylcholine as an endogenous regulator of proliferation and differentiation of Lgr5-positive stem cells in mice

Non‐neuronal acetylcholine (ACh) is predicted to function as a local cell signaling molecule. However, the physiological significance of the synthesis of non‐neuronal ACh in the intestine remains unclear. Here, experiments using crypt–villus organoids that lack nerve and immune cells in culture led us to suggest that endogenous ACh is synthesized in the intestinal epithelium to evoke growth and differentiation of the organoids through activation of muscarinic ACh receptors (mAChRs). The extracts of the cultured organoids showed a noticeable capacity for ACh synthesis that was sensitive to a potent inhibitor of choline acetyltransferase. Imaging MS revealed endogenous ACh localized in the epithelial layer in mouse small intestinal epithelium in vivo, suggesting that there are non‐neuronal resources of ACh. Treatment of organoids with carbachol downregulated the growth of organoids and the expression of marker genes for epithelial cells. On the other hand, antagonists for mAChRs enhanced the growth and differentiation of organoids, indicating the involvement of mAChRs in regulating the proliferation and differentiation of Lgr5‐positive stem cells. Collectively, our data provide evidence that endogenous ACh released from intestinal epithelium maintains homeostasis of intestinal epithelial cell growth and differentiation via mAChRs in mice.

Physical properties of mesenchymal stem cells are coordinated by the perinuclear actin cap.

Mesenchymal stem cells (MSCs) have been extensively investigated for their applications in regenerative medicine. Successful use of MSCs in cell-based therapies will rely on the ability to effectively identify their properties and functions with a relatively non-destructive methodology. In this study, we measured the surface stiffness and thickness of rat MSCs with atomic force microscopy and clarified their relation at a single-cell level. The role of the perinuclear actin cap in regulating the thickness, stiffness, and proliferative activity of these cells was also determined by using several actin cytoskeleton-modifying reagents. This study has helped elucidate a possible link between the physical properties and the physiological function of the MSCs, and the corresponding regulatory role of the actin cytoskeleton.

Actin-based biomechanical features of suspended normal and cancer cells

The mechanical features of individual cells have been regarded as unique indicators of their states, which could constantly change in accordance with cellular events and diseases. Particularly, cancer progression was characterized by the disruption and/or reorganization of actin filaments causing mechanical changes. Thus, mechanical characterization of cells could become an effective cytotechnological approach for early detection of cancer. To develop mechanical cytotechnology, it would be necessary to clarify the mechanical properties in various cell adhesion states. In this study, we investigated the surface mechanical behavior of cancer and normal cells in the adherent and suspended states using atomic force microscopy. Adherent normal stromal cells showed high surface stiffness due to developed actin cap structures on their apical surface, whereas cancer cells did not have developed filamentous actin structures, and their surface stiffness was low. Upon cell detachment from the substrate, filamentous actin structures of adherent normal stromal cells reorganized to the cortical region and their surface stiffness decreased consequently however, the stiffness of suspended normal cells remained higher than that of cancer cells. These suspended state actin structures were similar, regardless of the cell type. Furthermore, the mechanical responses of the cancer and normal stromal cells to perturbation of the actin cytoskeleton were different, suggesting distinct regulatory mechanisms for actin cytoskeleton in cancer and normal cells in both adherent and suspended states. Therefore, cancer cells possess specific mechanical and actin cytoskeleton features different from normal stromal cells.