Faculty Profiles - Shitamukai Atsunori

Papers - Shitamukai Atsunori

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Truncated radial glia as a common precursor in the late corticogenesis of gyrencephalic mammals Reviewed

Merve Bilgic, Quan Wu, Taeko Suetsugu, Atsunori Shitamukai, Yuji Tsunekawa, Tomomi Shimogori, Mitsutaka Kadota, Osamu Nishimura, Shigehiro Kuraku, Hiroshi Kiyonari, Fumio Matsuzaki

eLife 2023.10

Language：English Publishing type：Research paper (scientific journal) Publisher：eLife Sciences Publications, Ltd

The diversity of neural stem cells is a hallmark of the cerebral cortex development in gyrencephalic mammals, such as Primates and Carnivora. Among them, ferrets are a good model for mechanistic studies. However, information on their neural progenitor cells (NPC), termed radial glia (RG), is limited. Here, we surveyed the temporal series of single-cell transcriptomes of progenitors regarding ferret corticogenesis and, found a conserved diversity and temporal trajectory between human and ferret NPC, despite the large timescale difference. We found truncated RG (tRG) in ferret cortical development, a progenitor subtype previously described in humans. The combination of in silico and in vivo analyses identified that tRG differentiate into both ependymal and astrogenic cells. Via transcriptomic comparison, we predict that this is also the case in humans. Our findings suggest that tRG plays a role in the formation of adult ventricles, thereby providing the architectural bases for brain expansion.

DOI： 10.7554/elife.91406.2

Advanced Techniques Using In Vivo Electroporation to Study the Molecular Mechanisms of Cerebral Development Disorders Reviewed

Chen Yang, Atsunori Shitamukai, Shucai Yang, Ayano Kawaguchi

International Journal of Molecular Sciences 24 ( 18 ) 14128 - 14128 2023.9

Publishing type：Research paper (scientific journal) Publisher：MDPI AG

The mammalian cerebral cortex undergoes a strictly regulated developmental process. Detailed in situ visualizations, imaging of these dynamic processes, and in vivo functional gene studies significantly enhance our understanding of brain development and related disorders. This review introduces basic techniques and recent advancements in in vivo electroporation for investigating the molecular mechanisms underlying cerebral diseases. In utero electroporation (IUE) is extensively used to visualize and modify these processes, including the forced expression of pathological mutants in human diseases; thus, this method can be used to establish animal disease models. The advent of advanced techniques, such as genome editing, including de novo knockout, knock-in, epigenetic editing, and spatiotemporal gene regulation, has further expanded our list of investigative tools. These tools include the iON expression switch for the precise control of timing and copy numbers of exogenous genes and TEMPO for investigating the temporal effects of genes. We also introduce the iGONAD method, an improved genome editing via oviductal nucleic acid delivery approach, as a novel genome-editing technique that has accelerated brain development exploration. These advanced in vivo electroporation methods are expected to provide valuable insights into pathological conditions associated with human brain disorders.

DOI： 10.3390/ijms241814128

Notch1 and Notch2 collaboratively maintain radial glial cells in mouse neurogenesis Reviewed International journal

Shun Mase, Atsunori Shitamukai, Quan Wu, Mitsuru Morimoto, Thomas Gridley, Fumio Matsuzaki

Neuroscience Research 170 122 - 132 2021.9

Endfoot regeneration restricts radial glial state and prevents translocation into the outer subventricular zone in early mammalian brain development. Reviewed International journal

Ikumi Fujita*, Atsunori Shitamukai*, Fumiya Kusumoto*, Shun Mase, Taeko Suetsugu, Ayaka Omori, Kagayaki Kato, Takaya Abe, Go Shioi, Daijiro Konno, Fumio Matsuzaki, *equal contribution

Nature cell biology 22 ( 1 ) 26 - 37 2020.1

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media {LLC}

Neural stem cells, called radial glia, maintain epithelial structure during the early neocortical development. The prevailing view claims that when radial glia first proliferate, their symmetric divisions require strict spindle orientation; its perturbation causes precocious neurogenesis and apoptosis. Here, we show that despite this conventional view, radial glia at the proliferative stage undergo normal symmetric divisions by regenerating an apical endfoot even if it is lost by oblique divisions. We found that the Notch-R-Ras-integrin β1 pathway promotes the regeneration of endfeet, whose leading edge bears ectopic adherens junctions and the Par-polarity complex. However, this regeneration ability gradually declines during the subsequent neurogenic stage and hence oblique divisions induce basal translocation of radial glia to form the outer subventricular zone, a hallmark of the development of the convoluted brain. Our study reveals that endfoot regeneration is a temporally changing cryptic property, which controls the radial glial state and its shift is essential for mammalian brain size expansion.

DOI： 10.1038/s41556-019-0436-9

Lzts1 controls both neuronal delamination and outer radial glial-like cell generation during mammalian cerebral development Reviewed International journal

T. Kawaue, A. Shitamukai, A. Nagasaka, Y. Tsunekawa, T. Shinoda, K. Saito, R. Terada, M. Bilgic, T. Miyata, F. Matsuzaki, A. Kawaguchi

Nature Communications 10 ( 1 ) 2019.12

Reconstruction of Par-dependent polarity in apolar cells reveals a dynamic process of cortical polarization. Reviewed International journal

Kalyn Kono, Shigeki Yoshiura, Ikumi Fujita, Yasushi Okada, Atsunori Shitamukai, Tatsuo Shibata, Fumio Matsuzaki

eLife 8 2019.6

Language：English Publishing type：Research paper (scientific journal) Publisher：{eLife} Sciences Publications, Ltd

Cellular polarization is fundamental for various biological processes. The Par network system is conserved for cellular polarization. Its core complex consists of Par3, Par6, and aPKC. However, the general dynamic processes that occur during polarization are not well understood. Here, we reconstructed Par-dependent polarity using non-polarized Drosophila S2 cells expressing all three components endogenously in the cytoplasm. The results indicated that elevated Par3 expression induces cortical localization of the Par-complex at the interphase. Its asymmetric distribution goes through three steps: emergence of cortical dots, development of island-like structures with dynamic amorphous shapes, repeating fusion and fission, and polarized clustering of the islands. Our findings also showed that these islands contain a meshwork of unit-like segments. Furthermore, Par-complex patches resembling Par-islands exist in Drosophila mitotic neuroblasts. Thus, this reconstruction system provides an experimental paradigm to study features of the assembly process and structure of Par-dependent cell-autonomous polarity.

DOI： 10.7554/eLife.45559

Developing a de novo targeted knock-in method based on in utero electroporation into the mammalian brain. Reviewed International journal

Yuji Tsunekawa, Raymond Kunikane Terhune, Ikumi Fujita, Atsunori Shitamukai, Taeko Suetsugu, Fumio Matsuzaki

Development (Cambridge, England) 143 ( 17 ) 3216 - 22 2016.9

Language：English Publishing type：Research paper (scientific journal)

Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues.

DOI： 10.1242/dev.136325

Cell Division Modes and Cleavage Planes of Neural Progenitors during Mammalian Cortical Development. Reviewed International journal

Fumio Matsuzaki, Atsunori Shitamukai

Cold Spring Harbor perspectives in biology 7 ( 9 ) a015719 2015.9

Authorship：Lead author Language：English Publishing type：Part of collection (book)

During mammalian brain development, neural progenitor cells undergo symmetric proliferative divisions followed by asymmetric neurogenic divisions. The division mode of these self-renewing progenitors, together with the cell fate of their progeny, plays critical roles in determining the number of neurons and, ultimately, the size of the adult brain. In the past decade, remarkable progress has been made toward identifying various types of neuronal progenitors. Recent technological advances in live imaging and genetic manipulation have enabled us to link dynamic cell biological events to the molecular mechanisms that control the asymmetric divisions of self-renewing progenitors and have provided a fresh perspective on the modes of division of these progenitors. In addition, comparison of progenitor repertoires between species has provided insight into the expansion and the development of the complexity of the brain during mammalian evolution.

DOI： 10.1101/cshperspect.a015719

Novel and robust transplantation reveals the acquisition of polarized processes by cortical cells derived from mouse and human pluripotent stem cells. Reviewed International journal

Fumiaki Nagashima, Ikuo K Suzuki, Atsunori Shitamukai, Haruko Sakaguchi, Misato Iwashita, Taeko Kobayashi, Shigenobu Tone, Kazunori Toida, Pierre Vanderhaeghen, Yoichi Kosodo

Stem cells and development 23 ( 18 ) 2129 - 42 2014.9

Language：English Publishing type：Research paper (scientific journal) Publisher：MARY ANN LIEBERT, INC

Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apico-basal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.

DOI： 10.1089/scd.2013.0251

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging. Reviewed International journal

Togo Shimozawa, Kazuo Yamagata, Takefumi Kondo, Shigeo Hayashi, Atsunori Shitamukai, Daijiro Konno, Fumio Matsuzaki, Jun Takayama, Shuichi Onami, Hiroshi Nakayama, Yasuhito Kosugi, Tomonobu M Watanabe, Katsumasa Fujita, Yuko Mimori-Kiyosue

Proceedings of the National Academy of Sciences of the United States of America 110 ( 9 ) 3399 - 404 2013.2

Language：English

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.

DOI： 10.1073/pnas.1216696110

Amplification of progenitors in the mammalian telencephalon includes a new radial glial cell type. Reviewed International journal

Gregor-Alexander Pilz, Atsunori Shitamukai, Isabel Reillo, Emilie Pacary, Julia Schwausch, Ronny Stahl, Jovica Ninkovic, Hugo J Snippert, Hans Clevers, Leanne Godinho, Francois Guillemot, Victor Borrell, Fumio Matsuzaki, Magdalena Götz

Nature communications 4 2125 - 2125 2013

Language：English

The mechanisms governing the expansion of neuron number in specific brain regions are still poorly understood. Enlarged neuron numbers in different species are often anticipated by increased numbers of progenitors dividing in the subventricular zone. Here we present live imaging analysis of radial glial cells and their progeny in the ventral telencephalon, the region with the largest subventricular zone in the murine brain during neurogenesis. We observe lineage amplification by a new type of progenitor, including bipolar radial glial cells dividing at subapical positions and generating further proliferating progeny. The frequency of this new type of progenitor is increased not only in larger clones of the mouse lateral ganglionic eminence but also in cerebral cortices of gyrated species, and upon inducing gyrification in the murine cerebral cortex. This implies key roles of this new type of radial glia in ontogeny and phylogeny.

DOI： 10.1038/ncomms3125

Control of asymmetric cell division of mammalian neural progenitors. Invited

Atsunori Shitamukai, Fumio Matsuzaki

Development, growth & differentiation 54 ( 3 ) 277 - 86 2012.4

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal)

Although the vertebrate brain commonly stems from the neuroepithelial tube, the size and complexity of the pseudostratified organization of the brain have drastically expanded during mammalian evolution, resulting in the formation of a highly folded cortex. Developmental controls of neural progenitor divisions underlie these events. In this review, we introduce recent progress in understanding the control of proliferation and differentiation of neural progenitors from a structural point of view. We particularly shed light on the roles of epithelial structure and mitotic spindle orientation in the generation of various types of neural progenitors.

DOI： 10.1111/j.1440-169X.2012.01345.x

Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors. Reviewed International journal

Atsunori Shitamukai, Daijiro Konno, Fumio Matsuzaki

The Journal of neuroscience : the official journal of the Society for Neuroscience 31 ( 10 ) 3683 - 95 2011.3

Authorship：Lead author Language：English

Radial glia cells function as neural stem cells in the developing brain and generate self-renewing and differentiating daughter cells by asymmetric cell divisions. During these divisions, the apical process or basal process of the elongated epithelial structure is asymmetrically partitioned into daughter cells, depending on developmental contexts. However, in mammalian neurogenesis, the relationship between these subcellular structures and self-renewability is largely unknown. We induced oblique cleavages of radial glia cells to split the apical and basal processes into two daughters, and investigated the fate and morphology of the daughters in slice cultures. We observed that the more basal daughter cell that inherits the basal process self-renews outside of the ventricular zone (VZ), while the more apical daughter cell differentiates. These self-renewing progenitors, termed "outer VZ progenitors," retain the basal but not the apical process, as recently reported for the outer subventricular zone (OSVZ) progenitors in primates (Fietz et al., 2010; Hansen et al., 2010); to self-renew, they require clonal Notch signaling between sibling cells. We also found a small endogenous population of outer VZ progenitors in the mouse embryonic neocortex, consistent with a low frequency of oblique radial glia divisions. Our results describe the general role of the basal process in the self-renewal of neural progenitors and implicate the loss of the apical junctions during oblique divisions as a possible mechanism for generating OSVZ progenitors. We propose that mouse outer VZ progenitors, induced by oblique cleavages, provide a model to study both progenitor self-renewal and OSVZ progenitors.

DOI： 10.1523/JNEUROSCI.4773-10.2011

Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis. Reviewed International journal

Daijiro Konno, Go Shioi, Atsunori Shitamukai, Asako Mori, Hiroshi Kiyonari, Takaki Miyata, Fumio Matsuzaki

Nature cell biology 10 ( 1 ) 93 - 101 2008.1

Language：English

During mammalian development, neuroepithelial cells function as mitotic progenitors, which self-renew and generate neurons. Although spindle orientation is important for such polarized cells to undergo symmetric or asymmetric divisions, its role in mammalian neurogenesis remains unclear. Here we show that control of spindle orientation is essential in maintaining the population of neuroepithelial cells, but dispensable for the decision to either proliferate or differentiate. Knocking out LGN, (the G protein regulator), randomized the orientation of normally planar neuroepithelial divisions. The resultant loss of the apical membrane from daughter cells frequently converted them into abnormally localized progenitors without affecting neuronal production rate. Furthermore, overexpression of Inscuteable to induce vertical neuroepithelial divisions shifted the fate of daughter cells. Our results suggest that planar mitosis ensures the self-renewal of neuroepithelial progenitors by one daughter inheriting both apical and basal compartments during neurogenesis.

DOI： 10.1038/ncb1673

Ras/cAMP-dependent protein kinase (PKA) regulates multiple aspects of cellular events by phosphorylating the Whi3 cell cycle regulator in budding yeast. Reviewed International journal

Masaki Mizunuma, Ryohei Tsubakiyama, Takafumi Ogawa, Atsunori Shitamukai, Yoshifumi Kobayashi, Tomomi Inai, Kazunori Kume, Dai Hirata

The Journal of biological chemistry 288 ( 15 ) 10558 - 66 2013.4

Language：English

The Start/G1 phase in the cell cycle is an important period during which cells determine their developmental fate, onset of mitotic progression, or the switch to developmental stages in response to both external and internal signals. In the budding yeast Saccharomyces cerevisiae, Whi3, a negative regulator of the G1 cyclins, has been identified as a positive regulator of cell size control and is involved in the regulation of Start. However, the regulatory pathway of Whi3 governing the response to multiple signals remains largely unknown. Here, we show that Whi3 is phosphorylated by the Ras/cAMP-dependent protein kinase (PKA) and that phosphorylation of Ser-568 in Whi3 by PKA plays an inhibitory role in Whi3 function. Phosphorylation of Whi3 by PKA led to its decreased interaction with CLN3 G1 cyclin mRNA and was required for the promotion of G1/S progression. Furthermore, we demonstrate that the phosphomimetic S568D mutation of Whi3 prevented the developmental fate switch to sporulation or invasive growth. Thus, PKA modulated the function of Whi3 by phosphorylation, thus implicating PKA-mediated modulation of Whi3 in multiple cellular events.

DOI： 10.1074/jbc.M112.402214

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging (vol 110, pg 3399, 2013) Reviewed

Shimozawa Togo, Yamagata Kazuo, Kondo Takefumi, Hayashi Shigeo, Shitamukai Atsunori, Konno Daijiro, Matsuzaki Fumio, Takayama Jun, Onami Shuichi, Nakayama Hiroshi, Kosugi Yasuhito, Watanabe Tomonobu M, Fujita Katsumasa, Mimori-Kiyosue Yuko

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 ( 15 ) 6240 2013.4

Evidence of antagonistic regulation of restart from G(1) delay in response to osmotic stress by the Hog1 and Whi3 in budding yeast. Reviewed International journal

Masaki Mizunuma, Takafumi Ogawa, Tetsuya Koyama, Atsunori Shitamukai, Ryohei Tsubakiyama, Tadamasa Komaruyama, Toshinaga Yamaguchi, Kazunori Kume, Dai Hirata

Bioscience, biotechnology, and biochemistry 77 ( 10 ) 2002 - 7 2013

Language：English

Hog1 of Saccharomyces cerevisiae is activated by hyperosmotic stress, and this leads to cell-cycle delay in G1, but the mechanism by which cells restart from G1 delay remains elusive. We found that Whi3, a negative regulator of G1 cyclin, counteracted Hog1 in the restart from G1 delay caused by osmotic stress. We have found that phosphorylation of Ser-568 in Whi3 by RAS/cAMP-dependent protein kinase (PKA) plays an inhibitory role in Whi3 function. In this study we found that the phosphomimetic Whi3 S568D mutant, like the Δwhi3 strain, slightly suppressed G1 delay of Δhog1 cells under osmotic stress conditions, whereas the non-phosphorylatable S568A mutation of Whi3 caused prolonged G1 arrest of Δhog1 cells. These results indicate that Hog1 activity is required for restart from G1 arrest under osmotic stress conditions, whereas Whi3 acts as a negative regulator for this restart mechanism.

DOI： 10.1271/bbb.130260

Structural basis for self-renewal of neural progenitors in cortical neurogenesis. Reviewed International journal

Go Shioi, Daijiro Konno, Atsunori Shitamukai, Fumio Matsuzaki

Cerebral cortex (New York, N.Y. : 1991) 19 Suppl 1 i55-61 - 61 2009.7

Language：English

In mammalian brain development, neuroepithelial cells act as progenitors that produce self-renewing and differentiating cells. Recent technical advances in live imaging and gene manipulation now enable us to investigate how neural progenitors generate the 2 different types of cells with unprecedented accuracy and resolution, shedding new light on the roles of epithelial structure in cell fate decisions and also on the plasticity of neurogenesis.

DOI： 10.1093/cercor/bhp042

Identification of Tup1 and Cyc8 mutations defective in the responses to osmotic stress. Reviewed International journal

Yoshifumi Kobayashi, Tomomi Inai, Masaki Mizunuma, Ichitaro Okada, Atsunori Shitamukai, Dai Hirata, Tokichi Miyakawa

Biochemical and biophysical research communications 368 ( 1 ) 50 - 5 2008.3

Language：English

In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general transcriptional corepressor. This repression is mediated by recruitment of the Tup1-Cyc8 complex to target promoters through sequence-specific DNA-binding proteins such as Sko1, which mediates the HOG pathway-dependent regulation. We identified tup1 and cyc8 mutant alleles as the suppressor of osmo-sensitivity of the hog1Delta strain. In these mutants, although the expression of the genes under the control of DNA-binding proteins other than Sko1 was apparently normal, the Sko1-regulated genes GRE2 and AHP1 were derepressed under non-stress conditions, suggesting that the Tup1 and Cyc8 mutant proteins were specifically defective in the repression of the Sko1-dependent genes. Chromatin immunoprecipitation analyses of the GRE2 promoter in the mutants demonstrated that the Sko1-Tup1-Cyc8 complex was localized to the promoter, together with Gcn5/SAGA, suggesting that the erroneous recruitment of SAGA to the promoter led to the derepression.

DOI： 10.1016/j.bbrc.2008.01.033

Effect of ethanol on cell growth of budding yeast: genes that are important for cell growth in the presence of ethanol. Reviewed International journal

Shunsuke Kubota, Ikuko Takeo, Kazunori Kume, Muneyoshi Kanai, Atsunori Shitamukai, Masaki Mizunuma, Tokichi Miyakawa, Hitoshi Shimoi, Haruyuki Iefuji, Dai Hirata

Bioscience, biotechnology, and biochemistry 68 ( 4 ) 968 - 72 2004.4

Language：English Publishing type：Research paper (scientific journal)

The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.

DOI： 10.1271/bbb.68.968

Evidence for antagonistic regulation of cell growth by the calcineurin and high osmolarity glycerol pathways in Saccharomyces cerevisiae. Reviewed International journal

Atsunori Shitamukai, Dai Hirata, Shinya Sonobe, Tokichi Miyakawa

The Journal of biological chemistry 279 ( 5 ) 3651 - 61 2004.1

Authorship：Lead author Language：English

Because Ca(2+) signaling of budding yeast, through the activation of calcineurin and the Mpk1/Slt2 mitogen-activated protein kinase cascade, performs redundant function(s) in the events essential for growth, the simultaneous deletion of both these pathways (Delta cnb1 Delta mpk1) leads to lethality. A PTC4 cDNA that encodes a protein phosphatase belonging to the PP2C family was obtained as a high dosage suppressor of the lethality of Delta cnb1 Delta mpk1 strain. Overexpression of PTC4 led to a decrease in the high osmolarity-induced Hog1 phosphorylation, and HOG1 deletion remarkably suppressed the synthetic lethality, indicating an antagonistic role of the high osmolarity glycerol (HOG) pathway and the Ca(2+) signaling pathway in growth regulation. The calcineurin-Crz1 pathway was required for the down-regulation of the HOG pathway. Analysis of the time course of actin polarization, bud formation, and the onset of mitosis in synchronous cell cultures demonstrated that calcineurin negatively regulates actin polarization at the bud site, whereas the HOG pathway positively regulates bud formation at a later step after actin has polarized.

DOI： 10.1074/jbc.M306098200

酵母の特殊な変異形質を利用するCa^<2+>シグナル伝達に作用する薬剤のポジティブスクリーニング Invited

下向敦範, 水沼正樹, 平田大, 高橋英俊, 宮川都吉

日本農芸化学会誌 76 ( 8 ) 734 - 735 2002.8

酵母の特殊な変異形質を利用するCa2+シグナル伝達に作用する薬剤のポジティブスクリーニング Invited

下向敦範, 水沼正樹, 平田大, 高橋英俊, 宮川都吉

化学と生物(日本農芸化学会誌) 76 38 - 39 2002

酵母を使った生理活性物質の新規スクリーニング法 Invited

平田大, 下向敦範, 水沼正樹, 宮川都吉

生物工学会誌 79 ( 3 ) 79 - 79 2001

A positive screening for drugs that specifically inhibit the Ca2+-signaling activity on the basis of the growth promoting effect on a yeast mutant with a peculiar phenotype. Reviewed International journal

A Shitamukai, M Mizunuma, D Hirata, H Takahashi, T Miyakawa

Bioscience, biotechnology, and biochemistry 64 ( 9 ) 1942 - 6 2000.9

Authorship：Lead author Language：English

An inappropriate activation of a signaling pathway in yeast often has a deleterious physiological effect and causes various defects, including growth defects. In a certain genetic background (deltazds1) of Saccharomyces cerevisiae, the cell-cycle progression in G2 is specifically blocked in the medium with CaCl2 by the hyperactivation of the Ca2+-signaling pathways. Here, we developed a novel drug screening procedure designed to detect the active compounds that specifically attenuate the Ca2+-signaling activity on the basis of the ability to abrogate the growth defect of the cells suffering from the hyperactivated Ca2+ signal. Using known calcineurin inhibitors as model compounds, we have established the screening conditions for the drugs that suppress the Ca2+-induced growth inhibition. An indicator strain with an increased drug sensitivity was constructed with a syr1/erg3 null mutation.

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