Soumyadip Pal, Takaya Ishihara, Daiki Setoyama, Lin Chang Chen, Kenta Onoue, Shigenobu Yonemura, Emi Ogasawara and Naotada Ishihara : Non-canonical role of natural quinones in mitochondrial nucleoid organization for maintaining respiration and protecting cardiac function, Journal of Biochemistry, 179, 1, 5-20, 2026.
(要約)
Mitochondria contain their own DNA (mtDNA), which is essential for respiratory function. Multiple copies of mtDNA are assembled into dot-like structures called nucleoids. Nucleoids move dynamically within mitochondria, and their size and distribution are influenced by mitochondrial membrane fission and fusion. However, the molecular mechanisms and their pathophysiological significance, particularly in vivo, remain largely unknown. Here, we identify a novel role for ubiquinone, as well as natural quinones lacking electron-carrying capacity, in the organization of nucleoids and respiratory complexes, independent of their conventional roles. These quinones facilitate the association and packaging of mtDNA on the cardiolipin-enriched mitochondrial inner membrane. This quinone-dependent maintenance of nucleoids protects against mitochondrial dysfunction and heart failure induced by the anticancer drug doxorubicin. Our RNAi screen identifies a set of genes involved in mitochondrial diseases that exhibit nucleoid deformation, suggesting a novel therapeutic approach targeting mitochondrial nucleoids for various pathological conditions associated with mitochondrial dysfunction.
Quan Gan, Sachiko Tsukita and Shigenobu Yonemura : Force-dependent vinculin binding of α-catenin accelerates adherens junction formation, Cell Structure and Function, 51, 1, 23-35, 2026.
(要約)
Adherens junctions (AJs) mediate cell-cell adhesion and mechanical coupling in epithelial tissues. During AJ formation, punctate AJs (punctum adherens; PA) initially appear and subsequently transition into linear AJs or zonula adherens (ZAs). The mechanosensitive interaction of α-catenin with its binding partners-actin filaments and vinculin-is thought to act as a key switch that stabilizes AJs under tension. However, the physiological role of α-catenin's force sensitivity during the early stages of AJ formation remains unclear. Here, we analyzed α-catenin mutants with altered force sensitivity: Insensitive mutant L344P lacking vinculin binding, and Hypersensitive mutant L378P binding vinculin constitutively. Using calcium-switch assays combined with fluorescence and electron microscopy, we found that cells expressing insensitive α-catenin exhibited persistent, elongated PA-like structures corresponding to lateral associations of cellular protrusions from opposing cells, accompanied by delayed ZA formation. In contrast, cells expressing the hypersensitive mutant rapidly formed ZAs, possibly bypassing the PA stage. Similar phenotypes were observed in vinculin-knockout cells, indicating that the defects in Insensitive mutants result from the lack of vinculin recruitment to α-catenin. Based on these findings, we propose a model in which clusters of the cadherin-catenin complex (CCC) along actin filaments on opposing protrusions serve as initial adhesion sites. As protrusions shorten through actomyosin contraction, CCC clusters move toward the protrusion tips along actin filaments, where stretched α-catenin recruits vinculin to reinforce the adhesion, leading to PA formation. Thus, α-catenin's force sensitivity is crucial for smooth and timely AJ assembly, ensuring proper epithelial morphogenesis by coupling intercellular adhesion with cytoskeletal tension.Key words: α-catenin, vinculin, adherens junction, actin filament, force sensitivity.
Ami Banno, Kana Mizuno, Mizuki Sakamoto, Sota Komatsubara, Kenjiro Shirane, Katsuhiko Hayashi, Nobuhiko Hamazaki, Koshi Imami, Shigenobu Yonemura and Takashi Ishiuchi : A simple, efficient, and scalable method to generate oocyte-like cells in vitro, Life Science Alliance, 9, 2, 2025.
(要約)
Understanding the molecular basis of oocyte identity and function is essential not only for basic biology but also for clinical applications, as it is closely linked to female infertility. However, technical challenges remain in advancing this understanding, mainly because of the difficulty in obtaining a sufficient number of oocytes. In this study, through refining previously reported three-dimensional protocols, we established a two-dimensional culture method that efficiently generates oocyte-like cells, referred to as mini-oocytes, from mouse embryonic stem cells. This method requires minimal labor, does not rely on supporting somatic cells, and leverages a transcription factor-mediated approach for oocyte-like cell generation. Our transcriptome and proteome analyses revealed significant similarities between in vitro-derived mini-oocytes and in vivo oocytes, despite their relatively smaller size. Furthermore, we demonstrated the utility of mini-oocytes for investigating oocyte-specific molecular features through a small-scale knockout screen targeting the subcortical maternal complex. Given the simplicity, efficiency, and scalability of the mini-oocyte induction method, it offers a practical platform for conducting experiments that are otherwise challenging with in vivo oocytes.
A Bhat, R Berthoz, S Vecchio Lo, C Spiegelhalter, Shigenobu Yonemura, O Pertz and D Riveline : Myosin cluster dynamics determines epithelial wound ring constriction., iScience, 28, 8, 113030, 2025.
Erkhembayar Shinebaatar, Junko Morimoto, Rinna Koga, Nam Thanh Nguyen, Yuki Sasaki, Shigenobu Yonemura, Hidetaka Kosako and Koji Yasutomo : Proteasome dysfunction in T cells causes immunodeficiency via cell cycle disruption and apoptosis., International Immunology, 37, 8, 493-505, 2025.
(要約)
Proteasomes are essential molecular complexes that regulate intracellular protein homeostasis by selectively degrading ubiquitinated proteins. Genetic mutations in proteasome subunits lead to proteasome-associated autoinflammatory syndromes (PRAAS) characterized by autoinflammation, partial progressive lipodystrophy, and, in certain cases, immunodeficiency. However, the molecular mechanisms by which proteasome dysfunction results in these phenotypes remain unclear. Here, we established a mouse model carrying a mutation in β5i (encoded by Psmb8) along with T-cell-specific β5 (encoded by Psmb5) deficiency (KIKO mice). The KIKO mice presented severe loss of mature T cells in the spleen but not in the thymus, with reduced proteasome activity leading to the accumulation of ubiquitinated proteins. The CD4+ T cells of KIKO mice presented impaired proliferative activity with cell cycle arrest in the G0/G1 phase following T cell receptor (TCR) engagement. T cells from KIKO mice underwent rapid cell death through apoptosis, as treatment of T cells with the caspase inhibitor Z-Val-Ala-Asp(Ome)-fluoromethylketone (Z-VAD-FMK) rescued cell viability. Moreover, proteasome dysfunction induced apoptosis in T cells without affecting either mitochondrial functions or endoplasmic reticulum (ER) stress responses. Thus, our data provide insight into the molecular mechanisms underlying not only immunodeficiency in PRAAS patients but also T-cell deficiency associated with other disorders.
Mikiya Watanabe, Takayuki Yamada, Ya Hung Tu, Taro Chaya, Satoko Okayama, Kenta Onoue, Shigenobu Yonemura, Chieko Koike, Masao Tachibana, Takahisa Furukawa and Michiko Mandai : Graft-derived horizontal cells contribute to host-graft synapses in degenerated retinas after retinal organoid transplantation., Stem Cell Reports, 20, 7, 102545, 2025.
(要約)
Stem cell-derived retinal organoid (RO) transplantation is a promising approach for treating retinal degenerative diseases such as retinitis pigmentosa. Photoreceptors (PRs) in RO sheets form synaptic connections with host bipolar cells (BCs) and improve visual function in animal models of advanced retinal degeneration. However, the contribution of horizontal cells (HCs), which are critical for PR synapse formation in the developing retina, to host-graft synapse formation remains unclear. In this study, we used HC-depleted retinal degeneration mice (rd1) and Islet1-/- genome-edited ROs (gROs) that contain HCs but not rod BCs and showed that host HC deficiency did not alter the number of host BC-graft PR synapses after transplantation into rd1, while the restored light sensitivity was enhanced in the context of host HCs. These findings indicate that graft-derived HCs alone can support host-graft synapse formation, whereas the presence of host HCs facilitates functional recovery after gRO transplantation.
Dinh Loc Nguyen, Huong Ly Nguyen, Xuan Dat Dao, Tsuyoshi Hattori, Mika Takarada-Iemata, Hiroshi Ishii, Takashi Tamatani, Hiroshi Kawasaki, Yohei Shinmyo, Kenta Onoue, Shigenobu Yonemura, Jun Zhang, Masato Miyake, Seiichi Oyadomari, Kazutoshi Mori and Osamu Hori : Impact of ATF6 deletion on the embryonic brain development, iScience, 28, 6, 2025.
Yasuaki Iwama, Yasuko Sugase-Miyamoto, Kenta Onoue, Hirofumi Uyama, Keiji Matsuda, Kazuko Hayashi, Ryutaro Akiba, Tomohiro Masuda, Satoshi Yokota, Shigenobu Yonemura, Kohji Nishida, Masayo Takahashi, Yasuo Kurimoto and Michiko Mandai : Transplantation of human pluripotent stem cell-derived retinal sheet in a primate model of macular hole, Stem Cell Reports, 19, 11, 1524-1533, 2024.
(要約)
Macular hole (MH) is a retinal break involving the fovea that causes impaired vision. Although advances in vitreoretinal surgical techniques achieve >90% MH closure rate, refractory cases still exist. For such cases, autologous retinal transplantation is an optional therapy showing good anatomic success, but visual improvement is limited and peripheral visual field defects are inevitable after graft harvesting. Here, using a non-human primate model, we evaluated whether human embryonic stem cell-derived retinal organoid (RO) sheet transplantation can be an effective option for treating MH. After transplantation, MH was successfully closed by continuous filling of the MH space with the RO sheet, resulting in improved visual function, although no host-graft synaptic connections were confirmed. Mild xeno-transplantation rejection was controlled by additional focal steroid injections and rod/cone photoreceptors developed in the graft. Overall, our findings suggest pluripotent stem cell-derived RO sheet transplantation as a practical option for refractory MH treatment.
Maiko Sakai, Kohta Ohnishi, Masashi Masuda, Erika Harumoto, Teppei Fukuda, Aika Ohnishi, Shunsuke Ishii, Hirokazu Ohminami, Hisami Yamanaka-Okumura, Kazuto Ohashi, Eisuke Itakura, Kazuki Horikawa, Shigenobu Yonemura, Taichi Hara and Yutaka Taketani : Modulations of the mTORC2-GATA3 axis by an isorhamnetin activated endosomal-lysosomal system of the J774.1 macrophage-like cell line, Journal of Clinical Biochemistry and Nutrition, 75, 1, 24-32, 2024.
(キーワード)
endocytosis / GATA3 / isorhamnetin / mTORC2 / protein degradation
H Nishida, AB Albero, K Onoue, Y Ikegawa, S Sulekh, U Sakizli, Y Minami, Shigenobu Yonemura, YC Wang and SK Yoo : Necrosensor: a genetically encoded fluorescent sensor for visualizing necrosis in Drosophila., Biology Open, 13, 1, bio060104, 2023.
(要約)
Historically, necrosis has been considered a passive process, which is induced by extreme stress or damage. However, recent findings of necroptosis, a programmed form of necrosis, shed a new light on necrosis. It has been challenging to detect necrosis reliably in vivo, partly due to the lack of genetically encoded sensors to detect necrosis. This is in stark contrast with the availability of many genetically encoded biosensors for apoptosis. Here we developed Necrosensor, a genetically encoded fluorescent sensor that detects necrosis in Drosophila, by utilizing HMGB1, which is released from the nucleus as a damage-associated molecular pattern (DAMP). We demonstrate that Necrosensor is able to detect necrosis induced by various stresses in multiple tissues in both live and fixed conditions. Necrosensor also detects physiological necrosis that occurs during spermatogenesis in the testis. Using Necrosensor, we discovered previously unidentified, physiological necrosis of hemocyte progenitors in the hematopoietic lymph gland of developing larvae. This work provides a new transgenic system that enables in vivo detection of necrosis in real time without any intervention.
A Kira, I Tatsutomi, K Saito, M Murata, I Hattori, H Kajita, N Muraki, Y Oda, S Satoh, Y Tsukamoto, S Kimura, K Onoue, Shigenobu Yonemura, S Arakawa, H Kato, T Hirashima and K Kawane : Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion., Developmental Cell, 58, 14, 1282-1298, 2023.
(要約)
Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. Here, we report a conserved execution mechanism of apoptotic cell extrusion. We found extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells.
K Fukuda, T Shimi, C Shimura, T Ono, T Suzuki, K Onoue, S Okayama, H Miura, I Hiratani, K Ikeda, Y Okada, N Dohmae, Shigenobu Yonemura, A Inoue, H Kimura and Y Shinkai : Epigenetic plasticity safeguards heterochromatin configuration in mammals., Nucleic Acids Research, 51, 12, 6190-6207, 2023.
(要約)
Heterochromatin is a key architectural feature of eukaryotic chromosomes critical for cell type-specific gene expression and genome stability. In the mammalian nucleus, heterochromatin segregates from transcriptionally active genomic regions and exists in large, condensed, and inactive nuclear compartments. However, the mechanisms underlying the spatial organization of heterochromatin need to be better understood. Histone H3 lysine 9 trimethylation (H3K9me3) and lysine 27 trimethylation (H3K27me3) are two major epigenetic modifications that enrich constitutive and facultative heterochromatin, respectively. Mammals have at least five H3K9 methyltransferases (SUV39H1, SUV39H2, SETDB1, G9a and GLP) and two H3K27 methyltransferases (EZH1 and EZH2). In this study, we addressed the role of H3K9 and H3K27 methylation in heterochromatin organization using a combination of mutant cells for five H3K9 methyltransferases and an EZH1/2 dual inhibitor, DS3201. We showed that H3K27me3, which is normally segregated from H3K9me3, was redistributed to regions targeted by H3K9me3 after the loss of H3K9 methylation and that the loss of both H3K9 and H3K27 methylation resulted in impaired condensation and spatial organization of heterochromatin. Our data demonstrate that the H3K27me3 pathway safeguards heterochromatin organization after the loss of H3K9 methylation in mammalian cells.
Takayuki Onai, Toshihiro Aramaki, Akira Takai, Kisa Kakiguchi and Shigenobu Yonemura : Cranial cartilages: Players in the evolution of the cranium during evolution of the chordates in general and of the vertebrates in particular., Evolution & Development, 25, 3, 197-208, 2023.
(要約)
The present contribution is chiefly a review, augmented by some new results on amphioxus and lamprey anatomy, that draws on paleontological and developmental data to suggest a scenario for cranial cartilage evolution in the phylum chordata. Consideration is given to the cartilage-related tissues of invertebrate chordates (amphioxus and some fossil groups like vetulicolians) as well as in the two major divisions of the subphylum Vertebrata (namely, agnathans, and gnathostomes). In the invertebrate chordates, which can be considered plausible proxy ancestors of the vertebrates, only a viscerocranium is present, whereas a neurocranium is absent. For this situation, we examine how cartilage-related tissues of this head region prefigure the cellular cartilage types in the vertebrates. We then focus on the vertebrate neurocranium, where cyclostomes evidently lack neural-crest derived trabecular cartilage (although this point needs to be established more firmly). In the more complex gnathostome, several neural-crest derived cartilage types are present: namely, the trabecular cartilages of the prechordal region and the parachordal cartilage the chordal region. In sum, we present an evolutionary framework for cranial cartilage evolution in chordates and suggest aspects of the subject that should profit from additional study.
Aya Tentaku, Shusaku Kurisu, Kurumi Sejima, Toshiki Nagao, Akira Takahashi and Shigenobu Yonemura : Proximal deposition of collagen IV by fibroblasts contributes to basement membrane formation by colon epithelial cells invitro., The FEBS Journal, 289, 23, 7466-7485, 2022.
(要約)
The basement membrane (BM) underlying epithelial tissue is a thin layer of extracellular matrix that governs tissue integrity and function. Epithelial BMs are generally assembled using BM components secreted from two origins: epithelium and stroma. Although denovo BM formation involves self-assembly processes of large proteins, it remains unclear how stroma-derived macromolecules are transported and assembled, specifically in the BM region. In this study, we established an invitro co-culture model of BM formation in which DLD-1 human colon epithelial cells were cultured on top of collagen I gel containing human embryonic OUMS-36T-2 fibroblasts as stromal cells. A distinct feature of our system is represented by OUMS-36T-2 cells which are almost exclusively responsible for synthesis of collagen IV, a major BM component. Exploiting this advantage, we found that collagen IV incorporation was significantly impaired in culture conditions where OUMS-36T-2 cells were not allowed to directly contact DLD-1 cells. Soluble collagen IV, once diluted in the culture medium, did not accumulate in the BM region efficiently. Live imaging of fluorescently tagged collagen IV revealed that OUMS-36T-2 cells deposited collagen IV aggregates directly onto the basal surface of DLD-1 cells. Collectively, these results indicate a novel mode of collagen IV deposition in which fibroblasts proximal to epithelial cells exclusively contribute to collagen IV assembly during BM formation.
Ryosuke Nishimura, kagayaki Kato, M Saida, Y Kamei, M Takeda, H Miyoshi, Yutaka Yamagata, Yu Amano and Shigenobu Yonemura : Appropriate tension sensitivity of α-catenin ensures rounding morphogenesis of epithelial spheroids., Cell Structure and Function, 47, 2, 55-73, 2022.
(要約)
The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding α-catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of α-catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of α-catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that increased tension sensitivity of α-catenin affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive α-catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that the appropriate tension sensitivity of α-catenin is crucial for the coordinated round morphogenesis.Key words: α-catenin, vinculin, adherens junction, morphogenesis, mechanotransduction.
M Mori, T Yao, T Mishina, H Endoh, M Tanaka, N Yonezawa, Y Shimamoto, Shigenobu Yonemura, K Yamagata, TS Kitajima and M Ikawa : RanGTP and the actin cytoskeleton keep paternal and maternal chromosomes apart during fertilization., The Journal of Cell Biology, 220, 10, 2021.
(要約)
Zygotes require two accurate sets of parental chromosomes, one each from the mother and the father, to undergo normal embryogenesis. However, upon egg-sperm fusion in vertebrates, the zygote has three sets of chromosomes, one from the sperm and two from the egg. The zygote therefore eliminates one set of maternal chromosomes (but not the paternal chromosomes) into the polar body through meiosis, but how the paternal chromosomes are protected from maternal meiosis has been unclear. Here we report that RanGTP and F-actin dynamics prevent egg-sperm fusion in proximity to maternal chromosomes. RanGTP prevents the localization of Juno and CD9, egg membrane proteins that mediate sperm fusion, at the cell surface in proximity to maternal chromosomes. Following egg-sperm fusion, F-actin keeps paternal chromosomes away from maternal chromosomes. Disruption of these mechanisms causes the elimination of paternal chromosomes during maternal meiosis. This study reveals a novel critical mechanism that prevents aneuploidy in zygotes.
Hiroko Saito, F Matsukawa-Usami, T Fujimori, T Kimura, T Ide, T Yamamoto, T Shibata, Kenta Onoue, Satoko Okayama, Shigenobu Yonemura, Kazuyo Misaki, Y Soba, Y Kakui, M Sato, M Toya and Masatoshi Takeichi : Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating., Molecular Biology of the Cell, 32, 20, ar12, 2021.
(要約)
Motile cilia of multiciliated epithelial cells undergo synchronized beating to produce fluid flow along the luminal surface of various organs. Each motile cilium consists of an axoneme and a basal body (BB), which are linked by a "transition zone" (TZ). The axoneme exhibits a characteristic 9+2 microtubule arrangement important for ciliary motion, but how this microtubule system is generated is not yet fully understood. Here we show that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a protein that can stabilize the minus-end of a microtubule, concentrates at multiple sites of the cilium-BB complex, including the upper region of the TZ or the axonemal basal plate (BP) where the central pair of microtubules (CP) initiates. CAMSAP3 dysfunction resulted in loss of the CP and partial distortion of the BP, as well as the failure of multicilia to undergo synchronized beating. These findings suggest that CAMSAP3 plays pivotal roles in the formation or stabilization of the CP by localizing at the basal region of the axoneme and thereby supports the coordinated motion of multicilia in airway epithelial cells.
Shotaro Sakakibara, Kiyohito Mizutani, Ayumu Sugiura, Ayuko Sakane, Takuya Sasaki, Shigenobu Yonemura and Yoshimi Takai : Afadin regulates actomyosin organization through αE-catenin at adherens junctions, The Journal of Cell Biology, 219, 5, e201907079, 2020.
(要約)
Actomyosin-undercoated adherens junctions are critical for epithelial cell integrity and remodeling. Actomyosin associates with adherens junctions through αE-catenin complexed with β-catenin and E-cadherin in vivo; however, in vitro biochemical studies in solution showed that αE-catenin complexed with β-catenin binds to F-actin less efficiently than αE-catenin that is not complexed with β-catenin. Although a "catch-bond model" partly explains this inconsistency, the mechanism for this inconsistency between the in vivo and in vitro results remains elusive. We herein demonstrate that afadin binds to αE-catenin complexed with β-catenin and enhances its F-actin-binding activity in a novel mechanism, eventually inducing the proper actomyosin organization through αE-catenin complexed with β-catenin and E-cadherin at adherens junctions.
Shotaro Sakakibara, Kiyohito Mizutani, Ayumu Sugiura, Ayuko Sakane, Takuya Sasaki, Shigenobu Yonemura and Yoshimi Takai : Afadin regulates actomyosin organization through αE-catenin at adherens junctions., The Journal of Cell Biology, 219, 5, 2020.
(要約)
Actomyosin-undercoated adherens junctions are critical for epithelial cell integrity and remodeling. Actomyosin associates with adherens junctions through αE-catenin complexed with β-catenin and E-cadherin in vivo; however, in vitro biochemical studies in solution showed that αE-catenin complexed with β-catenin binds to F-actin less efficiently than αE-catenin that is not complexed with β-catenin. Although a "catch-bond model" partly explains this inconsistency, the mechanism for this inconsistency between the in vivo and in vitro results remains elusive. We herein demonstrate that afadin binds to αE-catenin complexed with β-catenin and enhances its F-actin-binding activity in a novel mechanism, eventually inducing the proper actomyosin organization through αE-catenin complexed with β-catenin and E-cadherin at adherens junctions.
S Konishi, T Yano, H Tanaka, T Mizuno, H Kanoh, K Tsukita, T Namba, A Tamura, Shigenobu Yonemura, S Gotoh, H Matsumoto, T Hirai and S Tsukita : Vinculin is critical for the robustness of the epithelial cell sheet paracellular barrier for ions., Life Science Alliance, 2, 4, 2019.
(要約)
The paracellular barrier function of tight junctions (TJs) in epithelial cell sheets is robustly maintained against mechanical fluctuations, by molecular mechanisms that are poorly understood. Vinculin is an adaptor of a mechanosensory complex at the adherens junction. Here, we generated vinculin KO Eph4 epithelial cells and analyzed their confluent cell-sheet properties. We found that vinculin is dispensable for the basic TJ structural integrity and the paracellular barrier function for larger solutes. However, vinculin is indispensable for the paracellular barrier function for ions. In addition, TJs stochastically showed dynamically distorted patterns in vinculin KO cell sheets. These KO phenotypes were rescued by transfecting full-length vinculin and by relaxing the actomyosin tension with blebbistatin, a myosin II ATPase activity inhibitor. Our findings indicate that vinculin resists mechanical fluctuations to maintain the TJ paracellular barrier function for ions in epithelial cell sheets.
Toshiya Ando, Sayaka Sekine, Sachi Inagaki, Kazuyo Misaki, Laurent Badel, Hiroyuki Moriya, Mustafa M. Sami, Yuki Itakura, Takahiro Chihara, Hokto Kazama, Shigenobu Yonemura and Shigeo Hayashi : Nanopore Formation in the Cuticle of an Insect Olfactory Sensillum., Current Biology, 29, 9, 1512-1520.e6, 2019.
(要約)
Nanometer-level patterned surface structures form the basis of biological functions, including superhydrophobicity, structural coloration, and light absorption [1-3]. In insects, the cuticle overlying the olfactory sensilla has multiple small (50- to 200-nm diameter) pores [4-8], which are supposed to function as a filter that admits odorant molecules, while preventing the entry of larger airborne particles and limiting water loss. However, the cellular processes underlying the patterning of extracellular matrices into functional nano-structures remain unknown. Here, we show that cuticular nanopores in Drosophila olfactory sensilla originate from a curved ultrathin film that is formed in the outermost envelope layer of the cuticle and secreted from specialized protrusions in the plasma membrane of the hair forming (trichogen) cell. The envelope curvature coincides with plasma membrane undulations associated with endocytic structures. The gore-tex/Osiris23 gene encodes an endosomal protein that is essential for envelope curvature, nanopore formation, and odor receptivity and is expressed specifically in developing olfactory trichogen cells. The 24-member Osiris gene family is expressed in cuticle-secreting cells and is found only in insect genomes. These results reveal an essential requirement for nanopores for odor reception and identify Osiris genes as a platform for investigating the evolution of surface nano-fabrication in insects.
Sayaka Wakayama, Taiyu Ito, Yuko Kamada, Shigenobu Yonemura, Masatoshi Oosa, Satoshi Kishigami and Teruhiko Wakayama : Tolerance of the freeze-dried mouse sperm nucleus to temperatures ranging from -196°C to 150°C., Scientific Reports, 9, 1, 5719, 2019.
(要約)
It has long been believed that tolerance against extreme environments is possible only for 'lower' groups, such as archaea, bacteria or tardigrades, and not for more 'advanced' species. Here, we demonstrated that the mammalian sperm nucleus also exhibited strong tolerance to cold and hot temperatures. When mouse spermatozoa were freeze-dried (FD), similar to the anhydrobiosis of Tardigrades, all spermatozoa were ostensibly dead after rehydration. However, offspring were obtained from recovered FD sperm nuclei, even after repeated treatment with conditions from liquid nitrogen to room temperature. Conversely, when FD spermatozoa were heated at 95 °C, although the birth rate was decreased with increasing duration of the treatment, offspring were obtained even for FD spermatozoa that had been heat-treated for 2 h. This period was improved up to 6 h when glucose was replaced with trehalose in the freeze-drying medium, and the resistance temperature was extended up to 150 °C for short periods of treatment. Randomly selected offspring grew into healthy adults. Our results suggest that, when considering the sperm nucleus/DNA as the material that is used as a blueprint of life, rather than cell viability, a significant tolerance to extreme temperatures is present even in 'higher' species, such as mammals.
Masatsune Tsujioka, Taro QP Uyeda, Yoshiaki Iwadate, Hitesh Patel, Keitaro Shibata, Tenji Yumoto and Shigenobu Yonemura : Actin-binding domains mediate the distinct distribution of two Dictyostelium Talins through different affinities to specific subsets of actin filaments during directed cell migration., PLoS ONE, 14, 4, e0214736, 2019.
(要約)
Although the distinct distribution of certain molecules along the anterior or posterior edge is essential for directed cell migration, the mechanisms to maintain asymmetric protein localization have not yet been fully elucidated. Here, we studied a mechanism for the distinct localizations of two Dictyostelium talin homologues, talin A and talin B, both of which play important roles in cell migration and adhesion. Using GFP fusion, we found that talin B, as well as its C-terminal actin-binding region, which consists of an I/LWEQ domain and a villin headpiece domain, was restricted to the leading edge of migrating cells. This is in sharp contrast to talin A and its C-terminal actin-binding domain, which co-localized with myosin II along the cell posterior cortex, as reported previously. Intriguingly, even in myosin II-null cells, talin A and its actin-binding domain displayed a specific distribution, co-localizing with stretched actin filaments. In contrast, talin B was excluded from regions rich in stretched actin filaments, although a certain amount of its actin-binding region alone was present in those areas. When cells were sucked by a micro-pipette, talin B was not detected in the retracting aspirated lobe where acto-myosin, talin A, and the actin-binding regions of talin A and talin B accumulated. Based on these results, we suggest that talin A predominantly interacts with actin filaments stretched by myosin II through its C-terminal actin-binding region, while the actin-binding region of talin B does not make such distinctions. Furthermore, talin B appears to have an additional, unidentified mechanism that excludes it from the region rich in stretched actin filaments. We propose that these actin-binding properties play important roles in the anterior and posterior enrichment of talin B and talin A, respectively, during directed cell migration.
Takuma Shinozuka, Ritsuko Takada, Chosei Yoshida, Shigenobu Yonemura and Shinji Takada : Wnt produced by stretched roof-plate cells is required for the promotion of cell proliferation around the central canal of the spinal cord, Development, 146, 2, dev159343, 2019.
(要約)
Cell morphology changes dynamically during embryogenesis, and these changes create new interactions with surrounding cells, some of which are presumably mediated by intercellular signaling. However, the effects of morphological changes on intercellular signaling remain to be fully elucidated. In this study, we examined the effect of morphological changes in Wnt-producing cells on intercellular signaling in the spinal cord. After mid-gestation, roof-plate cells stretched along the dorsoventral axis in the mouse spinal cord, resulting in new contact at their tips with the ependymal cells that surround the central canal. Wnt1 and Wnt3a were produced by the stretched roof-plate cells and delivered to the cell process tip. Whereas Wnt signaling was activated in developing ependymal cells, Wnt activation in dorsal ependymal cells, which were close to the stretched roof plate, was significantly suppressed in embryos with roof plate-specific conditional knockout of , which encodes a factor that is essential for Wnt secretion. Furthermore, proliferation of these cells was impaired in conditional knockout mice during development and after induced spinal cord injury in adults. Therefore, morphological changes in Wnt-producing cells appear to generate new Wnt signal targets.
Noboru Ishiyama, Rifu Sarpal, Megan N. Wood, Samantha K Garrick, Tadateru Nishikawa, Hanako Hayashi, Anna B Kobb, Annette S. Flozak, Alex Yemelyanov, Rodrigo Fernandez-Gonzalez, Shigenobu Yonemura, Deborah E. Neckband, Cara J. Gottardi, Ulrich Tepass and Mitsuhiko Ikura : Force-dependent allostery of the α-catenin actin-binding domain controls adherens junction dynamics and functions, Nature Communications, 9, 1, 5121, 2018.
(要約)
α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis.
Chun-Chun Cheng, Ko Tsutsui, Toru Taguchi, Noriko Sansen, Asako Nakagawa, Kisa Kakiguchi, Shigenobu Yonemura, Chiharu Tanegashima, Sean D Keeley, Hiroshi Kiyonari, Yasuhide Furuta, Yasuko Tomono, Fiona M Watt and Hironobu Fujiwara : Hair follicle epidermal stem cells define a niche for tactile sensation, eLife, 7, e38883, 2018.
(要約)
The heterogeneity and compartmentalization of stem cells is a common principle in many epithelia, and is known to function in epithelial maintenance, but its other physiological roles remain elusive. Here we show transcriptional and anatomical contributions of compartmentalized epidermal stem cells in tactile sensory unit formation in the mouse hair follicle. Epidermal stem cells in the follicle upper-bulge, where mechanosensory lanceolate complexes innervate, express a unique set of extracellular matrix (ECM) and neurogenesis-related genes. These epidermal stem cells deposit an ECM protein called EGFL6 into the collar matrix, a novel ECM that tightly ensheathes lanceolate complexes. EGFL6 is required for the proper patterning, touch responses, and αv integrin-enrichment of lanceolate complexes. By maintaining a quiescent original epidermal stem cell niche, the old bulge, epidermal stem cells provide anatomically stable follicle-lanceolate complex interfaces, irrespective of the stage of follicle regeneration cycle. Thus, compartmentalized epidermal stem cells provide a niche linking the hair follicle and the nervous system throughout the hair cycle.
Akira Honda, Tomoko Kita, Shri Vidhya Seshadri, Kazuyo Misaki, Jamal Ahmed, John E. Ladbury, Guy P. Richardson, Shigenobu Yonemura and Raj K. Lather : FGFR1-mediated protocadherin-15 loading mediates cargo specificity during intraflagellar transport in inner ear hair-cell kinocilia., Proceedings of the National Academy of Sciences of the United States of America, 115, 33, 8388-8393, 2018.
(要約)
The mechanosensory hair cells of the inner ear are required for hearing and balance and have a distinctive apical structure, the hair bundle, that converts mechanical stimuli into electrical signals. This structure comprises a single cilium, the kinocilium, lying adjacent to an ensemble of actin-based projections known as stereocilia. Hair bundle polarity depends on kinociliary protocadherin-15 (Pcdh15) localization. Protocadherin-15 is found only in hair-cell kinocilia, and is not localized to the primary cilia of adjacent supporting cells. Thus, Pcdh15 must be specifically targeted and trafficked into the hair-cell kinocilium. Here we show that kinocilial Pcdh15 trafficking relies on cell type-specific coupling to the generic intraflagellar transport (IFT) transport mechanism. We uncover a role for fibroblast growth factor receptor 1 (FGFR1) in loading Pcdh15 onto kinociliary transport particles in hair cells. We find that on activation, FGFR1 binds and phosphorylates Pcdh15. Moreover, we find a previously uncharacterized role for clathrin in coupling this kinocilia-specific cargo with the anterograde IFT-B complex through the adaptor, DAB2. Our results identify a modified ciliary transport pathway used for Pcdh15 transport into the cilium of the inner ear hair cell and coordinated by FGFR1 activity.
(キーワード)
Adaptor Proteins, Vesicular Transport / Animals / Cadherins / Chick Embryo / Clathrin / Flagella / Hair Cells, Auditory, Inner / Mice / Phosphorylation / Protein Precursors / Protein Transport / Receptor, Fibroblast Growth Factor, Type 1
So Goto, Akishi Onishi, Kazuyo Misaki, Shigenobu Yonemura, Sunao Sugita, Hiromi Ito, Yoko Ohigashi, Masatsugu Ema, Hirokazu Sakaguchi, Kohl Nishida and Masayo Takahashi : Neural retina-specific Aldh1a1 controls dorsal choroidal vascular development via Sox9 expression in retinal pigment epithelial cells, eLife, 7, 2018.
(要約)
VEGF secreted from retinal pigment epithelial (RPE) cells is responsible for the choroidal vascular development; however, the molecular regulatory mechanism is unclear. We found that mice showed choroidal hypoplasia with insufficient vascularization in the dorsal region, although Aldh1a1, an enzyme that synthesizes retinoic acids (RAs), is expressed in the dorsal neural retina, not in the RPE/choroid complex. The level of VEGF in the RPE/choroid was significantly decreased in mice, and RA-dependent enhancement of VEGF was observed in primary RPE cells. An RA-deficient diet resulted in dorsal choroidal hypoplasia, and simple RA treatment of pregnant females suppressed choroid hypoplasia in their offspring. We also found downregulation of Sox9 in the dorsal neural retina and RPE of mice and RPE-specific disruption of Sox9 phenocopied choroidal development. These results suggest that RAs produced by Aldh1a1 in the neural retina directs dorsal choroidal vascular development via Sox9 upregulation in the dorsal RPE cells to enhance RPE-derived VEGF secretion.
Yoshinori Hirano, Yu Amano, Shigenobu Yonemura and Toshio Takoshima : The force-sensing device region of alpha-catenin in an intrinsically disordered segment in the absence of intramolecular stabilization of the auto inhibitory form., Genes to Cells, 23, 5, 370-385, 2018.
(要約)
Mechanotransduction by α-catenin facilitates the force-dependent development of adherens junctions (AJs) by recruiting vinculin to reinforce actin anchoring of AJs. The α-catenin mechanotransducing action is facilitated by its force-sensing device region that autoinhibits the vinculin-binding site 1 (VBS1). Here, we report the high-resolution structure of the force-sensing device region of α-catenin, which shows the autoinhibited form comprised of helix bundles E, F and G. The cryptic VBS1 is embedded into helix bundle E stabilized by direct interactions with the autoinhibitory region forming helix bundles F and G. Our molecular dissection study showed that helix bundles F and G are stable in solution in each isolated form, whereas helix bundle E that contains VBS1 is unstable and intrinsically disordered in solution in the isolated form. We successfully identified key residues mediating the autoinhibition and produced mutated α-catenins that display variable force sensitivity and autoinhibition. Using these mutants, we demonstrate both in vitro and in vivo that, in the absence of this stabilization, the helix bundle containing VBS1 would adopt an unfolded form, thus exposing VBS for vinculin binding. We provide evidence for importance of mechanotransduction with the intrinsic force sensitivity for vinculin recruitment to adherens junctions of epithelial cell sheets with mutated α-catenins.
Satoshi Iraha, Hung-Ya Tu, Suguru Yamasaki, Takahiro Kagawa, Motohito Goto, Riichi Takahashi, Takehito Watanabe, Sunao Sugita, Shigenobu Yonemura, Genshiro A. Sunagawa, Take Matsuyama, Momo Fujii, Atsushi Kuwahara, Akiyoshi Kishino, Nosh Koide, Mototsugu Eiraku, Hidenobu Tanihara, Masayo Takahashi and Michiko Mandai : Establishment of Immunodeficient Retinal Degeneration Model Mice and Functional Maturation of Human ESC-Derived Retinal Sheets after Transplantation, Stem Cell Reports, 10, 3, 1059-1074, 2018.
(要約)
Increasing demand for clinical retinal degeneration therapies featuring human ESC/iPSC-derived retinal tissue and cells warrants proof-of-concept studies. Here, we established two mouse models of end-stage retinal degeneration with immunodeficiency, NOG-rd1-2J and NOG-rd10, and characterized disease progress and immunodeficient status. We also transplanted human ESC-derived retinal sheets into NOG-rd1-2J and confirmed their long-term survival and maturation of the structured graft photoreceptor layer, without rejection or tumorigenesis. We recorded light responses from the host ganglion cells using a multi-electrode array system; this result was consistent with whole-mount immunostaining suggestive of host-graft synapse formation at the responding sites. This study demonstrates an application of our mouse models and provides a proof of concept for the clinical use of human ESC-derived retinal sheets.
koichiro Maki, Sung-Woong Han, Yoshinori Hirano, Shigenobu Yonemura, Toshio Takoshima and Taiji Adachi : Real-time TIRF observation of vinculin recruitment to stretched alpha-catenin by AFM, Scientific Reports, 8, 1, 1575, 2018.
(要約)
Adherens junctions (AJs) adaptively change their intensities in response to intercellular tension; therefore, they integrate tension generated by individual cells to drive multicellular dynamics, such as morphogenetic change in embryos. Under intercellular tension, α-catenin, which is a component protein of AJs, acts as a mechano-chemical transducer to recruit vinculin to promote actin remodeling. Although in vivo and in vitro studies have suggested that α-catenin-mediated mechanotransduction is a dynamic molecular process, which involves a conformational change of α-catenin under tension to expose a cryptic vinculin binding site, there are no suitable experimental methods to directly explore the process. Therefore, in this study, we developed a novel system by combining atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF). In this system, α-catenin molecules (residues 276-634; the mechano-sensitive M-M domain), modified on coverslips, were stretched by AFM and their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, were observed simultaneously, in real time, using TIRF. We applied a physiologically possible range of tensions and extensions to α-catenin and directly observed its vinculin recruitment. Our new system could be used in the fields of mechanobiology and biophysics to explore functions of proteins under tension by coupling biomechanical and biochemical information.
(キーワード)
Animals / Fluorometry / Mice / Microscopy, Atomic Force / Protein Binding / Vinculin / alpha Catenin
Liqing Liu, Kentaro Suzuki, Eunice Chun, Aki Murashima, Yuki Sato, Naomi Nakagata, Toshihiko Fujimori, Shigenobu Yonemura, Wanzhong He and Gen Yamada : Androgen Regulates Dimorphic F-Actin Assemblies in the Genital Organogenesis, Sexual Development, 11, 4, 190-202, 2017.
(要約)
Impaired androgen activity induces defective sexual differentiation of the male reproductive tract, including hypospadias, an abnormal formation of the penile urethra. Androgen signaling in the urethral mesenchyme cells (UMCs) plays essential roles in driving dimorphic urethral development. However, cellular events for sexual differentiation remain virtually unknown. In this study, histological analyses, fluorescent staining, and transmission electron microscopy (TEM) were performed to reveal the cellular dimorphisms of UMCs. F-actin dynamics and migratory behaviors of UMCs were further analyzed by time-lapse imaging. We observed a prominent accumulation of F-actin with poorly assembled extracellular matrix (ECM) in female UMCs. In contrast, thin fibrils of F-actin co-aligning with the ECM through membrane receptors were identified in male UMCs. Processes for dimorphic F-actin assemblies were temporally identified during an androgen-regulated masculinization programming window and spatially distributed in several embryonic reproductive tissues. Stage-dependent modulation of the F-actin sexual patterns by androgen in UMCs was also demonstrated by time-lapse analysis. Moreover, androgen regulates coordinated migration of UMCs. These results suggest that androgen signaling regulates the assembly of F-actin from cytoplasmic accumulation to membranous fibrils. Such alteration appears to promote the ECM assembly and the mobility of UMCs, contributing to male type genital organogenesis.
Miki Takeuchi, Chikako Inoue, Akiko Goshima, Yusuke Nagao, Koichi Shimizu, Hiroki Miyamoto, Takashi Shimizu, Hisashi Hashimoto, Shigenobu Yonemura, Atsuo Kawahara, Yutaka Hirata, Masayuki Yoshida and Masahiko Hibi : Medaka and zebrafish contactin1 mutants as a model for understanding neural circuits for motor coordination, Genes to Cells, 22, 8, 723-741, 2017.
(要約)
A spontaneous medaka ro mutant shows abnormal wobbling and rolling swimming behaviors. By positional cloning, we mapped the ro locus to a region containing the gene encoding Contactin1b (Cntn1b), which is an immunoglobulin (Ig)-superfamily domain-containing membrane-anchored protein. The ro mutant had a deletion in the cntn1b gene that introduced a premature stop codon. Furthermore, cntn1b mutants generated by the CRISPR/Cas9 system and trans-heterozygotes of the CRISPR mutant allele and ro had abnormal swimming behavior, indicating that the cntn1b gene was responsible for the ro-mutant phenotype. We also established zebrafish cntn1a and cntn1b mutants by transcription activator-like effector nucleases (TALENs). Zebrafish cntn1b but not cntn1a mutants showed abnormal swimming behaviors similar to those in the ro mutant, suggesting that Cntn1b plays a conserved role in the formation or function of the neural circuits that control swimming in teleosts. Although Cntn1-deficient mice have abnormal cerebellar neural circuitry, there was no apparent histological abnormality in the cerebellum of medaka or zebrafish cntn1b mutants. The medaka cntn1b mutants had defective optokinetic response (OKR) adaptation and abnormal rheotaxis (body positioning relative to water flow). Medaka and zebrafish cntn1b mutants are effective models for studying the neural circuits involved in motor learning and motor coordination.
Toshihiro Akiyama, Akihito Inoko, Yuichi Kaji, Shigenobu Yonemura, Kisa Kakiguchi, Hiroki Segawa, Kei Ishitsuka, Masaki Yoshida, Osamu Numata, Philippe Leproux, Vincent Couderc, Tetsuro Oshika and Hideaki Kano : SHG-specificity of cellular Rootletin filaments enables naïve imaging with universal conservation., Scientific Reports, 7, 39967, 2017.
(要約)
Despite growing demand for truly naïve imaging, label-free observation of cilium-related structure remains challenging, and validation of the pertinent molecules is correspondingly difficult. In this study, in retinas and cultured cells, we distinctively visualized Rootletin filaments in rootlets in the second harmonic generation (SHG) channel, integrated in custom coherent nonlinear optical microscopy (CNOM) with a simple, compact, and ultra-broadband supercontinuum light source. This SHG signal was primarily detected on rootlets of connecting cilia in the retinal photoreceptor and was validated by colocalization with anti-Rootletin staining. Transfection of cells with Rootletin fragments revealed that the SHG signal can be ascribed to filaments assembled from the R234 domain, but not to cross-striations assembled from the R123 domain. Consistent with this, Rootletin-depleted cells lacked SHG signal expected as centrosome linker. As a proof of concept, we confirmed that similar fibrous SHG was observed even in unicellular ciliates. These findings have potential for broad applications in clinical diagnosis and biophysical experiments with various organisms.
T Otani, Y Ogura, K Misaki, T Maeda, A Kimpara, S Yonemura, S. Hayashi and Shigenobu Yonemura : IKK inhibits PKC to promote Fascin-dependent actin bundling., Development, 143, 20, 3806-3816, 2016.
(要約)
Signaling molecules have pleiotropic functions and are activated by various extracellular stimuli. Protein kinase C (PKC) is activated by diverse receptors, and its dysregulation is associated with diseases including cancer. However, how the undesired activation of PKC is prevented during development remains poorly understood. We have previously shown that a protein kinase, IKKϵ, is active at the growing bristle tip and regulates actin bundle organization during Drosophila bristle morphogenesis. Here, we demonstrate that IKKϵ regulates the actin bundle localization of a dynamic actin cross-linker, Fascin. IKKϵ inhibits PKC, thereby protecting Fascin from inhibitory phosphorylation. Excess PKC activation is responsible for the actin bundle defects in IKKϵ-deficient bristles, whereas PKC is dispensable for bristle morphogenesis in wild-type bristles, indicating that PKC is repressed by IKKϵ in wild-type bristle cells. These results suggest that IKKϵ prevents excess activation of PKC during bristle morphogenesis.
(キーワード)
Actins / Animals / Carrier Proteins / Drosophila / Drosophila Proteins / I-kappa B Kinase / Microfilament Proteins / Phosphorylation / Protein Kinase C / Signal Transduction
M Yoshida, E Kajikawa, D Yamamoto, D Kurokawa, S Yonemura, K Kobayashi, H Kiyonari, S. Aizawa and Shigenobu Yonemura : Conserved and Divergent Expression Patterns of Markers of Axial Development in the laboratory opossum, Monodelphis domestica., Developmental Dynamics, 245, 12, 1176-1188, 2016.
Y Iwasaki, S Sugita, M Mandai, S Yonemura, A Onishi, S Ito, M Mochizuki, K Ohno-Matsui, M. Takahashi and Shigenobu Yonemura : Differentiation/Purification Protocol for Retinal Pigment Epithelium from Mouse Induced Pluripotent Stem Cells as a Research Tool., PLoS ONE, 11, 7, e0158282, 2016.
(要約)
To establish a novel protocol for differentiation of retinal pigment epithelium (RPE) with high purity from mouse induced pluripotent stem cells (iPSC). Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated. We obtained iPS-RPE at high purity (approximately 98%). The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation. We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE.
M Yoshida, E Kajikawa, D Kurokawa, M Noro, T Iwai, S Yonemura, K Kobayashi, H Kiyonari, S. Aizawa and Shigenobu Yonemura : Conserved and Divergent Expression Patterns of Markers of Axial Development in Reptilian Embryos: Chinese Soft-shell Turtle and Madagascar Ground Gecko., Developmental Biology, 415, 1, 122-142, 2016.
(要約)
The processes of development leading up to gastrulation have been markedly altered during the evolution of amniotes, and it is uncertain how the mechanisms of axis formation are conserved and diverged between mouse and chick embryos. To assess the conservation and divergence of these mechanisms, this study examined gene expression patterns during the axis formation process in Chinese soft-shell turtle and Madagascar ground gecko preovipositional embryos. The data suggest that NODAL signaling, similarly to avian embryos but in contrast to eutherian embryos, does not have a role in epiblast and hypoblast development in reptilian embryos. The posterior marginal epiblast (PME) is the initial molecular landmark of axis formation in reptilian embryos prior to primitive plate development. Ontogenetically, PME may be the precursor of the primitive plate, and phylogenetically, Koller's sickle and posterior marginal zone in avian development may have been derived from the PME. Most of the genes expressed in the mouse anterior visceral endoderm (AVE genes), especially signaling antagonist genes, are not expressed in the hypoblast of turtle and gecko embryos, though they are expressed in the avian hypoblast. This study proposes that AVE gene expression in the hypoblast and the visceral endoderm could have been independently established in avian and eutherian lineages, similar to the primitive streak that has been independently acquired in these lineages.
A Onodera, K Yayama, A Tanaka, H Morosawa, T Furuta, N Takeda, K Kakiguchi, S Yonemura, I Yanagihara, Y Tsutsumi, Y. Kawai and Shigenobu Yonemura : Amorphous nanosilica particles evoke vascular relaxation through PI3K/Akt/eNOS signaling., Fundamental & Clinical Pharmacology, 30, 5, 419-428, 2016.
Y Otowa, K Moriwaki, K Sano, M Shirakabe, S Yonemura, M Shibuya, J Rossant, T Suda, Y Kakeji, M. Hirashima and Shigenobu Yonemura : Flt1/VEGFR1 heterozygosity causes transient embryonic edema., Scientific Reports, 6, 27186, 2016.
(要約)
Vascular endothelial growth factor-A is a major player in vascular development and a potent vascular permeability factor under physiological and pathological conditions by binding to a decoy receptor Flt1 and its primary receptor Flk1. In this study, we show that Flt1 heterozygous (Flt1(+/-)) mouse embryos grow up to adult without life-threatening abnormalities but exhibit a transient embryonic edema around the nuchal and back regions, which is reminiscent of increased nuchal translucency in human fetuses. Vascular permeability is enhanced and an intricate infolding of the plasma membrane and huge vesicle-like structures are seen in Flt1(+/-) capillary endothelial cells. Flk1 tyrosine phosphorylation is elevated in Flt1(+/-) embryos, but Flk1 heterozygosity does not suppress embryonic edema caused by Flt1 heterozygosity. When Flt1 mutants are crossed with Aspp1(-/-) mice which exhibit a transient embryonic edema with delayed formation and dysfunction of lymphatic vessels, only 5.7% of Flt1(+/-); Aspp1(-/-) mice survive, compared to expected ratio (25%). Our results demonstrate that Flt1 heterozygosity causes a transient embryonic edema and can be a risk factor for embryonic lethality in combination with other mutations causing non-lethal vascular phenotype.
Y Higashiguchi, K Katsuta, T Minegishi, S Yonemura, A Urasaki, N. Inagaki and Shigenobu Yonemura : Identification of a shootin1 isoform expressed in peripheral tissues., Cell and Tissue Research, 366, 1, 75-87, 2016.
(要約)
Shootin1 is a brain-specific cytoplasmic protein involved in neuronal polarity formation and axon outgrowth. It accumulates at the leading edge of axonal growth cones, where it mediates the mechanical coupling between F-actin retrograde flow and cell adhesions as a clutch molecule, thereby producing force for axon outgrowth. In this study, we report a novel splicing isoform of shootin1 which is expressed not only in the brain but also in peripheral tissues. We have renamed the brain-specific shootin1 as shootin1a and termed the novel isoform as shootin1b. Immunoblot and immunohistochemical analyses with a shootin1b-specific antibody revealed that shootin1b is distributed in various mouse tissues including the lung, liver, stomach, intestines, spleen, pancreas, kidney and skin. Interestingly, shootin1b immunoreactivity was widely detected in epithelial cells that constitute simple and stratified epithelia; in some cells, it colocalized with E-cadherin and cortactin at cell-cell contact sites. Shootin1b also localized in dendritic cells in the spleen. These results suggest that shootin1b may function in various peripheral tissues including epithelial cells.
(キーワード)
Aging / Amino Acid Sequence / Animals / Antibody Specificity / Cadherins / Cell Communication / Cortactin / Immunohistochemistry / Mice, Inbred C57BL / Nerve Tissue Proteins / Organ Specificity / Protein Isoforms / Protein Transport / Rats / Tissue Distribution
N Shibata, M Kashima, T Ishiko, O Nishimura, L Rouhana, K Misaki, S Yonemura, K Saito, H Siomi, MC Siomi, K. Agata and Shigenobu Yonemura : Inheritance of a Nuclear PIWI from Pluripotent Stem Cells by Somatic Descendants Ensures Differentiation by Silencing Transposons in Planarian., Developmental Cell, 37, 3, 226-237, 2016.
(要約)
Differentiation of pluripotent stem cells (PSCs) requires transposon silencing throughout the process. PIWIs, best known as key factors in germline transposon silencing, are also known to act in somatic differentiation of planarian PSCs (neoblasts). However, how PIWIs control the latter process remains elusive. Here, using Dugesia japonica, we show that a nuclear PIWI, DjPiwiB, was bound to PIWI-interacting RNAs (generally key mediators of PIWI-dependent transposon silencing), and was detected in not only neoblasts but also their descendant somatic cells, which do not express piwi. In contrast, cytoplasmic DjPiwiA and DjPiwiC were detected only in neoblasts, in accord with their transcription there. DjPiwiB was indispensable for regeneration, but dispensable for transposon silencing in neoblasts. However, transposons were derepressed at the onset of differentiation in DjPiwiB-knockdown planarians. Thus, DjPiwiB appears to be inherited by descendant somatic cells of neoblasts to ensure transposon silencing in those cells, which are unable to produce PIWI proteins.
(キーワード)
Animals / Argonaute Proteins / Base Sequence / Cell Differentiation / Cell Nucleus / DNA Transposable Elements / Gene Silencing / Immunohistochemistry / Inheritance Patterns / Models, Biological / Planarians / Pluripotent Stem Cells / RNA, Small Interfering
K Maki, SW Han, Y Hirano, S Yonemura, T Hakoshima, T. Adachi and Shigenobu Yonemura : Mechano-adaptive sensory mechanism of -catenin under tension., Scientific Reports, 6, 24878, 2016.
(要約)
The contractile forces in individual cells drive the tissue processes, such as morphogenesis and wound healing, and maintain tissue integrity. In these processes, α-catenin molecule acts as a tension sensor at cadherin-based adherens junctions (AJs), accelerating the positive feedback of intercellular tension. Under tension, α-catenin is activated to recruit vinculin, which recruits actin filaments to AJs. In this study, we revealed how α-catenin retains its activated state while avoiding unfolding under tension. Using single-molecule force spectroscopy employing atomic force microscopy (AFM), we found that mechanically activated α-catenin fragment had higher mechanical stability than a non-activated fragment. The results of our experiments using mutated and segmented fragments showed that the key intramolecular interactions acted as a conformational switch. We also found that the conformation of α-catenin was reinforced by vinculin binding. We demonstrate that α-catenin adaptively changes its conformation under tension to a stable intermediate state, binds to vinculin, and finally settles into a more stable state reinforced by vinculin binding. Our data suggest that the plastic characteristics of α-catenin, revealed in response to both mechanical and biochemical cues, enable the functional-structural dynamics at the cellular and tissue levels.
Miyuki Takeuchi, Ichirou Karahara, Naoko Kajimura, Akio Takaoka, Kazuyoshi Murata, Kazuyo Misaki, Shigenobu Yonemura, Andrew L. Staehelin and Yoshinobu Mineyuki : Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells, Molecular Biology of the Cell, 27, 11, 1809-1820, 2016.
(要約)
The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs.
H Inaba, H Goto, K Kasahara, K Kumamoto, Shigenobu Yonemura, A Inoko, S Yamano, H Wanibuchi, D He, N Goshima, T Kiyono, S Hirotsune and M Inagaki : Ndel1 suppresses ciliogenesis in proliferating cells by regulating the trichoplein-Aurora A pathway., The Journal of Cell Biology, 212, 4, 409-423, 2016.
(要約)
Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein-Aurora A pathway to inhibit primary cilia assembly.
C Ozone, H Suga, M Eiraku, T Kagoshima, Shigenobu Yonemura, N Takata, Y Oiso, T Tsuji and Y Sasai : Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells., Nature Communications, 7, 10351, 2016.
(要約)
Anterior pituitary is critical for endocrine systems. Its hormonal responses to positive and negative regulators are indispensable for homeostasis. For this reason, generating human anterior pituitary tissue that retains regulatory hormonal control in vitro is an important step for the development of cell transplantation therapy for pituitary diseases. Here we achieve this by recapitulating mouse pituitary development using human embryonic stem cells. We find that anterior pituitary self-forms in vitro following the co-induction of hypothalamic and oral ectoderm. The juxtaposition of these tissues facilitated the formation of pituitary placode, which subsequently differentiated into pituitary hormone-producing cells. They responded normally to both releasing and feedback signals. In addition, after transplantation into hypopituitary mice, the in vitro-generated corticotrophs rescued physical activity levels and survival of the hosts. Thus, we report a useful methodology for the production of regulator-responsive human pituitary tissue that may benefit future studies in regenerative medicine.
H Shirai, K Matsushita, A Kuwahara, Shigenobu Yonemura, T Nakano, J Assawachananount, T Kimura, K Saito, H Terasaki, M Eiraku, Y Sarai and Y Takahashi : Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration., Proceedings of the National Academy of Sciences of the United States of America, 113, 1, E81-E90, 2016.
(要約)
Retinal transplantation therapy for retinitis pigmentosa is increasingly of interest due to accumulating evidence of transplantation efficacy from animal studies and development of techniques for the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells into retinal tissues or cells. In this study, we aimed to assess the potential clinical utility of hESC-derived retinal tissues (hESC-retina) using newly developed primate models of retinal degeneration to obtain preparatory information regarding the potential clinical utility of these hESC-retinas in transplantation therapy. hESC-retinas were first transplanted subretinally into nude rats with or without retinal degeneration to confirm their competency as a graft to mature to form highly specified outer segment structure and to integrate after transplantation. Two focal selective photoreceptor degeneration models were then developed in monkeys by subretinal injection of cobalt chloride or 577-nm optically pumped semiconductor laser photocoagulation. The utility of the developed models and a practicality of visual acuity test developed for monkeys were evaluated. Finally, feasibility of hESC-retina transplantation was assessed in the developed monkey models under practical surgical procedure and postoperational examinations. Grafted hESC-retina was observed differentiating into a range of retinal cell types, including rod and cone photoreceptors that developed structured outer nuclear layers after transplantation. Further, immunohistochemical analyses suggested the formation of host-graft synaptic connections. The findings of this study demonstrate the clinical feasibility of hESC-retina transplantation and provide the practical tools for the optimization of transplantation strategies for future clinical applications.
Michio Yoshida, Eriko Kajikawa, Daisuke Kurokawa, Tomoyuki Tokunaga, Akira Onishi, Shigenobu Yonemura, Kensaku Kobayashi, Hiroshi Kiyonari and Shinichi Aizawa : Conserved and divergent expression patterns of markers of axial development in eutherian mammals, Developmental Dynamics, 245, 1, 67-86, 2015.
(要約)
Mouse embryos are cup shaped, but most nonrodent eutherian embryos are disk shaped. Extraembryonic ectoderm (ExEc), which may have essential roles in anterior-posterior (A-P) axis formation in mouse embryos, does not develop in many eutherian embryos. To assess A-P axis formation in eutherians, comparative analyses were made on rabbit, porcine, and Suncus embryos. All embryos examined expressed Nodal initially throughout epiblast and visceral endoderm; its expression became restricted to the posterior region before gastrulation. Anterior visceral endoderm (AVE) genes were expressed in Otx2-positive visceral endoderm, with Dkk1 expression being most anterior. The mouse pattern of AVE formation was conserved in rabbit embryos, but had diverged in porcine and Suncus embryos. No structure that was molecularly equivalent to Bmp-positive ExEc, existed in rabbit or pig embryos. In Suncus embryos, A-P axis was determined at prehatching stage, and these embryos attached to uterine wall at future posterior side. Nodal, but not Bmp, functions in epiblast and visceral endoderm development may be conserved in eutherians. AVE functions may also be conserved, but the pattern of its formation has diverged among eutherians. Roles of BMP and NODAL gradients in AVE formation seem to have been established in a subset of rodents.
K Shinohara, D Chen, T Nishida, K Misaki, Shigenobu Yonemura and H Hamada : Absence of Radial Spokes in Mouse Node Cilia Is Required for Rotational Movement but Confers Ultrastructural Instability as a Trade-Off., Developmental Cell, 35, 2, 236-246, 2015.
(要約)
Determination of left-right asymmetry in mouse embryos is established by a leftward fluid flow that is generated by clockwise rotation of node cilia. How node cilia achieve stable unidirectional rotation has remained unknown, however. Here we show that brief exposure to the microtubule-stabilizing drug paclitaxel (Taxol) induces randomly directed rotation and changes the ultrastructure of node cilia. In vivo observations and a computer simulation revealed that a regular 9+0 arrangement of doublet microtubules is essential for stable unidirectional rotation of node cilia. The 9+2 motile cilia of the airway, which manifest planar beating, are resistant to Taxol treatment. However, the airway cilia of mice lacking the radial spoke head protein Rsph4a undergo rotational movement instead of planar beating, are prone to microtubule rearrangement, and are sensitive to Taxol. Our results suggest that the absence of radial spokes allows node cilia to rotate unidirectionally but, as a trade-off, renders them ultrastructurally fragile.
(キーワード)
Animals / Body Patterning / Cilia / Embryo, Mammalian / Embryonic Development / Mice / Microtubules / Paclitaxel
M Takeuchi, S Yamaguchi, Shigenobu Yonemura, K Kakiguchi, Y Sato, T Higashiyama, T Shimizu and M Habi : Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish., PLoS Genetics, 11, e1005587, 2015.
(要約)
Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.
K Nakanishi, K Kakiguchi, Shigenobu Yonemura, A Nakano and N Morishima : Transient Ca2+ depletion from the endoplasmic reticulum is critical for skeletal myoblast differentiation., The FASEB journal, 29, 5, 2137-2149, 2015.
(要約)
Endoplasmic reticulum (ER) stress is a cellular condition in which unfolded proteins accumulate in the ER because of various but specific causes. Physiologic ER stress occurs transiently during myoblast differentiation, and although its cause remains unknown, it plays a critical role in myofiber formation. To examine the mechanism underlying ER stress, we monitored ER morphology during differentiation of murine myoblasts. Novel ER-derived structures transiently appeared prior to myoblast fusion both in vitro and in vivo. Electron microscopy studies revealed that these structures consisted of pseudoconcentric ER cisternae with narrow lumens. Similar structures specifically formed by pharmacologically induced ER Ca(2+) depletion, and inhibition of ER Ca(2+) efflux channels in differentiating myoblasts considerably suppressed ER-specific deformation and ER stress signaling. Thus, we named the novel structures stress-activated response to Ca(2+) depletion (SARC) bodies. Prior to SARC body formation, stromal interaction molecule 1 (STIM1), an ER Ca(2+) sensor protein, formed ER Ca(2+) depletion-specific clusters. Furthermore, myoblast differentiation manifested by myoblast fusion did not proceed under the same conditions as inhibition of ER Ca(2+) depletion. Altogether, these observations suggest that ER Ca(2+) depletion is a prerequisite for myoblast fusion, causing both physiologic ER stress signaling and SARC body formation.
H Nagai, M Sezaki, K Kakiguchi, Y Nakaya, HC Lee, R Lather, T Sasanami, JK Han, Shigenobu Yonemura and G Sheng : Cellular analysis of cleavage-stage chick embryos reveals hidden conservation in vertebrate early development., Development, 142, 7, 1279-1286, 2015.
(要約)
Birds and mammals, phylogenetically close amniotes with similar post-gastrula development, exhibit little conservation in their post-fertilization cleavage patterns. Data from the mouse suggest that cellular morphogenesis and molecular signaling at the cleavage stage play important roles in lineage specification at later (blastula and gastrula) stages. Very little is known, however, about cleavage-stage chick embryos, owing to their poor accessibility. This period of chick development takes place before egg-laying and encompasses several fundamental processes of avian embryology, including zygotic gene activation (ZGA) and blastoderm cell-layer increase. We have carried out morphological and cellular analyses of cleavage-stage chick embryos covering the first half of pre-ovipositional development, from Eyal-Giladi and Kochav stage (EGK-) I to EGK-V. Scanning electron microscopy revealed remarkable subcellular details of blastomere cellularization and subgerminal cavity formation. Phosphorylated RNA polymerase II immunostaining showed that ZGA in the chick starts at early EGK-III during the 7th to 8th nuclear division cycle, comparable with the time reported for other yolk-rich vertebrates (e.g. zebrafish and Xenopus). The increase in the number of cell layers after EGK-III is not a direct consequence of oriented cell division. Finally, we present evidence that, as in the zebrafish embryo, a yolk syncytial layer is formed in the avian embryo after EGK-V. Our data suggest that several fundamental features of cleavage-stage development in birds resemble those in yolk-rich anamniote species, revealing conservation in vertebrate early development. Whether this conservation lends morphogenetic support to the anamniote-to-amniote transition in evolution or reflects developmental plasticity in convergent evolution awaits further investigation.
Shigenobu Yonemura : Differential sensitivity of epithelial cells to extracellular matrix in polarity establishment, Plos One, 9, 11, e112922, 2014.
(要約)
Establishment of apical-basal polarity is crucial for epithelial sheets that form a compartment in the body, which function to maintain the environment in the compartment. Effects of impaired polarization are easily observed in three-dimensional (3-D) culture systems rather than in two-dimensional (2-D) culture systems. Although the mechanisms for establishing the polarity are not completely understood, signals from the extracellular matrix (ECM) are considered to be essential for determining the basal side and eventually generating polarity in the epithelial cells. To elucidate the common features and differences in polarity establishment among various epithelial cells, we analyzed the formation of epithelial apical-basal polarity using three cell lines of different origin: MDCK II cells (dog renal tubules), EpH4 cells (mouse mammary gland), and R2/7 cells (human colon) expressing wild-type α-catenin (R2/7 α-Cate cells). These cells showed clear apical-basal polarity in 2-D cultures. In 3-D cultures, however, each cell line displayed different responses to the same ECM. In MDCK II cells, spheroids with a single lumen formed in both Matrigel and collagen gel. In R2/7 α-Cate cells, spheroids showed similar apical-basal polarity as that seen in MDCK II cells, but had multiple lumens. In EpH4 cells, the spheroids displayed an apical-basal polarity that was opposite to that seen in the other two cell types in both ECM gels, at least during the culture period. On the other hand, the three cell lines showed the same apical-basal polarity both in 2-D cultures and in 3-D cultures using the hanging drop method. The three lines also had similar cellular responses to ECM secreted by the cells themselves. Therefore, appropriate culture conditions should be carefully determined in advance when using various epithelial cells to analyze cell polarity or 3-D morphogenesis.
Yoshinori Sato, Kenji Hayashi, Yoshiko Amano, Mikiko Takahashi, Shigenobu Yonemura, Ikuko Hayashi, Hiroko Hirose, Shigeo Ohno and Atsushi Suzuki : MTCL1 crosslinks and stabilizes non-centrosomal microtubules on the Golgi membrane, Nature Communications, 5, 5266, 2014.
(要約)
Recent studies have revealed the presence of a microtubule subpopulation called Golgi-derived microtubules that support Golgi ribbon formation, which is required for maintaining polarized cell migration. CLASPs and AKAP450/CG-NAP are involved in their formation, but the underlying molecular mechanisms remain unclear. Here, we find that the microtubule-crosslinking protein, MTCL1, is recruited to the Golgi membranes through interactions with CLASPs and AKAP450/CG-NAP, and promotes microtubule growth from the Golgi membrane. Correspondingly, MTCL1 knockdown specifically impairs the formation of the stable perinuclear microtubule network to which the Golgi ribbon tethers and extends. Rescue experiments demonstrate that besides its crosslinking activity mediated by the N-terminal microtubule-binding region, the C-terminal microtubule-binding region plays essential roles in these MTCL1 functions through a novel microtubule-stabilizing activity. These results suggest that MTCL1 cooperates with CLASPs and AKAP450/CG-NAP in the formation of the Golgi-derived microtubules, and mediates their development into a stable microtubule network.
Yoshikazu Tsukasaki, Naoyuki Miyazaki, Atsushi Matsumoto, Shigenori Nagae, Shigenobu Yonemura, Takuji Tanoue, Kenji Iwasaki and Masatoshi Takeichi : Giant cadherins Fat and Dachsous self-bend to organize properly spaced intercellular junctions, Proceedings of the National Academy of Sciences of the United States of America, 111, 45, 16011-16016, 2014.
(要約)
The cadherins Fat and Dachsous regulate cell polarity and proliferation via their heterophilic interactions at intercellular junctions. Their ectodomains are unusually large because of repetitive extracellular cadherin (EC) domains, which raises the question of how they fit in regular intercellular spaces. Cadherins typically exhibit a linear topology through the binding of Ca(2+) to the linker between the EC domains. Our electron-microscopic observations of mammalian Fat4 and Dachsous1 ectodomains, however, revealed that, although their N-terminal regions exhibit a linear configuration, the C-terminal regions are kinked with multiple hairpin-like bends. Notably, certain EC-EC linkers in Fat4 and Dachsous1 lost Ca(2+)-binding amino acids. When such non-Ca(2+)-binding linkers were substituted for a normal linker in E-cadherin, the mutant E-cadherins deformed more extensively than the wild-type molecule. To simulate cadherin structures with non-Ca(2+)-binding linkers, we used an elastic network model and confirmed that bent configurations can be generated by deformation of non-Ca(2+)-binding linkers. These findings suggest that Fat and Dachsous self-bend due to the loss of Ca(2+)-binding amino acids from specific EC-EC linkers, and can therefore adapt to confined spaces.
Kousuke Kasahara, Yoshitaka Kawakami, Tohru Kiyono, Shigenobu Yonemura, Yoshifumi Kawamura, Saho Era, Fumio Matsuzaki, Naoki Goshima and Masaki Inagaki : Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension, Nature Communications, 5, 5081, 2014.
(要約)
Primary cilia are microtubule-based sensory organelles that organize numerous key signals during developments and tissue homeostasis. Ciliary microtubule doublet, named axoneme, is grown directly from the distal end of mother centrioles through a multistep process upon cell cycle exit; however, the instructive signals that initiate these events are poorly understood. Here we show that ubiquitin-proteasome machinery removes trichoplein, a negative regulator of ciliogenesis, from mother centrioles and thereby causes Aurora-A inactivation, leading to ciliogenesis. Ciliogenesis is blocked if centriolar trichoplein is stabilized by treatment with proteasome inhibitors or by expression of non-ubiquitylatable trichoplein mutant (K50/57R). Started from two-stepped global E3 screening, we have identified KCTD17 as a substrate-adaptor for Cul3-RING E3 ligases (CRL3s) that polyubiquitylates trichoplein. Depletion of KCTD17 specifically arrests ciliogenesis at the initial step of axoneme extension through aberrant trichoplein-Aurora-A activity. Thus, CRL3-KCTD17 targets trichoplein to proteolysis to initiate the axoneme extension during ciliogenesis.
Xiaorei Sai, Shigenobu Yonemura and K. Raj Ladher : Junctionally restricted RhoA activity is necessary for apical constriction during phase 2 inner ear placode invagination, Developmental Biology, 394, 2, 206-216, 2014.
(要約)
After induction, the inner ear is transformed from a superficially located otic placode into an epithelial vesicle embedded in the mesenchyme of the head. Invagination of this epithelium is biphasic: phase 1 involves the expansion of the basal aspect of the otic cells, and phase 2, the constriction of their apices. Apical constriction is important not only for otic invagination, but also the invagination of many other epithelia; however, its molecular basis is still poorly understood. Here we show that phase 2 otic morphogenesis, like phase 1 morphogenesis, results from the activation of myosin-II. However unlike the actin depolymerising activity observed basally, active myosin-II results in actomyosin contractility. Myosin-II activation is triggered by the accumulation of the planar cell polarity (PCP) core protein, Celsr1 in apical junctions (AJ). Apically polarized Celsr1 orients and recruits the Rho Guanine exchange factor (GEF) ArhGEF11 to apical junctions, thus restricting RhoA activity to the junctional membrane where it activates the Rho kinase ROCK. We suggest that myosin-II and RhoA activation results in actomyosin dependent constriction in an apically polarised manner driving otic epithelium invagination.
Juthaporn Assawachananont, Michiko Mandai, Satoshi Okamoto, Chikako Yamada, Mototsugu Eiraku, Shigenobu Yonemura, Yoshiki Sasai and Masayo Takahashi : Transplantation of embryonic and induced pluripotent stem cell-derived 3D retinal sheets into retinal degenerative mice, Stem Cell Reports, 2, 5, 662-674, 2014.
(要約)
In this article, we show that mouse embryonic stem cell- or induced pluripotent stem cell-derived 3D retinal tissue developed a structured outer nuclear layer (ONL) with complete inner and outer segments even in an advanced retinal degeneration model (rd1) that lacked ONL. We also observed host-graft synaptic connections by immunohistochemistry. This study provides a "proof of concept" for retinal sheet transplantation therapy for advanced retinal degenerative diseases.
Takehiko Ueyama, Hirofumi Sakaguchi, Takashi Nakamura, Akihiro Goto, Shigefumi Morioka, Aya Shimizu, Kazuki Nakao, Yoshitaka Hishikawa, Yuzuru Ninoyu, Hidetoshi Kassai, Shiro Suetsugu, Takehiko Koji, Bernd Fritzsch, Shigenobu Yonemura, Yasuo Hisa, Michiyuki Matsuda, Atsu Aiba and Naoaki Saito : Maintenance of stereocilia and apical junctional complexes by Cdc42 in cochlear hair cells, Journal of Cell Science, 127, 9, 2040-2052, 2014.
(要約)
Cdc42 is a key regulator of dynamic actin organization. However, little is known about how Cdc42-dependent actin regulation influences steady-state actin structures in differentiated epithelia. We employed inner ear hair-cell-specific conditional knockout to analyze the role of Cdc42 in hair cells possessing highly elaborate stable actin protrusions (stereocilia). Hair cells of Atoh1-Cre;Cdc42(flox/flox) mice developed normally but progressively degenerated after maturation, resulting in progressive hearing loss particularly at high frequencies. Cochlear hair cell degeneration was more robust in inner hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP-Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells from transgenic mice expressing a Cdc42-FRET biosensor indicated Cdc42 presence and activation at stereociliary membranes and AJCs in cochlear hair cells. Cdc42-knockdown in MDCK cells produced phenotypes similar to those of Cdc42-deleted hair cells, including abnormal microvilli and disrupted AJCs, and downregulated actin turnover represented by enhanced levels of phosphorylated cofilin. Thus, Cdc42 influenced the maintenance of stable actin structures through elaborate tuning of actin turnover, and maintained function and viability of cochlear hair cells.
Kinya Yasui, Takao Kaji, R. Arseniy Morov and Shigenobu Yonemura : Development of oral and branchial muscles in lancelet larvae of Branchiostoma japonicum, Journal of Morphology, 275, 4, 465-477, 2013.
(要約)
The perforated pharynx has generally been regarded as a shared characteristic of chordates. However, there still remains phylogenetic ambiguity between the cilia-driven system in invertebrate chordates and the muscle-driven system in vertebrates. Giant larvae of the genus Asymmetron were reported to develop an orobranchial musculature similar to that of vertebrates more than 100 years ago. This discovery might represent an evolutionary link for the chordate branchial system, but few investigations of the lancelet orobranchial musculature have been completed since. We studied staged larvae of a Japanese population of Branchiostoma japonicum to characterize the developmental property of the orobranchial musculature. The larval mouth and the unpaired primary gills develop well-organized muscles. These muscles function only as obturators of the openings without antagonistic system. As the larval mouth enlarged posteriorly to the level of the ninth myomere, the oral musculature was fortified accordingly without segmental patterning. In contrast, the iterated branchial muscles coincided with the dorsal myomeric pattern before metamorphosis, but the pharynx was remodeled dynamically irrespective of the myomeric pattern during metamorphosis. The orobranchial musculature disappeared completely during metamorphosis, and adult muscles in the oral hood and velum, as well as on the pterygial coeloms developed independently. The lancelet orobranchial musculature is apparently a larval adaptation to prevent harmful intake. However, vestigial muscles appeared transiently with the secondary gill formation suggest a bilateral ancestral state of muscular gills, and a segmental pattern of developing branchial muscles without neural crest and placodal contributions is suggestive of a precursor of vertebrate branchiomeric pattern.
Makoto Matsuyama, Hiroki Tanaka, Akihito Inoko, Hidemasa Goto, Shigenobu Yonemura, Kyoko Kobori, Yuko Hayashi, Eisaku Kondo, Shigeyoshi Itohara, Ichiro Izawa and Masaki Inagaki : Defect of mitotic vimentin phosphorylation causes microophthalmia and cataract via aneuploidy and senescence in lens epithelial cells, Journal of Biological Chemistry, 288, 50, 35626-35635, 2013.
(要約)
Vimentin, a type III intermediate filament (IF) protein, is phosphorylated predominantly in mitosis. The expression of a phosphorylation-compromised vimentin mutant in T24 cultured cells leads to cytokinetic failure, resulting in binucleation (multinucleation). The physiological significance of intermediate filament phosphorylation during mitosis for organogenesis and tissue homeostasis was uncertain. Here, we generated knock-in mice expressing vimentin that have had the serine sites phosphorylated during mitosis substituted by alanine residues. Homozygotic mice (VIM(SA/SA)) presented with microophthalmia and cataracts in the lens, whereas heterozygotic mice (VIM(WT/SA)) were indistinguishable from WT (VIM(WT/WT)) mice. In VIM(SA/SA) mice, lens epithelial cell number was not only reduced but the cells also exhibited chromosomal instability, including binucleation and aneuploidy. Electron microscopy revealed fiber membranes that were disorganized in the lenses of VIM(SA/SA), reminiscent of similar characteristic changes seen in age-related cataracts. Because the mRNA level of the senescence (aging)-related gene was significantly elevated in samples from VIM(SA/SA), the lens phenotype suggests a possible causal relationship between chromosomal instability and premature aging.
Junichi Ikenouchi, Megumi Hirata, Shigenobu Yonemura and Masato Umeda : Sphingomyelin clustering is essential for the formation of microvilli, Journal of Cell Science, 126, 16, 3585-3592, 2013.
(要約)
Cellular architectures require regulated mechanisms to correctly localize the appropriate plasma membrane lipids and proteins. Microvilli are dynamic filamentous-actin-based protrusions of the plasma membrane that are found in the apical membrane of epithelial cells. However, it remains poorly understood how their formation is regulated. In the present study, we found that sphingomyelin clustering underlies the formation of microvilli. Clustering of sphingomyelin is required for the co-clustering of the sialomucin membrane protein podocalyxin-1 at microvilli. Podocalyxin-1 recruits ezrin/radixin/moesin (ERM)-binding phosphoprotein-50 (EBP50; also known as NHERF1), which recruits ERM proteins and phosphatidylinositol 4-phosphate 5-kinase β (PIP5Kβ). Thus, clustering of PIP5Kβ leads to local accumulation of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], which enhances the accumulation of ERM family proteins and induces the formation of microvilli. The present study revealed novel interactions between sphingomyelin and the cytoskeletal proteins from which microvilli are formed, and it clarified the physiological importance of the chemical properties of sphingomyelin that facilitate cluster formation.
Seham Ebrahim, Tomoki Fujita, A. Bryan Millis, Elliott Kozin, Xuefei Ma, Sachiyo Kawamoto, A. Michelle Baird, Michael Davidson, Shigenobu Yonemura, Yasuo Hisa, Anne Mary Conti, S. Robert Adelstein, Hirofumi Sakaguchi and Bechara Kachar : NMII forms a contractile transcellular sarcomeric network to regulate apical cell junctions and tissue geometry, Current Biology, 23, 8, 731-736, 2013.
(要約)
Nonmuscle myosin II (NMII) is thought to be the master integrator of force within epithelial apical junctions, mediating epithelial tissue morphogenesis and tensional homeostasis. Mutations in NMII are associated with a number of diseases due to failures in cell-cell adhesion. However, the organization and the precise mechanism by which NMII generates and responds to tension along the intercellular junctional line are still not known. We discovered that periodic assemblies of bipolar NMII filaments interlace with perijunctional actin and α-actinin to form a continuous belt of muscle-like sarcomeric units (∼400-600 nm) around each epithelial cell. Remarkably, the sarcomeres of adjacent cells are precisely paired across the junctional line, forming an integrated, transcellular contractile network. The contraction/relaxation of paired sarcomeres concomitantly impacts changes in apical cell shape and tissue geometry. We show differential distribution of NMII isoforms across heterotypic junctions and evidence for compensation between isoforms. Our results provide a model for how NMII force generation is effected along the junctional perimeter of each cell and communicated across neighboring cells in the epithelial organization. The sarcomeric network also provides a well-defined target to investigate the multiple roles of NMII in junctional homeostasis as well as in development and disease.
(キーワード)
Actin Cytoskeleton / Actinin / Actins / Animals / Heterocyclic Compounds, 4 or More Rings / Intercellular Junctions / Mice / Microscopy, Fluorescence / Myosin Heavy Chains / Myosin Type II / Organ of Corti / Rats
Asuka Takeishi, Erina Kuranaga, Ayako Tonoki, Kazuyo Misaki, Shigenobu Yonemura, Hirotaka Kanuka and Masayuki Miura : Homeostatic Epithelial Renewal in the Gut Is Required for Dampening a Fatal Systemic Wound Response in Drosophila, Cell Reports, 3, 3, 919-930, 2013.
(要約)
Effective defense responses involve the entire organism. To maintain body homeostasis after tissue damage, a systemic wound response is induced in which the response of each tissue is tightly orchestrated to avoid incomplete recovery or an excessive, damaging response. Here, we provide evidence that in the systemic response to wounding, an apoptotic caspase pathway is activated downstream of reactive oxygen species in the midgut enterocytes (ECs), cells distant from the wound site, in Drosophila. We show that a caspase-pathway mutant has defects in homeostatic gut cell renewal and that inhibiting caspase activity in fly ECs results in the production of systemic lethal factors after wounding. Our results indicate that wounding remotely controls caspase activity in ECs, which activates the tissue stem cell regeneration pathway in the gut to dampen the dangerous systemic wound reaction.
S. Vassil Vassilev, Michiko Mandai, Shigenobu Yonemura and Masatoshi Takeichi : Loss of N-Cadherin from the endothelium causes stromal edema and epithelial dysgenesis in the mouse cornea, Investigative Ophthalmology and Visual Science, 53, 11, 7183-7193, 2012.
(要約)
We analyzed the role of N-cadherin in maintaining proper architecture and function of corneal endothelium. To achieve specific removal of N-cadherin from corneal endothelium, we bred mice carrying a floxed N-cadherin gene with those expressing the Cre-recombinase gene under the control of P0 promoter. The corneal structure was analyzed by immunostaining for cell junction proteins as well as by electron microscopy. The apoptotic status was assessed by TUNEL staining. The permeability of corneal endothelium was evaluated using fluorescein dye. Removal of endothelial N-cadherin led to the appearance of opacity in the adult corneas. All corneal layers exhibited histological defects: The apical junctional complex (AJC) in corneal endothelium was disorganized, losing the continuity in tight junctions. Collagen fibrils were rearranged in the stroma. The corneal epithelium showed decreased thickness and TUNEL staining revealed increased central areas of apoptosis. Fluorescein dye injection in the anterior chamber confirmed an increased permeability of the endothelial layer. Developmental analysis indicated that, although N-cadherin was lost during embryonic stages, the AJC was maintained normally until early postnatal stages, probably due to the presence of other cadherins at these developmental stages. The junctional defects in endothelial cells, however, became obvious by postnatal day 21 (P21), although stromal and epithelial phenotypes were clearly detectable only in the adult eyes. N-cadherin is essential for maintaining proper structure of corneal endothelial AJCs from late postnatal to adult stages. Its ablation leads to increased endothelial permeability and corneal edema in mature eyes.
Masatsune Tsujioka, Shigehiko Yumura, Kei Inouye, Hitesh Patel, Masahiro Ueda and Shigenobu Yonemura : Talin couples the actomyosin cortex to the plasma membrane during rear retraction and cytokinesis, Proceedings of the National Academy of Sciences of the United States of America, 109, 32, 12992-12997, 2012.
(要約)
Contraction of the cortical actin cytoskeleton underlies both rear retraction in directed cell migration and cytokinesis. Here, we show that talin, a central component of focal adhesions, has a major role in these processes. We found that Dictyostelium talin A colocalized with myosin II in the rear of migrating cells and the cleavage furrow. During directed cell migration, talin A-null cells displayed a long thin tail devoid of actin filaments, whereas additional depletion of SibA, a transmembrane adhesion molecule that binds to talin A, reverted this phenotype, suggesting a requirement of the link between actomyosin and SibA by talin A for rear retraction. Disruptions of talin A also resulted in detachment of the actomyosin contractile ring from the cell membrane and concomitant regression of the cleavage furrow under certain conditions. The C-terminal actin-binding domain (ABD) of talin A exhibited a localization pattern identical to that of full-length talin A. The N-terminal FERM domain was found to bind phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] in vitro. In vivo, however, PtdIns(4,5)P2, which is known to activate talin, is believed to be enriched in the rear of migrating cells and the cleavage furrow in Dictyostelium. From these results, we propose that talin A activated by PtdIns(4,5)P2 in the cell posterior or cleavage furrow links actomyosin cytoskeleton to adhesion molecules or other membrane proteins, and that the force is transmitted through these links to retract the tail during cell migration or to cause efficient ingression of the equator during cytokinesis.
(キーワード)
Actomyosin / Blotting, Western / Cell Membrane / Cell Movement / Cytokinesis / Dictyostelium / Immunoprecipitation / Microscopy, Fluorescence / Microscopy, Phase-Contrast / Myosin Type II / Phosphatidylinositol 4,5-Diphosphate / Phosphatidylinositol Phosphates / Talin / Time-Lapse Imaging
Tokushige Nakano, Satoshi Ando, Nozomu Takata, Masako Kawada, Keiko Muguruma, Kiyotoshi Sekiguchi, Koichi Saito, Shigenobu Yonemura, Mototsugu Eiraku and Yoshiki Sasai : Self-formation of optic cups and storable stratified neural retina from human ESCs, Cell Stem Cell, 10, 6, 771-785, 2012.
(要約)
In this report, we demonstrate that an optic cup structure can form by self-organization in human ESC culture. The human ESC-derived optic cup is much larger than the mouse ESC-derived one, presumably reflecting the species differences. The neural retina in human ESC culture is thick and spontaneously curves in an apically convex manner, which is not seen in mouse ESC culture. In addition, human ESC-derived neural retina grows into multilayered tissue containing both rods and cones, whereas cone differentiation is rare in mouse ESC culture. The accumulation of photoreceptors in human ESC culture can be greatly accelerated by Notch inhibition. In addition, we show that an optimized vitrification method enables en bloc cryopreservation of stratified neural retina of human origin. This storage method at an intermediate step during the time-consuming differentiation process provides a versatile solution for quality control in large-scale preparation of clinical-grade retinal tissues.
Akihito Inoko, Makoto Matsuyama, Hidemasa Goto, Yuki Ohmuro-Matsuyama, Yuko Hayashi, Masato Enomoto, Miho Ibi, Takeshi Urano, Shigenobu Yonemura, Tohru Kiyono, Ichiro Izawa and Masaki Inagaki : Trichoplein and Aurora A block aberrant primary cilia assembly in proliferating cells, Journal of Cell Biology, 197, 3, 391-405, 2012.
(要約)
The primary cilium is an antenna-like organelle that modulates differentiation, sensory functions, and signal transduction. After cilia are disassembled at the G0/G1 transition, formation of cilia is strictly inhibited in proliferating cells. However, the mechanisms of this inhibition are unknown. In this paper, we show that trichoplein disappeared from the basal body in quiescent cells, whereas it localized to mother and daughter centrioles in proliferating cells. Exogenous expression of trichoplein inhibited primary cilia assembly in serum-starved cells, whereas ribonucleic acid interference-mediated depletion induced primary cilia assembly upon cultivation with serum. Trichoplein controlled Aurora A (AurA) activation at the centrioles predominantly in G1 phase. In vitro analyses confirmed that trichoplein bound and activated AurA directly. Using trichoplein mutants, we demonstrate that the suppression of primary cilia assembly by trichoplein required its ability not only to localize to centrioles but also to bind and activate AurA. Trichoplein or AurA knockdown also induced G0/G1 arrest, but this phenotype was reversed when cilia formation was prevented by simultaneous knockdown of IFT-20. These data suggest that the trichoplein-AurA pathway is required for G1 progression through a key role in the continuous suppression of primary cilia assembly.
Yuichi Yanagihashi, Tadao Usui, Yasushi Izumi, Shigenobu Yonemura, Motoyuki Sumida, Shoichiro Tsukita, Tadashi Uemura and Mikio Furuse : Snakeskin, a membrane protein associated with smooth septate junctions, is required for intestinal barrier function in Drosophila, Journal of Cell Science, 125, 8, 1980-1990, 2012.
(要約)
Septate junctions (SJs) are the membrane specializations observed between epithelial cells in invertebrates. SJs play a crucial role in epithelial barrier function by restricting the free diffusion of solutes through the intercellular space. In arthropod species, two morphologically different types of SJs have been described: pleated septate junctions (pSJs) and smooth septate junctions (sSJs), which are specific to ectodermal and endodermal epithelia, respectively. In contrast to the recent identification of pSJ-related proteins, the molecular constituents of sSJs are mostly unknown. Here, we report the discovery of a new sSJ-specific membrane protein, designated 'Snakeskin' (Ssk). Ssk is highly concentrated in sSJs in the Drosophila midgut and Malpighian tubules. Lack of Ssk expression is embryonically lethal in Drosophila and results in defective sSJ formation accompanied by abnormal morphology of midgut epithelial cells. We also show that the barrier function of the midgut to a fluorescent tracer is impaired in ssk-knockdown larvae. These results suggest that Ssk is required for the intestinal barrier function in Drosophila.
Yuuki Obata, Daisuke Takahashi, Masashi Ebisawa, Kisa Kakiguchi, Shigenobu Yonemura, Toshi Jinnohara, Takashi Kanaya, Yumiko Fujimura, Masumi Ohmae, Koji Hase and Hiroshi Ohno : Epithelial cell-intrinsic Notch signaling plays an essential role in the maintenance of gut immune homeostasis, Journal of Immunology, 188, 5, 2427-2436, 2012.
(要約)
Intestinal epithelial cells (IECs) have important functions as the first line of defense against diverse microorganisms on the luminal surface. Impaired integrity of IEC has been implicated in increasing the risk for inflammatory disorders in the gut. Notch signaling plays a critical role in the maintenance of epithelial integrity by regulating the balance of secretory and absorptive cell lineages, and also by facilitating epithelial cell proliferation. We show in this article that mice harboring IEC-specific deletion of Rbpj (RBP-J(ΔIEC)), a transcription factor that mediates signaling through Notch receptors, spontaneously develop chronic colitis characterized by the accumulation of Th17 cells in colonic lamina propria. Intestinal bacteria are responsible for the development of colitis, because their depletion with antibiotics prevented the development of colitis in RBP-J(ΔIEC) mice. Furthermore, bacterial translocation was evident in the colonic mucosa of RBP-J(ΔIEC) mice before the onset of colitis, suggesting attenuated epithelial barrier functions in these mice. Indeed, RBP-J(ΔIEC) mice displayed increase in intestinal permeability after rectal administration of FITC-dextran. In addition to the defect in physical barrier, loss of Notch signaling led to arrest of epithelial cell turnover caused by downregulation of Hes1, a transcriptional repressor of p27(Kip1) and p57(Kip2). Thus, epithelial cell-intrinsic Notch signaling ensures integrity and homeostasis of IEC, and this mechanism is required for containment of intestinal inflammation.
Hidetaka Suga, Taisuke Kadoshima, Maki Minaguchi, Masatoshi Ohgushi, Mika Soen, Tokushige Nakano, Nozomu Takata, Takafumi Wataya, Keiko Muguruma, Hiroyuki Miyoshi, Shigenobu Yonemura, Yutaka Oiso and Yoshiki Sasai : Self-formation of functional adenohypophysis in three-dimensional culture, Nature, 480, 7375, 57-62, 2011.
(要約)
The adenohypophysis (anterior pituitary) is a major centre for systemic hormones. At present, no efficient stem-cell culture for its generation is available, partly because of insufficient knowledge about how the pituitary primordium (Rathke's pouch) is induced in the embryonic head ectoderm. Here we report efficient self-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mouse embryonic stem (ES) cells. ES cells were stimulated to differentiate into non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers within the aggregate, and treated with hedgehog signalling. Self-organization of Rathke's-pouch-like three-dimensional structures occurred at the interface of these two epithelia, as seen in vivo, and various endocrine cells including corticotrophs and somatotrophs were subsequently produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone and, when grafted in vivo, these cells rescued the systemic glucocorticoid level in hypopituitary mice. Thus, functional anterior pituitary tissue self-forms in ES cell culture, recapitulating local tissue interactions.
Reiko Tajiri, Kazuyo Misaki, Shigenobu Yonemura and Shigeo Hayashi : Joint morphology in the insect leg: Evolutionary history inferred from Notch loss-of-function phenotypes in Drosophila, Development, 138, 21, 4621-4626, 2011.
(要約)
Joints permit efficient locomotion, especially among animals with a rigid skeleton. Joint morphologies vary in the body of individual animals, and the shapes of homologous joints often differ across species. The diverse locomotive behaviors of animals are based, in part, on the developmental and evolutionary history of joint morphogenesis. We showed previously that strictly coordinated cell-differentiation and cell-movement events within the epidermis sculpt the interlocking ball-and-socket joints in the adult Drosophila tarsus (distal leg). Here, we show that the tarsal joints of various insect species can be classified into three types: ball-and-socket, side-by-side and uniform. The last two probably result from joint formation without the cell-differentiation step, the cell-movement step, or both. Similar morphological variations were observed in Drosophila legs when Notch function was temporarily blocked during joint formation, implying that the independent acquisition of cell differentiation and cell movement underlay the elaboration of tarsal joint morphologies during insect evolution. These results provide a framework for understanding how the seemingly complex morphology of the interlocking joint could have developed during evolution by the addition of simple developmental modules: cell differentiation and cell movement.
Ichi Ken Wada, Kazuyoshi Itoga, Teruo Okano, Shigenobu Yonemura and Hiroshi Sasaki : Hippo pathway regulation by cell morphology and stress fibers, Development, 138, 18, 3907-3914, 2011.
(要約)
The Hippo signaling pathway plays an important role in regulation of cell proliferation. Cell density regulates the Hippo pathway in cultured cells; however, the mechanism by which cells detect density remains unclear. In this study, we demonstrated that changes in cell morphology are a key factor. Morphological manipulation of single cells without cell-cell contact resulted in flat spread or round compact cells with nuclear or cytoplasmic Yap, respectively. Stress fibers increased in response to expanded cell areas, and F-actin regulated Yap downstream of cell morphology. Cell morphology- and F-actin-regulated phosphorylation of Yap, and the effects of F-actin were suppressed by modulation of Lats. Our results suggest that cell morphology is an important factor in the regulation of the Hippo pathway, which is mediated by stress fibers consisting of F-actin acting upstream of, or on Lats, and that cells can detect density through their resulting morphology. This cell morphology (stress-fiber)-mediated mechanism probably cooperates with a cell-cell contact (adhesion)-mediated mechanism involving the Hippo pathway to achieve density-dependent control of cell proliferation.
Miki Tanaka-Okamoto, Keiko Hori, Hiroyoshi Ishizaki, Yu Itoh, Sachiko Onishi, Shigenobu Yonemura, Yoshimi Takai and Jun Miyoshi : Involvement of afadin in barrier function and homeostasis of mouse intestinal epithelia, Journal of Cell Science, 124, 13, 2231-2240, 2011.
(要約)
Afadin interacts with the cytoplasmic region of nectins, which are immunoglobulin-like cell adhesion molecules at adherens junctions, and links them to the actin cytoskeleton. Afadin regulates activities of cells in culture such as directional motility, proliferation and survival. We used Cre-loxP technology to generate mice conditionally lacking afadin specifically in the intestinal epithelia after birth. The loss of afadin caused increased paracellular permeability in the intestinal mucosa and enhanced susceptibility to the tissue destruction induced by dextran sulfate sodium. The junctional architecture of the intestinal epithelia appeared to be preserved, whereas the deficiency of afadin caused the mislocalization of nectin-2 and nectin-3 from adherens junctions to basolateral membrane domains but not that of other components of apical junctions. By contrast, such phenotypic changes were undetected in mice lacking nectin-2, nectin-3 or both. These findings suggest that afadin plays crucial roles, independently of the role as the nectin-afadin module, in barrier function and homeostasis of the intestinal epithelia once the epithelial structure has been established.
Leticia Sansores-Garcia, Wouter Bossuyt, Ichi Ken Wada, Shigenobu Yonemura, Chunyao Tao, Hiroshi Sasaki and Georg Halder : Modulating F-actin organization induces organ growth by affecting the Hippo pathway, EMBO Journal, 30, 12, 2325-2335, 2011.
(要約)
The Hippo tumour suppressor pathway is a conserved signalling pathway that controls organ size. The core of the Hpo pathway is a kinase cascade, which in Drosophila involves the Hpo and Warts kinases that negatively regulate the activity of the transcriptional coactivator Yorkie. Although several additional components of the Hippo pathway have been discovered, the inputs that regulate Hippo signalling are not fully understood. Here, we report that induction of extra F-actin formation, by loss of Capping proteins A or B, or caused by overexpression of an activated version of the formin Diaphanous, induced strong overgrowth in Drosophila imaginal discs through modulating the activity of the Hippo pathway. Importantly, loss of Capping proteins and Diaphanous overexpression did not significantly affect cell polarity and other signalling pathways, including Hedgehog and Decapentaplegic signalling. The interaction between F-actin and Hpo signalling is evolutionarily conserved, as the activity of the mammalian Yorkie-orthologue Yap is modulated by changes in F-actin. Thus, regulators of F-actin, and in particular Capping proteins, are essential for proper growth control by affecting Hippo signalling.
IKK-related kinases are key regulators of innate immunity and oncogenesis. While their effects on transcription are well characterized, their cytoplasmic functions remain poorly understood. Drosophila IKK-related kinase, IKKϵ, regulates cytoskeletal organization and cell elongation. Here, we demonstrate that IKKϵ is activated locally at the tip of growing mechanosensory bristles and regulates the rapid shuttling of recycling endosomes, independent of its roles in F-actin organization and caspase signaling. IKKϵ regulates the localization of recycling endosome regulators Rab11 and Dynein and phosphorylates their adaptor molecule, Nuclear fallout (Nuf). Nuf's negative regulation by IKKϵ suggests that local activation of IKKϵ inhibits Dynein on incoming recycling endosomes, converting them for outward transport. Mammalian IKK-related kinases also regulate the recycling endosomes' distribution by phosphorylating the Nuf homolog Rab11-FIP3. Our results establish an evolutionarily conserved function of IKK-related kinases in regulating recycling endosome dynamics and point to a key role of endosome dynamics in cell morphogenesis.
Mai Yamamoto, Ryoko Morita, Takamasa Mizoguchi, Hiromi Matsuo, Miho Isoda, Tohru Ishitani, B. Ajay Chitnis, Kunihiro Matsumoto, Gage J. Crump, Katsuto Hozumi, Shigenobu Yonemura, Koichi Kawakami and Motoyuki Itoh : Mib-Jag1-Notch signalling regulates patterning and structural roles of the notochord by controlling cell-fate decisions, Development, 137, 15, 2527-2537, 2010.
(要約)
In the developing embryo, cell-cell signalling is necessary for tissue patterning and structural organization. During midline development, the notochord plays roles in the patterning of its surrounding tissues while forming the axial structure; however, how these patterning and structural roles are coordinated remains elusive. Here, we identify a mechanism by which Notch signalling regulates the patterning activities and structural integrity of the notochord. We found that Mind bomb (Mib) ubiquitylates Jagged 1 (Jag1) and is essential in the signal-emitting cells for Jag1 to activate Notch signalling. In zebrafish, loss- and gain-of-function analyses showed that Mib-Jag1-Notch signalling favours the development of non-vacuolated cells at the expense of vacuolated cells in the notochord. This leads to changes in the peri-notochordal basement membrane formation and patterning surrounding the muscle pioneer cells. These data reveal a previously unrecognized mechanism regulating the patterning and structural roles of the notochord by Mib-Jag1-Notch signalling-mediated cell-fate determination.
Atsuo Iida, Kazuya Sakaguchi, Kiyoaki Sato, Hidetoshi Sakurai, Daigo Nishimura, Aya Iwaki, Miki Takeuchi, Makoto Kobayashi, Kazuyo Misaki, Shigenobu Yonemura, Atsuo Kawahara and Atsuko Sehara-Fujisawa : Metalloprotease-dependent onset of blood circulation in Zebrafish, Current Biology, 20, 12, 1110-1116, 2010.
(要約)
The primitive blood circulation requires intravascular plasma flow. However, it remains unclear whether the onset of earliest blood circulation is dependent solely on establishment of a functional circulatory organ or whether it also requires active processes inherent in blood cells. In this study, we present novel mechanisms for the onset of blood circulation by monitoring fluorescently labeled blood precursors and blood vessels in zebrafish. The earliest blood circulation occurs synchronously. This synchrony is achieved by the retention of erythroid precursors on the lumen of the vasculature after their invasion from the subaortic region, and then by simultaneous release of these precursors into the flow. Morphological and biochemical analyses suggest that the onset of blood circulation accompanies disruption of blood cell-to-vessel adhesion and requires metalloprotease-dependent processes. ADAM8, a member of the a disintegrin and metalloprotease (ADAM) family, mediates the onset of blood circulation. In ADAM8-depleted embryos, erythroid cells fail to detach from the vascular lumen and stagnate. Expression of a protease-defective ADAM8 in erythroid cells causes dominant-negative effects on blood circulation, suggesting cell-autonomous roles of ADAM8. Based on these findings, we propose that the first erythroid cells require both flow-dependent passive and proteolysis-dependent active processes to enter the circulation.
Reiko Tajiri, Kazuyo Misaki, Shigenobu Yonemura and Shigeo Hayashi : Dynamic shape changes of ECM-producing cells drive morphogenesis of ball-and-socket joints in the fly leg, Development, 137, 12, 2055-2063, 2010.
(要約)
Animal body shape is framed by the skeleton, which is composed of extracellular matrix (ECM). Although how the body plan manifests in skeletal morphology has been studied intensively, cellular mechanisms that directly control skeletal ECM morphology remain elusive. In particular, how dynamic behaviors of ECM-secreting cells, such as shape changes and movements, contribute to ECM morphogenesis is unclear. Strict control of ECM morphology is crucial in the joints, where opposing sides of the skeleton must have precisely reciprocal shapes to fit each other. Here we found that, in the development of ball-and-socket joints in the Drosophila leg, the two sides of ECM form sequentially. We show that distinct cell populations produce the 'ball' and the 'socket', and that these cells undergo extensive shape changes while depositing ECM. We propose that shape changes of ECM-producing cells enable the sequential ECM formation to allow the morphological coupling of adjacent components. Our results highlight the importance of dynamic cell behaviors in precise shaping of skeletal ECM architecture.
Shigenobu Yonemura, Yuko Wada, Toshiyuki Watanabe, Akira Nagafuchi and Mai Shibata : α-Catenin as a tension transducer that induces adherens junction development, Nature Cell Biology, 12, 6, 533-542, 2010.
(要約)
Adherens junctions (AJs), which are organized by adhesion proteins and the underlying actin cytoskeleton, probably sense pulling forces from adjacent cells and modulate opposing forces to maintain tissue integrity, but the regulatory mechanism remains unknown at the molecular level. Although the possibility that alpha-catenin acts as a direct linker between the membrane and the actin cytoskeleton for AJ formation and function has been minimized, here we show that alpha-catenin recruits vinculin, another main actin-binding protein of AJs, through force-dependent changes in alpha-catenin conformation. We identified regions in the alpha-catenin molecule that are required for its force-dependent binding of vinculin by introducing mutant alpha-catenin into cells and using in vitro binding assays. Fluorescence recovery after photobleaching analysis for alpha-catenin mobility and the existence of an antibody recognizing alpha-catenin in a force-dependent manner further supported the notion that alpha-catenin is a tension transducer that translates mechanical stimuli into a chemical response, resulting in AJ development.
Takaaki Sokabe, Tomoko Fukumi-Tominaga, Shigenobu Yonemura, Atsuko Mizuno and Makoto Tominaga : The TRPV4 channel contributes to intercellular junction formation in keratinocytes, Journal of Biological Chemistry, 285, 24, 18749-18758, 2010.
(要約)
Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca(2+) dynamics. We found that TRPV4 interacts with beta-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca(2+)-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca(2+) increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin.
Tomohiro Haruta, Rahul Warrior, Shigenobu Yonemura and Hiroki Oda : The proximal half of the Drosophila E-cadherin extracellular region is dispensable for many cadherin-dependent events but required for ventral furrow formation, Genes to Cells, 15, 3, 193-208, 2010.
(要約)
The formation of the ventral furrow during Drosophila gastrulation is driven by coordinated apical constriction. Cell-cell adhesion is thought to regulate apical constriction, but the mechanisms are poorly understood. DE-cadherin, an epithelial classic cadherin, has in its membrane-proximal extracellular region a suite of domains absent from vertebrate/urochordate classic cadherins. We constructed DEΔP, a DE-cadherin derivative that lacks the membrane-proximal half of the extracellular region but retains the entire cytoplasmic domain and still exhibits strong cell-cell binding ability. The extracellular region of DEΔP consists of only cadherin repeats, mimicking vertebrate/urochordate classic cadherins. In animals lacking DE-cadherin, DEΔP organized adherens junction assembly and functioned fully in many cadherin-dependent processes, including oogenesis. Embryos in which DE-cadherin was entirely replaced by DEΔP established the blastoderm epithelium but failed to form a ventral furrow. Apical constrictions were initiated relatively normally but subsequently decelerated. These were then followed by catastrophic disruption of the junctional network. Our results suggest that although the membrane-proximal half of the DE-cadherin extracellular region is dispensable for many developmental events, it is essential for efficient and robust apical constriction during ventral furrow formation.
Hideaki Nojima, Sophie Rothhämel, Takashi Shimizu, Hee Cheol Kim, Shigenobu Yonemura, L. Florence Marlow and Masahiko Hibi : Syntabulin, a motor protein linker, controls dorsal determination, Development, 137, 6, 923-933, 2010.
(要約)
In amphibian and teleost embryos, the dorsal determinants (DDs) are believed to be initially localized to the vegetal pole and then transported to the prospective dorsal side of the embryo along a microtubule array. The DDs are known to activate the canonical Wnt pathway and thereby promote the expression of genes that induce the dorsal organizer. Here, by identifying the locus of the maternal-effect ventralized mutant tokkaebi, we show that Syntabulin, a linker of the kinesin I motor protein, is essential for dorsal determination in zebrafish. We found that syntabulin mRNA is transported to the vegetal pole during oogenesis through the Bucky ball (Buc)-mediated Balbiani body-dependent pathway, which is necessary for establishment of animal-vegetal (AV) oocyte polarity. We demonstrate that Syntabulin is translocated from the vegetal pole in a microtubule-dependent manner. Our findings suggest that Syntabulin regulates the microtubule-dependent transport of the DDs, and provide evidence for the link between AV and dorsoventral axis formation.
(キーワード)
Animals / Animals, Genetically Modified / Body Patterning / Cell Polarity / Cells, Cultured / Cloning, Molecular / Embryo, Nonmammalian / Female / Humans / Male / Mice / Mice, Inbred BALB C / Microtubule-Associated Proteins / Molecular Motor Proteins / Protein Transport / RNA, Messenger, Stored / Zebrafish / Zebrafish Proteins
Makoto Ikeya, Kumi Fukushima, Masako Kawada, Sachiko Onishi, Yasuhide Furuta, Shigenobu Yonemura, Toshio Kitamura, Tetsuya Nosaka and Yoshiki Sasai : Cv2, functioning as a pro-BMP factor via twisted gastrulation, is required for early development of nephron precursors, Developmental Biology, 337, 2, 405-414, 2009.
(要約)
The fine-tuning of BMP signals is critical for many aspects of complex organogenesis. In this report, we show that the augmentation of BMP signaling by a BMP-binding secreted factor, Crossveinless2 (Cv2), is essential for the early embryonic development of mammalian nephrons. In the Cv2-null mouse, the number of cap condensates (clusters of nephron progenitors, which normally express Cv2) was decreased, and the condensate cells exhibited a reduced level of aggregation. In these Cv2(-/-) condensates, the level of phosphorylated Smad1 (pSmad1) was substantially lowered. The loss of a Bmp7 allele in the Cv2(-/-) mouse enhanced the cap condensate defects and further decreased the level of pSmad1 in this tissue. These observations indicated that Cv2 has a pro-BMP function in early nephrogenesis. Interestingly, the renal defects of the Cv2(-/-) mutant were totally suppressed by a null mutation of Twisted gastrulation (Tsg), which encodes another BMP-binding factor, showing that Cv2 exerts its pro-BMP nephrogenic function Tsg-dependently. By using an embryonic kidney cell line, we presented experimental evidence showing that Cv2 enhances pro-BMP activity of Tsg. These findings revealed the molecular hierarchy between extracellular modifiers that orchestrate local BMP signal peaks in the organogenetic microenvironment.
Takashi Ishiuchi, Kazuyo Misaki, Shigenobu Yonemura, Masatoshi Takeichi and Takuji Tanoue : Mammalian Fat and Dachsous cadherins regulate apical membrane organization in the embryonic cerebral cortex, Journal of Cell Biology, 185, 6, 959-967, 2009.
(要約)
Compartmentalization of the plasma membrane in a cell is fundamental for its proper functions. In this study, we present evidence that mammalian Fat4 and Dachsous1 cadherins regulate the apical plasma membrane organization in the embryonic cerebral cortex. In neural progenitor cells of the cortex, Fat4 and Dachsous1 were concentrated together in a cell-cell contact area positioned more apically than the adherens junction (AJ). These molecules interacted in a heterophilic fashion, affecting their respective protein levels. We further found that Fat4 associated and colocalized with the Pals1 complex. Ultrastructurally, the apical junctions of the progenitor cells comprised the AJ and a stretch of plasma membrane apposition extending apically from the AJ, which positionally corresponded to the Fat4-Dachsous1-positive zone. Depletion of Fat4 or Pals1 abolished this membrane apposition. These results highlight the importance of the Fat4-Dachsous1-Pals1 complex in organizing the apical membrane architecture of neural progenitor cells.
Yosuke Horikoshi, Atsushi Suzuki, Tomoyuki Yamanaka, Kazunori Sasaki, Keiko Mizuno, Hajime Sawada, Shigenobu Yonemura and Shigeo Ohno : Interaction between PAR-3 and the aPKC-PAR-6 complex is indispensable for apical domain development of epithelial cells, Journal of Cell Science, 122, 10, 1595-1606, 2009.
(要約)
The evolutionarily conserved polarity proteins PAR-3, atypical protein kinase C (aPKC) and PAR-6 critically regulate the apical membrane development required for epithelial organ development. However, the molecular mechanisms underlying their roles remain to be clarified. We demonstrate that PAR-3 knockdown in MDCK cells retards apical protein delivery to the plasma membrane, and eventually leads to mislocalized apical domain formation at intercellular regions in both two-dimensional and three-dimensional culture systems. The defects in PAR-3 knockdown cells are efficiently rescued by wild-type PAR-3, but not by a point mutant (S827/829A) that lacks the ability to interact with aPKC, indicating that formation of the PAR-3-aPKC-PAR-6 complex is essential for apical membrane development. This is in sharp contrast with tight junction maturation, which does not necessarily depend on the aPKC-PAR-3 interaction, and indicates that the two fundamental processes essential for epithelial polarity are differentially regulated by these polarity proteins. Importantly, highly depolarized cells accumulate aPKC and PAR-6, but not PAR-3, on apical protein-containing vacuoles, which become targeted to PAR-3-positive primordial cell-cell contact sites during the initial stage of the repolarization process. Therefore, formation of the PAR-3-aPKC-PAR-6 complex might be required for targeting of not only the aPKC-PAR-6 complex but also of apical protein carrier vesicles to primordial junction structures.
Ichi Ken Takeuchi, Yoshiro Nakano, Utako Kato, Mizuho Kaneda, Masako Aizu, Wakae Awano, Shigenobu Yonemura, Shigeki Kiyonaka, Yasuo Mori, Daisuke Yamamoto and Masato Umeda : Changes in temperature preferences and energy homeostasis in dystroglycan mutants, Science, 323, 5922, 1740-1743, 2009.
(要約)
Temperature affects the physiology, behavior, and evolution of organisms. We conducted mutagenesis and screens for mutants with altered temperature preference in Drosophila melanogaster and identified a cryophilic (cold-seeking) mutant, named atsugari (atu). Reduced expression of the Drosophila ortholog of dystroglycan (DmDG) induced tolerance to cold as well as preference for the low temperature. A sustained increase in mitochondrial oxidative metabolism caused by the reduced expression of DmDG accounted for the cryophilic phenotype of the atu mutant. Although most ectothermic animals do not use metabolically produced heat to regulate body temperature, our results indicate that their thermoregulatory behavior is closely linked to rates of mitochondrial oxidative metabolism and that a mutation in a single gene can induce a sustained change in energy homeostasis and the thermal responses.
(キーワード)
Adenosine Triphosphate / Animals / Animals, Genetically Modified / Body Temperature Regulation / Calcium / Cold Temperature / Drosophila Proteins / Drosophila melanogaster / Dystroglycans / Energy Metabolism / Homeostasis / Mitochondria / Mutant Proteins / Mutation / Oxygen Consumption / Phenotype / Pyruvate Dehydrogenase Complex / Temperature
C-terminal Src kinase (Csk) is a non-receptor type of tyrosine kinase, and serves as an essential negative regulator of Src family tyrosine kinases (SFKs) in vertebrates. However, analyses of Csk and SFKs from primitive animals suggest that the Csk-mediated mechanisms regulating SFK activity might diverge between evolutional branches, different tissues or SFK family members. We examined in vivo roles of CSK-1, a Caenorhabditis elegans orthologue of Csk, by generating animals lacking csk-1 function. Although some csk-1 mutants died during embryogenesis, the majority of mutants died during the first stage of larval development. In csk-1 mutants, the function of pharyngeal muscles, the major site of CSK-1 expression, was severely damaged. The pumping of pharyngeal grinder cells became arrhythmic, causing disabled feeding. Electron microscopy showed that pharyngeal muscle filaments were disorientated in the csk-1 mutants. These indicate that CSK-1 is crucial for proper organization of pharyngeal muscles. However, the growth arrest phenotype in csk-1 mutants could not be suppressed by src-1 and/or src-2 mutation, and SRC-1 was not significantly activated in the csk-1 mutants. These results suggest that CSK-1 has an essential function in organization of pharyngeal muscle filaments that does not require C. elegans SFKs.
Mototsugu Eiraku, Kiichi Watanabe, Mami Matsuo-Takasaki, Masako Kawada, Shigenobu Yonemura, Michiru Matsumura, Takafumi Wataya, Ayaka Nishiyama, Keiko Muguruma and Yoshiki Sasai : Self-Organized Formation of Polarized Cortical Tissues from ESCs and Its Active Manipulation by Extrinsic Signals, Cell Stem Cell, 3, 5, 519-532, 2008.
(要約)
Here, we demonstrate self-organized formation of apico-basally polarized cortical tissues from ESCs using an efficient three-dimensional aggregation culture (SFEBq culture). The generated cortical neurons are functional, transplantable, and capable of forming proper long-range connections in vivo and in vitro. The regional identity of the generated pallial tissues can be selectively controlled (into olfactory bulb, rostral and caudal cortices, hem, and choroid plexus) by secreted patterning factors such as Fgf, Wnt, and BMP. In addition, the in vivo-mimicking birth order of distinct cortical neurons permits the selective generation of particular layer-specific neurons by timed induction of cell-cycle exit. Importantly, cortical tissues generated from mouse and human ESCs form a self-organized structure that includes four distinct zones (ventricular, early and late cortical-plate, and Cajal-Retzius cell zones) along the apico-basal direction. Thus, spatial and temporal aspects of early corticogenesis are recapitulated and can be manipulated in this ESC culture.
Ichirou Karahara, Jinsuke Suda, Hiroshi Tahara, Etsuo Yokota, Teruo Shimmen, Kazuyo Misaki, Shigenobu Yonemura, Andrew Lucas Staehelin and Yoshinobu Mineyuki : The preprophase band is a localized center of clathrin-mediated endocytosis in late prophase cells of the onion cotyledon epidermis, Plant Journal, 57, 5, 819-831, 2008.
(要約)
The preprophase band (PPB) marks the site on the plant cell cortex where the cell plate will fuse during the final stage of cytokinesis. Recent studies have shown that several cytoskeletal proteins are depleted at the PPB site, but the processes that bring about these changes are still unknown. We have investigated the membrane systems associated with the PPB regions of epidermal cells of onion cotyledons by means of serial thin sections and electron tomograms. In contrast with specimens preserved by chemical fixatives, our high-pressure frozen cells demonstrated the presence of large numbers of clathrin-coated pits and vesicles in the PPB regions. The vesicles were of two types: clathrin-coated and structurally related, non-coated vesicles. Quantitative analysis of the data revealed that the number of clathrin-coated pits and vesicles is higher in the PPB regions than outside of these regions. Immunofluorescent microscopy using anti-plant clathrin-antibody confirmed this result. In contrast, no differences in secretory activities were observed. We postulate that the removal of membrane proteins by endocytosis plays a role in the formation of PPB 'memory' structures.
(キーワード)
Clathrin-coated pits / Clathrin-coated vesicles / Electron tomography / Endocytosis / Preprophase band of microtubules
Masahiko Sugimoto, Akihito Inoko, Takashi Shiromizu, Masanori Nakayama, Peng Zou, Shigenobu Yonemura, Yuko Hayashi, Ichiro Izawa, Mikio Sasoh, Yukitaka Uji, Kozo Kaibuchi, Tohru Kiyono and Masaki Inagaki : The keratin-binding protein Albatross regulates polarization of epithelial cells, Journal of Cell Biology, 183, 1, 19-28, 2008.
(要約)
The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell-cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.
Mariko Hirano, Shigeru Hashimoto, Shigenobu Yonemura, Hisataka Sabe and Shinichi Aizawa : EPB41L5 functions to post-transcriptionally regulate cadherin and integrin during epithelial-mesenchymal transition, Journal of Cell Biology, 182, 6, 1217-1230, 2008.
(要約)
EPB41L5 belongs to the band 4.1 superfamily. We investigate here the involvement of EPB41L5 in epithelial-mesenchymal transition (EMT) during mouse gastrulation. EPB41L5 expression is induced during TGFbeta-stimulated EMT, whereas silencing of EPB41L5 by siRNA inhibits this transition. In EPB41L5 mutants, cell-cell adhesion is enhanced, and EMT is greatly impaired during gastrulation. Moreover, cell attachment, spreading, and mobility are greatly reduced by EPB41L5 deficiency. Gene transcription regulation during EMT occurs normally at the mRNA level; EPB41L5 siRNA does not affect either the decrease in E-cadherin or the increase in integrin expression. However, at the protein level, the decrease in E-cadherin and increase in integrin are inhibited in both EPB41L5 siRNA-treated NMuMG cells and mutant mesoderm. We find that EPB41L5 binds p120ctn through its N-terminal FERM domain, inhibiting p120ctn-E-cadherin binding. EPB41L5 overexpression causes E-cadherin relocalization into Rab5-positive vesicles in epithelial cells. At the same time, EPB41L5 binds to paxillin through its C terminus, enhancing integrin/paxillin association, thereby stimulating focal adhesion formation.
Shinji Yamamoto, Osamu Nishimura, Kazuyo Misaki, Michiru Nishita, Yasuhiro Minami, Shigenobu Yonemura, Hiroshi Tarui and Hiroshi Sasaki : Cthrc1 Selectively Activates the Planar Cell Polarity Pathway of Wnt Signaling by Stabilizing the Wnt-Receptor Complex, Developmental Cell, 15, 1, 23-36, 2008.
(要約)
Vertebrate Wnt proteins activate several distinct pathways. Intrinsic differences among Wnt ligands and Frizzled (Fzd) receptors, and the availability of pathway-specific coreceptors, LRP5/6, and Ror2, affect pathway selection. Here, we show that a secreted glycoprotein, Cthrc1, is involved in selective activation of the planar cell polarity (PCP) pathway by Wnt proteins. Although Cthrc1 null mutant mice appeared normal, the introduction of a heterozygous mutation of a PCP gene, Vangl2, resulted in abnormalities characteristic of PCP mutants. In HEK293T cells, Cthrc1 activated the PCP pathway but suppressed the canonical pathway. Cell-surface-anchored Cthrc1 bound to Wnt proteins, Fzd proteins, and Ror2 and enhanced the interaction of Wnt proteins and Fzd/Ror2 by forming the Cthrc1-Wnt-Fzd/Ror2 complex. Consistent with this, Ror2 mutant mice also showed PCP-related abnormalities in the inner ear. These results suggest that Cthrc1 is a Wnt cofactor protein that selectively activates the Wnt/PCP pathway by stabilizing ligand-receptor interaction.
Tomohiro Yamauchi, Takefumi Ishidao, Teruaki Nomura, Toshie Shinagawa, Yasunori Tanaka, Shigenobu Yonemura and Shunsuke Ishii : A B-Myb complex containing clathrin and filamin is required for mitotic spindle function, EMBO Journal, 27, 13, 1852-1862, 2008.
(要約)
B-Myb is one member of the vertebrate Myb family of transcription factors and is ubiquitously expressed. B-Myb activates transcription of a group of genes required for the G2/M cell cycle transition by forming the dREAM/Myb-MuvB-like complex, which was originally identified in Drosophila. Mutants of zebrafish B-myb and Drosophila myb exhibit defects in cell cycle progression and genome instability. Although the genome instability caused by a loss of B-Myb has been speculated to be due to abnormal cell cycle progression, the precise mechanism remains unknown. Here, we have purified a B-Myb complex containing clathrin and filamin (Myb-Clafi complex). This complex is required for normal localization of clathrin at the mitotic spindle, which was previously reported to stabilize kinetochore fibres. The Myb-Clafi complex is not tightly associated with the mitotic spindles, suggesting that this complex ferries clathrin to the mitotic spindles. Thus, identification of the Myb-Clafi complex reveals a previously unrecognized function of B-Myb that may contribute to its role in chromosome stability, possibly, tumour suppression.
Toshihiro Uesaka, Mayumi Nagashimada, Shigenobu Yonemura and Hideki Enomoto : Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice, Journal of Clinical Investigation, 118, 5, 1890-1898, 2008.
(要約)
Mutations in the RET gene are the primary cause of Hirschsprung disease (HSCR), or congenital intestinal aganglionosis. However, how RET malfunction leads to HSCR is not known. It has recently been shown that glial cell line-derived neurotrophic factor (GDNF) family receptor alpha1 (GFRalpha1), which binds to GDNF and activates RET, is essential for the survival of enteric neurons. In this study, we investigated Ret regulation of enteric neuron survival and its potential involvement in HSCR. Conditional ablation of Ret in postmigratory enteric neurons caused widespread neuronal death in the colon, which led to colonic aganglionosis. To further examine this finding, we generated a mouse model for HSCR by reducing Ret expression levels. These mice recapitulated the genetic and phenotypic features of HSCR and developed colonic aganglionosis due to impaired migration and successive death of enteric neural crest-derived cells. Death of enteric neurons was also induced in the colon, where reduction of Ret expression was induced after the period of enteric neural crest cell migration, indicating that diminished Ret expression directly affected the survival of colonic neurons. Thus, enteric neuron survival is sensitive to RET dosage, and cell death is potentially involved in the etiology of HSCR.
Hiroyuki Nakajima, Shigenobu Yonemura, Masayuki Murata, Nobuhiro Nakamura, Helen Piwnica-Worms and Eisuke Nishida : Myt1 protein kinase is essential for Golgi and ER assembly during mitotic exit, Journal of Cell Biology, 181, 1, 89-103, 2008.
(要約)
Myt1 was originally identified as an inhibitory kinase for Cdc2 (Cdk1), the master engine of mitosis, and has been thought to function, together with Wee1, as a negative regulator of mitotic entry. In this study, we report an unexpected finding that Myt1 is essential for Golgi and endoplasmic reticulum (ER) assembly during telophase in mammalian cells. Our analyses reveal that both cyclin B1 and cyclin B2 serve as targets of Myt1 for proper Golgi and ER assembly to occur. Thus, our results show that Myt1-mediated suppression of Cdc2 activity is not indispensable for the regulation of a broad range of mitotic events but is specifically required for the control of intracellular membrane dynamics during mitosis.
Masatsune Tsujioka, Kunito Yoshida, Akira Nagasaki, Shigenobu Yonemura, Annette Müller-Taubenberger and Q.P. Taro Uyeda : Overlapping functions of the two talin homologues in Dictyostelium, Eukaryotic Cell, 7, 5, 906-916, 2008.
(要約)
Talin is a cytoskeletal protein involved in constructing and regulating focal adhesions in animal cells. The cellular slime mold Dictyostelium discoideum has two talin homologues, talA and talB, and earlier studies have characterized the single knockout mutants. talA(-) cells show reduced adhesion to the substrates and slightly impaired cytokinesis leading to a high proportion of multinucleated cells in the vegetative stage, while the development is normal. In contrast, talB(-) cells are characterized by reduced motility in the developmental stage, and they are arrested at the tight-mound stage. Here, we created and analyzed a double mutant with a disruption of both talA and talB. Defects in adhesion to the substrates, cytokinesis, and development were more severe in cells with a disruption of both talA and talB. The talA(-) talB(-) cells failed to attach to the substrates in the vegetative stage, exhibited a higher proportion of multinucleated cells than talA(-) cells, and showed more-reduced motility during the development and an earlier developmental arrest than talB(-) cells at the loose-mound stage. Moreover, overexpression of either talA or talB compensated for the loss of the other talin, respectively. The analysis of talA(-) talB(-) cells also revealed that talin was required for the formation of paxillin-rich adhesion sites and that there was another adhesion mechanism which is independent of talin in the developmental stage. This is the first study demonstrating overlapping functions of two talin homologues, and our data further indicate the importance of talin.
(キーワード)
Animals / Cell Cycle / Cell Movement / Cytokinesis / Dictyostelium / Focal Adhesions / Green Fluorescent Proteins / Life Cycle Stages / Oligopeptides / Peptides / Protein Isoforms / Recombinant Fusion Proteins / Talin
Nagatoki Kinoshita, Noriaki Sasai, Kazuyo Misaki and Shigenobu Yonemura : Apical accumulation of Rho in the neural plate is important for neural plate cell shape change and neural tube formation, Molecular Biology of the Cell, 19, 5, 2289-2299, 2008.
(要約)
Although Rho-GTPases are well-known regulators of cytoskeletal reorganization, their in vivo distribution and physiological functions have remained elusive. In this study, we found marked apical accumulation of Rho in developing chick embryos undergoing folding of the neural plate during neural tube formation, with similar accumulation of activated myosin II. The timing of accumulation and biochemical activation of both Rho and myosin II was coincident with the dynamics of neural tube formation. Inhibition of Rho disrupted its apical accumulation and led to defects in neural tube formation, with abnormal morphology of the neural plate. Continuous activation of Rho also altered neural tube formation. These results indicate that correct spatiotemporal regulation of Rho is essential for neural tube morphogenesis. Furthermore, we found that a key morphogenetic signaling pathway, the Wnt/PCP pathway, was implicated in the apical accumulation of Rho and regulation of cell shape in the neural plate, suggesting that this signal may be the spatiotemporal regulator of Rho in neural tube formation.
W. Thomas Gould, Shigenobu Yonemura, W. Ronald Oppenheim, Shiho Ohmori and Hideki Enomoto : The neurotrophic effects of glial cell line-derived neurotrophic factor on spinal motoneurons are restricted to fusimotor subtypes, Journal of Neuroscience, 28, 9, 2131-2146, 2008.
(要約)
Glial cell line-derived neurotrophic factor (GDNF) regulates multiple aspects of spinal motoneuron (MN) development, including gene expression, target selection, survival, and synapse elimination, and mice lacking either GDNF or its receptors GDNF family receptor alpha1 (GFRalpha1) and Ret exhibit a 25% reduction of lumbar MNs at postnatal day 0 (P0). Whether this loss reflects a generic trophic role for GDNF and thus a reduction of all MN subpopulations, or a more restricted role affecting only specific MN subpopulations, such as those innervating individual muscles, remains unclear. We therefore examined MN number and innervation in mice in which Ret, GFRalpha1, or GDNF was deleted and replaced by reporter alleles. Whereas nearly all hindlimb muscles exhibited normal gross innervation, intrafusal muscle spindles displayed a significant loss of innervation in most but not all muscles at P0. Furthermore, we observed a dramatic and restricted loss of small myelinated axons in the lumbar ventral roots of adult mice in which the function of either Ret or GFRalpha1 was inactivated in MNs early in development. Finally, we demonstrated that the period during which spindle-innervating MNs require GDNF for survival is restricted to early neonatal development, because mice in which the function of Ret or GFRalpha1 was inactivated after P5 failed to exhibit denervation of muscle spindles or MN loss. Therefore, although GDNF influences several aspects of MN development, the survival-promoting effects of GDNF during programmed cell death are mostly confined to spindle-innervating MNs.
Eiichi Takaki, Mitsuaki Fujimoto, Takashi Nakahari, Shigenobu Yonemura, Yoshihiko Miyata, Naoki Hayashida, Kaoru Yamamoto, B. Richard Vallee, Tsuyoshi Mikuriya, Kazuma Sugahara, Hiroshi Yamashita, Sachiye Inouye and Akira Nakai : Heat shock transcription factor 1 is required for maintenance of ciliary beating in mice, Journal of Biological Chemistry, 282, 51, 37285-37292, 2007.
(要約)
Heat shock transcription factors (HSFs) maintain protein homeostasis through regulating expression of heat shock proteins, especially in stressed conditions. In addition, HSFs are involved in cellular differentiation and development by regulating development-related genes, as well as heat shock genes. Here, we showed chronic sinusitis and mild hydrocephalus in postnatal HSF1-null mice, which are associated with impaired mucociliary clearance and cerebrospinal flow, respectively. Analysis of ciliary beating revealed that the amplitude of the beating was significantly reduced, and ciliary beat frequencies were lower in the respiratory epithelium, ependymal cells, oviduct, and trachea of HSF1-null mice than those of wild-type mice. Cilia possess a common axonema structure composed of microtubules of alpha- and beta-tubulin. We found a marked reduction in alpha- and ciliary betaiv-tubulin in the HSF1-null cilia, which is developmentally associated with reduced Hsp90 expression in HSF1-null mice. Treatment of the respiratory epithelium with geldanamycin resulted in rapid reduction of ciliary beating in a dose-dependent manner. Furthermore, Hsp90 was physically associated with ciliary betaiv-tubulin, and Hsp90 stabilizes tubulin polymerization in vitro. These results indicate that HSF1 is required to maintain ciliary beating in postnatal mice, probably by regulating constitutive expression of Hsp90 that is important for tubulin polymerization.
Toshihiro Uesaka, Sanjay Jain, Shigenobu Yonemura, Yasuo Uchiyama, Jeffrey Milbrandt and Hideki Enomoto : Conditional ablation of GFRα1 in postmigratory eneric neurons triggers unconventional neuronal death in the colon and causes Hirschsprungs's disease phenotype, Development, 134, 11, 2171-2181, 2007.
(要約)
The regulation of neuronal survival and death by neurotrophic factors plays a central role in the sculpting of the nervous system, but the identity of survival signals for developing enteric neurons remains obscure. We demonstrate here that conditional ablation of GFRalpha1, the high affinity receptor for GDNF, in mice during late gestation induces rapid and widespread neuronal death in the colon, leading to colon aganglionosis reminiscent of Hirschsprung's disease. Enteric neuron death induced by GFRalpha1 inactivation is not associated with the activation of common cell death executors, caspase-3 or -7, and lacks the morphological hallmarks of apoptosis, such as chromatin compaction and mitochondrial pathology. Consistent with these in vivo observations, neither caspase inhibition nor Bax deficiency blocks death of colon-derived enteric neurons induced by GDNF deprivation. This study reveals an essential role for GFRalpha1 in the survival of enteric neurons and suggests that caspase-independent death can be triggered by abolition of neurotrophic signals.
Toshiyuki Watanabe, Hiroshi Hosoya and Shigenobu Yonemura : Regulation of myosin II dynamics by phosphorylation and dephosphorylation of its light chain in epithelial cells, Molecular Biology of the Cell, 18, 2, 605-616, 2006.
(要約)
Nonmuscle myosin II, an actin-based motor protein, plays an essential role in actin cytoskeleton organization and cellular motility. Although phosphorylation of its regulatory light chain (MRLC) is known to be involved in myosin II filament assembly and motor activity in vitro, it remains unclear exactly how MRLC phosphorylation regulates myosin II dynamics in vivo. We established clones of Madin Darby canine kidney II epithelial cells expressing MRLC-enhanced green fluorescent protein or its mutants. Time-lapse imaging revealed that both phosphorylation and dephosphorylation are required for proper dynamics of myosin II. Inhibitors affecting myosin phosphorylation and MRLC mutants indicated that monophosphorylation of MRLC is required and sufficient for maintenance of stress fibers. Diphosphorylated MRLC stabilized myosin II filaments and was distributed locally in regions of stress fibers where contraction occurs, suggesting that diphosphorylation is involved in the spatial regulation of myosin II assembly and contraction. We further found that myosin phosphatase or Zipper-interacting protein kinase localizes to stress fibers depending on the activity of myosin II ATPase.
(キーワード)
Adenosine Triphosphatases / Animals / Apoptosis Regulatory Proteins / Calcium-Calmodulin-Dependent Protein Kinases / Cells, Cultured / Death-Associated Protein Kinases / Dogs / Epithelial Cells / Mutation / Myosin Light Chains / Myosin Type II / Myosin-Light-Chain Phosphatase / Phosphorylation / Protein Serine-Threonine Kinases / Stress Fibers
Yoshifumi Yamaguchi, Shigenobu Yonemura and Shinji Takada : Grainyhead-related transcription factor is required for duct maturation in the salivary gland and the kidney of the mouse, Development, 133, 23, 4737-4748, 2006.
(要約)
Duct epithelial structure is an essential feature of many internal organs, including exocrine glands and the kidney. The ducts not only mediate fluid transfer but also help to maintain homeostasis. For instance, fluids and solutes are resorbed from or secreted into the primary fluid flowing through the lumen of the ducts in the exocrine glands and kidneys. The molecular mechanism underlying the functional maturation of these ducts remains largely unknown. Here, we show that a grainyhead-related transcription factor, CP2-like 1 (CP2L1), is required for the maturation of the ducts of the salivary gland and kidney. In the mouse, Cp2l1 is specifically expressed in the developing ducts of a number of exocrine glands, including the salivary gland, as well as in those of the kidney. In Cp2l1-deficient mice, the expression of genes directly involved in functional maturation of the ducts was specifically reduced in both the salivary gland and kidney, indicating that Cp2l1 is required for the differentiation of duct cells. Furthermore, the composition of saliva and urine was abnormal in these mice. These results indicate that Cp2l1 expression is required for normal duct development in both the salivary gland and kidney.
Atsushi Wada, Kagayaki Kato, F. Makiko Uwo, Shigenobu Yonemura and Shigeo Hayashi : Specialized extraembryonic cells connect embryonic and extraembryonic epidermis in response to Dpp during dorsal closure in Drosophila, Developmental Biology, 301, 2, 340-349, 2006.
(要約)
Dorsal closure in Drosophila embryogenesis involves expansion of the dorsal epidermis, followed by closure of the opposite epidermal edges. This process is driven by contractile force generated by an extraembryonic epithelium covering the yolk syncytium known as the amnioserosa. The secreted signaling molecule Dpp is expressed in the leading edge of the dorsal epidermis and is essential for dorsal closure. We found that the outermost row of amnioserosa cells (termed pAS) maintains a tight basolateral cell-cell adhesion interface with the leading edge of dorsal epidermis throughout the dorsal closure process. pAS was subject to altered cell motility in response to Dpp emanating from the dorsal epidermis, and this response was essential for dorsal closure. alphaPS3 and betaPS integrin subunits accumulated in the interface between pAS and dorsal epidermis, and were both required for dorsal closure. Looking at alphaPS3, type I Dpp receptor, and JNK mutants, we found that pAS cell motility was altered and pAS and dorsal epidermis adhesion failed under the mechanical stress of dorsal closure, suggesting that a Dpp-mediated mechanism connects the squamous pAS to the columnar dorsal epidermis to form a single coherent epithelial layer.
Yuka Miyake, Naoko Inoue, Koji Nishimura, Nagatoki Kinoshita, Hiroshi Hosoya and Shigenobu Yonemura : Actomyosin tension is required for correct recruitment of adherens junction components and zonula occludens formation, Experimental Cell Research, 312, 9, 1637-1650, 2006.
(要約)
The adherens junction (AJ) densely associated with actin filaments is a major cell-cell adhesion structure. To understand the importance of actin filament association in AJ formation, we first analyzed punctate AJs in NRK fibroblasts where one actin cable binds to one AJ structure unit. The accumulation of AJ components such as the cadherin/catenin complex and vinculin, as well as the formation of AJ-associated actin cables depended on Rho activity. Inhibitors for the Rho target, ROCK, which regulates myosin II activity, and for myosin II ATPase prevented the accumulation of AJ components, indicating that myosin II activity is more directly involved than Rho activity. Depletion of myosin II by RNAi showed similar results. The inhibition of myosin II activity in polarized epithelial MTD-1A cells affected the accumulation of vinculin to circumferential AJ (zonula adherens). Furthermore, correct zonula occludens (tight junction) formation along the apicobasal axis that requires cadherin activity was also impaired. Although MDCK cells which are often used as typical epithelial cells do not have a typical zonula adherens, punctate AJs formed dependently on myosin II activity by inducing wound closure in a MDCK cell sheet. These findings suggest that tension generated by actomyosin is essential for correct AJ assembly.
Yuko Shimada, Shigenobu Yonemura, Hiroyuki Ohkura, David Strutt and Tadashi Uemura : Polarized transport of Frizzled along the planar microtubule arrays in Drosophila wing epithelium, Developmental Cell, 10, 2, 209-222, 2006.
(要約)
Cells in a variety of developmental contexts sense extracellular cues that are given locally on their surfaces, and subsequently amplify the initial signal to achieve cell polarization. Drosophila wing cells acquire planar polarity along the proximal-distal (P-D) axis, in which the amplification of the presumptive cue involves assembly of a multiprotein complex that spans distal and proximal boundaries of adjacent cells. Here we pursue the mechanisms that place one of the components, Frizzled (Fz), at the distal side. Intracellular particles of GFP-tagged Fz moved preferentially toward distal boundaries before Fz::GFP and other components were tightly localized at the P/D cortex. Arrays of microtubules (MTs) were approximately oriented along the P-D axis and these MTs contributed to the formation of the cortical complex. Furthermore, there appeared to be a bias in the P-D MTs, with slightly more plus ends oriented distally. The hypothesis of polarized vesicular trafficking of Fz is discussed.
Yukako Nishimura and Shigenobu Yonemura : Centralspindlin regulates ECT2 and RhoA accumulation at the equatorial cortex during cytokinesis, Journal of Cell Science, 119, 1, 104-114, 2005.
(要約)
During determination of the cell division plane, an actomyosin contractile ring is induced at the equatorial cell cortex by signals from the mitotic apparatus and contracts to cause cleavage furrow progression. Although the small GTPase RhoA is known to regulate the progression, probably by controlling actin filament assembly and enhancing actomyosin interaction, any involvement of RhoA in division plane determination is unknown. In this study, using a trichloroacetic acid (TCA) fixation protocol we recently developed, we show that RhoA accumulates at the equatorial cortex before furrow initiation and continues to concentrate at the cleavage furrow during cytokinesis. We also demonstrate that both Rho activity and microtubule organization are required for RhoA localization and proper furrowing. Selective disruption of microtubule organization revealed that both astral and central spindle microtubules can recruit RhoA at the equatorial cortex. We find that centralspindlin and ECT2 are required for RhoA localization and furrowing. Centralspindlin is localized both to central spindle microtubules and at the tips of astral microtubules near the equatorial cortex and recruits ECT2. Positional information for division plane determination from microtubules is transmitted to the cell cortex to organize actin cytoskeleton through a mechanism involving these proteins.
Eiichi Takaki, Mitsuaki Fujimoto, Kazuma Sugahara, Takashi Nakahari, Shigenobu Yonemura, Yasunori Tanaka, Naoki Hayashida, Sachiye Inouye, Tsuyoshi Takemoto, Hiroshi Yamashita and Akira Nakai : Maintenance of olfactory neurogenesis requires HSF1, a major heat shock transcription factor in mice, Journal of Biological Chemistry, 281, 8, 4931-4937, 2005.
(要約)
Heat shock transcription factors (HSFs) play roles not only in heat shock response but also in development of the reproductive organs, brain, and lens. Here, we analyzed sensory organs and found abnormalities of the olfactory epithelium in adult HSF1-null mice, which is developmentally related to the lens. The olfactory epithelium was normal until postnatal 3 weeks but was not maintained later than 4 weeks in HSF1-null mice. The olfactory epithelium was atrophied with increased cell death of olfactory sensory neurons. Analysis of the epithelium revealed that induction of HSP expression and reduction of LIF expression are lacking in adult HSF1-null mice. We found that DNA binding activity of HSF1 is induced in the olfactory epithelium later than 4 weeks and that HSF1 binds directly to Lif gene and inhibits its expression. HSF4 has opposing effects on LIF expression and olfactory neurogenesis. These data indicate that HSF1 is required for the precise expression of Hsp and cytokine genes that is obligatory for maintenance of olfactory neurogenesis in adult mice and suggest that stress-related processes are involved in its maintenance.
Satoshi Komiya, Masayuki Shimizu, Junichi Ikenouchi, Shigenobu Yonemura, Takeshi Matsui, Yoshitaka Fukunaga, Huijie Liu, Fumio Endo, Shoichiro Tsukita and Akira Nagafuchi : Apical membrane and junctional complex formation during simple epithelial cell differentiation of F9 cells, Genes to Cells, 10, 11, 1065-1080, 2005.
(要約)
Epithelium formation is a common event in animal morphogenesis. It has been reported that F9 cells differentiate into visceral endoderm-like epithelial cells when cell aggregates are cultured in the presence of retinoic acid. The present investigation set out to determine whether this in vitro model could be used under monolayer culture conditions, which is suitable for a detailed analysis of epithelial differentiation. We performed comparative gene expression analyses of F9 cells grown under aggregate and monolayer culture conditions prior to and following treatment with retinoic acid. Under these conditions, induction in the expression of differentiation marker genes was confirmed, even in monolayer cultures. Junctional complex and apical membrane formation, both of which are characteristic of epithelial cells, were also observed under monolayer culture conditions. Because of the merit of monolayer culture condition, we found that apical membrane and junctional complex formation are strictly regulated during epithelial differentiation. It was also revealed that F9 cells differentiated into epithelial cells predominantly on the fourth and fifth day following retinoic acid induction. These results showed that a monolayer culture of F9 cells represents a viable in vitro model that can be employed to elucidate mechanisms pertaining to epithelium formation.
Chiharu Kimura-Yoshida, Hiroshi Nakano, Daiji Okamura, Kazuki Nakao, Shigenobu Yonemura, A. Jose Belo, Shinichi Aizawa, Yasuhisa Matsui and Isao Matsuo : Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm, Developmental Cell, 9, 5, 639-650, 2005.
(要約)
The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-catenin localization is impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.
(キーワード)
Animals / Body Patterning / Cell Movement / Embryonic Development / Endoderm / Gene Expression Regulation, Developmental / Intercellular Signaling Peptides and Proteins / Mice / Mice, Knockout / Mice, Transgenic / Models, Biological / Otx Transcription Factors / Signal Transduction / Viscera / Wnt Proteins / beta Catenin
Takashi Shimizu, Taijiro Yabe, Osamu Muraoka, Shigenobu Yonemura, Shinsuke Aramaki, Kohei Hatta, Ki Young Bae, Hideaki Nojima and Masahiko Hibi : E-cadherin is required for gastrulation cell movements in zebrafish, Mechanisms of Development, 122, 6, 747-763, 2005.
(要約)
E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish.
Hideki Enomoto, Inna Hughes, Judith Golden, H. Robert Baloh, Shigenobu Yonemura, O. Robert Heuckeroth, M. Eugene Johnson and Jeffrey Milbrandt : GFRα1 expression in cells lacking ret is dispensable for organogenesis and nerve regeneration, Neuron, 44, 4, 623-636, 2004.
(要約)
The GDNF family ligands signal through a receptor complex composed of a ligand binding subunit, GFRalpha, and a signaling subunit, the RET tyrosine kinase. GFRalphas are expressed not only in RET-expressing cells, but also in cells lacking RET. A body of evidence suggests that RET-independent GFRalphas are important for (1) modulation of RET signaling in a non-cell-autonomous fashion (trans-signaling) and (2) regulation of NCAM function. To address the physiological significance of these roles, we generated mice specifically lacking RET-independent GFRalpha1. These mice exhibited no deficits in regions where trans-signaling has been implicated in vitro, including enteric neurons, motor neurons, kidney, and regenerating nerves. Furthermore, no abnormalities were found in the olfactory bulb, which requires proper NCAM function for its formation and is putatively a site of GDNF-GFRalpha-NCAM signaling. Thus RET-independent GFRalpha1 is dispensable for organogenesis and nerve regeneration in vivo, indicating that trans-signaling and GFRalpha-dependent NCAM signaling play a minor role physiologically.
Mitsuaki Fujimoto, Hanae Izu, Keisuke Seki, Ken Fukuda, Teruo Nishida, Ichi Shu Yamada, Kanefusa Kato, Shigenobu Yonemura, Sachiye Inouye and Akira Nakai : HSF4 is required for normal cell growth and differentiation during mouse lens development, EMBO Journal, 23, 21, 4297-4306, 2004.
(要約)
The heat shock transcription factor (HSF) family consists of three members in mammals and regulates expression of heat shock genes via a heat shock element. HSF1 and HSF2 are required for some developmental processes, but it is unclear how they regulate these processes. To elucidate the mechanisms of developmental regulation by HSFs, we generated mice in which the HSF4 gene is mutated. HSF4-null mice had cataract with abnormal lens fiber cells containing inclusion-like structures, probably due to decreased expression of gamma-crystallin, which maintains protein stability. Furthermore, we found increased proliferation and premature differentiation of the mutant lens epithelial cells, which is associated with increased expression of growth factors, FGF-1, FGF-4, and FGF-7. Unexpectedly, HSF1 competed with HSF4 for the expression of FGFs not only in the lens but also in other tissues. These findings reveal the lens-specific role of HSF4, which activates gamma-crystallin genes, and also indicate that HSF1 and HSF4 are involved in regulating expression of growth factor genes, which are essential for cell growth and differentiation.
Shigenobu Yonemura, Kazuyo Hirao-Minakuchi and Yukako Nishimura : Rho localization in cells and tissues, Experimental Cell Research, 295, 2, 300-314, 2004.
(要約)
Rho family small GTPases regulate cytoskeletal organization. Although their spatiotemporal activities appear to be important for cellular morphogenesis, there has been little characterization of the localization of Rho family GTPases in cells and tissues. Here we show precise localization of Rho subfamily proteins in mammalian cultured cells and tissues through evaluation of anti-Rho antibodies and fixation protocols. Although Rho is not a structural protein but functions as a switching molecule, it often localizes at several distinct domains or structures of cells. In cultured epithelial cells, Rho was highly accumulated at lateral membranes. However, in fibroblastic cells, Rho appeared to be distributed evenly in the cytoplasm. Rho concentration at the cleavage furrow at cytokinesis was generally observed. In A431 cells, Rho translocation from the cytoplasm to elongating microvilli at the apical membrane within 30 s after EGF stimulation was clearly demonstrated. Also, Myc- or GFP-tagged RhoA did not always reflect the localization of endogenous Rho, indicating a drawback of protein-tagging methods for localization research. In mouse tissues, Rho localization differed depending on cell type, probably reflecting the functional differences of each cell type.
During mitosis, the mitotic spindle, a bipolar structure composed of microtubules (MTs) and associated motor proteins, segregates sister chromatids to daughter cells. Initially some MTs emanating from one centrosome attach to the kinetochore at the centromere of one of the duplicated chromosomes. This attachment allows rapid poleward movement of the bound chromosome. Subsequent attachment of the sister kinetochore to MTs growing from the other centrosome results in the bi-orientation of the chromosome, in which interactions between kinetochores and the plus ends of MTs are formed and stabilized. These processes ensure alignment of chromosomes during metaphase and their correct segregation during anaphase. Although many proteins constituting the kinetochore have been identified and extensively studied, the signalling responsible for MT capture and stabilization is unclear. Small GTPases of the Rho family regulate cell morphogenesis by organizing the actin cytoskeleton and regulating MT alignment and stabilization. We now show that one member of this family, Cdc42, and its effector, mDia3, regulate MT attachment to kinetochores.
Keisuke Hamada, Toshiyuki Shimizu, Shigenobu Yonemura, Shoichiro Tsukita, Sachiko Tsukita and Toshio Hakoshima : Structural basis of adhesion-molecule recognition by ERM proteins revealed by the crystal structure of the radixin-ICAM-2 complex, EMBO Journal, 22, 3, 502-514, 2003.
(要約)
ERM (ezrin/radixin/moesin) proteins recognize the cytoplasmic domains of adhesion molecules in the formation of the membrane-associated cytoskeleton. Here we report the crystal structure of the radixin FERM (4.1 and ERM) domain complexed with the ICAM-2 cytoplasmic peptide. The non-polar region of the ICAM-2 peptide contains the RxxTYxVxxA sequence motif to form a beta-strand followed by a short 3(10)-helix. It binds the groove of the phosphotyrosine-binding (PTB)-like subdomain C mediated by a beta-beta association and several side-chain interactions. The binding mode of the ICAM-2 peptide to the FERM domain is distinct from that of the NPxY motif-containing peptide binding to the canonical PTB domain. Mutation analyses based on the crystal structure reveal the determinant elements of recognition and provide the first insights into the physical link between adhesion molecules and ERM proteins.
Shojiro Kikuchi, Masaki Hata, Kanehisa Fukumoto, Yukari Yamane, Takeshi Matsui, Atsushi Tamura, Shigenobu Yonemura, Hisakazu Yamagishi, Dietrich Keppler, Shoichiro Tsukita and Sachiko Tsukita : Radixin deficiency causes conjugated hyperbilirubinemia with loss of Mrp2 from bile canalicular membranes, Nature Genetics, 31, 3, 320-325, 2002.
(要約)
The ezrin-radixin-moesin (ERM) family of proteins crosslink actin filaments and integral membrane proteins. Radixin (encoded by Rdx) is the dominant ERM protein in the liver of wildtype mice and is concentrated at bile canalicular membranes (BCMs). Here we show that Rdx(-/-) mice are normal at birth, but their serum concentrations of conjugated bilirubin begin to increase gradually around 4 weeks, and they show mild liver injury after 8 weeks. This phenotype is similar to human conjugated hyperbilirubinemia in Dubin-Johnson syndrome, which is caused by mutations in the multidrug resistance protein 2 (MRP2, gene symbol ABCC2), although this syndrome is not associated with overt liver injury. In wildtype mice, Mrp2 concentrates at BCMs to secrete conjugated bilirubin into bile. In the BCMs of Rdx(-/-) mice, Mrp2 is decreased compared with other BCM proteins such as dipeptidyl peptidase IV (CD26) and P-glycoproteins. In vitro binding studies show that radixin associates directly with the carboxy-terminal cytoplasmic domain of human MRP2. These findings indicate that radixin is required for secretion of conjugated bilirubin through its support of Mrp2 localization at BCMs.
Shigenobu Yonemura, Takeshi Matsui, Shoichiro Tsukita and Sachiko Tsukita : Rho-dependent and -indenpendent activation mechanisms of ezrin/radixin/moesin proteins: An essential role for polyphosphoinositides in vivo, Journal of Cell Science, 115, 12, 2569-2580, 2002.
(要約)
Ezrin/radixin/moesin (ERM) proteins crosslink actin filaments to plasma membranes and are involved in the organization of the cortical cytoskeleton, especially in the formation of microvilli. ERM proteins are reported to be activated as crosslinkers in a Rho-dependent manner and are stabilized when phosphorylated at their C-terminal threonine residue to create C-terminal threonine-phosphorylated ERM proteins (CPERMs). Using a CPERM-specific mAb, we have shown, in vivo, that treatment with C3 transferase (a Rho inactivator) or staurosporine (a protein kinase inhibitor) leads to the dephosphorylation of CPERMs, the translocation of ERM proteins from plasma membranes to the cytoplasm and microvillar breakdown. We further elucidated that ERM protein activation does not require C-terminal phosphorylation in A431 cells stimulated with epidermal growth factor. In certain types of kidney-derived cells such as MDCK cells, however, ERM proteins appear to be activated in the absence of Rho activation and remain active without C-terminal phosphorylation. Interestingly, microinjection of an aminoglycoside antibiotic, neomycin, which binds to polyphosphoinositides, such as phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)], affected the activation of ERM proteins regardless of cell type. These findings not only indicate the existence of a Rho-independent activation mechanism of ERM proteins but also suggest that both Rho-dependent and -independent activation of ERM proteins require a local elevation of PtdIns(4,5)P(2) concentration in vivo.
(キーワード)
ERM protein / Microvilli / Neomycin / Ptdlns(4,5)P2 / Rho
Junji Fujikura, Eiji Yamato, Shigenobu Yonemura, Kiminori Hosoda, Shinji Masui, Kazuwa Nakao, Ichi Jun Miyazaki and Hitoshi Niwa : Differentiation of embryonic stem cells is induced by GATA factors, Genes and Development, 16, 7, 784-789, 2002.
(要約)
Extraembryonic endoderm (ExE) is differentiated from the inner cell mass of the late blastocyst-stage embryo to form visceral and parietal endoderm, both of which have an important role in early embryogenesis. The essential roles of Gata-6 and Gata-4 on differentiation of visceral endoderm have been identified by analyses of knockout mice. Here we report that forced expression of either Gata-6 or Gata-4 in embryonic stem (ES) cells is sufficient to induce the proper differentiation program towards ExE. We believe that this is the first report of a physiological differentiation event induced by the ectopic expression of a transcription factor in ES cells.
M. Eda, Shigenobu Yonemura, T. Kato, N. Watanabe, T. Ishizaki, P. Madaule and S. Narumiya : Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividing cells, Journal of Cell Science, 114, 18, 3273-3284, 2001.
(要約)
Citron-kinase (Citron-K) is a Rho effector working in cytokinesis. It is enriched in cleavage furrow, but how Rho mobilizes Citron-K remains unknown. Using anti-Citron antibody and a Citron-K Green Fluorescence Protein (GFP)-fusion, we monitored its localization in cell cycle. We have found: (1) Citron-K is present as aggregates in interphase cells, disperses throughout the cytoplasm in prometaphase, translocates to cell cortex in anaphase and accumulates in cleavage furrow in telophase; (2) Rho colocalizes with Citron-K in the cortex of ana- to telophase cells and the two proteins are concentrated in the cleavage furrow and to the midbody; (3) inactivation of Rho by C3 exoenzyme does not affect the dispersion of Citron-K in prometaphase, but prevented its transfer to the cell cortex, and Citron-K stays in association with the midzone spindles of C3 exoenzyme-treated cells. To clarify further the mechanism of the Rho-mediated transfer and concentration of Citron-K in cleavage furrow, we expressed active Val14RhoA in interphase cells expressing GFP-Citron-K. Val14RhoA expression transferred Citron-K to the ventral cortex of interphase cells, where it formed band-like structures in a complex with Rho. This structure was localized at the same plane as actin stress fibers, and they exclude each other. Disruption of F-actin abolished the band and dispersed the Citron-K-Rho-containing patches throughout the cell cortex. Similarly, in dividing cells, a structure composed of Rho and Citron-K in cleavage furrow excludes cortical actin cytoskeleton, and disruption of F-actin disperses Citron-K throughout the cell cortex. These results suggest that Citron-K is a novel type of a passenger protein, which is dispersed to the cytoplasm in prometaphase and associated with midzone spindles by a Rho-independent signal. Rho is then activated, binds to Citron-K and translocates it to cell cortex, where the complex is then concentrated in the cleavage furrow by the action of actin cytoskeleton beneath the equator of dividing cells.
Takeshi Matsui, Shigenobu Yonemura, Shoichiro Tsukita and Sachiko Tsukita : Activation of ERM proteins in vivo by Rho involves phosphatidylinositol 4-phosphate 5-kinase and not ROCK kinases, Current Biology, 9, 21, 1259-1262, 1999.
(要約)
When activated, ERM (ezrin, radixin, moesin) proteins are recruited to the plasma membrane, with concomitant carboxy-terminal threonine phosphorylation, where they crosslink actin filaments to the plasma membrane to form microvilli (reviewed in [1] [2] [3] [4] [5]). Here, we report that, when NIH3T3 or HeLa cells were transfected with a constitutively active mutant of the small GTPase RhoA (V14RhoA), microvilli were induced and the level of carboxy-terminal threonine-phosphorylated ERM proteins (CPERM) [6] [7] increased approximately 30-fold. This increase was not observed following transfection of constitutively active forms of two other Rho-family GTPases, Rac1 and Cdc42, or of a direct effector of Rho, Rho-kinase (also known as ROKalpha or ROCK-II) [8] [9] [10]. The V14RhoA-induced phosphorylation of ERM proteins was not suppressed by Y-27632, a specific inhibitor of ROCK kinases including Rho-kinase [11]. Overexpression of another direct effector of Rho, phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) type Ialpha [12] [13] [14], but not a kinase-inactive mutant [15], increased approximately sixfold the level of CPERM, and induced microvilli. Together with the previous finding that the PI4P5K product phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates ERM proteins in vitro [16], our data suggest that PIP(2), and not ROCK kinases, is involved in the RhoA-dependent activation of ERM proteins in vivo. The active state of ERM proteins is maintained through threonine phosphorylation by as yet undetermined kinases, leading to microvillus formation.
Noriyuki Sonoda, Mikio Furuse, Hiroyuki Sasaki, Shigenobu Yonemura, Jun Katahira, Yasuhiko Horiguchi and Shoichiro Tsukita : Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier, Journal of Cell Biology, 147, 1, 195-204, 1999.
(要約)
Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.
Shigenobu Yonemura, Sachiko Tsukita and Shoichiro Tsukita : Direct involvement of ezrin/radixin/moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins, Journal of Cell Biology, 145, 7, 1497-1509, 1999.
(要約)
Ezrin/radixin/moesin (ERM) proteins have been thought to play a central role in the organization of cortical actin-based cytoskeletons including microvillar formation through cross-linking actin filaments and integral membrane proteins such as CD43, CD44, and ICAM-2. To examine the functions of these ERM-binding membrane proteins (ERMBMPs) in cortical morphogenesis, we overexpressed ERMBMPs (the extracellular domain of E-cadherin fused with the transmembrane/cytoplasmic domain of CD43, CD44, or ICAM-2) in various cultured cells. In cultured fibroblasts such as L and CV-1 cells, their overexpression significantly induced microvillar elongation, recruiting ERM proteins and actin filaments. When the ERM-binding domains were truncated from these molecules, their ability to induce microvillar elongation became undetectable. In contrast, in cultured epithelial cells such as MTD-1A and A431 cells, the overexpression of ERMBMPs did not elongate microvilli. However, in the presence of EGF, overexpression of ERMBMPs induced remarkable microvillar elongation in A431 cells. These results indicated that ERMBMPs function as organizing centers for cortical morphogenesis by organizing microvilli in collaboration with activated ERM proteins. Furthermore, immunodetection with a phosphorylated ERM-specific antibody and site-directed mutagenesis suggested that ERM proteins phosphorylated at their COOH-terminal threonine residue represent activated ERM proteins.
Yuhko Ando-Akatsuka, Shigenobu Yonemura, Masahiko Itoh, Mikio Furuse and Shoichiro Tsukita : Differential behavior of E-cadherin and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity, Journal of Cellular Physiology, 179, 2, 115-125, 1999.
(要約)
At the initial stage of cell-cell contact of epithelial cells, primordial spot-like junctions are formed at the tips of thin cellular protrusions radiating from adjacent cells, where E-cadherin and ZO-1 are precisely coconcentrated (Yonemura et al., 1995, J. Cell Sci. 108:127-142). In fully polarized epithelial cells, E-cadherin and ZO-1 are completely sorted into belt-like adherens junctions (AJ) and tight junctions (TJ), respectively. Here we examined the behavior of occludin, an integral membrane protein consisting of TJ, during the establishment of epithelial cell polarity. Using confocal immunofluorescence microscopy, we quantitatively compared the spatial relationship of occludin/ZO-1 with that of E-cadherin/ZO-1 during epithelial cellular polarization by replating or wounding cultured mouse epithelial cells (MTD1-A). At the initial stage of cell-cell contact, E-cadherin and ZO-1 appeared to be simultaneously recruited to the primordial form of spot-like junctions at the tips of cellular processes which showed no concentration of occludin. Then, as cellular polarization proceeded, occludin was gradually accumulated at the ZO-1-positive spot-like junctions to form belt-like TJ, and in a complementary manner E-cadherin was sorted out from the ZO-1-positive spot-like junctions to form belt-like AJ. The molecular mechanism of TJ/AJ formation during epithelial cellular polarization is discussed with special reference to the roles of ZO-1.
Ken Hayashi, Shigenobu Yonemura, Takeshi Matsui, Sachiko Tsukita and Shoichiro Tsukita : Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues. Application of a novel fixation protocol using trichloroacetic acid (TCA) as a fixative, Journal of Cell Science, 112, 8, 1149-1158, 1999.
(要約)
Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.
Yoshinori Doi, Masahiko Itoh, Shigenobu Yonemura, Satoru Ishihara, Hiroshi Takano, Tetsuo Noda, Shoichiro Tsukita and Sachiko Tsukita : Normal development of mice and unimpaired cell adhesion/cell motility/actin-based cytoskeleton without compensatory up-regulation of ezrin or radixin in moesin gene knockout, Journal of Biological Chemistry, 274, 4, 2315-2321, 1999.
(要約)
Ezrin/radixin/moesin (ERM) proteins are general cross-linkers between the plasma membrane and actin filaments. Because their expression is regulated in a tissue-specific manner, each ERM protein has been proposed to have unique functions. On the other hand, experiments at the cellular level and in vitro have suggested their functional redundancy. To assess the possible unique functions of ERM proteins in vivo, the moesin gene located on the X chromosome was disrupted by gene targeting in embryonic stem cells. Male mice hemizygous for the mutation as well as homozygous females were completely devoid of moesin but developed normally and were fertile, with no obvious histological abnormalities in any of the tissues examined. In the tissues of the mutant mice, moesin completely disappeared without affecting the expression levels or subcellular distribution of ezrin and radixin. Also, in platelets, fibroblasts, and mast cells isolated from moesin-deficient mice, targeted disruption of the moesin gene did not affect their ERM-dependent functions, i.e. platelet aggregation, stress fiber/focal contact formation of fibroblasts, and microvillar formation of mast cells, without compensatory up-regulation of ezrin or radixin. These findings favor the notion that ERM proteins are functionally redundant at the cellular as well as the whole body level.
Shigenobu Yonemura, Motohiro Hirao, Yoshinori Doi, Nobuyuki Takahashi, Takahisa Kondo, Sachiko Tsukita and Shoichiro Tsukita : Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2, Journal of Cell Biology, 140, 4, 885-895, 1998.
(要約)
CD44 has been identified as a membrane-binding partner for ezrin/radixin/moesin (ERM) proteins, plasma membrane/actin filament cross-linkers. ERM proteins, however, are not necessarily colocalized with CD44 in tissues, but with CD43 and ICAM-2 in some types of cells. We found that glutathione-S-transferase fusion proteins with the cytoplasmic domain of CD43 and ICAM-2, as well as CD44, bound to moesin in vitro. The regions responsible for the in vitro binding of CD43 and CD44 to moesin were narrowed down to their juxta-membrane 20-30-amino acid sequences in the cytoplasmic domain. These sequences and the cytoplasmic domain of ICAM-2 (28 amino acids) were all characterized by the positively charged amino acid clusters. When E-cadherin chimeric molecules bearing these positively charged amino acid clusters of CD44, CD43, or ICAM-2 were expressed in mouse L fibroblasts, they were co-concentrated with ERM proteins at microvilli, whereas those lacking these clusters were diffusely distributed on the cell surface. The specific binding of ERM proteins to the juxta-membrane positively charged amino acid clusters of CD44, CD43, and ICAM-2 was confirmed by immunoprecipitation and site-directed mutagenesis. From these findings, we conclude that ERM proteins bind to integral membrane proteins bearing a positively charged amino acid cluster in their juxta-membrane cytoplasmic domain.
Takeshi Matsui, Masato Maeda, Yoshinori Doi, Shigenobu Yonemura, Mutsuki Amano, Kozo Kaibuchi, Sachiko Tsukita and Shoichiro Tsukita : Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association, Journal of Cell Biology, 140, 3, 647-657, 1998.
(要約)
The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and approximately 30 and approximately 100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase-dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase-dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.
Takahisa Kondo, Kosei Takeuchi, Yoshinori Doi, Shigenobu Yonemura, Shigekazu Nagata, Shoichiro Tsukita and Sachiko Tsukita : ERM (ezrin/radixin/moesin)-based molecular mechanism of microvillar breakdown at an early stage of apoptosis, Journal of Cell Biology, 139, 3, 749-758, 1997.
(要約)
Breakdown of microvilli is a common early event in various types of apoptosis, but its molecular mechanism and implications remain unclear. ERM (ezrin/radixin/moesin) proteins are ubiquitously expressed microvillar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and function as general actin filament/plasma membrane cross-linkers to form microvilli. Immunofluorescence microscopic and biochemical analyses revealed that, at the early phase of Fas ligand (FasL)-induced apoptosis in L cells expressing Fas (LHF), ERM proteins translocate from the plasma membranes of microvilli to the cytoplasm concomitant with dephosphorylation. When the FasL-induced dephosphorylation of ERM proteins was suppressed by calyculin A, a serine/threonine protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins was blocked. The interleukin-1beta-converting enzyme (ICE) protease inhibitors suppressed the dephosphorylation as well as the cytoplasmic translocation of ERM proteins. These findings indicate that during FasL-induced apoptosis, the ICE protease cascade was first activated, and then ERM proteins were dephosphorylated followed by their cytoplasmic translocation, i.e., microvillar breakdown. Next, to examine the subsequent events in microvillar breakdown, we prepared DiO-labeled single-layered plasma membranes with the cytoplasmic surface freely exposed from FasL-treated or nontreated LHF cells. On single-layered plasma membranes from nontreated cells, ERM proteins and actin filaments were densely detected, whereas those from FasL-treated cells were free from ERM proteins or actin filaments. We thus concluded that the cytoplasmic translocation of ERM proteins is responsible for the microvillar breakdown at an early phase of apoptosis and that the depletion of ERM proteins from plasma membranes results in the gross dissociation of actin-based cytoskeleton from plasma membranes. The physiological relevance of this ERM protein-based microvillar breakdown in apoptosis will be discussed.
Sachiko Tsukita, Shigenobu Yonemura and Shoichiro Tsukita : ERM (ezrin/radixin/moesin) family: From cytoskeleton to signal transduction, Current Opinion in Cell Biology, 9, 1, 70-75, 1997.
(要約)
The ERM family consists of three closely related proteins, ezrin, radixin, and moesin, that are thought to work as cross-linkers between plasma membranes and actin-based cytoskeletons. Recent analyses of the structure and functions of ERM proteins have revealed that these molecules are involved not only in cytoskeletal organization but also in signal transduction. Furthermore, identification of the neurofibromatosis type 2 tumour suppressor, which shows striking sequence similarity to ERM proteins, has increased interest in this family.
Motohiro Hirao, Naruki Sato, Takahisa Kondo, Shigenobu Yonemura, Morito Monden, Takuya Sasaki, Yoshimi Takai, Shoichiro Tsukita and Sachiko Tsukita : Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: Possible involvement of phosphatidylinositol turnover and rho-dependent signaling pathway, Journal of Cell Biology, 135, 1, 37-51, 1996.
(要約)
The ERM proteins, ezrin, radixin, and moesin, are involved in the actin filament/plasma membrane interaction as cross-linkers. CD44 has been identified as one of the major membrane binding partners for ERM proteins. To examine the CD44/ERM protein interaction in vitro, we produced mouse ezrin, radixin, moesin, and the glutathione-S-transferase (GST)/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) by means of recombinant baculovirus infection, and constructed an in vitro assay for the binding between ERM proteins and the cytoplasmic domain of CD44. In this system, ERM proteins bound to GST-CD44cyt with high affinity (Kd of moesin was 9.3 +/- 1.6nM) at a low ionic strength, but with low affinity at a physiological ionic strength. However, in the presence of phosphoinositides (phosphatidylinositol [PI], phosphatidylinositol 4-monophosphate [4-PIP], and phosphatidylinositol 4.5-bisphosphate [4,5-PIP2]), ERM proteins bound with a relatively high affinity to GST-CD44cyt even at a physiological ionic strength: 4,5-PIP2 showed a marked effect (Kd of moesin in the presence of 4,5-PIP2 was 9.3 +/- 4.8 nM). Next, to examine the regulation mechanism of CD44/ERM interaction in vivo, we reexamined the immunoprecipitated CD44/ERM complex from BHK cells and found that it contains Rho-GDP dissociation inhibitor (GDI), a regulator of Rho GTPase. We then evaluated the involvement of Rho in the regulation of the CD44/ERM complex formation. When recombinant ERM proteins were added and incubated with lysates of cultured BHK cells followed by centrifugation, a portion of the recombinant ERM proteins was recovered in the insoluble fraction. This binding was enhanced by GTP gamma S and markedly suppressed by C3 toxin, a specific inhibitor of Rho, indicating that the GTP form of Rho in the lysate is required for this binding. A mAb specific for the cytoplasmic domain of CD44 also markedly suppressed this binding, identifying most of the binding partners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, in living BHK cells treated with C3 toxin, most insoluble ERM proteins moved to soluble compartments in the cytoplasm, leaving CD44 free from ERM. These findings indicate that Rho regulates the CD44/ERM complex formation in vivo and that the phosphatidylinositol turnover may be involved in this regulation mechanism.
Yuhko Ando-Akatsuka, Mitinori Saitou, Tetsuaki Hirase, Masashi Kishi, Akira Sakakibara, Masahiko Itoh, Shigenobu Yonemura, Mikio Furuse and Shoichiro Tsukita : Interspecies diversity of the occludin sequence: CDNA cloning of human, mouse, dog, and rat-kangaroo homologues, Journal of Cell Biology, 133, 1, 43-47, 1996.
(要約)
Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions (Furuse, M., T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 123:1777-1788). To analyze and modulate the functions of tight junctions, it would be advantageous to know the mammalian homologues of occludin and their genes. Here we describe the nucleotide sequences of full length cDNAs encoding occludin of rat-kangaroo (potoroo), human, mouse, and dog. Rat-kangaroo occludin cDNA was prepared from RNA isolated from PtK2 cell culture, using a mAb against chicken occludin, whereas the others were amplified by polymerase chain reaction based on the sequence found around the human neuronal apoptosis inhibitory protein gene. The amino acid sequences of the three mammalian (human, murine, and canine) occludins were very closely related to each other (approximately 90% identity), whereas they diverged considerably from those of chicken and rat-kangaroo (approximately 50% identity). Implications of these data and novel experimental options in cell biological research are discussed.
Noriko Tokai, Akiko Fujimoto-Nishiyama, Yoko Toyoshima, Shigenobu Yonemura, Shoichiro Tsukita, Junichiro Inoue and Tadashi Yamamoto : Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle, EMBO Journal, 15, 3, 457-467, 1996.
(要約)
Microtubule-associated motor proteins are thought to be involved in spindle formation and chromosome movements in mitosis/meiosis. We have molecularly cloned cDNAs for a gene that codes for a novel member of the kinesin family of proteins. Nucleotide sequencing reveals that the predicted gene product is a 73 kDa protein and is related to some extent to the Drosophila node gene product, which is involved in chromosomal segregation during meiosis. A sequence similar to the microtubule binding motor domain of kinesin is present in the N-terminal half of the protein, and its ability to bind to microtubules is demonstrated. Furthermore we show that its C-terminal half contains a putative nuclear localization signal similar to that of Jun and is able to bind to DNA. Accordingly, the protein was termed Kid (kinesin-like DNA binding protein). Indirect immunofluorescence studies show that Kid colocalizes with mitotic chromosomes and that it is enriched in the kinetochore at anaphase. Thus, we propose that Kid might play a role(s) in regulating the chromosomal movement along microtubules during mitosis.
(キーワード)
Chromosome segregation / Kinesin family / Kinetochore / Mitosis
Hiroshi Takeda, Akira Nagafuchi, Shigenobu Yonemura, Sachiko Tsukita, Jürgen Behrens, Walter Birchmeier and Shoichiro Tsukita : V-src kinase shifts the cadherin-based cell adhesion from the strong to the weak state and β catenin is not required for the shift, Journal of Cell Biology, 131, 6 II, 1839-1847, 1995.
(要約)
The elevation of tyrosine phosphorylation level is thought to induce the dysfunction of cadherin through the tyrosine phosphorylation of beta catenin. We evaluated this assumption using two cell lines. First, using temperature-sensitive v-src-transfected MDCK cells, we analyzed the modulation of cadherin-based cell adhesion by tyrosine phosphorylation. Cell aggregation and dissociation assays at nonpermissive and permissive temperatures indicated that elevation of the tyrosine phosphorylation does not totally affect the cell adhesion ability of cadherin but shifts it from a strong to a weak state. The tyrosine phosphorylation levels of beta catenin, ZO-1, ERM (ezrin/radixin/moesin), but not alpha catenin, vinculin, and alpha-actinin, were elevated in the weak state. To evaluate the involvement of the tyrosine phosphorylation of beta catenin in this shift of cadherin-based cell adhesion, we introduced v-src kinase into L fibroblasts expressing the cadherin-alpha catenin fusion protein, in which beta catenin is not involved in cell adhesion. The introduction of v-src kinase in these cells shifted their adhesion from a strong to a weak state. These findings indicated that the tyrosine phosphorylation of beta catenin is not required for the strong-to-weak state shift of cadherin-based cell adhesion, but that the tyrosine phosphorylation of other junctional proteins, ERM, ZO-1 or unidentified proteins is involved.
Junji Sagara, Sachiko Tsukita, Shigenobu Yonemura, Shoichiro Tsukita and Akihiko Kawai : Cellular actin-binding ezrin-radixin-moesin (ERM) family proteins are incorporated into the rabies virion and closely associated with viral envelope proteins in the cell, Virology, 206, 1, 485-494, 1995.
(要約)
Cellular ezrin-radixin-moesin (ERM) family proteins, members of the actin-binding proteins of the band 4.1 superfamily, were detected in the virions of enveloped viruses, such as rabies, vesicular stomatitis, Newcastle disease, and influenza viruses. To elucidate the mechanism of ERM protein incorporation, we investigated possible association of ERM proteins with viral components in rabies virus-infected BHK-21 cells. Double immunofluorescence studies demonstrated that the ERM proteins are concentrated in the microvilli, where the colocalized viral G protein was also seen. Viral G protein expressed in the G cDNA-transfected COS-7 cells also displayed similar distributions to those seen in the virus-infected cells. Both the ERM and viral envelope proteins were coprecipitated by anti-viral G antibody from lysates of the virus-infected cells, while the anti-ERM antibody coprecipitated viral G and ERM proteins. These observations suggest that the ERM proteins are closely associated with viral envelope proteins in the cell, which would be involved in the selective incorporation of cellular actin into the virion.
Shigenobu Yonemura, Masahiko Itoh, Akira Nagafuchi and Shoichiro Tsukita : Cell-to-cell adherens junction formation and actin filament organization: Similarities and differences between non-polarized fibroblasts and polarized epithelial cells, Journal of Cell Science, 108, 1, 127-142, 1995.
(要約)
Cadherin has an intimate spatial relationship with actin filaments (AF) in various types of cells, forming the cell-to-cell adherens junction (AJ). We compared the AJ/AF relationship between non-polarized fibroblasts (NRK cells) and polarized epithelial cells (MTD-1A cells). E/P-cadherin, alpha-catenin, ZO-1 and vinculin were localized with reference to AF in these cells using laser scan microscopy as well as conventional light and electron microscopy. NRK cells adhered to each other at the tips of thin cellular processes, where spot-like AJ were formed, where P-cadherin, alpha-catenin, ZO-1 and vinculin were concentrated. Some stress-fiber-like AF bundles ran axially in these processes and terminated at spot-like AJ on their tips. At the electron microscopic level these spot-like AJ were seen as aggregates of small 'units' of AJ, where AF were densely and perpendicularly associated with the plasma membrane. In MTD-1A cells, the AJ/AF relationship was investigated during the cell polarization process after replating or wounding. At the early stage, the AJ/AF relationship was quite similar to that in NRK cells. As polarization proceeded, the spot-like AJs were gradually fused side by side with the concomitant shortening of the associated stress-fiber-like AF bundles. Finally, the belt-like AJ was established, which was lined with circumferential AF bundles. The similarities and differences in the AJ/AF relationship between non-polarized fibroblasts and polarized epithelial cells are discussed.
Mikio Furuse, Masahiko Itoh, Tetsuaki Hirase, Akira Nagaftichi, Shigenobu Yonemura, Sachiko Tsukita and Shoichiro Tsukita : Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions, Journal of Cell Biology, 127, 6 I, 1617-1626, 1994.
(要約)
Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.
Kosei Takeuchi, Naruki Sato, Hideko Kasahara, Noriko Funayama, Akira Nagafuchi, Shigenobu Yonemura, Sachiko Tsukita and Shoichiro Tsukita : Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members, Journal of Cell Biology, 125, 6, 1371-1384, 1994.
(要約)
To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.
T. Kishino, T. Ariga, H. Soejima, T. Tamura, T. Ohta, Y. Jinno, Shigenobu Yonemura, N. Sato, S. Tsukita, S. Tsukita, Y. Sakiyama and N. Niikawa : Assignment of the human moesin gene (MSN) to chromosome region Xq11.2 → q12, Cytogenetics and Cell Genetics, 66, 3, 167-169, 1994.
(要約)
The human moesin gene (MSN) was mapped to the long arm of the X chromosome. PCR products for the moesin gene cDNA were used as probes to isolate their corresponding cosmid clones. Fluorescence in situ hybridization (FISH) with two of the isolated cosmid probes showed signals at Xq11.2-->q12, whereas four other cosmids showed FISH signals on chromosome 5. Southern blot hybridization, using a PCR product corresponding to the 3' region of the moesin gene cDNA as a probe (probe-3), on one of the two cosmids that produced signals on the X chromosome gave 5.7- and 3.5-kb HindIII fragments. Further Southern hybridization of the DNA from XY, XX, and XXXXX individuals using probe-3 revealed a gene-dose effect of the X chromosome on the size of a 3.5-kb and a 3.0-kb HindIII fragment; in contrast, an invariant 9.8-kb band was present in the DNA of all individuals tested. Sequencing of an exon-intron border revealed that the two cosmids had predicted sequences. These results indicated that the two cosmids contained MSN, and it was consequently assigned to human chromosome region Xq11.2-->q12. These results strongly suggest that MSN may be removed from candidacy for Wiskott-Aldrich syndrome, which has been putatively mapped to Xp11.3-->p11.22.
(キーワード)
Chromosome Mapping / DNA, Complementary / Female / Genetic Linkage / Humans / In Situ Hybridization, Fluorescence / Male / Microfilament Proteins / Proteins / Wiskott-Aldrich Syndrome / X Chromosome
Mikio Furuse, Tetsuaki Hirase, Masahiko Itoh, Akira Nagafuchi, Shigenobu Yonemura, Sachiko Tsukita and Shoichiro Tsukita : Occludin: A novel integral membrane protein localizing at tight junctions, Journal of Cell Biology, 123, 6 PART 2, 1777-1788, 1993.
(要約)
Recently, we found that ZO-1, a tight junction-associated protein, was concentrated in the so called isolated adherens junction fraction from the liver (Itoh, M., A. Nagafuchi, S. Yonemura, T. Kitani-Yasuda, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 121:491-502). Using this fraction derived from chick liver as an antigen, we obtained three monoclonal antibodies specific for a approximately 65-kD protein in rats. This antigen was not extractable from plasma membranes without detergent, suggesting that it is an integral membrane protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this approximately 65-kD membrane protein was exclusively localized at tight junctions of both epithelial and endothelial cells: at the electron microscopic level, the labels were detected directly over the points of membrane contact in tight junctions. To further clarify the nature and structure of this membrane protein, we cloned and sequenced its cDNA. We found that the cDNA encoded a 504-amino acid polypeptide with 55.9 kDa. A search of the data base identified no proteins with significant homology to this membrane protein. A most striking feature of its primary structure was revealed by a hydrophilicity plot: four putative membrane-spanning segments were included in the NH2-terminal half. This hydrophilicity plot was very similar to that of connexin, an integral membrane protein in gap junctions. These findings revealed that an integral membrane protein localizing at tight junctions is now identified, which we designated as "occludin."
(キーワード)
Amino Acid Sequence / Animals / Antibodies, Monoclonal / Base Sequence / Brain / Chickens / DNA, Complementary / Epithelial Cells / Epithelium / Intercellular Junctions / Intestines / Liver / Membrane Proteins / Molecular Sequence Data / Myocardium / Occludin / Organ Specificity / Phosphoproteins / Protein Conformation / Protein Structure, Secondary / Restriction Mapping / Zonula Occludens-1 Protein
Masahiko Itoh, Akira Nagafuchi, Shigenobu Yonemura, Takako Kitani-Yasuda, Sachiko Tsukita and Shoichiro Tsukita : The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy, Journal of Cell Biology, 121, 3, 491-502, 1993.
(要約)
We previously identified a 220-kD constitutive protein of the plasma membrane undercoat which colocalizes at the immunofluorescence microscopic level with cadherins and occurs not only in epithelial M., S. Yonemura, A. Nagafuchi, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). To clarify the nature and possible functions of this protein, we cloned its full-length cDNA and sequenced it. Unexpectedly, we found mouse 220-kD protein to be highly homologous to rat protein ZO-1, only a part of which had been already sequenced. This relationship was confirmed by immunoblotting with anti-ZO-1 antibody. As protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, we analyzed the distribution of cadherins and the 220-kD protein by ultrathin cryosection immunoelectron microscopy. We found that in non-epithelial cells lacking tight junctions cadherins and the 220-kD protein colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions cadherins and the 220-kD protein are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, which tight junctions are not so well developed, the 220-kD protein is detected not only in the tight junction zone but also at adherens junctions. Furthermore, we show in mouse L cells transfected with cDNAs encoding N-, P-, E-cadherins that cadherins interact directly or indirectly with the 220-kD protein. Possible functions of the 220-kD protein (ZO-1) are discussed with special reference to the molecular mechanism for adherens and tight junction formation.
Shigenobu Yonemura, Akira Nagafuchi, Naruki Sato and Shoichiro Tsukita : Concentration of an integral membrane protein, CD43 (leukosialin, sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeletons, Journal of Cell Biology, 120, 2, 437-449, 1993.
(要約)
In leukocytes such as thymocytes and basophilic leukemia cells, a glycosilated integral membrane protein called CD43 (leukosialin or sialophorin), which is defective in patients with Wiskott-Aldrich syndrome, was highly concentrated in the cleavage furrow during cytokinesis. Not only at the mitotic phase but also at interphase, CD43 was precisely colocalized with ezrin-radixin-moesin family members. (ERM), which were previously reported to play an important role in the plasma membrane-actin filament association in general. At the electron microscopic level, throughout the cell cycle, both CD43 and ERM were tightly associated with microvilli, providing membrane attachment sites for actin filaments. We constructed a cDNA encoding a chimeric molecule consisting of the extracellular domain of mouse E-cadherin and the transmembrane/cytoplasmic domain of rat CD43, and introduced it into mouse L fibroblasts lacking both endogenous CD43 and E-cadherin. In dividing transfectants, the chimeric molecules were concentrated in the cleavage furrow together with ERM, and both proteins were precisely colocalized throughout the cell cycle. Furthermore, using this transfection system, we narrowed down the domain responsible for the CD43-concentration in the cleavage furrow. Based on these findings, we conclude that CD43 is concentrated in the cleavage furrow through the direct or indirect interaction of its cytoplasmic domain with ERM and actin filaments.
Shigenobu Yonemura : A mechanism of mechanotransduction at the cell-cell interface: Emergence of α-catenin as the center of a force-balancing mechanism for morphogenesis in multicellular organisms, Bioessays, 33, 10, 732-736, 2011.
In homeostatic epithelial tissues, the basement membrane appears to be a quiet, motionless structure. However, during embryonic development and tissue regeneration, the basement membrane dramatically changes its distribution and shows a variety of dynamics such as compositional transition and physicochemical alterations. Recently, it has been pointed out that the shape and function of epithelial tissues is greatly influenced by the way of formation and arrangement of the basement membrane. Here, we outline the current understanding of the roles of the basement membrane dynamics in epithelial morphogenesis, and briefly introduce our approach to visualize the movement of basement membrane components.
Shigenobu Yonemura : Actin filament association at adherens junctions, Journal of Medical Investigation, 64, 1-2, 14-19, 2017.
(要約)
The adherens junction (AJ) is a cadherin-based and actin filament associated cell-to-cell junction. AJs can contribute to tissue morphogenesis and homeostasis and their association with actin filaments is crucial for the functions. There are three types of AJs in terms of the mode of actin filament/AJ association. Among many actin-binding proteins associated with AJs, α-catenin is one of the most important actin filament/AJ linkers that functions in all types of AJs. Although α-catenin in cadherin-catenin complex appears to bind to actin filaments within cells, it fails to bind to actin filaments in vitro mysteriously. Recent report revealed that α-catenin in the complex can bind to actin filaments in vitro when forces are applied to the filament. In addition to force-sensitive vinculin binding, α-catenin has another force-sensitive property of actin filament-binding. Elucidation of its significance and the molecular mechanism is indispensable for understanding AJ formation and maintenance during tissue morphogenesis, function and repair. J. Med. Invest. 64: 14-19, February, 2017.
Shigenobu Yonemura : Cadherin-actin interactions at adherens junctions, Current Opinion in Cell Biology, 23, 5, 515-522, Jul. 2011.
(要約)
The adherens junction (AJ) is a major cell-cell junction that mediates cell recognition, adhesion, morphogenesis, and tissue integrity. Although AJs transmit forces generated by actomyosin from one cell to another, AJs have long been considered as an area where signal transduction from cadherin ligation takes place through cell adhesion. Through the efforts to understand embryonic or cellular morphogenesis, dynamic interactions between the AJ and actin filaments have become crucial issues to be addressed since actin association is essential for AJ development, remodeling and function. Here, I provide an overview of cadherin-actin interaction from morphological aspects and of possible molecular mechanisms revealed by recent studies.
Sachiko Tsukita, Shigenobu Yonemura and Shoichiro Tsukita : ERM proteins: Head-to-tail regulation of actin-plasma membrane interaction, Trends in Biochemical Sciences, 22, 2, 53-58, Feb. 1997.
(要約)
ERM (ezrin/radixin/moesin) proteins crosslink actin filaments with plasma membranes. The carboxyl termini of these proteins bind actin filaments, while the amino termini bind plasma membranes using a binding partner, such as CD44. Specific signals activate ERM proteins to bind actin filaments and the plasma membrane; these include phosphoinositides and/or phosphorylation mechanisms, which might be located downstream from the Rho-dependent pathway.
S. Tsukita, S. Tsukita, A. Nagafuchi, 米村 重信 : Molecular biology of intercellular junctions, Seikagaku the Journal of Japanese Biochemical Society, 67, 4, 279-289, 1995年4月.
S. Tsukita, S. Tsukita, A. Nagafuchi and Shigenobu Yonemura : Possible involvement of adherens junction plaque proteins in tumorigenesis and metastasis., Princess Takamatsu Symposia, 24, 38-50, 1994.
(要約)
The cell-to-cell adherens junction is a site for cadherin-mediated adhesion where actin filaments are densely associated with the plasma membrane through its well developed plasmalemmal undercoat, a "plaque" structure. Recently, we succeeded in isolating the cell-to-cell adherens junctions from rat liver, and in identifying some novel AJ plaque constituents, including src-like tyrosine kinases, radixin, alpha-catenin, and a 220kD protein. The application of genetic engineering techniques to AJ plaque proteins has recently generated a wealth of novel observations, leading to the speculation that these proteins are involved in tumorigenesis and metastasis. This paper reviews these findings and discusses some functions of the AJ plaque proteins in normal cells, as well as in tumorigenesis and metastasis.
Shigenobu Yonemura : Adjusting tension sensitivity of a-catenin for epithelial morphogenesis, The 51st NIPS Symposium Frontiers in Epithelial Cell Biology, Okazaki, Aichi, Japan, Dec. 2021.
2.
Shigenobu Yonemura : Adjusting Tension sensitivity of -catenin for epithelial morphogenesis, The 51st NIPS International Symposium "Frontiers in Epithelial Cell Biology", Dec. 2021.
3.
Shigenobu Yonemura : Force-sensing device in alpha-catenin,, Mechanobiology, from molecules to tissue, Jun. 2016.
4.
Y Enomoto, Y Inoue, Shigenobu Yonemura and T Apache : Three-dimensional vertex simulation on smooth surface maintenance of growing epithelial tissue based on intercellular mechano-feedback, Biophysical Society 60th Annual Meeting, Los Angeles, California, USA, Feb. 2016.
Ichi Ken Wada, Kazuyoshi Itoga, Teruo Okano, Shigenobu Yonemura and Hiroshi Sasaki : Analysis for effect of cell shape on hippo signaling pathway using micro-fabricated cell culture platform, Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences Microtas 2012, 1051-1053, Jan. 2012.
栗栖 修作, Callista Chan Ying Yi, 米村 重信 : Molecular stoichiometry of mechanosensory proteins at adherens junction, and its implication in epithelial collective cell migration, Joint Meeting of JSCB 77th and JSDB 58th, 2025年7月.
2.
甘 泉, 米村 重信 : Force Sensitivity of α-Catenin Regulates Junction Formation Process in Epithelial Cells, Joint Meeting of JSCB 77th and JSDB 58th, 2025年7月.
3.
Li Shuntao, Shusaku Kurisu and Shigenobu Yonemura : G12/13 family of G protein is essential for epithelial apical junction network formation, Joint Meeting of JSCB 77th and JSDB 58th, Jul. 2025.
4.
浅野 千帆莉, 柴田 桂太朗, 米村 重信 : Visualization and Control of the Mechanical Network Driving Collective Cell Migration, Joint Meeting of JSCB 77th and JSDB 58th, 2025年7月.
5.
Yi Chan Ying Callista, Shusaku Kurisu and Shigenobu Yonemura : Force Sensitivity of α-Catenin Regulates Branching Tubulogenesis in MDCK II Cells, oint Meeting of JSCB 77th and JSDB 58th, Jul. 2025.
6.
Keitaro Shibata and Shigenobu Yonemura : Directional control of Collective Cell Migration by CD44 Dependent on Hyaluronic Acid, Joint Meeting of JSCB 77th and JSDB 58th, Jul. 2025.
Yin Chan Callista, Shusaku Kurisu and Shigenobu Yonemura : Force sensitivity in alpha-catenin mutants governs tubulogenesis variability in MDCK II cells., 第9回日本メカノバイオロジー学会学術総会, Mar. 2025.
Shusaku Kurisu and Shigenobu Yonemura : The roles of cortical actin rings in the establishment of epithelial cell polarity, The 126th Annual Meeting of The Japanese Association of Anatomists, The 98th Annual Meeting of The Physiological Society of Japan, Mar. 2021.
本田 尚三, 名黒 功, 米村 重信 : Analyses of SH3BP4, a novel candidate gene, found by genome-wide RNAi screening for apicobasal polarityrelated factors, 第72回日本細胞生物学会大会, 2020年6月.
32.
米村 重信, 栗栖 修作，米村 重信 : Protein quanti cation at epithelial cell-cell junctions reveals a differential distribution of the actin-associated components, 第72回日本細胞生物学会大会, 2020年6月.
Shozo Honda, Isao Naguro and Shigenobu Yonemura : Development of the RNAi screening system for apicobasal polarity factors, Joint Annual Meeting of JSDB and JSCB, 70, 3, 12, Jun. 2018.
(要約)
Epithelial cells have apicobasal polarity, which shows the asymmetric distributions of proteins between apical and basolateral surface within single cell that produce asymmetric cellular function to maintain homeostasis in the body. Therefore, apicobasal polarity is essential for multicellular organisms and its disturbance causes various diseases including cancer. Some apicobasal polarity factors have been identified in model animals through genetic studies. However, the comprehensive molecular system remains to be clarified. RNAi screening for apicobasal polarity using culturable epithelial cells has not been performed because of the robustness of epithelial sheets that protects cells from disturbance of the polarity factors. Here, we develop a screening system for apicobasal polarity factors using α-catenin-deficient R2/7 cells that are not capable of forming cell-cell adhesion. Intriguingly, R2/7 cells form the apicobasal polarity: aPKC, SCRIB and ZO-1 were localized to apical, basolateral and the boundary region, respectively. In particular, ZO-1 was observed as ring just below plasma membrane. Knockdown of known apical factors shrank the ZO-1 ring, whereas knockdown of known basolateral factors enlarged the ring. Therefore, the ZO-1 ring in R2/7 would be a good marker for sensitive detection of the change in apicobasal polarity. We established an algorithm for the detection and estimation of ZO-1 area that was ensured as acceptable assay (Z´-factor>0.5). Then we performed the screening using siRNA library (18,152 genes) and high-throughput image analysis. Using bioinformatic analyses, we could extract 1,263 and 422 candidate genes showing shrinking and enlarging type, respectively. Now, we are executing biochemical analyses for novel molecules to clarify comprehensive molecular system of the apicobasal polarity.
40.
Shusaku Kurisu and Shigenobu Yonemura : Determination of protein composition at epithelial cell-cell junctions by CRISPR/Cas9-mediated fluorescent protein knockin, Joint Annual Meeting of 70th JSCB and 51st JSDB co-sponsored by APDBN, Jun. 2018.