Researcher Profile and Settings

Affiliation

• Faculty of Advanced Life Science　Functional Life Sciences　Molecular and Cellular Biology

Job Title

• Associate Professor

URL

https://altair.sci.hokudai.ac.jp/uehara_lab/

J-Global ID

• 201501030050228535

Research Interests

• Ploidy   Ploidy   多倍体   一倍体   Spindle   Motor proteins   Mitosis   Cytokinesis   Centrosome   細胞質分裂   endoplasmic reticulum stress   微小管   自己組織化   細胞分裂   生細胞観察   紡錘体   中央紡錘体   分子モーター   

Research Areas

• Life sciences / Cell biology

Educational Organization

•  Bachelor's degree program　School of Science
•  Master's degree program　Graduate School of Life Science
•  Doctoral (PhD) degree program　Graduate School of Life Science

Academic & Professional Experience

• 2017/11 - Today Hokkaido Univ. Faculty of Adv. Life Sci. Associate Prof. (PI)
• 2017/11 - Today Hokkaido University Faculty of Advanced Life Science
• 2013/12 - 2017/10 Hokkaido University
• 2013/02 - 2013/12 The University of Tokyo
• 2008/04 - 2013/01 Nagoya University

Education

• 2003/04 - 2008/03  東京大学 大学院
• 2006/06 - 2006/08  米国海洋生物学研究所 生理学コース
• 2001/04 - 2003/03  The University of Tokyo  College of Arts and Sciences  Department of Life and Cognitive Sciences
• 1999/04 - 2001/03  The University of Tokyo  College of Arts and Sciences

Association Memberships

• 米国細胞生物学会   JAPAN SOCIETY FOR CELL BIOLOGY   日本分子生物学会   

Research Activities

Published Papers

• Cell shape instability during cytokinesis in tetraploid HCT116 cells.

  Takahiro Yamamoto, Ryota Uehara

  Biochemical and biophysical research communications 678 39 - 44 2023/08/18 [Refereed]
   

  Tetraploidy is a hallmark of broad cancer types, but it remains largely unknown which aspects of cellular processes are influenced by tetraploidization in human cells. Here, we found that tetraploid HCT116 cells manifested severe cell shape instability during cytokinesis, unlike their diploid counterparts. The cell shape instability accompanied the formation of protrusive deformation at the cell poles, indicating ectopic contractile activity of the cell cortex. While cytokinesis regulators such as RhoA and anillin correctly accumulated at the equatorial cortex, myosin II was over-accumulated at the cell poles, specifically in tetraploid cells. Suppression of myosin II activity by Y27632 treatment restored smooth cell shape in tetraploids during cytokinesis, indicating dysregulation of myosin II as a primary cause of the cell shape instability in the tetraploid state. Our results demonstrate a new aspect of the dynamic cellular process profoundly affected by tetraploidization in human cells, which provides a clue to molecular mechanisms of tetraploidy-driven pathogenic processes.
• Tetraploidy-linked sensitization to CENP-E inhibition in human cells.

  Koya Yoshizawa, Akira Matsura, Masaya Shimada, Sumire Ishida-Ishihara, Fuyu Sato, Takahiro Yamamoto, Kan Yaguchi, Eiji Kawamoto, Taruho Kuroda, Kazuya Matsuo, Nobuyuki Tamaoki, Ryuichi Sakai, Yasuhito Shimada, Mithilesh Mishra, Ryota Uehara

  Molecular oncology 2023/01/23 [Refereed]
   

  Tetraploidy is a hallmark of cancer cells, and tetraploidy-selective cell growth suppression is a potential strategy for targeted cancer therapy. However, how tetraploid cells differ from normal diploids in their sensitivity to anti-proliferative treatments remains largely unknown. In this study, we found that tetraploid cells are significantly more susceptible to inhibitors of a mitotic kinesin (CENP-E) than diploids. Treatment with a CENP-E inhibitor preferentially diminished the tetraploid cell population in a diploid-tetraploid co-culture at optimum conditions. Live imaging revealed that a tetraploidy-linked increase in unsolvable chromosome misalignment caused substantially longer mitotic delay in tetraploids than in diploids upon moderate CENP-E inhibition. This time gap of mitotic arrest resulted in cohesion fatigue and subsequent cell death, specifically in tetraploids, leading to tetraploidy-selective cell growth suppression. In contrast, the microtubule-stabilizing compound paclitaxel caused tetraploidy-selective suppression through the aggravation of spindle multipolarization. We also found that treatment with a CENP-E inhibitor had superior generality to paclitaxel in its tetraploidy selectivity across a broader spectrum of cell lines. Our results highlight the unique properties of CENP-E inhibitors in tetraploidy-selective suppression and their potential use in the development of tetraploidy-targeting interventions in cancer.
• Ultrastructural Characteristics of Finger-Like Membrane Protrusions in Cell Competition.

  Tomoko Kamasaki, Ryota Uehara, Yasuyuki Fujita

  Microscopy (Oxford, England) 2022/04/08 [Refereed]
   

  A small number of oncogenic mutated cells sporadically arise within the epithelial monolayer. Newly emerging Ras- or Src-transformed epithelial cells are often apically eliminated during competitive interaction between normal and transformed cells. Our recent electron microscopy (EM) analyses revealed that characteristic finger-like membrane protrusions are formed at the interface between normal and RasV12-transformed cells via the cdc42-FBP17 pathway, potentially playing a positive role in intercellular recognition during apical extrusion. However, the spatial distribution and ultrastructural characteristics of finger-like protrusions remain unknown. In this study, we performed both X-Y and X-Z EM analyses of finger-like protrusions during the apical extrusion of RasV12-transformed cells. Quantification of the distribution and widths of the protrusions showed comparable results between the X-Y and X-Z sections. Finger-like protrusions were observed throughout the cell boundary between normal and RasV12 cells, except for apicalmost tight junctions. In addition, a non-cell-autonomous reduction in protrusion widths was observed between RasV12 cells and surrounding normal cells under the mix culture condition. In the finger-like protrusions, intercellular adhesions via thin electron-dense plaques were observed, implying that immature and transient forms of desmosomes, adherens junctions, or unknown weak adhesions were distributed. Interestingly, unlike RasV12-transformed cells, Src-transformed cells form fewer evident protrusions, and FBP17 in Src cells is dispensable for apical extrusion. Collectively, these results suggest that the dynamic reorganization of intercellular adhesions via finger-like protrusions may positively control cell competition between normal and RasV12-transformed cells. Furthermore, our data indicate a cell context-dependent diversity in the modes of apical extrusion.
• Low-invasive 5D visualization of mitotic progression by two-photon excitation spinning-disk confocal microscopy.

  Takafumi Kamada, Kohei Otomo, Takashi Murata, Kaito Nakata, Shota Hiruma, Ryota Uehara, Mitsuyasu Hasebe, Tomomi Nemoto

  Scientific reports 12 (1) 809 - 809 2022/01/17 [Refereed]
   

  Non-linear microscopy, such as multi-photon excitation microscopy, offers spatial localities of excitations, thereby achieving 3D cross-sectional imaging with low phototoxicity even in thick biological specimens. We had developed a multi-point scanning two-photon excitation microscopy system using a spinning-disk confocal scanning unit. However, its severe color cross-talk has precluded multi-color simultaneous imaging. Therefore, in this study, we introduced a mechanical switching system to select either of two NIR laser light pulses and an image-splitting detection system for 3- or 4-color imaging. As a proof of concept, we performed multi-color fluorescent imaging of actively dividing human HeLa cells and tobacco BY-2 cells. We found that the proposed microscopy system enabled time-lapse multi-color 3D imaging of cell divisions while avoiding photodamage. Moreover, the application of a linear unmixing method to the 5D dataset enabled the precise separation of individual intracellular components in multi-color images. We thus demonstrated the versatility of our new microscopy system in capturing the dynamic processes of cellular components that could have multitudes of application.
• Visualization of DNA replication in single chromosome by stable isotope labeling.

  Kosuke Nagata, Ken-Ichi Bajo, Hideyuki Mitomo, Ryosuke Fujita, Ryota Uehara, Kuniharu Ijiro, Hisayoshi Yurimoto

  Cell structure and function 46 (2) 95 - 101 2021/09/25 [Refereed]
   

  Among the inheritance of cellular components during cell division, deoxyribonucleic acid (DNA) and its condensate (chromosome) are conventionally visualized using chemical tag-labeled nucleotide analogs. However, associated mutagenesis with nucleotide analogs in the visualization of chromosomes is cause for concern. This study investigated the efficiency of using stable isotope labels in visualizing the replicating cultured human cell-chromosomes, in the absence of analog labels, at a high spatial resolution of 100 nm. The distinct carbon isotope ratio between sister chromatids reflected the semi-conservative replication of individual DNA strands through cell cycles and suggested the renewal of histone molecules in daughter chromosomes. Thus, this study provides a new, powerful approach to trace and visualize cellular components with stable-isotope labeling.Key Words: stable isotope, chromosome replication, semi-conservative replication, imaging, mass spectrometry.
• FBP17-mediated finger-like membrane protrusions in cell competition between normal and RasV12-transformed cells.

  Tomoko Kamasaki, Yumi Miyazaki, Susumu Ishikawa, Kazuya Hoshiba, Keisuke Kuromiya, Nobuyuki Tanimura, Yusuke Mori, Motosuke Tsutsumi, Tomomi Nemoto, Ryota Uehara, Shiro Suetsugu, Toshiki Itoh, Yasuyuki Fujita

  iScience 24 (9) 102994 - 102994 2021/09/24 [Refereed]
   

  At the initial stage of carcinogenesis, cell competition often occurs between newly emerging transformed cells and the neighboring normal cells, leading to the elimination of transformed cells from the epithelial layer. For instance, when RasV12-transformed cells are surrounded by normal cells, RasV12 cells are apically extruded from the epithelium. However, the underlying mechanisms of this tumor-suppressive process still remain enigmatic. We first show by electron microscopic analysis that characteristic finger-like membrane protrusions are projected from both normal and RasV12 cells at their interface. In addition, FBP17, a member of the F-BAR proteins, accumulates in RasV12 cells, as well as surrounding normal cells, which plays a positive role in the formation of finger-like protrusions and apical elimination of RasV12 cells. Furthermore, cdc42 acts upstream of these processes. These results suggest that the cdc42/FBP17 pathway is a crucial trigger of cell competition, inducing "protrusion to protrusion response" between normal and RasV12-transformed cells.
• Molecular basis of functional exchangeability between ezrin and other actin-membrane associated proteins during cytokinesis.

  Guang Yang, Shota Hiruma, Akira Kitamura, Masataka Kinjo, Mithilesh Mishra, Ryota Uehara

  Experimental cell research 403 (2) 112600 - 112600 2021/04/13 [Refereed]
   

  The mechanism that mediates the interaction between the contractile ring and the plasma membrane during cytokinesis remains elusive. We previously found that ERM (Ezrin/Radixin/Moesin) proteins, which usually mediate cellular pole contraction, become over-accumulated at the cell equator and support furrow ingression upon the loss of other actin-membrane associated proteins, anillin and supervillin. In this study, we addressed the molecular basis of the exchangeability between ezrin and other actin-membrane associated proteins in mediating cortical contraction during cytokinesis. We found that depletion of anillin and supervillin caused over-accumulation of the membrane-associated FERM domain and actin-binding C-terminal domain (C-term) of ezrin at the cleavage furrow, respectively. This finding suggests that ezrin differentially shares its binding sites with these proteins on the actin cytoskeleton or inner membrane surface. Using chimeric mutants, we found that ezrin C-term, but not the FERM domain, can substitute for the corresponding anillin domains in cytokinesis and cell proliferation. On the other hand, either the membrane-associated or the actin/myosin-binding domains of anillin could not substitute for the corresponding ezrin domains in controlling cortical blebbing at the cell poles. Our results highlight specific designs of actin- or membrane-associated moieties of different actin-membrane associated proteins with limited exchangeability, which enables them to support diverse cortical activities on the shared actin-membrane interface during cytokinesis.
• Mevalonate pathway-mediated ER homeostasis is required for haploid stability in human somatic cells.

  Kan Yaguchi, Kimino Sato, Koya Yoshizawa, Daisuke Mikami, Kohei Yuyama, Yasuyuki Igarashi, Gabor Banhegyi, Eva Margittai, Ryota Uehara

  Cell structure and function 2020/12/22 [Refereed]
   

  The somatic haploidy is unstable in diplontic animals, but cellular processes determining haploid stability remain elusive. Here, we found that inhibition of mevalonate pathway by pitavastatin, a widely used cholesterol-lowering drug, drastically destabilized the haploid state in HAP1 cells. Interestingly, cholesterol supplementation did not restore haploid stability in pitavastatin-treated cells, and cholesterol inhibitor U18666A did not phenocopy haploid destabilization. These results ruled out the involvement of cholesterol in haploid stability. Besides cholesterol perturbation, pitavastatin induced endoplasmic reticulum (ER) stress, the suppression of which by a chemical chaperon significantly restored haploid stability in pitavastatin-treated cells. Our data demonstrate the involvement of the mevalonate pathway in the stability of the haploid state in human somatic cells through managing ER stress, highlighting a novel link between ploidy and ER homeostatic control. Key words: Haploid, ER stress, Mevalonate, pathway.
• Uncoupling of DNA Replication and Centrosome Duplication Cycles Is a Primary Cause of Haploid Instability in Mammalian Somatic Cells

  Koya Yoshizawa, Kan Yaguchi, Ryota Uehara

  Frontiers in Cell and Developmental Biology 8 721  2020/07 [Refereed][Not invited]
  
• Photoswitchable CENP-E Inhibitor Enabling the Dynamic Control of Chromosome Movement and Mitotic Progression.

  Noushaba Nusrat Mafy, Kazuya Matsuo, Shota Hiruma, Ryota Uehara, Nobuyuki Tamaoki

  Journal of the American Chemical Society 142 (4) 1763 - 1767 2020/01/29 [Refereed][Not invited]
   

  Interfering with mitosis is a potential cancer therapy strategy. However, the lack of controllability of antimitotic drugs in cell growth suppression causes severe side effects and limits their clinical utility. Herein, we developed an azobenzene-based photoswitchable inhibitor of CENP-E, a mitotic kinesin required for chromosome transportation. The new inhibitor enabled reversible photoswitching of CENP-E activity with ∼10-fold change in IC50 between cis and trans photoisomerization states both in vitro and in living cells. It also enabled repeatable photoswitching of CENP-E-dependent chromosome congression and hence mitotic progression with UV/vis light illumination cycles. Using this technique, we could specify the exact process of mitotic progression in which CENP-E plays an indispensable role. Our data demonstrate the power of a photochemical approach for highly controllable mitotic interference as well as for discovery of precise molecular functions in dynamic cellular processes.
• Ploidy-dependent change in cyclin D2 expression and sensitization to cdk4/6 inhibition in human somatic haploid cells.

  Kan Yaguchi, Takahiro Yamamoto, Masaya Shimada, Rina Sugimoto, Kiminori Nakamura, Tokiyoshi Ayabe, Ryota Uehara

  Biochemical and biophysical research communications 504 (1) 231 - 237 0006-291X 2018/09/26 [Refereed][Not invited]
   

  Near-haploidy is observed in certain cancer types, but ploidy-dependent alterations in gene regulation in the haploid state remain elusive. Here, by comparative transcriptome analysis between human isogenic haploid and diploid cell lines, we found lowering of cyclin D2 level in haploids. Acute genome duplication in haploids restored cyclin D2 expression to diploid level, indicating that the regulation of cyclin D2 expression is directly linked to ploidy. Downstream pathways of cyclin D2, such as Rb phosphorylation and p27 sequestration remained intact in haploids, suggesting that they adapt to lowered cyclin D level. Interestingly, however, haploid cells were more susceptible to cdk4/6 inhibition compared to diploids. Our finding indicates feasibility of selective growth suppression of haploid cells based on ploidy-linked gene regulation.
• Uncoordinated centrosome cycle underlies the instability of non-diploid somatic cells in mammals.

  Kan Yaguchi, Takahiro Yamamoto, Ryo Matsui, Yuki Tsukada, Atsuko Shibanuma, Keiko Kamimura, Toshiaki Koda, Ryota Uehara

  The Journal of cell biology 217 (7) 2463 - 2483 0021-9525 2018/07/02 [Refereed][Not invited]
   

  In animals, somatic cells are usually diploid and are unstable when haploid for unknown reasons. In this study, by comparing isogenic human cell lines with different ploidies, we found frequent centrosome loss specifically in the haploid state, which profoundly contributed to haploid instability through subsequent mitotic defects. We also found that the efficiency of centriole licensing and duplication changes proportionally to ploidy level, whereas that of DNA replication stays constant. This caused gradual loss or frequent overduplication of centrioles in haploid and tetraploid cells, respectively. Centriole licensing efficiency seemed to be modulated by astral microtubules, whose development scaled with ploidy level, and artificial enhancement of aster formation in haploid cells restored centriole licensing efficiency to diploid levels. The ploidy-centrosome link was observed in different mammalian cell types. We propose that incompatibility between the centrosome duplication and DNA replication cycles arising from different scaling properties of these bioprocesses upon ploidy changes underlies the instability of non-diploid somatic cells in mammals.
• Tetraploidy-associated centrosome overduplication in mouse early embryos.

  Kan Yaguchi, Takahiro Yamamoto, Ryo Matsui, Masaya Shimada, Atsuko Shibanuma, Keiko Kamimura, Toshiaki Koda, Ryota Uehara

  Communicative & integrative biology 11 (4) e1526605 - e1526605-4 2018 [Refereed][Not invited]
   

  Recently, we observed that tetraploidization of certain types of human cancer cells resulted in upregulation of centrosome duplication cycles and chronic generation of the extra centrosome. Here, we investigated whether tetraploidy-linked upregulation of centrosome duplication also occurs in non-cancer cells using tetraploidized parthenogenetic mouse embryos. Cytokinesis blockage at early embryonic stage before de novo centriole biogenesis provided the unique opportunity in which tetraploidization can be induced without transient doubling of centrosome number. The extra numbers of the centrioles and the centrosomes were observed more frequently in tetraploidized embryos during the blastocyst stage than in their diploid counterparts, demonstrating the generality of the newly found tetraploidy-driven centrosome overduplication in mammalian non-cancer systems.
• Dynamics and function of ERM proteins during cytokinesis in human cells.

  Shota Hiruma, Tomoko Kamasaki, Kohei Otomo, Tomomi Nemoto, Ryota Uehara

  FEBS letters 591 (20) 3296 - 3309 0014-5793 2017/10 [Refereed][Not invited]
   

  The molecular mechanism that governs cytoskeleton-membrane interaction during animal cytokinesis remains elusive. Here, we investigated the dynamics and functions of ERM (Ezrin/Radixin/Moesin) proteins during cytokinesis in human cultured cells. We found that ezrin is recruited to the cleavage furrow through its membrane-associated domain in a cholesterol-dependent but largely Rho-independent manner. While ERMs are dispensable for furrow ingression, they play a pivotal role in contractile activity of the polar cortex. Notably, when anillin and supervillin are codepleted, ERMs increasingly accumulate at the cleavage furrow and substantially contribute to the furrow ingression. These results reveal a supportive role of ERMs in cortical activities during cytokinesis, and also provide insight into the selective mechanism that preferentially associates cytokinesis-relevant proteins with the division site.
• Augmin shapes the anaphase spindle for efficient cytokinetic furrow ingression and abscission.

  Ryota Uehara, Tomoko Kamasaki, Shota Hiruma, Ina Poser, Kinya Yoda, Junichiro Yajima, Daniel W Gerlich, Gohta Goshima

  Molecular biology of the cell 27 (5) 812 - 27 1939-4586 2016/03/01 [Refereed][Not invited]
   

  During anaphase, distinct populations of microtubules (MTs) form by either centrosome-dependent or augmin-dependent nucleation. It remains largely unknown whether these different MT populations contribute distinct functions to cytokinesis. Here we show that augmin-dependent MTs are required for the progression of both furrow ingression and abscission. Augmin depletion reduced the accumulation of anillin, a contractile ring regulator at the cell equator, yet centrosomal MTs were sufficient to mediate RhoA activation at the furrow. This defect in contractile ring organization, combined with incomplete spindle pole separation during anaphase, led to impaired furrow ingression. During the late stages of cytokinesis, astral MTs formed bundles in the intercellular bridge, but these failed to assemble a focused midbody structure and did not establish tight linkage to the plasma membrane, resulting in furrow regression. Thus augmin-dependent acentrosomal MTs and centrosomal MTs contribute to nonredundant targeting mechanisms of different cytokinesis factors, which are required for the formation of a functional contractile ring and midbody.
• 比べてみよう:細胞ダイナミクスの共通性と独自性 動物細胞における中央紡錘体の形成・形態制御機構

  上原 亮太, 釜崎 とも子, 依田 欣哉, 五島 剛太

  日本細胞生物学会大会講演要旨集 (一社)日本細胞生物学会 66回 91 - 91 2014/05
• Aurora B and Kif2A control microtubule length for assembly of a functional central spindle during anaphase.

  Ryota Uehara, Yuki Tsukada, Tomoko Kamasaki, Ina Poser, Kinya Yoda, Daniel W Gerlich, Gohta Goshima

  The Journal of cell biology 202 (4) 623 - 36 0021-9525 2013/08/19 [Refereed][Not invited]
   

  The central spindle is built during anaphase by coupling antiparallel microtubules (MTs) at a central overlap zone, which provides a signaling scaffold for the regulation of cytokinesis. The mechanisms underlying central spindle morphogenesis are still poorly understood. In this paper, we show that the MT depolymerase Kif2A controls the length and alignment of central spindle MTs through depolymerization at their minus ends. The distribution of Kif2A was limited to the distal ends of the central spindle through Aurora B-dependent phosphorylation and exclusion from the spindle midzone. Overactivation or inhibition of Kif2A affected interchromosomal MT length and disorganized the central spindle, resulting in uncoordinated cell division. Experimental data and model simulations suggest that the steady-state length of the central spindle and its symmetric position between segregating chromosomes are predominantly determined by the Aurora B activity gradient. On the basis of these results, we propose a robust self-organization mechanism for central spindle formation.
• 細胞分裂131年目の真実:分子から動態へ 動物と植物におけるオーグミンの分裂期スピンドル形成への寄与

  中岡由貴, 上原亮太

  細胞工学 32 (3) 0287-3796 2013
• Ultrastructural Insights into Augmin-Dependent Microtubule Nucleation in the Spindle

  釜崎とも子, 上原亮太

  顕微鏡 48 (2 (Web)) 1349-0958 2013
• An inducible RNA interference system in Physcomitrella patens reveals a dominant role of augmin in phragmoplast microtubule generation.

  Yuki Nakaoka, Tomohiro Miki, Ryuta Fujioka, Ryota Uehara, Akiko Tomioka, Chikashi Obuse, Minoru Kubo, Yuji Hiwatashi, Gohta Goshima

  The Plant cell 24 (4) 1478 - 93 1532-298X 2012/04 [Refereed][Not invited]
   

  Mitosis is a fundamental process of eukaryotic cell proliferation. However, the molecular mechanisms underlying mitosis remain poorly understood in plants partly because of the lack of an appropriate model cell system in which loss-of-function analyses can be easily combined with high-resolution microscopy. Here, we developed an inducible RNA interference (RNAi) system and three-dimensional time-lapse confocal microscopy in the moss Physcomitrella patens that allowed in-depth phenotype characterization of the moss genes essential for cell division. We applied this technique to two microtubule regulators, augmin and γ-tubulin complexes, whose mitotic roles remain obscure in plant cells. Live imaging of caulonemal cells showed that they proceed through mitosis with continual generation and self-organization of acentrosomal microtubules. We demonstrated that augmin plays an important role in γ-tubulin localization and microtubule generation from prometaphase to cytokinesis. Most evidently, microtubule formation in phragmoplasts was severely compromised after RNAi knockdown of an augmin subunit, leading to incomplete expansion of phragmoplasts and cytokinesis failure. Knockdown of the γ-tubulin complex affected microtubule formation throughout mitosis. We conclude that postanaphase microtubule generation is predominantly stimulated by the augmin/γ-tubulin machinery in moss and further propose that this RNAi system serves as a powerful tool to dissect the molecular mechanisms underlying mitosis in land plants.
• Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase.

  Ryota Uehara, Gohta Goshima

  The Journal of cell biology 191 (2) 259 - 67 0021-9525 2010/10/18 [Refereed][Not invited]
   

  The central spindle forms between segregating chromosomes during anaphase and is required for cytokinesis. Although anaphase-specific bundling and stabilization of interpolar microtubules (MTs) contribute to formation of the central spindle, it remains largely unknown how these MTs are prepared. Using live imaging of MT plus ends and an MT depolymerization and regrowth assay, we show that de novo MT generation in the interchromosomal region during anaphase is important for central spindle formation in human cells. Generation of interchromosomal MTs and subsequent formation of the central spindle occur independently of preanaphase MTs or centrosomal MT nucleation but require augmin, a protein complex implicated in nucleation of noncentrosomal MTs during preanaphase. MTs generated in a hepatoma up-regulated protein (HURP)-dependent manner during anaphase also contribute to central spindle formation redundantly with preanaphase MTs. Based on these results, a new model for central spindle assembly is proposed.
• Determinants of myosin II cortical localization during cytokinesis.

  Ryota Uehara, Gohta Goshima, Issei Mabuchi, Ronald D Vale, James A Spudich, Eric R Griffis

  Current biology : CB 20 (12) 1080 - 5 1879-0445 2010/06/22 [Refereed][Not invited]
   

  Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear. By analyzing the spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments and that the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages: (1) turnover of thick filaments throughout the cell cycle, (2) myosin heavy chain-based control of myosin assembly at the metaphase-anaphase transition, and (3) redistribution and/or activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering preassembled filaments to the equatorial zone via cortical flow. We conclude that multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.
• 細胞分裂・染色体分配の新常識:鍵を握る動原体の制御 分裂期スピンドル形成における微小管生成機構

  上原亮太, 五島剛太

  細胞工学 29 (9) 0287-3796 2010
• [Mitotic spindle formation mediated by augmin protein complex].

  Ryota Uehara, Gohta Goshima

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 54 (14) 1850 - 5 0039-9450 2009/11 [Refereed][Not invited]
  
• The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells.

  Ryota Uehara, Ryu-suke Nozawa, Akiko Tomioka, Sabine Petry, Ronald D Vale, Chikashi Obuse, Gohta Goshima

  Proceedings of the National Academy of Sciences of the United States of America 106 (17) 6998 - 7003 0027-8424 2009/04/28 [Refereed][Not invited]
   

  The mitotic spindle is constructed from microtubules (MTs) nucleated from centrosomes, chromosome proximal regions, and preexisting spindle MTs. Augmin, a recently identified protein complex, is a critical factor in spindle MT-based MT generation in Drosophila S2 cells. Previously, we identified one subunit of human augmin. Here, by using mass spectrometry, we identified the full human augmin complex of 8 subunits and show that it interacts with the gamma-tubulin ring complex (gamma-TuRC). Unlike augmin-depleted S2 cells, in which the defect in spindle-mediated MT generation is mostly compensated by centrosomal MTs, augmin knockdown alone in HeLa cells triggers the spindle checkpoint, reduces tension on sister kinetochores, and severely impairs metaphase progression. Human augmin knockdown also reduces the number of central spindle MTs during anaphase and causes late-stage cytokinesis failure. A link between augmin and gamma-TuRC is likely critical for these functions, because a gamma-TuRC mutant that attenuates interaction with augmin does not restore function in vivo. These results demonstrate that MT generation mediated by augmin and gamma-TuRC is critical for chromosome segregation and cytokinesis in human cells.
• In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesis.

  Ryota Uehara, Hiroshi Hosoya, Issei Mabuchi

  Cell motility and the cytoskeleton 65 (2) 100 - 15 0886-1544 2008/02 [Refereed][Not invited]
   

  Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs.

Conference Activities & Talks

• A novel link between ploidy level and centrosome homeostasis in mammalian somatic cells  [Invited]

  Ryota Uehara

  Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India  2018/02
• Molecular mechanism of the contractile ring-the plasma membrane interaction during cytokinesis in human cells  [Invited]

  Hiruma Shota, Uehara Ryota

  69th JSCB meeting symposium  2017/06
• Novel link between ploidy level and centrosome homeostasis in mammalian somatic cells  [Not invited]

  Yaguchi, K, Matsui, R, Tsukada, Y, Koda, T, Uehara, R

  Cold Spring Harber Asia Meeting “Cilia and Centrosomes”  2017/04
• 動物細胞における中央紡錘体の形成・形態制御機構  [Invited]

  上原亮太, 釜崎とも子, 依田欣也, 五島剛太

  第66回日本細胞生物学会大会 シンポジウム  2014/06
• 細胞生物学的手法と数理モデルを用いた細胞質分裂制御の解析  [Invited]

  上原亮太, 塚田祐基

  第6回 定量生物学会年会  2013/11
• Aurora BとKif2Aによる微小管の長さ制御を介した細胞質分裂制御の仕組み  [Invited]

  上原亮太, 塚田祐基, 釜崎とも子, 五島剛太

  第65回 日本細胞生物学会大会 シンポジウム  2013/06
• Central spindle formation through the regulation of microtubule depolymerization during cytokinesis.  [Not invited]

  Uehara, R, Tsukada, Y, Goshima, G

  The American society for cell biology 52th auunual meeting, Minisymposium  2012/12
• Cell division plane determination through regulation of MT depolymerization during cytokinesis.  [Not invited]

  Uehara, R, Tsukada, Y, Yoda, K, Gerlich, D. W, Goshima, G

  The 2012 FASEB Science Research Conference (Mitosis: Spindle Assembly and Function)  2012/08
• Human augmin complex is required for microtubule organization during mitosis and cytokinesis. Cytokinesis-dynamics of the contractile ring.  [Invited]

  Ryota Uehara

  JSPS Bilateral project Japan-Singapore seminar.  2009/06

MISC

• 動物細胞における中央紡錘体の形成・形態制御機構

  上原亮太, 釜崎とも子, 依田欣哉, 五島剛太  日本細胞生物学会大会要旨集  66th-  2014

Awards & Honors

• 2013/06 第65回日本細胞生物学会年会 若手優秀発表賞
   

  受賞者: 上原亮太
• 2012/08 米国実験生物学会年会（FASEB meeting）トラベルアウォード
   

  受賞者: 上原亮太
• 2010/12 名古屋大学GCOEシステム生命科学研究 若手顕彰
   

  受賞者: 上原亮太

Research Grants & Projects

• Principle of body malfunctioning through local cell division failure

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))

  Date (from‐to) : 2019/10 -2024/03 

  Author : 上原 亮太, 塚田 祐基, 松尾 和哉

   

  本研究では、生体内で起こる少数細胞の分裂異常が個体機能に及ぼす影響を明らかにし、分裂異常に起因した病態形成メカニズムを理解することを目的としている。独自の細胞イメージング技術と光応答性化合物の合成技術を駆使し、個体局所で細胞分裂の異常を任意の規模で誘導して、その細胞運命への影響をマルチスケールに追跡する実験系を構築し、これらをインドTata研究所生物科学科の脊椎動物発生学研究室が独自に保有するゼブラフィッシュ胚ゲノム改変・細胞分裂解析システムに導入することで、ゼブラフィッシュ胚で局所的に発生する少数細胞の分裂異常が胚発生や組織機能・恒常性に及ぼす影響を定量解析可能にする。2019年度は、動物細胞分裂において複製染色体の整列運動をつかさどるモーターたんぱく質のCenpEに対する光応答性阻害化合物を作成し、ヒト培養細胞を用いた性能評価によって、照射光波長依存的にすばやく細胞分裂の進行を阻害・解除できる制御系の構築を行った。この実験系の構築自体が非常に新規性の高い成果であることから、これを論文にまとめて有力米化学誌Journal of American Chemical Societyに発表した。その後、Tata研究所上記研究室とのインターネット会議を重ね、胚調整からマイクロインジェクションによる試薬導入、各種分裂マーカーを用いた評価系構築に関する綿密な技術指導の下、この細胞分裂の光制御技術をゼブラフィッシュ初期胚への導入を開始した。さらに、分担研究者との連携によって、胚局所の細胞分裂動態と、胚全体の形態変化の同時観察を可能にするためのマルチスケール撮像システムの構築も順調に進行している。
• 中心体複製チェックポイントの人工合成による安定一倍体細胞の樹立

  日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)

  Date (from‐to) : 2021/07 -2023/03 

  Author : 上原 亮太

   

  ゲノムを1セットのみもつ一倍体哺乳類細胞は、（ゲノムを2セットもつ通常の二倍体細胞に比べ）ゲノム編集による細胞機能改変を大幅に効率化する重要な遺伝学・細胞工学ツールである。しかし、一倍体哺乳類細胞は一般に著しいゲノム不安定性を呈し、短期間で二倍体化してしまう性質をもち、これが一倍体細胞技術の汎用性の妨げとなっている。我々は最近、哺乳類体細胞における一倍体状態が、細胞分裂期の染色体分配を司る中心体の複製反応の慢性的な遅延を引き起こし、中心体喪失による細胞分裂障害によってゲノム不安定性を引き起こすことを明らかにした。そこで本研究では、中心体複製の進行をモニターし、その遅延に応じて細胞周期の進行を一旦停止できる新規のチェックポイント機構を人工遺伝子合成技術により作出する。この人工遺伝子回路を一倍体細胞に組み込むことで中心体喪失、ひいては一倍体細胞特有のゲノム不安定性を解消することを目的としている。 2021年度は、中心体複製の開始（母中心小体からの娘中心小体の合成の開始）を検知して、人工のシグナル伝達回路をon/off制御可能にする人工センサー遺伝子群をコードするプラスミドをデザイン・作成した。現在、これらのプラスミドを安定発現する細胞株の取得に着手しており、随時細胞内機能を評価する段階に入った。 また、相補的アプローチとして、中心体喪失時に双極性紡錘体形成を制御する中心体非依存的な紡錘体形成経路の遺伝子制御系を人工的に補強することで、一倍体のゲノム安定性を改善する試みにも着手した。これまでに、紡錘体極への細胞分裂制御関連因子の集積を司る複数の遺伝子の発現補強とノックアウトを組み合わせることで、一倍体細胞の分裂不安定性が劇的に改善し、一ヶ月強の長期培養においても一倍体状態を保持する細胞株の取得に成功した。
• Principle of chronic centrosome aberrations in whole-genome duplicated cells and its contribution to cellular heterogeneity

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2019/04 -2022/03 

  Author : Uehara Ryota

   

  In this study, we aimed to elucidate the principle and significance of centrosome hyperactivation associated with whole-genome duplication (WGD), a cellular abnormality common in 30% of cancers. We found that this phenomenon is caused by an increase in the accumulation of centrosomal proteins resulting from the doubling of the gene dosage of a centrosome scaffolding gene upon WGD. By developing a method to cancel this centrosome hyperactivation artificially, we found that this phenomenon does not play an adaptive role in the proliferation control of WGD cells. However, contrary to our initial expectation, we found that the centrosome hyperactivation made WGD cells more fragile in centrosomal structural homeostatic regulation, suggesting the feasibility of WGD cell-selective suppression through targeting their fragility.
• ガン形成を駆動する普遍的レアイベント「倍数性逆転」の発生原理に迫る

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2019/04 -2021/03 

  Author : 上原 亮太

   

  本研究では、細胞周期異常による染色体倍加に続いて発生する大規模な染色体喪失によって生体の監視を逃れて細胞が悪性化する「倍数性逆転」現象の発生プロセスの理解を目的とする。まず2019年度は、異なる経緯で発生する染色体倍加細胞で倍数性逆転が発生する過程を長期ライブイメージングで可視化することを試みた。興味深いことに、染色体倍加を引き起こす細胞周期異常の種類の違いによって、その後の倍数性逆転が起こる頻度に著しい違いがあることが明らかになった。このことから、従来一律に確率的かつ希少な現象と考えられてきた倍数性逆転が、特定の経緯で発生する倍数性異常においてより発生しやすい現象である可能性が示唆された。さらに、染色体倍加細胞の高解像ライブイメージングにより、染色体倍加とともに倍加された４つの中心体が、間期のうちに未知の仕組みでクラスター化し、それが原因となって次の分裂期初期に高確率で単極性の紡錘体を形成していることがわかった。また、光変色性の蛍光染色体マーカーを用いた高解像イメージングによって、この染色体倍加細胞における一時的な紡錘体の単極化が細胞分裂における染色体分配のランダムさに影響を及ぼす可能性があることがわかった。 また、倍数性逆転現象の詳細な観察のための、低倍率撮像による細胞系譜追跡と、高倍率撮像による細胞分裂動態の定量解析を同時実施可能なマルチスケール顕微鏡システムの光学系の構築を行った。現有の共焦点顕微鏡に、広視野低侵襲性の長期細胞追跡を可能にする独立光学系を追加設置した。これによって、上記の染色体倍加細胞に特徴的な中心体動態が、染色体倍加細胞の倍数性逆転の発生頻度や、その子孫細胞の長期的運命に及ぼす影響を１：１対応で解析可能にする準備が整った。
• Molecular mechanism of mitotic spindle adaptation to drastic chromosome number changes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2017/04 -2019/03 

  Author : Uehara Ryota, YAGUCHI Kan, YAMAMOTO Takahiro, SHIMADA Masaya

   

  Animal somatic cells are usually in the diploid state, containing two copies of genomes. Abnormal changes in ploidy state (ie. drastic changes in chromosome number) are hallmarks of cancer cells, but it remains largely unknown how cells can manage to survive with abnormal chromosome numbers. In this research, I aimed to elucidate the molecular mechanism through which mitotic and cell cycle regulatory systems can adapt to drastic chromosome number changes. Through cell imaging and comprehensive gene expression analyses, we found characteristic changes in mitotic centrosome structure and cyclin D expression upon ploidy changes, which potentially contribute to cellular adaptation to drastic chromosome number changes.
• Molecular mechanism of plasma membrane deformation during cell division.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

  Date (from‐to) : 2015/04 -2017/03 

  Author : Uehara Ryota, HIRUMA Shota

   

  Precisely controlled actin-membrane interactions are required for animal cytokinesis. We investigated the dynamics and functions of ERM (Ezrin/Radixin/Moesin) proteins in control of cytokinesis in human cells. We found that stable association of ezrin to the furrow depended on cholesterol, but not on RhoA. Depletion of ERMs significantly changed the kinetics of furrow ingression but did not drastically affect the accuracy of cytokinesis. Notably, however, in the background of anillin and supervillin co-depletion, ezrin became drastically accumulated at the furrow and substantially contributed to the furrow ingression activity. The ERM-driven cleavage furrow in the anillin- and supervillin-depleted cells was narrower than that in unperturbed cells, suggesting characteristic mechanical property of ERM in inducing cell deformation. These results provide insight into cooperative regulatory system of actin-membrane interaction featuring multiple linkers with distinct molecular properties.
• 細胞質分裂をつかさどる逆平行微小管超分子マシナリーが動く仕組み

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

  Date (from‐to) : 2013/04 -2015/03 

  Author : 上原 亮太

   

  細胞質分裂期の分裂位置制御は、分離染色体の中央に一過的に形成される後期紡錘体およびその中央部、細胞赤道面上に円板状に形成される微小管逆平行束マシナリー「ステムボディー」により制御されるが、後期紡錘体の形態およびステムボディーの位置制御の仕組みは理解されていない。昨年度の研究で明らかになった分裂期タンパク質リン酸化酵素Aurora Bを介した微小管脱重合因子Kif2Aの活性制御による分裂期後期紡錘体（ステムボディー微小管）の長さ制御機構の分子基盤を明らかにするため、Kif2Aの機能構造解析を実施し、ステムボディー長さ制御に必須の機能を果たすドメインを探索した。Kif2Aを含むキネシン13ファミリーで高度に保存されたN末端領域ドメインを欠損する変異体や保存アミノ酸に点変異を導入した変異体は中央紡錘体への局在が著しく抑制された。一方でN末端のみのトランケートは中央紡錘体の中央部に集積することを見出した。逆にKif2AのC末端側を欠損した変異体は後期紡錘体微小管全体に過剰集積して後期紡錘体の過剰短縮を引き起こすことがわかった。これらの結果から、Kif2Aは複数のドメインに依存した形で後期紡錘体の異なるモチーフへの集積が制御される複雑なメカニズムで後期紡錘体のサイズ制御に関与していることが明らかになった。 また、微小管動態制御異常により生じる分裂障害の詳細な原因を細胞学的に解析し、微小管配向の変化が細胞内シグナル配置と細胞の力学特性の双方に異常をもたらすことで重篤な分裂異常が引き起こされることを明らかにした。研究成果は現在学術誌に投稿準備中である。
• Spatial control of cytokinesis through regulation of microtubule dynamics

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2013/04 -2015/03 

  Author : UEHARA Ryota

   

  Precise control of cell division is essential for securing biological processes of heredity and homeostasis. However, molecular mechanisms of spatiotemporal regulation of cell division remain largely unknown. In this project, we looked into microtubule dynamics during cell division phase in human cultured cells, and aimed to elucidate their contributions to the determination of division sites within dividing cells. We found that Kif2A, a microtubule depolymerizing protein plays a pivotal role in microtubule length control within the central spindle, which segregates the two masses of the sister chromosomes in an appropriate distance. We also found that augmin, a regulator of central spindle microtubule generation mediates accumulation of cytokinesis regulators to the equatorial cortex, which is essential for efficient furrow ingression. Our results shed light on new mechanisms of cell division control through dynamic reorganizations of microtubules during cytokinesis.
• Molecular mechanism of the central spindle formation during cytokinesis

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity Start-up

  Date (from‐to) : 2010 -2011 

  Author : UEHARA Ryota

   

  The central spindle forms between segregating chromosomes during anaphase and is required for proper coupling of karyokinesis and cytokinesis. It remains largely unknown how this microtubule-based structure is prepared during anaphase. Using live imaging of MT plus ends and a MT depolymerization and regrowth assay, we show that de novo MT generation in the interchromosomal region during anaphase is important for central spindle formation in human cells. Generation of interchromosomal MTs and subsequent formation of the central spindle require augmin, a protein complex implicated in nucleation of noncentrosomal MTs during pre-anaphase. MTs generated in an HURP-dependent manner during anaphase also contribute to central spindle MT formation redundantly with pre-anaphase MTs. Based upon these results, I could established a new model for central spindle assembly.

Educational Activities

Teaching Experience