基本情報

所属

• 先端生命科学研究院　生命機能科学研究部門　分子細胞生物学分野

職名

• 准教授

学位

• 博士（学術）（東京大学 大学院 総合文化研究科）

ホームページURL

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

J-Global ID

• 201501030050228535

研究キーワード

• Ploidy   倍数性   多倍体   一倍体   Spindle   Motor proteins   Mitosis   Cytokinesis   中心体   細胞質分裂   小胞体ストレス   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   

研究分野

• ライフサイエンス / 細胞生物学

担当教育組織

•  学士課程　理学部
•  修士課程　生命科学院
•  博士課程　生命科学院

職歴

• 2017年11月 - 現在 Hokkaido Univ. Faculty of Adv. Life Sci. Associate Prof. (PI)
• 2017年11月 - 現在 北海道大学大学院先端生命科学研究院 准教授（PI)
• 2013年12月 - 2017年10月 北海道大学 特任助教（テニュアトラック・PI）
• 2013年02月 - 2013年12月 東京大学 助教
• 2008年04月 - 2013年01月 名古屋大学 博士研究員及び助教

学歴

• 2003年04月 - 2008年03月   東京大学 大学院   総合文化研究科 広域科学専攻   生命環境科学系
• 2006年06月 - 2006年08月   米国海洋生物学研究所 生理学コース
• 2001年04月 - 2003年03月   東京大学 教養学部 生命・認知科学科
• 1999年04月 - 2001年03月   東京大学 教養学部 理科II類

所属学協会

• 米国細胞生物学会   日本細胞生物学会   日本分子生物学会   

研究活動情報

論文

• Cell shape instability during cytokinesis in tetraploid HCT116 cells.

  Takahiro Yamamoto, Ryota Uehara

  Biochemical and biophysical research communications 678 39 - 44 2023年08月18日 [査読有り]
   

  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年01月23日 [査読有り]
   

  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年04月08日 [査読有り]
   

  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年01月17日 [査読有り]
   

  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年09月25日 [査読有り]
   

  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年09月24日 [査読有り]
   

  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年04月13日 [査読有り]
   

  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年12月22日 [査読有り]
   

  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年07月 [査読有り][通常論文]
  
• 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年01月29日 [査読有り][通常論文]
   

  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.

  Yaguchi K, Yamamoto T, Shimada M, Sugimoto R, Nakamura K, Ayabe T, Uehara R

  Biochemical and biophysical research communications 504 1 231 - 237 2018年09月26日 [査読有り][通常論文]
   

  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.

  Yaguchi K, Yamamoto T, Matsui R, Tsukada Y, Shibanuma A, Kamimura K, Koda T, Uehara R

  The Journal of cell biology 217 7 2463 - 2483 2018年07月02日 [査読有り][通常論文]
   

  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

  Yaguchi, K, Yamamoto, T, Matsui, R, Shimada, M, Shibanuma, A, Kamimura, K, Koda, T, Uehara, R

  Communicative & Integrative Biology 11 4 e1526605 - e1526605-4 2018年 [査読有り][通常論文]
   

  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.

  Hiruma S, Kamasaki T, Otomo K, Nemoto T, Uehara R

  FEBS letters 591 20 3296 - 3309 2017年10月 [査読有り][通常論文]
   

  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.

  Uehara R, Kamasaki T, Hiruma S, Poser I, Yoda K, Yajima J, Gerlich DW, Goshima G

  Molecular biology of the cell 27 5 812 - 27 2016年03月01日 [査読有り][通常論文]
   

  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 66回 91 - 91 (一社)日本細胞生物学会 2014年05月
• Aurora B and Kif2A control microtubule length for assembly of a functional central spindle during anaphase.

  Uehara R, Tsukada Y, Kamasaki T, Poser I, Yoda K, Gerlich DW, Goshima G

  The Journal of cell biology 202 4 623 - 36 2013年08月19日 [査読有り][通常論文]
   

  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 2013年
• 細胞骨格イメージング 紡錘体微小管の生成メカニズムに関する微細構造学的解析

  釜崎とも子, 上原亮太

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

  Nakaoka Y, Miki T, Fujioka R, Uehara R, Tomioka A, Obuse C, Kubo M, Hiwatashi Y, Goshima G

  The Plant cell 24 4 1478 - 93 2012年04月 [査読有り][通常論文]
   

  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.

  Uehara R, Goshima G

  The Journal of cell biology 191 2 259 - 67 2010年10月18日 [査読有り][通常論文]
   

  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.

  Uehara R, Goshima G, Mabuchi I, Vale RD, Spudich JA, Griffis ER

  Current biology : CB 20 12 1080 - 5 2010年06月22日 [査読有り][通常論文]
   

  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 2010年
• [Mitotic spindle formation mediated by augmin protein complex].

  Uehara R, Goshima G

  Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 54 14 1850 - 5 2009年11月 [査読有り][通常論文]
  
• The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells.

  Uehara R, Nozawa RS, Tomioka A, Petry S, Vale RD, Obuse C, Goshima G

  Proceedings of the National Academy of Sciences of the United States of America 106 17 6998 - 7003 2009年04月28日 [査読有り][通常論文]
   

  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.

  Uehara R, Hosoya H, Mabuchi I

  Cell motility and the cytoskeleton 65 2 100 - 15 2008年02月 [査読有り][通常論文]
   

  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.

講演・口頭発表等

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

  上原亮太

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

  比留間翔太, 上原亮太

  第69回日本細胞生物学会大会 シンポジウム 2017年06月 口頭発表（招待・特別）
• Novel link between ploidy level and centrosome homeostasis in mammalian somatic cells  [通常講演]

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

  Cold Spring Harber Asia Meeting “Cilia and Centrosomes” 2017年04月 口頭発表（一般）
• 動物細胞における中央紡錘体の形成・形態制御機構  [招待講演]

  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年06月 口頭発表（招待・特別）
• 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  [招待講演]

  上原亮太, 塚田祐基

  第6回 定量生物学会年会 2013年11月 口頭発表（招待・特別）
• 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  [招待講演]

  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年06月 口頭発表（招待・特別）
• Central spindle formation through the regulation of microtubule depolymerization during cytokinesis.  [通常講演]

  Uehara, R, Tsukada, Y, Goshima, G

  The American society for cell biology 52th auunual meeting, Minisymposium 2012年12月 口頭発表（一般）
• Cell division plane determination through regulation of MT depolymerization during cytokinesis.  [通常講演]

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

  The 2012 FASEB Science Research Conference (Mitosis: Spindle Assembly and Function) 2012年08月 口頭発表（一般）
• Human augmin complex is required for microtubule organization during mitosis and cytokinesis. Cytokinesis-dynamics of the contractile ring.  [招待講演]

  上原亮太

  JSPS Bilateral project Japan-Singapore seminar. 2009年06月 口頭発表（招待・特別）

その他活動・業績

• 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年

受賞

• 2013年06月 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年08月 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年12月 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

共同研究・競争的資金等の研究課題

• 少数細胞の分裂異常が個体機能を喪失させる原理の解明

  日本学術振興会：科学研究費助成事業 国際共同研究加速基金(国際共同研究強化(B))

  研究期間 : 2019年10月 -2024年03月 

  代表者 : 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)

  研究期間 : 2021年07月 -2023年03月 

  代表者 : 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。
• 染色体倍加に伴う慢性的な中心体異常の発生原理と細胞不均一性への寄与

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2019年04月 -2022年03月 

  代表者 : 上原 亮太, 塚田 祐基

   

  本研究では、申請者が最近発見したヒト体細胞における染色体倍加現象に伴う中心体の増大、機能亢進および複製亢進現象の発生原理と、それらが細胞形質変化に及ぼす影響を理解することを目的としている。2019年度はまず、染色体倍加細胞で見られる中心体増大の実態を理解するために、種々の染色体マーカーや微小管形成能評価アッセイを用いて、染色体倍加に伴い、細胞分裂期の中心体に具体的にどのような変化が起こっているかを調べた。ヒト大腸がん由来HCT116細胞の二倍体コントロールおよび染色体倍加細胞株の比較によって、染色体倍加に伴い中心体周辺物質の主要構成因子の中心体への集積がいずれも有意に増大し、微小管重合能も倍増していることを定量的に突き止めた。さらに、染色体倍加細胞における中心体増大の生理・病理的意義を検証するために、人為的に中心体増大現象を抑制することができる染色体倍加HCT116細胞株の確立を試みた。具体的には、中心体周辺物質の集積の足場を提供する中心体構成遺伝子の発現量を、オーキシンデグロンシステムでコンディショナルに半減できる細胞株を作成し、実際にオーキシン添加によって染色体倍加細胞における中心体サイズ、中心体周辺物質の集積、微小管形成能を、二倍体レベルにまで低減させることに成功した。興味深いことに、この人為的に中心体増大を抑制した染色体倍加細胞では、著しい細胞増殖能の低下が見られた。さらにライブイメージング解析により、この中心体増大抑制細胞株では、細胞死の頻度が増加し、他の細胞分裂擾乱処理への感受性が著しく増加することを見出した。このことから、新奇中心体増大現象は、染色体倍加細胞において、その生存性や増殖の堅牢性を担保する役割を果たす可能性が示唆された。
• 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

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2019年04月 -2021年03月 

  代表者 : 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。
• 紡錘体が極端な染色体数変動に順応する仕組みの解明

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2017年04月 -2019年03月 

  代表者 : 上原 亮太, 矢口 完, 山本 隆博, 島田 将矢

   

  通常体細胞は父母方ゲノムを1セットずつ持つ二倍体であるが、ガンでは二倍体を劇的に逸脱した異常細胞が見られる。本研究では細胞、とくに染色体分配を司る紡錘体がどのように極端な染色体数異常に順応して細胞増殖を可能にしているのかを解明することを目的とした。細胞イメージングおよび網羅的遺伝子発現解析から、染色体数の劇的な変化が分裂期中心体構造や細胞周期制御因子cyclin D遺伝子の発現を変化させることを突き止めた。さらに、それらの細胞制御経路の特異的抑制が正常二倍体状態から逸脱した細胞で選択的な増殖抑制を起こすことを見つけ、劇的な染色体数変化に細胞が順応する分子機構の一端を明らかにした。
• 細胞のくびれ運動を駆動する収縮環と細胞膜の相互作用メカニズムの解明

  日本学術振興会：科学研究費助成事業 挑戦的萌芽研究

  研究期間 : 2015年04月 -2017年03月 

  代表者 : 上原 亮太, 比留間 翔太

   

  細胞膜と細胞骨格の相互作用の正確な制御は動物細胞の細胞質分裂にとって必須であるが、その分子機構は明らかでない。本研究では種々の細胞骨格細胞膜結合性因子の分裂期細胞内動態、機能解析を通して、細胞質分裂を司る膜、細胞骨格相互作用機構の理解を目指した。我々はERMタンパク質が細胞質分裂期に他の膜結合タンパク質とは異なる特徴的な細胞内動態および局在依存性を持つことを発見した。ERMタンパク質の欠損は細胞質分裂の完了を阻害しなかったが、膜収縮運動の特性を変化させた。興味深いことに、他の膜因子の発現を抑制するとERMの分裂位置への集積が顕著に増加し、収縮運動により積極的に寄与するようになることがわかった。
• 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

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2013年04月 -2015年03月 

  代表者 : 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。
• 微小管動態制御による分裂位置制御メカニズム

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2013年04月 -2015年03月 

  代表者 : 上原 亮太

   

  細胞分裂の正確な制御は、生命の継承および恒常性の維持に必須であるが、その分子機構は明らかでない。本研究では、細胞質分裂が起こる細胞内位置の制御機構を明らかにするために、ヒト培養細胞を用いて、微小管形成因子をRNAi法によって特異的に機能阻害し、それにより生じる分裂障害を詳細に調べた。その結果、微小管脱重合因子Kif2Aが中央紡錘体微小管の長さ制御を通して、分離染色体間距離を適正に保つ機能を有すること、また微小管重合因子オーグミンによる中央紡錘体微小管形成が、細胞のくびれ運動を司るアクチン細胞骨格制御因子の集積に必須の機能を果たすことを明らかにした。
• 細胞質分裂期の中央紡錘体形成機構の研究

  日本学術振興会：科学研究費助成事業 研究活動スタート支援

  研究期間 : 2010年 -2011年 

  代表者 : 上原 亮太

   

  染色体分配と細胞質分裂の正確な時空間的連携は分裂期後期に形成される「中央紡錘体」によって制御されるが、その形成メカニズムは未解明であった。 本研究では分裂細胞内の微小管の形成過程を可視化するアッセイを開発し,中央紡錘体微小管形成が主に分離した染色体の間の領域の染色体近傍で行われることを突き止めた。分子細胞生物学的解析によって、該当領域に新規の微小管形成中心が存在することを明らかにし、その形成に携わる機能分子として、微小管制御因子のオーグミンおよびHURPを同定した。この新規の微小管重合経路を特異的に阻害すると中央紡錘体形成および細胞質分裂の完了における架橋切断反応が阻害されたことから,今回発見した微小管形成経路が機能的な中央紡錘体の形成に必須の役割を果たすことが分かった。

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