Research activity information

• Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics

  Hironori Inaba, Tsuyoshi Imasaki, Kazuhiro Aoyama, Shogo Yoshihara, Hiroko Takazaki, Takayuki Kato, Hidemasa Goto, Kaoru Mitsuoka, Ryo Nitta, Takao Nakata

  Jun. 2025, iScience

  Scientific journal


• Enhanced axonal transport in large vertebrates: KIF5A adaptations in giraffes and pythons

  Taketoshi Kambara, Lu Rao, Yosuke Yamagishi, Kazuho Ikeda, Daisuke Taniguchi, Tsuyoshi Imasaki, Hideki Shigematsu, Naoki Sakai, Arne Gennerich, Ryo Nitta, Yasushi Okada

  May 2025


• Dimerization of GAS2 mediates crosslinking of microtubules and F-actin

  Jiancheng An, Tsuyoshi Imasaki, Akihiro Narita, Shinsuke Niwa, Ryohei Sasaki, Tsukasa Makino, Ryo Nitta, Masahide Kikkawa

  Abstract The spectraplakin family protein GAS2 was originally identified as a growth arrest-specific protein, and recent studies have revealed its involvement in multiple cellular processes. Its dual interaction with actin filaments and microtubules highlights its essential role in cytoskeletal organization, such as cell division, apoptosis, and possibly tumorigenesis. However, the structural basis of cytoskeletal dynamics regulation by GAS2 remains unclear. In this study, we present cryo-electron microscopy structures of the GAS2 type 3 calponin homology domain (CH3) in complex with F-actin at 2.8 Å resolution, thus solving the first type CH3 domain structure bound to F-actin and confirming its actin-binding activity. We also provide the first near-atomic resolution cryo-EM structure of the GAS2-GAR domain bound to microtubules and identify conserved microtubule-binding residues. Our biochemical experiments show that GAS2 promotes microtubule nucleation and polymerization, and that its C-terminal region is essential for dimerization, bundling of both F-actin and microtubules, and microtubule nucleation. As mutations leading to expression of C-terminally truncated GAS2 have been linked to hearing loss, these findings suggest that the disruption of GAS2-dependent cytoskeletal organisation could underlie auditory dysfunction.

  Springer Science and Business Media LLC, Apr. 2025, The EMBO Journal

  Scientific journal


• Neuronal Populations Involved in Motor Function Show Prominent Expression of Sbno1 During Postnatal Brain Development

  Sunjidmaa Zolzaya, Dai Ihara, Munkhsoyol Erkhembaatar, Shinsuke Ochiai, Ayaka Isa, Mariko Nishibe, Jean-Pierre Bellier, Takahiro Shimizu, Satoshi Kikkawa, Ryo Nitta, Yu Katsuyama

  Jan. 2025, Journal of Developmental Biology

  Scientific journal


• Left atrial single-cell transcriptomics reveals amphiregulin as a surrogate marker for atrial fibrillation.

  Yuya Suzuki, Takuo Emoto, Shunsuke Sato, Takeshi Yoshida, Mitsuhiko Shoda, Hiromi Endoh, Manabu Nagao, Tomoyo Hamana, Taishi Inoue, Tomohiro Hayashi, Eriko Nitta, Hiroki Konishi, Kunihiko Kiuchi, Mitsuru Takami, Kimitake Imamura, Masayuki Taniguchi, Masatoshi Inoue, Toshihiro Nakamura, Yusuke Sonoda, Hiroyuki Takahara, Kazutaka Nakasone, Kyoko Yamamoto, Kenichi Tani, Hidehiro Iwai, Yusuke Nakanishi, Shogo Yonehara, Atsushi Murakami, Ryuji Toh, Takenao Ohkawa, Tomoyuki Furuyashiki, Ryo Nitta, Tomoya Yamashita, Ken-Ichi Hirata, Koji Fukuzawa

  Atrial fibrillation (AF) is strongly associated with strokes, heart failure, and increased mortality. This study aims to identify the monocyte-macrophage heterogeneity and interactions of these cells with non-immune cells, and to identify functional biomarkers in patients with AF. Therefore, we assess the single cell landscape of left atria (LA), using a combination of single cell and nucleus RNA-seq. Myeloid cells in LA tissue are categorized into five macrophage clusters, three monocyte clusters, and others. Cell-Chat analysis revealed that monocytes and IL1B+ macrophages send epidermal growth factor (EGF) signals to fibroblasts. Amphiregulin (AREG) is the most upregulated gene in monocytes and IL1B+ macrophages in the AF group, compared with healthy controls from other groups. Serum AREG levels are higher in patients with persistent AF. These data suggested that EGF signaling pathway could be a therapeutic target for AF and serum AREG levels provide an effective biomarker for predicting persistent AF.

  Dec. 2024, Communications biology, 7(1) (1), 1601 - 1601, English, International magazine

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Innervation of the human sternoclavicular joint

  Kenji Emura, Ryo Nitta, Takamitsu Arakawa

  Abstract The sternoclavicular joint (SCJ) functions as the basal joint of the entire upper limb and must move in the proper pattern for normal scapular motion. Afferent sensations from joints, such as proprioception and pain sensation, are important for maintaining the proper motion and condition of joints. Detailed anatomical data are useful for discussing injuries or surgeries that impair the afferent nerve to the SCJ. Nerve branches to SCJs were examined on 12 sides, and the subclavian nerve was investigated on six sides to clarify whether it innervates this joint. On seven of the 12 sides, the SCJ was innervated by two nerves, (1) a branch from the medial supraclavicular nerve that ran medially along the clavicle and (2) a branch from the lateral pectoral nerve that innervated the clavicular head and upper part of the sternocostal head of the pectoralis major. This branch ran medially behind the clavicular head of the pectoralis major and reached the SCJ. In the remaining five sides, the SCJ was innervated solely by the branch from the medial supraclavicular nerve. Subclavian nerves ended within the subclavius muscle or periosteum of the clavicle and were separate from the SCJs. Our data on the route of nerve branches to the SCJ suggest that injury or surgery, such as clavicle fracture or resection of the clavicular head of the pectoralis major for myocutaneous flap transfer, can impair the SCJ's afferent nerve supply.

  Wiley, Aug. 2024, Clinical Anatomy

  Scientific journal


• Cryo-electron tomography of the actin cytoskeleton and membrane organization during lamellipodia formation using optogenetics

  Hironori Inaba, Tsuyoshi Imasaki, Kazuhiro Aoyama, Shogo Yoshihara, Hiroko Takazaki, Takayuki Kato, Hidemasa Goto, Kaoru Mitsuoka, Ryo Nitta, Takao Nakata

  Abstract Lamellipodia are sheet-like cellular protrusions crucial for cell migration and endocytosis; their ultrastructure has been extensively studied using electron microscopy. However, the ultrastructure of the actin cytoskeleton during lamellipodia formation remains underexplored. Here, we employed the optogenetic tool PA-Rac1 combined with cryo-electron tomography (cryo-ET) to precisely control Rac1 activation and subsequent freezing via blue light irradiation. This approach enabled detailed structural analysis of newly formed lamellipodia in cells with intact plasma membranes. We successfully visualized lamellipodia with varying degrees of extension, representing different stages of lamellipodia formation. In minor extensions, several unbundled actin filaments formed “Minor protrusions” at several points along the leading edge. For moderately extended lamellipodia, cross-linked actin filaments formed small filopodia-like structures, termed “mini filopodia.” In the most extended lamellipodia, filopodia matured at multiple points along the leading edge, and the number of cross-linked actin filaments running nearly parallel to the leading edge increased throughout the lamellipodia. These observations suggest that actin polymerization begins in specific plasma membrane regions, forming mini filopodia that either mature into full filopodia or detach from the leading edge to form parallel filaments. This turnover of actin structures likely drives lamellipodial protrusion and stabilizes the base, providing new insights into the structural dynamics of the actin cytoskeleton and the mechanisms driving cell migration.

  Cold Spring Harbor Laboratory, Aug. 2024


• Structural transitions in kinesin minus-end directed microtubule motility

  Satoki Shibata, Matthew Y. Wang, Tsuyoshi Imasaki, Hideki Shigematsu, Yuanyuan Wei, Chacko Jobichen, Hajime Hagio, J. Sivaraman, Sharyn A. Endow, Ryo Nitta

  Abstract Kinesin motor proteins hydrolyze ATP to produce force for spindle assembly and vesicle transport, performing essential functions in cell division and motility, but the structural changes required for force generation are uncertain. We now report high-resolution structures showing new transitions in the kinesin mechanochemical cycle, including power stroke fluctuations upon ATP binding and a post-hydrolysis state with bound ADP + free phosphate. We find that rate-limiting ADP release occurs upon microtubule binding, accompanied by central β-sheet twisting, which triggers the power stroke – stalk rotation and neck mimic docking – upon ATP binding. Microtubule release occurs with β-strand-to-loop transitions, implying that β-strand refolding induces Pi release and the recovery stroke. The strained β-sheet during the power stroke and strand-to-loop transitions identify the β-sheet as the long-sought motor spring. Teaser Stalk rotation, β-sheet twisting and refolding, and neck mimic docking drive the reversed working stroke of kinesin-14 INTRODUCTION Kinesin family proteins couple ATP hydrolysis to microtubule binding, generating force to produce steps or displacements along microtubules. The mechanism by which kinesins and other cytoskeletal motor proteins produce force is not fully understood. A current hypothesis is that the motors contain a spring-like or elastic element that creates strain under load during nucleotide binding or release, followed by a strain-relieving conformational change that produces force and a working stroke of the motor. The spring has not yet been identified for any motor. The power stroke differs for different motors – it consists of neck linker docking for plus-end directed kinesin-1 or a swing of the helical stalk for minus-end directed kinesin-14. RATIONALE Despite considerable research, the molecular dynamics of the kinesin-14 power stroke are still obscure, impeded by the weak microtubule binding of the motor. We overcame the weak binding by introducing a point mutation into the motor that results in faster ATP hydrolysis than wild type and tighter microtubule binding, which enabled us to resolve the motor mode of action. We now present high-resolution cryo-electron microscopy (cryo-EM) and x-ray structures of key mechanochemical states across the full force-producing cycle of a kinesin dimeric motor. RESULTS The new structures represent five different nucleotide states – two pre-power stroke states, a fluctuating power stroke, and two post-power stroke states. The structures are both microtubule-attached and unattached. They show the motor trapped in previously unreported transition states and reveal new conformational changes involved in energy transduction. The new transition states include a transient state in which the power stroke fluctuates during ATP binding and a new state of a kinesin motor bound to ADP and free Pi prior to phosphate release. The conformational changes include the folding of the kinesin-14 neck mimic into a structure resembling the docked kinesin-1 neck linker, accompanying the power stroke, and previously unreported β-strand-to-loop transitions with stored free energy that potentially induce Pi release and drive the recovery stroke. We interpret the new structures in the context of the hypothesis that the central β-sheet undergoes distortional changes during the mechanochemical cycle that store and release free energy, functioning as the elusive spring of the motors. CONCLUSION The new structures show that force is produced by coupled movements of the helical stalk, central β-sheet, and neck mimic, and uncover structural changes during the power stroke that are conserved among kinesins and myosin. We find that kinesin-14 binds to a microtubule by one head during the mechanical cycle, undergoes rate-limiting ADP release, and changes in conformation during ATP binding and hydrolysis to produce force. Notably, kinesin-14 utilizes the same mechanical strategy for force production as other kinesins but couples the changes to a large swing of the stalk, an innovation derived from myosin that is not observed for kinesin-1 or other kinesin motors. Force is produced by rearranging the binding surfaces of the stalk, strand β1, helices ɑ4 and ɑ6, and the neck mimic, and by twisting and shortening strands of the central β-sheet. These structural changes produce a power stroke – rotation of the helical stalk accompanied by neck mimic docking – during the transition from the nucleotide-free to ATP-bound state, and a reverse stroke after phosphate release that reprimes the motor for the next microtubule binding interaction. Kinesin-14 force production New transition states and structural movements in a model for motor energy transduction and force production: β-sheet twisting stores free energy in the microtubule-bound nucleotide-free (NF) state. A fluctuating power stroke is produced in the ATP state with neck mimic docking in the ADP·Pi state, resembling the kinesin-1 neck linker. This is followed by β-strand-to-loop transitions in the microtubule-bound ADP + free Pi state. Finally, β-sheet refolding drives the recovery stroke for reversion to the ADP state.

  Cold Spring Harbor Laboratory, Jul. 2024


• Structural basis of Irgb6 inactivation by Toxoplasma gondii through the phosphorylation of switch I

  Hiromichi Okuma, Yumiko Saijo‐Hamano, Hiroshi Yamada, Aalaa Alrahman Sherif, Emi Hashizaki, Naoki Sakai, Takaaki Kato, Tsuyoshi Imasaki, Satoshi Kikkawa, Eriko Nitta, Miwa Sasai, Tadashi Abe, Fuminori Sugihara, Yoshimasa Maniwa, Hidetaka Kosako, Kohji Takei, Daron M. Standley, Masahiro Yamamoto, Ryo Nitta

  Abstract Irgb6 is a priming immune‐related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell‐autonomous immunity. However, the molecular mechanism by which immunity‐related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide‐free Irgb6‐T95D, resulting from a conformational change in the G‐domain that allosterically modified the PV membrane‐binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii‐induced allosteric inactivation mechanism of Irgb6.

  Wiley, Nov. 2023, Genes to Cells

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Amyloid deposition through endocytosis in vascular endothelial cells.

  Seiji Nishikage, Akira Fujisawa, Hiromi Endoh, Hirotaka Sakamoto, Tomohide Suzuki, Maki Kanzawa, Shinichi Ishii, Mitsumasa Okano, Eriko Nitta, Kimikazu Yakushijin, Hidesaku Asakura, Kandai Nozu, Ryo Nitta, Yoshio Katayama, Kazuhiko Sakaguchi

  No mechanistic lead is known for establishing AL amyloid deposits in organs. We here report an electron microscopic (EM) analysis in a case of intestinal AL amyloidosis before initiating treatment for amyloidosis. The dense deposits of amyloid fibrils are concentrated around the small blood vessels in the submucosal area in intestinal tissue. Surprisingly, we observed endothelial cells (ECs) of blood vessels containing plenty of endocytotic (pinocytotic) and transcytotic vesicles at the luminal side and above the basement membrane, indicating the one-way active trafficking of either immunoglobulin light chain or pre-assembled amyloid fibrils from the luminal side of ECs to the extraluminal area of ECs. Immunoelectron microscopy displayed that the immuno-gold signals were observed in the vascular cavity and the subendothelial area of amyloid deposits. However, there is no sign of immunoglobulin light chain in pinocytotic vesicles. Therefore, the intestinal ECs may actively pump out mainly the pre-assembled amyloid fibrils (not light chains) from the blood stream into the subendothelial area as a physiological function.

  Nov. 2023, Experimental hematology, English, International magazine

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• CD47 promotes peripheral T cell survival by preventing dendritic cell–mediated T cell necroptosis

  Satomi Komori, Yasuyuki Saito, Taichi Nishimura, Datu Respatika, Hiromi Endoh, Hiroki Yoshida, Risa Sugihara, Rie Iida-Norita, Tania Afroj, Tomoko Takai, Okechi S. Oduori, Eriko Nitta, Takenori Kotani, Yoji Murata, Yoriaki Kaneko, Ryo Nitta, Hiroshi Ohnishi, Takashi Matozaki

  Conventional dendritic cells (cDCs) are required for peripheral T cell homeostasis in lymphoid organs, but the molecular mechanism underlying this requirement has remained unclear. We here show that T cell–specific CD47-deficient ( Cd47^ΔT ) mice have a markedly reduced number of T cells in peripheral tissues. Direct interaction of CD47-deficient T cells with cDCs resulted in activation of the latter cells, which in turn induced necroptosis of the former cells. The deficiency and cell death of T cells in Cd47^ΔT mice required expression of its receptor signal regulatory protein α on cDCs. The development of CD4 ⁺ T helper cell–dependent contact hypersensitivity and inhibition of tumor growth by cytotoxic CD8 ⁺ T cells were both markedly impaired in Cd47^ΔT mice. CD47 on T cells thus likely prevents their necroptotic cell death initiated by cDCs and thereby promotes T cell survival and function.

  Proceedings of the National Academy of Sciences, Aug. 2023, Proceedings of the National Academy of Sciences, 120(33) (33)

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• The molecular mechanism of non-centrosomal microtubule nucleation

  Tsuyoshi Imasaki, Ryo Nitta

  Aug. 2023, Seikagaku, Japanese

  [Refereed][Invited]


• TEAD1 trapping by the Q353R–Lamin A/C causes dilated cardiomyopathy

  Shintaro Yamada, Toshiyuki Ko, Masamichi Ito, Tatsuro Sassa, Seitaro Nomura, Hiromichi Okuma, Mayuko Sato, Tsuyoshi Imasaki, Satoshi Kikkawa, Bo Zhang, Takanobu Yamada, Yuka Seki, Kanna Fujita, Manami Katoh, Masayuki Kubota, Satoshi Hatsuse, Mikako Katagiri, Hiromu Hayashi, Momoko Hamano, Norifumi Takeda, Hiroyuki Morita, Shuji Takada, Masashi Toyoda, Masanobu Uchiyama, Masashi Ikeuchi, Kiminori Toyooka, Akihiro Umezawa, Yoshihiro Yamanishi, Ryo Nitta, Hiroyuki Aburatani, Issei Komuro

  Mutations in the LMNA gene encoding Lamin A and C (Lamin A/C), major components of the nuclear lamina, cause laminopathies including dilated cardiomyopathy (DCM), but the underlying molecular mechanisms have not been fully elucidated. Here, by leveraging single-cell RNA sequencing (RNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), protein array, and electron microscopy analysis, we show that insufficient structural maturation of cardiomyocytes owing to trapping of transcription factor TEA domain transcription factor 1 (TEAD1) by mutant Lamin A/C at the nuclear membrane underlies the pathogenesis of Q353R -LMNA– related DCM. Inhibition of the Hippo pathway rescued the dysregulation of cardiac developmental genes by TEAD1 in LMNA mutant cardiomyocytes. Single-cell RNA-seq of cardiac tissues from patients with DCM with the LMNA mutation confirmed the dysregulated expression of TEAD1 target genes. Our results propose an intervention for transcriptional dysregulation as a potential treatment of LMNA -related DCM.

  American Association for the Advancement of Science (AAAS), Apr. 2023, Science Advances, 9(15) (15)

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation

  Hiroshi Yamada, Tadashi Abe, Hikaru Nagaoka, Eizo Takashima, Ryo Nitta, Masahiro Yamamoto, Kohji Takei

  Irgb6 is a member of interferon γ-induced immunity related GTPase (IRG), and one of twenty “effector” IRGs, which coordinately attack parasitophorous vacuole membrane (PVM), causing death of intracellular pathogen. Although Irgb6 plays a pivotal role as a pioneer in the process of PVM disruption, the direct effect of Irgb6 on membrane remained to be elucidated. Here, we utilized artificial lipid membranes to reconstitute Irgb6-membrane interaction in vitro, and revealed that Irgb6 directly deformed the membranes. Liposomes incubated with recombinant Irgb6 were drastically deformed generating massive tubular protrusions in the absence of guanine nucleotide, or with GMP-PNP. Liposome deformation was abolished by incubating with Irgb6-K275A/R371A, point mutations at membrane targeting residues. The membrane tubules generated by Irgb6 were mostly disappeared by the addition of GTP or GDP, which are caused by detachment of Irgb6 from membrane. Binding of Irgb6 to the membrane, which was reconstituted in vitro using lipid monolayer, was stimulated at GTP-bound state. Irgb6 GTPase activity was stimulated by the presence of liposomes more than eightfold. Irgb6 GTPase activity in the absence of membrane was also slightly stimulated, by lowering ionic strength, or by increasing protein concentration, indicating synergistic stimulation of the GTPase activity. These results suggest that membrane targeting of Irgb6 and resulting membrane deformation does not require GTP, but converting into GTP-bound state is crucial for detaching Irgb6 from the membrane, which might coincident with local membrane disruption.

  Frontiers Media SA, Sep. 2022, Frontiers in Cellular and Infection Microbiology, 12, 992198 - 992198, English, International magazine

  Scientific journal


• Structural model of microtubule dynamics inhibition by kinesin-4 from the crystal structure of KLP-12 –tubulin complex

  Shinya Taguchi, Juri Nakano, Tsuyoshi Imasaki, Tomoki Kita, Yumiko Saijo-Hamano, Naoki Sakai, Hideki Shigematsu, Hiromichi Okuma, Takahiro Shimizu, Eriko Nitta, Satoshi Kikkawa, Satoshi Mizobuchi, Shinsuke Niwa, Ryo Nitta

  Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of the inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

  eLife Sciences Publications, Ltd, Sep. 2022, eLife, 11, English, International magazine

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation

  Tsuyoshi Imasaki, Satoshi Kikkawa, Shinsuke Niwa, Yumiko Saijo-Hamano, Hideki Shigematsu, Kazuhiro Aoyama, Kaoru Mitsuoka, Takahiro Shimizu, Mari Aoki, Ayako Sakamoto, Yuri Tomabechi, Naoki Sakai, Mikako Shirouzu, Shinya Taguchi, Yosuke Yamagishi, Tomiyoshi Setsu, Yoshiaki Sakihama, Eriko Nitta, Masatoshi Takeichi, Ryo Nitta

  Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αβ-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.

  eLife Sciences Publications, Ltd, Jun. 2022, eLife, 11, English, International magazine

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6

  Yumiko Saijo-Hamano, Aalaa Alrahman Sherif, Ariel Pradipta, Miwa Sasai, Naoki Sakai, Yoshiaki Sakihama, Masahiro Yamamoto, Daron M Standley, Ryo Nitta

  The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. Irgb6 is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive because of lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane-binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.

  Life Science Alliance, {LLC}, Jan. 2022, Life Science Alliance, 5(1) (1), e202101149 - e202101149

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• クロマチンリモデリング因子BRMは造血幹細胞を静止期に維持する(The chromatin remodeling factor BRM maintains a quiescent state of the hematopoietic stem cells)

  宮地 洋希, 桐山 大輝, 川端 野乃子, 糸川 直樹, 小出 周平, 山下 真幸, 大島 基彦, 今崎 剛, 須田 年生, 岩間 厚志, 仁田 亮, 仁田 英里子

  (一社)日本血液学会, Sep. 2021, 日本血液学会学術集会, 83回, OS1 - 4, English


• 【細胞機能の構造生物学】細胞骨格の構造生物学 微小管結合タンパク質

  吉川 知志, 仁田 英里子, 今崎 剛, 仁田 亮

  ＜文献概要＞細胞はすべて固有の動的な形態を持ち,なかには運動するものもある。このような細胞の形態維持や運動の際の骨組みとなるのが,細胞骨格と呼ばれるタンパク質複合体である。細胞骨格は太さにより3種類に分類され,細いものからマイクロフィラメント,中間系フィラメント,そして最も太いものが微小管である。微小管は直径25nmの中空な管状線維であるが,αおよびβチューブリンから成るヘテロダイマーを基本単位として,それが長軸方向に連なってプロトフィラメントと呼ばれる細線維構造体を形成し,それが10-15本程度,螺旋状に結合することで管状の構造が形づくられている。長軸方向に交互に位置するα,βチューブリンの特性の違いにより微小管は方向性を有し,βチューブリンが露出する端はαβチューブリンヘテロダイマーが付加(重合)したり解離(脱重合)する速度が速くプラス端と呼ばれるのに対し,反対端は逆の性質を持ちマイナス端と呼ばれる。微小管は極めて動的な細胞骨格で,常に重合と脱重合を繰り返すことにより細胞形態の維持や変化を担い運動を制御するのみならず,細胞分裂や細胞内物質輸送においても極めて重要な役割を果たす。この微小管の重合と脱重合のバランスは多種多様なタンパク質により制御されており,なかでも微小管に直接結合するタンパク質群は微小管結合タンパク質(microtubule associated proteins;MAPs)と呼ばれる。MAPsは微小管への結合特性などから幾つかのグループに分類され,代表的なものは古典的(構造的)MAPs,分子モーター,プラス端結合タンパク質(+TIPs),マイナス端結合タンパク質(-TIPs)の4種類である(図1)。本稿ではこれらについて,筆者らの最近の成果を中心に概説したい。

  (公財)金原一郎記念医学医療振興財団, Aug. 2020, 生体の科学, 71(4) (4), 298 - 303, Japanese


• Kinesin-binding-triggered conformation switching of microtubules contributes to polarized transport.

  Tomohiro Shima, Manatsu Morikawa, Junichi Kaneshiro, Taketoshi Kambara, Shinji Kamimura, Toshiki Yagi, Hiroyuki Iwamoto, Sotaro Uemura, Hideki Shigematsu, Mikako Shirouzu, Taro Ichimura, Tomonobu M Watanabe, Ryo Nitta, Yasushi Okada, Nobutaka Hirokawa

  Kinesin-1, the founding member of the kinesin superfamily of proteins, is known to use only a subset of microtubules for transport in living cells. This biased use of microtubules is proposed as the guidance cue for polarized transport in neurons, but the underlying mechanisms are still poorly understood. Here, we report that kinesin-1 binding changes the microtubule lattice and promotes further kinesin-1 binding. This high-affinity state requires the binding of kinesin-1 in the nucleotide-free state. Microtubules return to the initial low-affinity state by washing out the binding kinesin-1 or by the binding of non-hydrolyzable ATP analogue AMPPNP to kinesin-1. X-ray fiber diffraction, fluorescence speckle microscopy, and second-harmonic generation microscopy, as well as cryo-EM, collectively demonstrated that the binding of nucleotide-free kinesin-1 to GDP microtubules changes the conformation of the GDP microtubule to a conformation resembling the GTP microtubule.

  Dec. 2018, The Journal of cell biology, 217(12) (12), 4164 - 4183, English, International magazine

  [Refereed]

  Scientific journal


• Structural insight into microtubule stabilization and kinesin inhibition by Tau family MAPs.

  Hideki Shigematsu, Tsuyoshi Imasaki, Chihiro Doki, Takuya Sumi, Mari Aoki, Tomomi Uchikubo-Kamo, Ayako Sakamoto, Kiyotaka Tokuraku, Mikako Shirouzu, Ryo Nitta

  The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these proteins, and the distribution of these variants regulates fundamental physiological phenomena in cells. In this study, using cryo-EM, we visualized the MAP4 microtubule complex with the molecular motor kinesin-1. MAP4 bound to the C-terminal domains of tubulins along the protofilaments stabilizes the longitudinal contacts of the microtubule. The strongest bond of MAP4 was found around the intertubulin-dimer interface such that MAP4 coexists on the microtubule with kinesin-1 bound to the intratubulin-dimer interface as well. MAP4, consisting of five repeats, further folds and accumulates above the intertubulin-dimer interface, interfering with kinesin-1 movement. Therefore, these cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.

  Dec. 2018, The Journal of cell biology, 217(12) (12), 4155 - 4163, English, International magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Recent progress in structural biology: lessons from our research history

  Ryo Nitta, Tsuyoshi Imasaki, Eriko Nitta

  Aug. 2018, Microscopy

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Structural insights into the altering function of CRMP2 by phosphorylation

  Takuya Sumi, Tsuyoshi Imasaki, Mari Aoki, Naoki Sakai, Eriko Nitta, Mikako Shirouzu, Ryo Nitta

  Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity by controlling microtubule dynamics. CRMP2 activity is regulated by semaphorin-induced phosphorylation at the C-terminal tail domain. Unphosphorylated CRMP2 induces effective axonal microtubule formation to give the axonal characteristics to a neurite, whereas phosphorylated CRMP2 leads to the apparently opposite effect, growth cone collapse. We have recently characterized the structural detail of CRMP2-induced axonal microtubule formation (Niwa et al. (2017) Sci. Rep., 7: 10681). CRMP2 forms the hetero-trimer with GTP-tubulin to induce effective axonal microtubule formation in the future axon. Phosphorylation of CRMP2 has been reported to decrease the affinity between CRMP2 and the microtubule, albeit the molecular mechanisms of how the phosphorylation of CRMP2 changes the structure to achieve distinct effects from unphosphorylated CRMP2 is not well understood. Here we performed a series of biochemical and structural analyses of phospho-mimic CRMP2. Phosphorylation of CRMP2 undergoes small conformational changes at the C-terminal tail with shifting the surface charge, which not only alters the interactions within the CRMP2 tetramer but also alters the interactions with GTP-tubulin. Consequently, phospho-mimic CRMP2 fails to form a hetero-trimer with GTP-tubulin, thus losing the ability to establish and maintain the axonal microtubules.

  Japan Society for Cell Biology, Feb. 2018, Cell Structure and Function, 43(1) (1), 15 - 23, English

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• キネシン型分子モーターの多彩な機能を支える分子構造基盤

  仁田亮

  2018, 顕微鏡, 53(2) (2), Japanese

  [Refereed][Invited]


• Structural basis for CRMP2-induced axonal microtubule formation

  Shinsuke Niwa, Fumio Nakamura, Yuri Tomabechi, Mari Aoki, Hideki Shigematsu, Takashi Matsumoto, Atsushi Yamagata, Shuya Fukai, Nobutaka Hirokawa, Yoshio Goshima, Mikako Shirouzu, Ryo Nitta

  Microtubule associated protein Collapsin response mediator protein 2 (CRMP2) regulates neuronal polarity in developing neurons through interactions with tubulins or microtubules. However, how CRMP2 promotes axonal formation by affecting microtubule behavior remains unknown. This study aimed to obtain the structural basis for CRMP2-tubulin/microtubule interaction in the course of axonogenesis. The X-ray structural studies indicated that the main interface to the soluble tubulin-dimer is the last helix H19 of CRMP2 that is distinct from the known C-terminal tail-mediated interaction with assembled microtubules. In vitro structural and functional studies also suggested that the H19-mediated interaction promoted the rapid formation of GTP-state microtubules directly, which is an important feature of the axon. Consistently, the H19 mutants disturbed axon elongation in chick neurons, and failed to authorize the structural features for axonal microtubules in Caenorhabditis elegans. Thus, CRMP2 induces effective axonal microtubule formation through H19-mediated interactions with a soluble tubulin-dimer allowing axonogenesis to proceed.

  NATURE PUBLISHING GROUP, Sep. 2017, SCIENTIFIC REPORTS, 7(1) (1), 10681, English

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• 分子モーター研究の新しい方向 キネシンによる微小管の構造変化

  島 知弘, 森川 真夏, 金城 純一, 神原 丈敏, 上村 慎治, 八木 俊樹, 岩本 裕之, 市村 垂生, 渡邉 朋信, 上村 想太郎, 仁田 亮, 岡田 康志, 廣川 信隆

  (一社)日本細胞生物学会, May 2017, 日本細胞生物学会大会講演要旨集, 69回, 39 - 39, Japanese

  [Refereed]


• Motility and microtubule depolymerization mechanisms of the Kinesin-8 motor, KIF19A

  Doudou Wang, Ryo Nitta, Manatsu Morikawa, Hiroaki Yajima, Shigeyuki Inoue, Hideki Shigematsu, Masahide Kikkawa, Nobutaka Hirokawa

  The kinesin-8 motor, KIF19A, accumulates at cilia tips and controls cilium length. Defective KIF19A leads to hydrocephalus and female infertility because of abnormally elongated cilia. Uniquely among kinesins, KIF19A possesses the dual functions of motility along ciliary microtubules and depolymerization of microtubules. To elucidate the molecular mechanisms of these functions we solved the crystal structure of its motor domain and determined its cryoelectron microscopy structure complexed with a microtubule. The features of KIF19A that enable its dual function are clustered on its microtubule-binding side. Unexpectedly, a destabilized switch II coordinates with a destabilized L8 to enable KIF19A to adjust to both straight and curved microtubule protofilaments. The basic clusters of L2 and L12 tether the microtubule. The long L2 with a characteristic acidic-hydrophobic-basic sequence effectively stabilizes the curved conformation of microtubule ends. Hence, KIF19A utilizes multiple strategies to accomplish the dual functions of motility and microtubule depolymerization by ATP hydrolysis.

  ELIFE SCIENCES PUBLICATIONS LTD, Sep. 2016, ELIFE, 5, English

  [Refereed]

  Scientific journal


• Structural Basis of Backwards Motion in Kinesin-1-Kinesin-14 Chimera: Implication for Kinesin-14 Motility

  Masahiko Yamagishi, Hideki Shigematsu, Takeshi Yokoyama, Masahide Kikkawa, Mitsuhiro Sugawa, Mari Aoki, Mikako Shirouzu, Junichiro Yajima, Ryo Nitta

  Kinesin-14 is a unique minus-end-directed microtubule-based motor. A swinging motion of a class-specific N-terminal neck helix has been proposed to produce minus-end directionality. However, it is unclear how swinging of the neck helix is driven by ATP hydrolysis utilizing the highly conserved catalytic core among all kinesins. Here, using a motility assay, we show that in addition to the neck helix, the conserved five residues at the C-terminal region in kinesin-14, namely the neck mimic, are necessary to give kinesin-1 an ability to reverse its directionality toward the minus end of microtubules. Our structural analyses further demonstrate that the C-terminal neck mimic, in cooperation with conformational changes in the catalytic core during ATP binding, forms a kinesin-14 bundle with the N-terminal neck helix to swing toward the minus end of microtubules. Thus, the neck mimic plays a crucial role in coupling the chemical ATPase reaction with the mechanical cycle to produce the minus-end-directed motility of kinesin-14.

  CELL PRESS, Aug. 2016, STRUCTURE, 24(8) (8), 1322 - 1334, English

  [Refereed]

  Scientific journal


• Motility and Microtubule Depolymerization Mechanisms of the Kinesin-8 motor, KIF19A.

  D. Wang, R. Nitta, M. Morikawa, H. Yajima, S. Inoue, H. Shigematsu, M. Kikkawa, N. Hirokawa

  AMER SOC CELL BIOLOGY, 2016, MOLECULAR BIOLOGY OF THE CELL, 27, English

  [Refereed]


• X-ray and Cryo-EM structures reveal mutual conformational changes of Kinesin and GTP-state microtubules upon binding

  Manatsu Morikawa, Hiroaki Yajima, Ryo Nitta, Shigeyuki Inoue, Toshihiko Ogura, Chikara Sato, Nobutaka Hirokawa

  The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the rigor conformation', where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules.

  WILEY-BLACKWELL, May 2015, EMBO JOURNAL, 34(9) (9), 1270 - 1286, English

  [Refereed]

  Scientific journal


• New simulated annealing approach considering helix bending applied to determine the 8.8 angstrom structure of 15-protofilament microtubules

  Toshihiko Ogura, Hiroaki Yajima, Ryo Nitta, Nobutaka Hirokawa, Chikara Sato

  The helix is an important motif in biological architectures. The helical structures of nanoscale proteins are principally determined by three-dimensional (3D) reconstruction from electron micrographs. However, bending or distortion of flexible helices and the low contrast of the images recorded by cryo-electron microscopy, prevent the analysis from reaching high resolution. We have developed a novel helical reconstruction method that overcomes these issues, and present the processing of microtubule images to demonstrate its application. Cropping long helical structures into small square pieces allows bending or distortion of the helices to be accounted for. The initial image-frames are automatically positioned assuming perfect helical symmetry. A simulated annealing (SA)-based algorithm is then used to adjust the framing. This is guided by the contrast of 2D averages, which serve as an accuracy index. After the initial 3D reconstruction, the position and orientation of each average image is iteratively adjusted to give the best match between the input average and the reprojection from the reconstruction. Finally, reconstructions from images recorded at different defocus values, are aligned and averaged to compensate the contrast transfer modulation and improve the resolution. The method successfully determined the structure of a 15-protofilament microtubule. The 8.8 angstrom resolution (7.8 angstrom using the 0.143 FSC criterion) attained allows differences between the alpha- and beta- tubulins to be discerned in the absence of a molecular landmark such as microtubule-associated proteins, for the first time by electron microscopy. The SA-based method is applicable to other helical protein complexes and in general to helical structures. (C) 2014 Elsevier Inc. All rights reserved.

  ACADEMIC PRESS INC ELSEVIER SCIENCE, Nov. 2014, JOURNAL OF STRUCTURAL BIOLOGY, 188(2) (2), 165 - 176, English

  [Refereed]

  Scientific journal


• キネシン型分子モーターの構造変化と動作機構

  仁田亮

  2014, 生物物理 (解説), 54(3) (3)

  [Refereed][Invited]


• Structural Basis for the ATP-Induced Isomerization of Kinesin

  Qing Chang, Ryo Nitta, Shigeyuki Inoue, Nobutaka Hirokawa

  Kinesin superfamily proteins (KIFs) are microtubule-based molecular motors driven by the energy derived from the hydrolysis of ATP. Previous studies have revealed that the ATP binding step is crucial both for the power stroke to produce motility and for the inter-domain regulation of ATPase activity to guarantee the processive movement of dimeric KIFs. Here, we report the first crystal structure of KIF4 complexed with the non-hydrolyzable ATP analog, AMPPNP (adenylyl imidodiphosphate), at 1.7 angstrom resolution. By combining our structure with previously solved KIF1A structures complexed with two ATP analogs, molecular snapshots during ATP binding reveal that the closure of the nucleotide-binding pocket during ATP binding is achieved by closure of the backdoor. Closure of the backdoor stabilizes two mobile regions, switch I and switch II, to generate the phosphate tube from which hydrolyzed phosphate is released. Through the stabilization of switch II, the local conformational change at the catalytic center is further relayed to the neck-linker element that fully docks to the catalytic core to produce the power stroke. Because the neck linker is a sole element that connects the partner heads in dimeric KIFs, this tight structural coordination between the catalytic center and neck linker enables inter-domain communication between the partner heads. This study also revealed the putative microtubule-binding site of KIF4, thus providing structural insights that describe the specific binding of KIF4 to the microtubule. (C) 2013 Elsevier Ltd. All rights reserved.

  ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, Jun. 2013, JOURNAL OF MOLECULAR BIOLOGY, 425(11) (11), 1869 - 1880, English

  [Refereed]

  Scientific journal


• Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy

  Hiroaki Yajima, Toshihiko Ogura, Ryo Nitta, Yasushi Okada, Chikara Sato, Nobutaka Hirokawa

  Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by beta-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the guanosine diphosphate (GDP) state. In this paper, we solved the cryoelectron microscopy (cryo-EM) structure of microtubule stabilized with a GTP analogue, guanylyl 5'-alpha,beta-methylenediphosphonate (GMPCPP), at 8.8-angstrom resolution by developing a novel cryo-EM image reconstruction algorithm. In contrast to the crystal structures of GTP-bound tubulin relatives such as gamma-tubulin and bacterial tubulins, significant changes were detected between GMPCPP and GDP-taxol microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings are consistent with the structural plasticity or lattice model and suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule-binding proteins, such as EB1 or kinesin.

  ROCKEFELLER UNIV PRESS, Aug. 2012, JOURNAL OF CELL BIOLOGY, 198(3) (3), 315 - 322, English

  [Refereed]

  Scientific journal


• 3H0900 Large Conformational Changes in Tubulin in the GTP- and GDPStates Microtubules Observed by Cryo Electron Microscopy(Cell Biology III:Cytoskeleton & Motility,Oral Presentation)

  Yajima Hiroaki, Ogura Toshihiko, Nitta Ryo, Okada Yasushi, Sato Chikara, Hirokawa Nobutaka

  The Biophysical Society of Japan General Incorporated Association, 2012, Seibutsu Butsuri, 52, S69, English


• The mechanisms of kinesin motor motility: lessons from the monomeric motor KIF1A

  Nobutaka Hirokawa, Ryo Nitta, Yasushi Okada

  Most kinesins move processively along microtubules by using energy derived from ATP hydrolysis. Almost all of the intermediate structures of this ATPase reaction cycle have been solved for the monomeric kinesin 3 family motor KIF1A. Based on this structural information, we propose a common mechanism of kinesin motility, focusing on the regulation of kinesin motility through their interaction with microtubules and by their 'neck-linker' region, which connects their motor domain to cargo and kinesin partner heads.

  NATURE PUBLISHING GROUP, Dec. 2009, NATURE REVIEWS MOLECULAR CELL BIOLOGY, 10(12) (12), 877 - 884, English

  [Refereed]


• 分子モーターキネシンのヌクレオチド交換過程の構造的基盤

  仁田 亮, 岡田 康志, 廣川 信隆

  (一社)日本解剖学会, Mar. 2009, 解剖学雑誌, 84(Suppl.) (Suppl.), 151 - 151, Japanese

  [Refereed]


• Structural model for strain-dependent microtubule activation of Mg-ADP release from kinesin

  Ryo Nitta, Yasushi Okada, Nobutaka Hirokawa

  Mg-ADP release is considered to be a crucial process for the regulation and motility of kinesin. To gain insight into the structural basis of this process, we solved the atomic structures of kinesin superfamily protein-1A (KIF1A) during and after Mg2+ release. On the basis of new structural and mutagenesis data, we propose a model mechanism for microtubule activation of Mg-ADP release from KIF1A. In our model, a specific interaction between loop L7 of KIF1A and beta-tubulin reconfigures the KIF1A active site by shifting the relative positions of switches I and II. This leads to the sequential release of a group of water molecules that sits over the Mg2+ in the active site, followed by Mg2+ and finally the ADP. We further propose that this set of events is linked to a strain-dependent docking of the neck linker to the motor core, which produces a two-step power stroke.

  NATURE PUBLISHING GROUP, Oct. 2008, NATURE STRUCTURAL & MOLECULAR BIOLOGY, 15(10) (10), 1067 - 1075, English

  [Refereed]

  Scientific journal


• Structural model for strain-dependent microtubule activation of Mg-ADP release from kinesin

  Ryo Nitta, Yasushi Okada, Nobutaka Hirokawa

  Mg-ADP release is considered to be a crucial process for the regulation and motility of kinesin. To gain insight into the structural basis of this process, we solved the atomic structures of kinesin superfamily protein-1A (KIF1A) during and after Mg2+ release. On the basis of new structural and mutagenesis data, we propose a model mechanism for microtubule activation of Mg-ADP release from KIF1A. In our model, a specific interaction between loop L7 of KIF1A and β-tubulin reconfigures the KIF1A active site by shifting the relative positions of switches I and II. This leads to the sequential release of a group of water molecules that sits over the Mg2+ in the active site, followed by Mg2+ and finally the ADP. We further propose that this set of events is linked to a strain-dependent docking of the neck linker to the motor core, which produces a two-step power stroke. © 2008 Nature Publishing Group.

  10, Oct. 2008, Nature Structural and Molecular Biology, 15(10) (10), 1067 - 1075, English

  [Refereed]

  Scientific journal


• Crystallization of the Mg-releasing intermediates of kinesin ATPase

  Ryo Nitta, Yasushi Okada, Nobutaka Hirokawa

  2008, Nature protoc.

  [Invited]


• 1SB56 Structural mechanism of ATP hydrolysis and coupled conformational changes in kinesin-type motor KIF1A

  Nitta R., Okada Y., Hirokawa N.

  The Biophysical Society of Japan General Incorporated Association, 2005, Seibutsu Butsuri, 45(0) (0), S12, Japanese

  [Refereed]


• KIF1A alternately uses two loops to bind microtubules.

  Ryo Nitta, Masahide Kikkawa, Yasushi Okada, Nobutaka Hirokawa

  The motor protein kinesin moves along microtubules, driven by adenosine triphosphate (ATP) hydrolysis. However, it remains unclear how kinesin converts the chemical energy into mechanical movement. We report crystal structures of monomeric kinesin KIF1A with three transition-state analogs: adenylyl imidodiphosphate (AMP-PNP), adenosine diphosphate (ADP)-vanadate, and ADP-AlFx (aluminofluoride complexes). These structures, together with known structures of the ADP-bound state and the adenylyl-(beta,gamma-methylene) diphosphate (AMP-PCP)-bound state, show that kinesin uses two microtubule-binding loops in an alternating manner to change its interaction with microtubules during the ATP hydrolysis cycle; loop L11 is extended in the AMP-PNP structure, whereas loop L12 is extended in the ADP structure. ADP-vanadate displays an intermediate structure in which a conformational change in two switch regions causes both loops to be raised from the microtubule, thus actively detaching kinesin.

  5684, Jul. 2004, Science (New York, N.Y.), 305(5684) (5684), 678 - 83, English, International magazine

  [Refereed]


• A common mechanism for microtubule destabilizers - M type kinesins stabilize curling of the protofilament using the class-specific neck and loops

  T Ogawa, R Nitta, Y Okada, N Hirokawa

  Unlike other kinesins, middle motor domain-type kinesins depolymerize the microtubule from its ends. To elucidate its mechanism, we solved the X-ray crystallographic structure of KIF2C, a murine member of this family. Three major class-specific features were identified. The class-specific N-terminal neck adopts a long and rigid helical structure extending out vertically into the interprotofilament groove. This structure explains its dual roles in targeting to the end of the microtubule and in destabilization of the lateral interaction of the protofilament. The loop L2 forms a unique finger-like structure, long and rigid enough to reach the next tubulin subunit to stabilize the peeling of the protofilament. The open conformation of the switch I loop could be reversed by the shift of the microtubule binding L8 loop, suggesting its role as the sensor to trigger ATP hydrolysis. Mutational analysis supports these structural implications.

  CELL PRESS, Feb. 2004, CELL, 116(4) (4), 591 - 602, English

  [Refereed]

  Scientific journal


• 2SA01 Atomic model of depolymerization mechanism of micro-tubules by a kinesin superfamily protein, KIF2C

  Okada Y.

  The Biophysical Society of Japan General Incorporated Association, 2004, Seibutsu Butsuri, 44(0) (0), S13, Japanese

  [Refereed]


• Primary cardiac angiosarcoma of the right atrium undiagnosed by transvenous endocardial tumor biopsy

  R Nitta, Y Sakomura, K Tanimoto, T Hidai, H Kasanuki, S Aomi, T Nishikawa

  A 50-year-old man was admitted with acute pericarditis, Echocardiography demonstrated a large mass on the right atrial free wall along with a pericardial effusion. We performed transvenous biopsy of this mass under transesophageal echocardiographic guidance. Though the biotome obtained the mass, the pathological findings were of organized thrombus. Two weeks later, a new precordial mass appeared around the left third rib and was suspected to be a metastasis. Incisional biopsy of this mass gave the diagnosis of angiosarcoma.

  JAPAN SOC INTERNAL MEDICINE, Dec. 1998, INTERNAL MEDICINE, 37(12) (12), 1023 - 1026, English

  [Refereed]

  Scientific journal

• MAPs: microtubule associated proteins

  吉川知志, 仁田英里子, 今崎剛, 仁田亮

  2020, 生体の科学, 71(4) (4)


• Mutual conformational changes of kinesin and GTP microtubule upon their binding.

  M. Morikawa, H. Yajima, R. Nitta, S. Inoue, T. Ogura, C. Sato, N. Hirokawa

  AMER SOC CELL BIOLOGY, Dec. 2014, MOLECULAR BIOLOGY OF THE CELL, 25, English

  Summary international conference


• クライオ電顕を用いたGTP型/GDP-taxol型微小管におけるtubulinの構造変化

  矢島孔明, 小椋俊彦, 仁田亮, 岡田康志, 佐藤主税, 廣川信隆

  2013, 日本解剖学会総会・全国学術集会講演プログラム・抄録集, 118th


• Conformational Changes in Tubulin in GMPCPP and GDP-taxol Microtubules Observed by Cryo Electron Microscopy

  H. Yajima, T. Ogura, R. Nitta, Y. Okada, C. Sato, N. Hirokawa

  AMER SOC CELL BIOLOGY, 2012, MOLECULAR BIOLOGY OF THE CELL, 23, English

  Summary international conference


• A common mechanism for microtubule destabilizers-M-Type kinesins stabilize curling of the protofilament using the class-specific neck and loops.

  T Ogawa, R Nitta, Y Okada, N Hirokawa

  AMER SOC CELL BIOLOGY, Nov. 2004, MOLECULAR BIOLOGY OF THE CELL, 15, 36A - 36A, English

  Summary international conference


• KIF1A alternately uses two loops to bind microtubules

  R Nitta, M Kikkawa, Y Okada, N Hirokawa

  AMER SOC CELL BIOLOGY, Nov. 2004, MOLECULAR BIOLOGY OF THE CELL, 15, 278A - 279A, English

  Summary international conference


• A common mechanism for microtubule destabilizers-M-type kinesins stabilize curling of the protofilament using the class-specific neck and loops

  Tadayuki Ogawa, Ryo Nitta, Yasushi Okada, Nobutaka Hirokawa

  JAPAN SOC CELL BIOLOGY, May 2004, CELL STRUCTURE AND FUNCTION, 29, 37 - 37, English

  Summary international conference


• Swinging movement of the back door of kinesin generates the alternating use of its two microtubule-binding loops

  Nitta R, M. Kikkawa, Y. Okada, N. Hirokawa

  2004, Science, 305, 678 - 683

• 微小管結合タンパク質

  吉川知志, 仁田英里子, 今崎剛, 仁田 亮

  Contributor, Aug. 2020


• Structural Lifescience

  仁田亮, 今崎剛

  Contributor, X-ray and cryo-EM visualize the motility of microtubule-based motors, 羊土社, Mar. 2020


• Cutting Edge of Molecular Cardiology

  Tsuyoshi Imasaki, Eriko Nitta, Ryo Nitta

  Contributor, 分子構造解析で何が見えるか, 南山堂, Apr. 2019


• Jikken Igaku

  Ryo Nitta

  Contributor, X-ray and cryo-EM visualize the motility of microtubule-based motors., 羊土社, May 2018


• Encyclopedia of Biophysics

  Ryo Nitta, Nobutaka Hirokawa

  Contributor, Fundamental Properties and Structure of Kinesin, Springer-Verlag Berlin Heidelberg, 2012


• 入門 構造生物学

  廣川信隆, 仁田亮

  Contributor, 生体分子モーター, 共立出版, Apr. 2010

• Cross-scale biology

  吉川 雅英, 稲葉 謙次, 水上 進, 田中 元雅, 仁田 亮, 西田 紀貴, 杉田 有治, 山本 林, 倉永 英里奈, 福間 剛士

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Transformative Research Areas (A), The University of Tokyo, 10 Sep. 2021 - 31 Mar. 2026

  本研究領域では、分子レベルからオルガネラ・細胞レベルまでの定量的クロススケール計測、特に細胞内における20-500 nm程度の大きさの「メゾ複雑体」の計測も可能にすることにより、どのように生命現象や病気の起源を決定するのかを分子レベルからオルガネラ・細胞レベルまでシームレスに解明することを目指している。 この目的のために、本領域では、分子レベルからメゾ複雑体を定量的に計測できるバーチャルな「クロススケール細胞計測センター」を創設し、総括班がその運営を担っている。このクロススケール観察には、最近急速に発展しつつあるクライオ電子線トモグラフィー（Cryo-ET, 吉川班）を中心に、超解像イメー ジング（水上班）、In-cell NMR（西田班）、In-cell AFM（福間班）を有機的に組み合わせ、また、複数の手法に使える標識の開発、実験データの 統合と解釈の為に大規模計算科学（杉田班）を用いている。また、メゾ複雑体を研究対象とする重要な生物学的課題を持つ研究グループが集まり、クロススケール細胞計測センターを通して緊密に連携する体制を整えている。 本研究領域のホームページを開設し、研究の目的、先端技術紹介、研究内容、成果等について広く一般に公開を行っており、SNSでの情報発信なども行っている。緊密な共同研究を推進するため、計画班代表による領域会議を開催し、また、本領域所属の研究者による班会議をハイブリッドで開催し、動画配信なども行った。


• クロススケール細胞内分子構造動態解析が解明する細胞骨格ネットワーク構築とその破綻

  仁田 亮

  日本学術振興会, 科学研究費助成事業 学術変革領域研究(A), 学術変革領域研究(A), 神戸大学, 10 Sep. 2021 - 31 Mar. 2026

  学術変革領域「クロススケール新生物学」の中で、本グループは細胞骨格ネットワーク構築とその破綻による疾病発症の分子機構を解明する。本領域で創設されるクロススケール細胞計測センターを活用して、3種の医学・生命科学的課題を遂行するとともに、技術開発へのフィードバックも進めている。 1)非中心体性微小管ネットワーク形成のクロススケール解析：in vitroで、CAMSAPによるLLPSの形成、LLPS内部での重合反応の進行、LLPSから微小管が放射状に伸長する過程を、クライオ電子顕微鏡、TIRF、AFM=高速分子動画を用いて解析している。HeLa細胞では、GFP-tubulinの安定発現株を導入し、微小管ネットワーク形成の時系列のキーフレーム、例えば相分離によるMTOC形成過程や、微小管が放射状に伸び る星状体形成過程の時間軸を共焦点顕微鏡を用いて解析しており、今後、クライオ電子線トモグラフィー解析へ持ち込む。 2)光遺伝学によるタイムラ プスクライオEM技術の開発：Rac1光スイッチによる葉状仮足形成におけるアクチンネットワークの構築過程を材料として、まずはCOS-7細胞を用いて、タイムラプスクライオEM技術を研究開発している。 3)分子から個体のクロススケール解析法の開発―医学応用へ：拡張型心筋症やパーキンソン病などを題材に、ヒトiPS細胞内の分子構造変化をクロススケール計測により捉え、さらに組織レベル・個体レベルへのクロススケール解析法も合わせて開発する。現在、iPS細胞の急速凍結・クライオCLEM観察の条件検討を行っている。


• Investigation of MTCL proteins which regulate assembly structures of microtubules based on the Golgi membrane.

  鈴木 厚, 仁田 亮

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Yokohama City University, 01 Apr. 2022 - 31 Mar. 2025


• Molecular mechanisms of non-centrosomal microtubule network formation

  仁田 亮

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, Apr. 2022 - Mar. 2025


• 臓器連関の包括的理解に基づく認知症関連疾患の克服

  高橋良輔

  内閣府, ムーンショット型研究開発制度, 2020 - 2024, Coinvestigator


• クライオ電子顕微鏡による細胞内分子構造解析法：微小管形成の場を原子レベルで捉える

  仁田 亮

  日本学術振興会, 科学研究費助成事業 挑戦的研究(萌芽), 挑戦的研究(萌芽), 神戸大学, 09 Jul. 2021 - 31 Mar. 2023

  本研究課題では、中心体非依存性微小管ネットワークの形成の場を題材に、クライオ電子顕微鏡法による細胞内分子構造解析法の整備を行っている。 まず、COS7細胞を用いて実験系を整備し、以下の行程を一通り確認した。目的タンパク質を蛍光標識した細胞をグリッド上で培養・急速凍結し、クライオ蛍光顕微鏡で標的分子のグリッド上でのマップを作成する。その後、クライオFIB- SEMを用いて標的部位をクライオ条件のまま薄く削り、クライオ電子線トモグラフィー撮影を行う。そして得られた画像を3次元再構成し、得られた3次元構造の解釈は深層学習またはテンプレートマッチングにより自動化する。 HeLa細胞へGFP-tubulinの安定発現株を導入し、微小管ネットワーク形成の時系列のキーフレーム、例えば相分離によるMTOC形成過程や、微小管が放射状に伸びる星状体形成過程の時間軸を共焦点顕微鏡を用いて解析している。今後、クライオ電子線トモグラフィー解析へ持ち込む。 また、三次元構造のテンプレートともなるCAMSAP2微小管複合体の原子レベルの構造を、クライオ電子顕微鏡単粒子解析法で決定した。


• 心筋メカノバイオロジー機構の解明による心不全治療法の開発

  小室一成

  日本医療研究開発機構, AMED-CREST, Oct. 2017 - Mar. 2023

  Competitive research funding


• Study on the regulatory mechanisms of microtubule organization by MTCL proteins.

  Atsushi Suzuki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (B), Yokohama City University, 01 Apr. 2019 - 31 Mar. 2022

  We have previously found the coiled-coil protein MTCL1, which stabilizes microtubules nucleated from the Golgi membrane. In this study, we newly analyzed an MTCL1 paralog, MTCL2, which preferentially acts on the perinuclear microtubules accumulated around the Golgi. MTCL2 associates with the Golgi membrane through the N-terminal coiled-coil region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown of MTCL2 significantly impaired microtubule accumulation around the Golgi as well as the compactness of the Golgi ribbon assembly structure. Together with several additional results, we concluded that MTCL2 promotes asymmetric microtubule organization by crosslinking microtubules on the Golgi membrane. We also suggested that this function of MTCL2 enables integration of the centrosomal and Golgi-associated microtubules on the Golgi membrane, supporting directional migration.


• 細胞の形態・極性制御を司る微小管ネットワーク形成の構造基盤

  仁田亮

  文部科学省：科学研究費補助金(基盤研究(B)), 2019 - 2021, Principal investigator

  Competitive research funding


• クライオ電子顕微鏡を用いた心筋リモデリングの分子基盤の解明

  仁田亮

  文部科学省, 挑戦的研究(萌芽), Jun. 2018 - Mar. 2020, Principal investigator

  Competitive research funding


• CAMSAPファミリータンパク質に着目した微小管重合制御の分子機構の解明

  仁田亮

  ひょうご科学技術協会, 学術研究助成, 2019, Principal investigator

  Competitive research funding


• クライオ電子顕微鏡を用いた心不全病態解明の新たなアプローチの構築

  仁田亮

  ブリストル・マイヤーズ スクイブ株式会社, 研究助成, 2018, Principal investigator

  Competitive research funding


• Correlative Structural Analysis of Microtubule Dynamics Regulation by CRMP2

  Nitta Ryo

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), 国立研究開発法人理化学研究所, 2015 - 2017, Principal investigator

  The aim of this project is to elucidate the molecular and structural mechanisms of CRMP2-regulated axon induction/repulsion. During the neuronal cell development, CRMP2 localizes the tips of the future axon in which CRMP2 forms the hetero-trimer with GTP-tubulin to add tubulins to the plus-ends of microtubules. This effectively induces the axonal microtubule formation, and thus the axon is effectively elongated. On the other hand, phosphorylation of CRMP2 decreases the affinity between CRMP2 and microtubule. It is driven by the small conformational changes at the C-terminal tail of CRMP2 with shifting the surface charges, which not only alter the interactions within the CRMP2 tetramer but also alter the interactions with GTP-tubulin. Consequently, phosphorylated CRMP2 fails to form a hetero-trimer with GTP-tubulin, thus losing the ability to establish and maintain the axonal microtubules.

  Competitive research funding


• CRMP2 による軸索誘導・反発の分子構 造基盤の解明

  仁田亮

  上原記念生命科学財団, 研究助成, 2017, Principal investigator

  Competitive research funding


• 微小管結合タンパク質 CRMP2 の軸索微 小管誘導・反発の分子機構

  仁田亮

  武田科学振興財団, 医学系研究奨励継続 助成, 2017, Principal investigator

  Competitive research funding


• クライオ電子顕微鏡による心筋リモ デリングの病理構造基盤の解明と新 規治療法開発に向けたシーズの創出

  仁田亮

  持田記念医学薬学振興財団, 研究助成, 2016, Principal investigator

  Competitive research funding


• Integrative biological research on function and regulation of Kinesin superfamily molecular motors

  HIROKAWA Nobutaka, TAKEI Yosuke, KANAI Yoshimitsu, OKADA Yasushi, TANAKA Yosuke, NITTA Ryo, HOMMA Noriko, MIKI Harukata, OGAWA Tadayuki, YAJIMA Hiroaki

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Specially Promoted Research, The University of Tokyo, 2011 - 2015

  Main accomplishment follows. 1) Functions and regulation of KIFs in cellular level and in memory and learning. : a) KIF17 regulates transcription of NR2B and KIF17 to enhance memory and learning. b) KIF17 unloads cargo containing NR2B by phosphorylation of KIF17 tail domain by CaMKIIa and regulatesmemory and learning. c) KIF1A is essential for hippocampal synaptogenesis and learning enhancement in an enriched environment. d) KIF3A loads cargo vesicles containing N cadherin via the phosphorylation by PKA and CaMKIIa by the activity dependent mechanism. 2) The mechanism of directional transport, axon vs dentrites: a) Preferential binding of KIF5 motor domain to GTP-tubulin -rich microtubules in the axon underlies polarlized cargo transport. 3) Function of KIFs at the whole body level and its relationship with diseases. a) KIF5A is essential for GABA_A receptor transport and KIF5A deletion causes epilepsy. b) KIF13A controls anxiety by transporting the serotonin type 1A receptor. c) KIF19A is a microtubule-depolymerizing kinesin for ciliary length control and its deletion causes hydrocephalus and female infertility. 4) New functions of KIFs as a factor in signaling cascades. a) Antioxidant signaling involving KIF12 is an intracellular target of nutrition excess in pancreatic beta cells.


• 微小管結合タンパク質 CRMP2 の軸索微 小管誘導・反発の分子機構

  仁田亮

  武田科学振興財団, 医学系研究奨励, 2015, Principal investigator

  Competitive research funding


• The Mechanism of Intracellular Transport and Kinesin Motors, KIFs : Structure, Function, Dynamics and Regulation

  Nobutaka HIROKAWA, 中田 隆夫, 金井 克光, 野田 泰子, 武井 陽介, 岡田 康志, Takao NAKATA, Yasuko NODA, Yoshimitsu KANAI, Yosuke TAKEI, Yasushi OKADA, Yosuke TANAKA, Ryo NITTA, Noriko HOMMA, Harukata MIKI, Tadayuki OGAWA, Hiroaki YAJIMA

  Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(特別推進研究), 特別推進研究, 東京大学, 2006 - 2010, Coinvestigator not use grants

  Intracellular transport is fundamental for cellular functions in cells in general. We studied this mechanism focusing on the kinesin superfamily proteins (KIFs). The primary structures of 13 new KIFs were solved. Using molecular cell biology and molecular genetics we revealed that KIF4 is a key molecule determining activity dependent survival or death of juvenile neurons, that KIF26A is fundamental for development of enteric nervous system by acting as a suppressor for GDNF/Ret signaling, and that KIF17 plays a significant role by not only transporting NDMA receptors, but also controlling transcription and translation of NR2B and KIF17 through CREB phosphorylation and ubiquitin-proteasome dependent degradation of NR2A. We solved atomic structures during Mg^<++> and water release from Mg^<++>ADP so that we solved almost all states during ATP hydrolysis which gives us strong bases to understand how KIFs move along microtubules.

  Competitive research funding


• Molecular mechanism of Intracellular Transport: Approaches from molecular Cell Biology, Structural Biology, and Molecular Genetics.

  Nobutaka HIROKAWA, 岡田 康志, 寺田 純雄, 瀬藤 光利, 中田 隆夫, Yoshimitsu KANAI, 竹田 扇, 野田 泰子, 武井 陽介, 川岸 将彦, 岡田 康志, 仁田 亮, 寺田 純雄, Yosuke TANAKA, Takao NAKATA, Yasuko NODA, Yosuke TAKEI

  Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(COE形成基礎研究費, 特別推進研究(COE)), COE形成基礎研究費, 特別推進研究(COE), 東京大学, 2001 - 2006, Coinvestigator not use grants

  Our research concerning the mechanism of intracellular transport elucidated followings using molecular cell biology, molecular genetics, biophysics and structural biology.1) Identification of all genes of kinesin superfamily proteins, KIFs in mammals such as human and mouse.2) KIF1B beta transport synaptic vesicle precursor and fundamental for neuronal function and survival and it is a responsible gene of a human hereditary neuropathy.3) KIF2A depolymerizes microtubules and suppress unnecessary extension of axonal branches thus important for brain wiring.4) KIF3 transports Ncadherin and beta-catenin from Golgi to plasma membrane and suppresses tumorigenesis by suppressing beta-catenins transfer into the nucleus and its function as a transcriptional factor for cell proliferation.5) KIF3 transports protein complexes in the cilia at the ventral node in early embryo. The cilia generate leftward nodal flow of extraembryonic fluid by rotation whose axis is tilted posteriorly. The nodal flow convey lipid-enriched nodal vesicular parcels released from node cells dependent on FGF signaling toward left and determine left-right asymmetry. Thus KIF3 is fundamental for important body planning.6) KIF5 transport AMPA type glutamate receptors in dendrites through the interaction between KIF5 tail and GRIP1 - GluR2.7) KIF5 also transport mRNAs with a large protein complexes related to RNAs in dendrites.8) KIF17 transport NMDA type glutamate receptors in dendrites through interaction between KI17tail and scaffolding protein complexes and this transport is shown to be important for higher brain function such as working and special memories.9) KIFC3 transports vesicles containing Anexin XIIIb to the apical membrane of epitherial cells, and plays a role in localization and integrity of the Golgi apparatus.10) As mechanisms for differential transport to axon vs dendrites we identified two mechanism. One is by the recognition of the difference of microtubules in the axon initial segment by motor domain and other is by control via binding of cargoes to the tail domain.11) We revealed that motor protein can move processively along microtubules as monomer by biased Brownian movement and also solved atomic structure of KIF1A motor domain of 5 different states during ATP hydrolysis, thus how conformational changes occur in the motor domain.12) We solved atomic structures of ADP and ATP like states of KIF2 and elucidated how KIF2 depolymerizes microtubules.

  Competitive research funding