Nobutaka Hirokawa

The University of Tokyo, Edo, Tōkyō, Japan

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Publications (301)3023.91 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: 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 Å resolution (7.8 Å using the 0.143 FSC criterion) attained allows differences between the α- and β- 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.
    Journal of Structural Biology 11/2014; · 3.37 Impact Factor
  • Wenxing Yang, Yosuke Tanaka, Miki Bundo, Nobutaka Hirokawa
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    ABSTRACT: Beta cell injury due to oxidative stress is a typical etiology of diabetes caused by nutritional excess, but its precise mechanism remains largely elusive. Here, we demonstrate that the microtubule motor KIF12 mediates an antioxidant cascade in beta cells as an intracellular target of excess fat intake or "lipotoxicity." KIF12 knockout mice suffer from hypoinsulinemic glucose intolerance due to increased beta cell oxidative stress. Using this model, we identified an antioxidant signaling cascade involving KIF12 as a scaffold for the transcription factor Sp1. The stabilization of nascent Sp1 appeared to be essential for proper peroxisomal function by enhancing Hsc70 expression, and the pharmacological induction of Hsc70 expression with teprenone counteracted the oxidative stress. Because KIF12 is transcriptionally downregulated by chronic exposure to fatty acids, this antioxidant cascade involving KIF12 and Hsc70 is proposed to be a critical target of nutritional excess in beta cells in diabetes.
    Developmental Cell 10/2014; 31(2):202-14. · 10.37 Impact Factor
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    Yoshimitsu Kanai, Daliang Wang, Nobutaka Hirokawa
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    ABSTRACT: Multifunctional low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) recognizes and internalizes a large number of diverse ligands, including LDL and factor VIII. However, little is known about the regulation of LRP1 endocytosis. Here, we show that a microtubule-based motor protein, KIF13B, in an unexpected and unconventional function, enhances caveolin-dependent endocytosis of LRP1. KIF13B was highly expressed in the liver and was localized on the sinusoidal plasma membrane of hepatocytes. KIF13B knockout (KO) mice showed elevated levels of serum cholesterol and factor VIII, and KO MEFs showed decreased uptake of LDL. Exogenous KIF13B, initially localized on the plasma membrane with caveolae, was translocated to the vesicles in the cytoplasm with LRP1 and caveolin-1. KIF13B bound to hDLG1 and utrophin, which, in turn, bound to LRP1 and caveolae, respectively. These linkages were required for the KIF13B-enhanced endocytosis of LRP1. Thus, we propose that KIF13B, working as a scaffold, recruits LRP1 to caveolae via LRP1-hDLG1-KIF13B-utrophin-caveolae linkage and enhances the endocytosis of LRP1.
    The Journal of Cell Biology 01/2014; · 9.69 Impact Factor
  • Nobutaka Hirokawa
    BMC Genomics 01/2014; 15(Suppl 2):O19. · 4.04 Impact Factor
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    ABSTRACT: Extensive axonal pruning and neuronal cell death are critical events for the development of the nervous system. Like neuronal cell death, axonal elimination occurs in discrete steps; however, the regulators of these processes remain mostly elusive. Here, we identify the kinesin superfamily protein 2A (KIF2A) as a key executor of microtubule disassembly and axonal breakdown during axonal pruning. Knockdown of Kif2a, but not other microtubule depolymerization or severing proteins, protects axonal microtubules from disassembly upon trophic deprivation. We further confirmed and extended this result to demonstrate that the entire degeneration process is delayed in neurons from the Kif2a knockout mice. Finally, we show that the Kif2a-null mice exhibit normal sensory axon patterning early during development, but abnormal target hyperinnervation later on, as they compete for limited skin-derived trophic support. Overall, these findings reveal a central regulatory mechanism of axonal pruning during development.
    Cell Reports 04/2013; · 7.21 Impact Factor
  • Shinsuke Niwa, Hironori Takahashi, Nobutaka Hirokawa
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    ABSTRACT: Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β-tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of β-tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of β-tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations.
    The EMBO Journal 03/2013; · 10.75 Impact Factor
  • Qing Chang, Ryo Nitta, Shigeyuki Inoue, Nobutaka Hirokawa
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    ABSTRACT: 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 Å 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. (226 words).
    Journal of Molecular Biology 03/2013; · 3.91 Impact Factor
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    ABSTRACT: Molecular motors are fundamental to neuronal morphogenesis and function. However, the extent to which molecular motors are involved in higher brain functions remains largely unknown. In this study, we show that mice deficient in the kinesin family motor protein KIF13A (Kif13a(-/-) mice) exhibit elevated anxiety-related behavioral phenotypes, probably because of a reduction in 5HT(1A) receptor (5HT(1A)R) transport. The cell-surface expression level of the 5HT(1A)R was reduced in KIF13A-knockdown neuroblastoma cells and Kif13a(-/-) hippocampal neurons. Biochemical analysis showed that the forkhead-associated (FHA) domain of KIF13A and an intracellular loop of the 5HT(1A)R are the interface between the motor and cargo vesicles. A minimotor consisting of the motor and FHA domains is able to transport 5HT(1A)R-carrying organelles in in vitro reconstitution assays. Collectively, our results suggest a role for this molecular motor in anxiety control.
    Cell Reports 02/2013; 3(2):509-19. · 7.21 Impact Factor
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    ABSTRACT: KIF5 (also known as kinesin-1) family members, consisting of KIF5A, KIF5B, and KIF5C, are microtubule-dependent molecular motors that are important for neuronal function. Among the KIF5s, KIF5A is neuron specific and highly expressed in the central nervous system. However, the specific roles of KIF5A remain unknown. Here, we established conditional Kif5a-knockout mice in which KIF5A protein expression was postnatally suppressed in neurons. Epileptic phenotypes were observed by electroencephalogram abnormalities in knockout mice because of impaired GABA(A) receptor (GABA(A)R)-mediated synaptic transmission. We also identified reduced cell surface expression of GABA(A)R in knockout neurons. Importantly, we identified that KIF5A specifically interacted with GABA(A)R-associated protein (GABARAP) that is known to be involved in GABA(A)R trafficking. KIF5A regulated neuronal surface expression of GABA(A)Rs via an interaction with GABARAP. These results provide an insight into the molecular mechanisms of KIF5A, which regulate inhibitory neural transmission.
    Neuron 12/2012; 76(5):945-961. · 15.77 Impact Factor
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    ABSTRACT: Cilia control homeostasis of the mammalian body by generating fluid flow. It has long been assumed that ciliary length-control mechanisms are essential for proper flow generation, because fluid flow generation is a function of ciliary length. However, the molecular mechanisms of ciliary length control in mammals remain elusive. Here, we suggest that KIF19A, a member of the kinesin superfamily, regulates ciliary length by depolymerizing microtubules at the tips of cilia. Kif19a(-/-) mice displayed hydrocephalus and female infertility phenotypes due to abnormally elongated cilia that cannot generate proper fluid flow. KIF19A localized to cilia tips, and recombinant KIF19A controlled the length of microtubules polymerized from axonemes in vitro. KIF19A had ATP-dependent microtubule-depolymerizing activity mainly at the plus end of microtubules. Our results indicated a molecular mechanism of ciliary length regulation in mammals, which plays an important role in the maintenance of the mammalian body.
    Developmental Cell 11/2012; · 10.37 Impact Factor
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    ABSTRACT: Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by β-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'-α,β-methylenediphosphonate (GMPCPP), at 8.8-Å resolution by developing a novel cryo-EM image reconstruction algorithm. In contrast to the crystal structures of GTP-bound tubulin relatives such as γ-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.
    The Journal of Cell Biology 07/2012; 198(3):315-22. · 9.69 Impact Factor
  • Xiling Yin, Xue Feng, Yosuke Takei, Nobutaka Hirokawa
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    ABSTRACT: Regulation of NMDA receptor trafficking is crucial to modulate neuronal communication. Ca(2+)/calmodulin-dependent protein kinase phosphorylates the tail domain of KIF17, a member of the kinesin superfamily, to control NMDA receptor subunit 2B (GluN2B) transport by changing the KIF17-cargo interaction in vitro. However, the mechanisms of regulation of GluN2B transport in vivo and its physiological significance are unknown. We generated transgenic mice carrying wild-type KIF17 (TgS), or KIF17 with S1029A (TgA) or S1029D (TgD) phosphomimic mutations in kif17(-/-) background. TgA/kif17(-/-) and TgD/kif17(-/-) mice exhibited reductions in synaptic NMDA receptors because of their inability to load/unload GluN2B onto/from KIF17, leading to impaired neuronal plasticity, CREB activation, and spatial memory. Expression of GFP-KIF17 in TgS/kif17(-/-) mouse neurons rescued the synaptic and behavioral defects of kif17(-/-) mice. These results suggest that phosphorylation-based regulation of NMDA receptor transport is critical for learning and memory in vivo.
    Journal of Neuroscience 04/2012; 32(16):5486-99. · 6.75 Impact Factor
  • Makoto Kondo, Yosuke Takei, Nobutaka Hirokawa
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    ABSTRACT: Environmental enrichment causes a variety of effects on brain structure and function. Brain-derived neurotrophic factor (BDNF) plays an important role in enrichment-induced neuronal changes; however, the precise mechanism underlying these effects remains uncertain. In this study, a specific upregulation of kinesin superfamily motor protein 1A (KIF1A) was observed in the hippocampi of mice kept in an enriched environment and, in hippocampal neurons in vitro, BDNF increased the levels of KIF1A and of KIF1A-mediated cargo transport. Analysis of Bdnf(+/-) and Kif1a(+/-) mice revealed that a lack of KIF1A upregulation resulted in a loss of enrichment-induced hippocampal synaptogenesis and learning enhancement. Meanwhile, KIF1A overexpression promoted synaptogenesis via the formation of presynaptic boutons. These findings demonstrate that KIF1A is indispensable for BDNF-mediated hippocampal synaptogenesis and learning enhancement induced by enrichment. This is a new molecular motor-mediated presynaptic mechanism underlying experience-dependent neuroplasticity.
    Neuron 02/2012; 73(4):743-57. · 15.77 Impact Factor
  • Nobutaka Hirokawa, Yosuke Tanaka, Yasushi Okada
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    ABSTRACT: The establishment of left-right asymmetry during development of vertebrate embryos depends on leftward flow in the nodal cavity. The flow is produced by the rotational movement of the posteriorly tilted nodal cilia. However, it remains poorly understood how the nodal cilia are tilted posteriorly, and how the directionality of the flow is translated into gene expression patterns in the embryo. Recent studies have identified signaling molecules involved in these processes. First, planar cell polarity signaling has been shown to be involved in the posterior positioning of the basal bodies of nodal cilia, which leads to the posterior tilting of their rotation axes. Second, identification of putative receptors and signaling molecules suggests a link between the signaling molecules delivered by the nodal flow, and downstream signaling in the cells surrounding the nodal cavity and the lateral plate mesoderm.
    Current opinion in cell biology 02/2012; 24(1):31-9. · 14.15 Impact Factor
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    ABSTRACT: Neuronal morphology is regulated by cytoskeletons. Kinesin superfamily protein 2A (KIF2A) depolymerizes microtubules (MTs) at growth cones and regulates axon pathfinding. The factors controlling KIF2A in neurite development remain totally elusive. Here, using immunoprecipitation with an antibody specific to KIF2A, we identified phosphatidylinositol 4-phosphate 5-kinase alpha (PIPKα) as a candidate membrane protein that regulates the activity of KIF2A. Yeast two-hybrid and biochemical assays demonstrated direct binding between KIF2A and PIPKα. Partial colocalization of the clusters of punctate signals for these two molecules was detected by confocal microscopy and photoactivated localization microscopy. Additionally, the MT-depolymerizing activity of KIF2A was enhanced in the presence of PIPKα in vitro and in vivo. PIPKα suppressed the elongation of axon branches in a KIF2A-dependent manner, suggesting a unique PIPK-mediated mechanism controlling MT dynamics in neuronal development.
    Proceedings of the National Academy of Sciences 01/2012; 109(5):1725-30. · 9.81 Impact Factor
  • Neuroscience Research 09/2011; 71. · 2.15 Impact Factor
  • Neuroscience Research 09/2011; 71. · 2.15 Impact Factor
  • Nobutaka Hirokawa
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    ABSTRACT: Cells transport and sort various proteins and lipids following synthesis as distinct types of membranous organelles and protein complexes to the correct destination at appropriate velocities. This intracellular transport is fundamental for cell morphogenesis, survival and functioning not only in highly polarized neurons but also in all types of cells in general. By developing quick-freeze electron microscopy (EM), new filamentous structures associated with cytoskeletons are uncovered. The characterization of chemical structures and functions of these new filamentous structures led us to discover kinesin superfamily molecular motors, KIFs. In this review, I discuss the identification of these new structures and characterization of their functions using molecular cell biology and molecular genetics. KIFs not only play significant roles by transporting various cargoes along microtubule rails, but also play unexpected fundamental roles on various important physiological processes such as learning and memory, brain wiring, development of central nervous system and peripheral nervous system, activity-dependent neuronal survival, development of early embryo, left-right determination of our body and tumourigenesis. Furthermore, by combining single-molecule biophysics with structural biology such as cryo-electrom microscopy and X-ray crystallography, atomic structures of KIF1A motor protein of almost all states during ATP hydrolysis have been determined and a common mechanism of motility has been proposed. Thus, this type of studies could be a good example of really integrative multidisciplinary life science in the twenty-first century.
    Journal of electron microscopy 08/2011; 60 Suppl 1:S63-92. · 1.63 Impact Factor
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    ABSTRACT: Polarized transport in neurons is fundamental for the formation of neuronal circuitry. A motor domain-containing truncated KIF5 (a kinesin-1) recognizes axonal microtubules, which are enriched in EB1 binding sites, and selectively accumulates at the tips of axons. However, it remains unknown what cue KIF5 recognizes to result in this selective accumulation. We found that axonal microtubules were preferentially stained by the anti-GTP-tubulin antibody hMB11. Super-resolution microscopy combined with EM immunocytochemistry revealed that hMB11 was localized at KIF5 attachment sites. In addition, EB1, which binds preferentially to guanylyl-methylene-diphosphate (GMPCPP) microtubules in vitro, recognized hMB11 binding sites on axonal microtubules. Further, expression of hMB11 antibody in neurons disrupted the selective accumulation of truncated KIF5 in the axon tips. In vitro studies revealed approximately threefold stronger binding of KIF5 motor head to GMPCPP microtubules than to GDP microtubules. Collectively, these data suggest that the abundance of GTP-tubulin in axonal microtubules may underlie selective KIF5 localization and polarized axonal vesicular transport.
    The Journal of Cell Biology 07/2011; 194(2):245-55. · 9.69 Impact Factor
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    Xiling Yin, Yosuke Takei, Mizuho A Kido, Nobutaka Hirokawa
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    ABSTRACT: Kinesin superfamily motor protein 17 (KIF17) is a candidate transporter of N-methyl-D-aspartate (NMDA) receptor subunit 2B (NR2B). Disruption of the murine kif17 gene inhibits NR2B transport, accompanied by decreased transcription of nr2b, resulting in a loss of synaptic NR2B. In kif17(-/-) hippocampal neurons, the NR2A level is also decreased because of accelerated ubiquitin-proteasome system-dependent degradation. Accordingly, NMDA receptor-mediated synaptic currents, early and late long-term potentiation, long-term depression, and CREB responses are attenuated in kif17(-/-) neurons, concomitant with a hippocampus-dependent memory impairment in knockout mice. In wild-type neurons, CREB is activated by synaptic inputs, which increase the levels of KIF17 and NR2B. Thus, KIF17 differentially maintains the levels of NR2A and NR2B, and, when synapses are stimulated, the NR2B/KIF17 complex is upregulated on demand through CREB activity. These KIF17-based mechanisms for maintaining NR2A/2B levels could underlie multiple phases of memory processes in vivo.
    Neuron 04/2011; 70(2):310-25. · 15.77 Impact Factor

Publication Stats

26k Citations
3,023.91 Total Impact Points


  • 1970–2014
    • The University of Tokyo
      • • Faculty & Graduate School of Medicine
      • • School of Medicine
      Edo, Tōkyō, Japan
  • 2005
    • National Taiwan University
      • College of Medicine
      Taipei, Taipei, Taiwan
  • 2004
    • Ludwig-Maximilians-University of Munich
      München, Bavaria, Germany
  • 1991
    • University of Texas Southwestern Medical Center
      • Medical School
      Dallas, TX, United States
  • 1982–1983
    • Washington University in St. Louis
      • Department of Anatomy and Neurobiology
      San Luis, Missouri, United States