Research outline

The cerebellar cortex and hippocampus are formed of clear layered cytoarchitectures that provide favorable systems for the study of the formation and function of neural circuitry. We have developed in vitro reconstructions of cell morphogenesis and migration during cerebellar and hippocampal development. We aim to discover novel phenomena and the underlying rules of brain formation using multidisciplinary approaches, including advanced microscopies, cell and molecular biology, and mechanobiology. Our main research directions can be summarized under the following three themes:




Mechanisms and principles of branch pattern formation of dendrites

Dendrite morphologies of CNS neurons are highly diverse, depending on cell type and function. The architecture of dendritic arbors critically affects the integration of neuronal inputs and propagation of chemical signals, and hence determines the connectivity of neurons. The question of how neurons acquire their appropriate morphology is a major issue in the study of neuronal development. In spite of the increasing number of molecular signals that have been identified as regulators of dendritic arborization patterns, the precise function of each molecule in the specific steps of branch dynamics largely remains elusive.

Cerebellar Purkinje cells develop intricate dendritic arbors with minimal branch overlap. We developed a method of long-term time-lapse observation of dendritic branch dynamics in growing Purkinje cells in culture. Using a combinatorial approach with quantitative image analyses and computer-aided simulation, we identified the fundamental rules of growth dynamics that govern the construction of the characteristic dendritic patterns in Purkinje cells(Fujishima et al., Development, 2012). We also demonstrated that a small change in actin dynamics led to a siginifant difference in dendrite growth dynamics and the untimate branch pattern in a mature neuron (Kawabata Galbraith et al., Cell Reports, 2018).

Time-lapse observation and computer-assisted simulation of dendrite formation in cultured Purkinje cells


長期タイムラプス観察により樹状突起形成過程を解析し、必要最小限のパラメータを抽出して モデル細胞を再構築した。

We have successfully visualized the dynamic motility of organelles, including the nucleus, centrosomes, mitochondria and Golgi apparatus, in developing neurons. We recently found that developing neurons actively transport mitochondria into growing dendrites to fuel ATP energy necessary for arbor formation. We also found that dendrites sense local ATP levels and tune their growth rates by slowing actin turnover to avoid overconsumption of the ATP necessary for cellular metabolism.

Super-resolutioin images of mitochondria dynamics in cultured hippocampus neuron


培養した海馬ニューロンの細胞質(緑)とミトコンドリア(マゼンダ、白)の動態を超解像顕微鏡(Zeiss Airy scan)を用いて観察した。

Neuronal dendrites tend to extend radially within the brain to form perpendicular contacts with the afferent axon fibers, which run horizontally. Such organization has been shown to maximize the number of potential anatomical connections, yet the mechanism of how neurons orient dendrites perpendicular to afferent axons is unknown. Using electrospun carbon nanofibers as an artificial scaffold, we cultivated cerebellar neurons and reproduced the perpendicular contact observed between Purkinje cell dendrites and the aligned granule cell axons in culture dishes. Utilizing this system, we seek to identify the molecular and mechanical bases underlying axon-dendrite wiring topology.





 我々は中枢神経系ニューロンの中でも際立って緻密な樹状突起を形成する小脳プルキンエ細胞を用い、分散培養下で樹状突起発達過程を1週間以上連続観察する系を確立し、樹状突起ダイナミクスの定量的解析と数理解析を用いてその形成原理を明らかにしました(Fujishima et al. 2012)。また、分化中の細胞骨格アクチン動態のわずかなズレが樹状突起ダイナミクスに影響し、成熟したニューロンの分岐の形を大きく変えてしまうことを証明しました(Kawabata Galbraith et al. 2018)。 また、発達中の樹状突起内でゴルジ体、ミトコンドリアなどのオルガネラの動態を可視化することにも成功しています。樹状突起発達に必要な大量のATPエネルギーが樹状突起局所のミトコンドリア活性で供給され、その供給レベルによりアクチン代謝と樹状突起伸長速度が制御されるしくみを明らかにしました。

脳神経回路では、軸索同士は束化して原則として脳表面に水平に並走する一方、樹状突起は脳表面に垂直な法線方向に展開して軸索束と直交し、格子状に配向する傾向があります。この直交座標は混線しにくい効率的な神経回路の構築に寄与すると考えられています(Cuntz, Front Neuroanat. 2012; Wedeen et al., Science 2012)。これまで突起伸展の誘導やシナプス結合特異性に関わる数多くの細胞間シグナル分子が同定されていますが、それらの分布と組み合わせのみでは神経線維間の厳密な接合トポロジーを説明できません。カーボンナノ繊維などの人工スキャフォールドを用いた培養系で軸索-樹状突起の直交接合を再構成し、背景にある細胞・力学機構を明らかにすることを目指しています。


Kawabata-Galbraith, K., Fujishima, K., Mizuno, H., Lee, S.J., Uemura, T., Sakimura, K., Mishina, M., Watanabe, N.  and Kengaku, M. (2018) MTSS1 regulation of actin-nucleating formin DAAM1 in dendritic filopodia determines final dendritic configuration of Purkinje cells. Cell Rep. 24(1):95-106.

Hatsukano, T., Kurisu, J., Fukumitsu, K., Fujishima, K. and Kengaku, M. (2017) Thyroid hormone induces PGC-1 α during dendritic outgrowth in mouse cerebellar Purkinje cells. Front Cell Neurosci. 11:133

Fukumitsu, K., Hatsukano, T., Yoshimura, A., Heuser, J., Fujishima, K. and Kengaku, M. (2016) Mitochondrial fission protein Drp1 regulates mitochondrial transport and dendritic arborization in cerebellar Purkinje cells. Mol Cell Neurosci. 71:56-65

Fukumitsu, K., Fujishima, K., Yoshimura, A., Wu, Y.K., Heuser, J. and Kengaku, M. (2015) Synergistic action of dendritic mitochondria and creatine kinase maintains ATP homeostasis and actin dynamics in growing neuronal dendrites. J. Neurosci. 35(14):5707- 5723.

Wu, Y.K., Fujishima, K. and Kengaku, M. (2015) Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated Hippocampal Cultures. PLoS ONE 10(2) e0118482.

Fujishima, K., Horie, R., Mochizuki, A. and Kengaku M. (2012) Principles of branch dynamics governing shape characteristics of cerebellar Purkinje cell dendrites. Development 139: 3442-3455.

Cellular and molecular dynamics of neuronal migration

Neurons are generated from neural stem cells in the germinal layer, and then migrate through the crowded neural tissues toward their specific sites of function within the cortex. Failure in neuronal migration may cause severe brain malformation and psychiatric disorders.

Cerebellar granule cells are the excitatory interneurons of the cerebellar cortex, which undergo significant migration during cortex formation. We have established a time-lapse imaging system for quantitative analyses of granule cell migration in organotypic cultures, which retain the cell architecture and environment of the cerebellar cortex. We have successfully visualized organelle dynamics in migrating neurons in the brain tissue and have proposed a novel model for neuronal migration (Umeshima et al., 2007, Umeshima et l., 2012).

Neuronal migration in cultured cerebellar tissue



Cytoskeletal dynamics of migratory granule cells in vitro



We have recently developed an in vitro system for analyses of motion dynamics of neuronal migration at a high spatio-temporal resolution using spinning-disc confocal microscopy. We have found that the migrating nucleus exhibits highly dynamic motion, including sharp deformation and rotation, suggesting the involvement of multiple motor systems (Wu et al., 2018). We now seek to visualize the force which drives migration, by quantitative measurement of the rheological properties of migrating neurons.




 小脳皮質の介在ニューロンである顆粒細胞は、発生過程で最も大規模に移動する細胞のひとつです。我々は生後発達中の小脳組織を器官培養し、皮質内を移動する顆粒細胞の動きを長時間観察するリアルタイムイメージング系を確立しています。このシステムを用い、組織内を移動するニューロンのオルガネラのダイナミクスの観察に世界に先駆けて成功しました(Umeshima et al., 2007Umeshima et al., 2012)。スピニングディスク型共焦点顕微鏡を用いてさらに時空間解像度を上げて観察すると、ニューロン核は前進のみならず回転や変形など、複数のモーター分子活性を示唆する複雑な動きを伴うことが明らかとなりました(Wu et al., 2018)。

Nuclear dynamics of migratory granule cells


培養下で移動する顆粒細胞の核(H2B:マゼンダ、白)と微小管(Dcx; 緑)を高時空間解像タイムラプスにより観察した。



Wu YK, Umeshima H, Kurisu J and Kengaku M. “Nesprins and opposing microtubule motors generate a point force that drives directional nuclear motion in migrating neurons.”
Development, 145, 10.1242/dev.158782. (2018)

Nakashima K, Umeshima H and Kengaku M. “Cerebellar granule cells are predominantly generated by terminal symmetric divisions of granule cell precursors.”
Dev Dyn, 244(6), 748-758 (2015)

Umeshima H and Kengaku M. “Differential roles of cyclin-dependent kinase 5 in tangential and radial migration of cerebellar granule cells.”
Mol Cell Neurosci, 52, 62-72 (2013)

Umeshima H, Hirano T and Kengaku M. “Microtubule-based nuclear movement occurs independently of cetrosome positioning in migrating neurons.”
Proc Natl Acad Sci U S A, 104, 16182-16187 (2007) Must Read Article by Faculty of 1000


New techniques based on light microscopy

Using advanced light microscopies, we aim to develop new techniques for image analysis of cell motility and molecular signals. In collaboration with other iCeMS laboratories, we also seek to develop nano-fabrication techniques for reconstruction of the chemical and mechanical environment in tissues with the desired topographies, by using fibers, sheets, and gels. The following list is a part of our ongoing research activities.

  • Traction force microscopy of migrating neurons
  • Identification of cytoskeletal organization in neurons by super-resolution microscopy
  • Reconstruction of 3D cell architectures of the brain using artificial scaffolds
  • Gene-switching using gold nano-rods



  • ニューロン移動の牽引力顕微鏡解析
  • ニューロン細胞骨格の超解像顕微鏡解析
  • 人工スキャフォールドを用いた脳皮質のin vitro再構築
  • 金ナノロッドを用いた遺伝子発現制御


Umeshima H, Nomura KI, Yoshikawa S, Hörning M, Tanaka M, Sakuma S, Arai F, Kaneko M and Kengaku M. “Local traction force in the proximal leading process triggers nuclear translocation during neuronal migration.”
Neurosci Res, 10.1016/j.neures.2018.04.001. (2018)

Nakatsuji H, Kawabata Galbraith K, Kurisu J, Imahori H, Murakami T and Kengaku M. “Surface chemistry for cytosolic gene delivery and photothermal transgene expression by gold nanorods.”
Sci. Rep, 7:4694 (2017)