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Kengaku Lab
iCeMS, Kyoto University

Research Outline

The cerebellar cortex is formed of a clear, three-layered cytoarchitecture that provides a favorable system for the study of the formation and function of neural circuitry. We have developed in vitro reconstructions of cell morphogenesis and migration during cerebellar 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:

  1. Mechanisms and principles of branch pattern formation of dendrites
  2. Cellular and molecular dynamics of neuronal migration
  3. Development of techniques based on light microscopy

1. 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. 2012).

We have successfully visualized the dynamic motility of organelles, including the nucleus, centrosomes, mitochondria and Golgi apparatus, in developing neurons (Wu et al, Fukumitsu et al. 2015). 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.

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.

2. 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, 2013).

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 now seek to visualize the force which drives migration, by quantitative measurement of the rheological properties of migrating neurons.

3. Development of techniques

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.