DEPARTMENT OF MOLECULAR PHYSIOLOGY

Ion channels, receptors and G proteins play critical roles for the excitability and its regulation of neurons. We focus on these molecules which enable brain function. From the biophysical point of view, we study structure-function relationships, regulation mechanisms and dynamic structural rearrangements of ion channels and receptors. We also plan to study the functional significance of specific features of ion channels and receptors in the brain function by making knock-in mice and by studying their abnormalities in the synaptic transmission and whole animal behavior.

Specific themes of research projects currently running are as follows

1. Structure-function relationship of inwardly rectifying K⁺ channels.
2. Molecular mechanisms and functional significance of the Ca²⁺/Gd³⁺ sensing function of metabotropic glutamate receptor.
3. Analysis of the dynamic structural rearrangements of ion channels and receptors by FRET measurement under evanescent field illumination.
4. Molecular mechanisms of the regulation of m- channel function by muscarinic stimulation.
5. Expression density dependent changes of the pore properties of ATP receptor channel, P2X₂.
6. Cell biological analysis of a novel large GTP binding protein, mOPA, which causes fragmentation of mitochondria.
7. cDNA cloning and functional analysis of RGS family, regulators of G protein signaling
8. Purification of ATP receptor channel protein towards structural analysis.
9. Functional analysis of the regulation of G protein coupled inwardly rectifying K⁺ channel by phosphorylation.

Fig. 1. Comparison of the structure of voltage-gated K channel and the inward rectifier K channel. (Kubo et al., Nature 1993)	Fig. 2. Ligand-induced dynamic rearrangement of the intracellular dimeric conformation of metabotropic glutamate receptor. (Tateyama et al., Nature Structural & Molecular Biology, 2004)
Fig. 3. Different roles of PIP2 and PKC during muscarinic inhibition of KCNQ/M channels. (Nakajo and Kubo, in preparation)	Fig. 4. Changes of the channel pore properties of ATP receptor channel P2X2 depending on the open channel density. (Fujiwara and Kubo., J. Physiol. 2004)
Fig. 5. mOPA1 protein localizes at mitochondria, and causes fragmentation of mitochondria. (Misaka et al., J. Biol. Chem., 2002)

Staff

During the course of formation of the mammalian central nervous system, neuroepithelial cells differentiate into various kinds of cells to make a fine three-dimensional network. Our goal is to understand genetic control over these processes. As a first step, we have cloned several genes that are specifically expressed in a certain type of brain cells and are investigating their role on cell fate determination. Neural cells are known to leave the ventricular zone after their commitment, and migrate towards destinations. While radial neuronal migration has been studied extensively in the developing cerebral and cerebellar cortices, mechanisms underlining tangential migration of neuronal and glial progenitors remains unclear. We are employing in ovo or in utero electroporation method to introduce exogenous genes in developing central nervous system, and studying mode and mechanisms of neural cell migration.

We are making use of hereditary mutant mice that exhibit abnormal development of the nervous system. We also use in situ hybridization and immunohistochemical technique to study cell lineages during development of the nervous system.

Neural stem cells, which are ultimate lineage precursors to all neurons and glia in the mammalian brain, are present not only in embryonic but also in adult brains, and contribute to adult neurogenesis. We are investigating molecular mechanisms underlying the generation, proliferation, maintenance, differentiation, and senescence of the neural stem cells, which will clarify their in vivo kinetics and function.

An automated system to analyze N-linked sugar chains was developed to study their biological roles during development and tumorigenesis.

New retroviral vectors are also constructed for efficient gene delivery, which will be used for cancer gene therapy.

In Utero Gene Transfer Systems to the Embryonic Mouse Brains
A) We established retrovirus producer cell lines that enable us to generate high titer viruses. The retrovirus carrying lacZ gene was injected into an embryonic mouse brain in utero. The mouse was analyzed at adult stage. Numerous infected cells were observed throughout the brain.

B) In utero electroporation was carried out for plasmid DNA transfer. Green fluorescent protein (GFP) expression vector was injected into lateral ventricle and electroporated in utero. The cells in the restricted region were observed to express GFP

C) Oligodendrocyte Development(Left) pMN domain which is the site of oligodendrogenesis Expression of Olig2 gene in embryonic day 12 spinal cord. Olig2 (purple) is expressed ventral ventricular zone called pMN domain.(Middle) Migrating oligodendrocyte progenitor Oligodendrocyte progenitor is double-stained by anti-GFP antibody (green) and O4 antibody (red). O4 is an oligodendrocyte lineage specific marker(Right) Myelinating oligodendrocyte Mature oligodendrocyte (green) is observed with extending processes toward several axons.

Staff

Cell signaling that generates proper cell responses to various stimuli is the essence of life. To understand its mechanism is one of the goals of life sciences. This division is aiming to elucidate the spatio-temporal regulation mechanisms underlying cell signaling, focusing on the dynamics of ion channels, cytoskeletons, and adhesion molecules by use of electrophysiological and advanced imaging techniques.

The subjects of research are,

(1) Cell signaling in response to mechanical stimuli:
Virtually every cell can properly respond to mechanical stimuli, e.g., electrical responses in the inner ear hair cells and cutaneous mechanoreceptors, disuse atrophy in muscle and bone under microgravity, or shear stress induced NO production in endothelial cells. However, its molecular mechanisms are largely unknown due to the ambiguity of mechanotransduction process in cells. We therefore focus on SA channels and cytoskeleton/focal adhesion complex as representative cell mechanosensors and investigate their roles in mechanosignaling through the development of innovative light microscopy and micro mechanical manipulation of the cell (Fig.1). A typical subject is stretch-induced shape remodeling, where endothelial cells align their long axis perpendicular to the stretch axis. This response includes many intriguing functions such as sensation of force direction or spatio-temporal integration of dynamics of stress fibers and focal adhesions during their rearrangement (Fig.2).

(2) Intracellular Ca²⁺ signaling:
When various mechanical stimuli, such as have induced by cell-cell interaction or stimuli induced by biological activators such as hormones, are given to a cell, the cell exhibits intracellular Ca²⁺ changes in response to them. The Ca²⁺ changes are modulated and processed on to the next signal pathways, leading to various significant cell functions. The process called intracellular Ca²⁺ signaling is one of the most significant and major signal transduction mechanisms in cells of almost all organisms. We use Ca²⁺ and Na⁺ imaging techniques, and electrophysiological methods to perform our experiments in addition to cellular manipulations such as microinjection of materials into cells. We presently focus on the Ca²⁺ signaling in stretched-induced or migrating cells to investigate the mechanisms aforementioned in (1). We also investigate the mechanisms of fertilization and oocyte maturation in mammals through the study of the Ca²⁺ oscillations and Ca²⁺ increase.

(3) Proton signaling:
Hydrogen ion (proton, H⁺) is an important signaling ion that determines pH and participates in a variety of biological responses, for instance bone remodeling, natural immunity, and pain sensation. H⁺-transferring molecules at the plasma membrane serve to regulate the pH environment dynamically. Voltage-gated H⁺ channels function as sensitive pH monitors and acid-secreting apparatuses, and have been cast as a key player in the processes of H⁺ signaling. The primary goal of this study is employing H⁺ channels to elucidate the mechanisms underlying H⁺ mobilization linked with cellular functions.

Fig.1: Diagram for mechanical stimulation of focal adhesions through stress fibers. Left: Fibronectin-coated glass beads connected to the basal focal adhesions via stress fibers. By displacing the bead, we can apply localized mechanical stimuli onto focal adhesions, while recording the surface dynamics of intracellular calcium and integrin by near field microscopy. Right: Projected side views of focal adhesions (top, green spots), stress fibers (middle, red strands), and their superimposition (bottom) in an endothelial cell .

Fig.2: Stretch-induced shape remodeling. Left: When subjected to uniaxial cyclic stretch, endothelial cells cultured on an elastic silicone membrane change their shape from cobble stone-like to spindle-like by aligning their long axis perpendicular to the stretch axis. Right: Dynamic rearrangement of focal adhesions (green spots) and stress fibers (orange strands) before (top) and after (bottom) remodeling.

Staff