H. STRUCTURAL BIOLOGY CENTER
H-a. Biological Macromolecules Laboratory - Makio Tokunaga Group

RESEARCH ACTIVITIES

(1) Single Molecule Imaging and Quantitative Analysis of Nuclear Transport in Cells using Highly Inclined and Laminated Optical sheet microscopy

Makio Tokunaga and Naoko Imamoto¹ (¹Cellular Dynamics Laboratory, Riken)

--What is a key to enter the nuclei through nuclear pores? This question has been answered by visualizing single molecules inside cells. Clear video images of single molecules translocating into the nuclei are obtained using novel fluorescence microscopy, Highly Inclined and Laminated Optical sheet (HILO) microscopy. Very little is known about the interactions between transport molecules and the assembled nuclear pore complex (NPC) because of its large supramolecular structure. Obtained single-molecule video images inside cells are very clear, therefore kinetic parameters of the molecular interactions in cells are obtained through quantitative analysis. We have discovered how many molecules interact, how strong the interactions are, and which molecular interaction is the open sesame to nuclear import. We have opened up a new way to quantify molecular dynamics and interactions at the single-cell level, which is 'open sesame' toward quantitative molecular and system biology.

(2) RNG105: A Novel Regulatory Protein in Neuronal RNA Granules Responsible for Stimulation-Dependent Local Translation

Nobuyuki Shiina, Kazumi Shinkura and Makio Tokunaga

--mRNA translocation and subsequent local translation in neuronal dendrites are important bases for long-term synaptic plasticity, but responsible molecules have not been fully identified. We previously identified RNG105 (RNA granule protein 105) as a component of RNA granules, which play central roles in the transport of mRNAs to the dendrites. The RNG105-localizing RNA granules contain mRNAs, such as CaM kinase II alpha, CREB and BDNF mRNAs, whose translational products play key roles in synaptic plasticity.
--In this year, we have found that RNG105 contains an RNA-binding motif, and shown that RNG105 is a regulatory protein for local translation in dendrites of hippocampal neurons. RNG105 has an ability to repress translation in vivo, consistent with the finding that the RNA granule is translationally arrested in the basal conditions. Dissociation of RNG105 from the RNA granules is induced by BDNF (brain-derived neurotrophic factor), a growth factor responsible for synaptic plasticity. The dissociation of RNG105 is significantly correlated with the induction of local translation of the mRNAs near the granules. These findings indicate that RNG105 is responsible for the local translational regulation in neuronal dendrites, and suggest its implication in the regulation of local synaptic plasticity in a stimulation-dependent manner.

(3) Single Hydrogen Bonds of DNA Base Pairs Detected in Unzipping Force by Intermolecular Force Microscopy

Michio Hiroshima¹ and Makio Tokunaga (¹Single Molecule Immunoimaging, Research Center for Allergy and Immunology, RIKEN)

--Single hydrogen bonds of DNA base pairs have been measured by unzipping double-stranded DNA oligomers with an intermolecular force microscope (IMF). To detect such ultrafine forces, high resolution of force as well as high accuracy in controlling the probe position is required. IFM has achieved the force resolution of subpico-newton using ultrasensitive cantilevers. The probe position is controlled with nanometer accuracy using a feedback system, which uses laser radiation pressure to reduce thermal fluctuation of the cantilever.
--Force vs. extension curves showed repeated force peaks of 10-15pN. Auto- or cross-correlation analysis and averaging of force curves were carried out to reduce noises in the force curve. The previous studies showed that the force for separating poly(G-C) DNA was 1.5 to 2 times stronger than that for poly(A-T). However, no difference was found in the force between individual G-C and A-T base pairs. The force curve of individual G-C and A-T pairs showed three and two peaks, respectively, which are assigned to single hydrogen bonds. The force is variable but the work is constant. The work to break single hydrogen bonds is 1.3 kB･T, in other words, about 1.3-fold of the thermal energy. This is the first report to detect the force of single hydrogen bonds in biological macromolecules.

(4) Mechanism for passive force generation of invertebrate connectin revealed by single molecule measurement

Michio Hiroshima¹, Atushi Fukuzawa², Koscak Maruyama², Sumiko Kimura² and Makio Tokunaga (¹Single Molecule Immunoimaging, Research Center for Allergy and Immunology, RIKEN, ²Department of Biology, Chiba University)

--Connectin is an elastic protein in muscle and is thought to keep the thick filament at the center of sarcomere or protect a myofibril from damages by extraordinary loads. Invertebrate connectin (I-connectin) is a 1960 kDa elastic protein linking the Z line to the tip of the myosin filament in the giant sarcomere of crayfish claw closer muscle. There are several extensible regions in I-connectin: two long PEVK regions, one unique sequence region and Ser-, Glu- and Lys-rich 68 residue-repeats called SEK repeats.
--The force measurements of the single recombinant polypeptide including SEK or PEVK regions was performed by intermolecular force microscopy (IFM). The force versus extension curves were well fit to the wormlike chain (WLC) model. The obtained persistence lengths were 0.38±0.1nm (n=63) of SEK peptide and 3.07±1.0nm (n=35) of PEVK peptide. The persistence lengths well explain the elastic behavior of SEK and PEVK regions in muscle fiber. The value of 0.38nm indicates that the SEK region is a random coil for full length. This is the first observation of an entropic elasticity of fully random coil region contributing to the physiological function of invertebrate connectin.

(5) Asymmetric nucleocytoplasmic transport revealed by a novel assay system using planner reconstituted nuclear envelope

Atsuhito Okonogi, Michio Hiroshima¹, Nobuyuki Shiina, Shingo Kose², Naoko Imamoto² and Makio Tokunaga (¹Single Molecule Immunoimaging, RCAI, Riken, ²Cellular Dynamics Laboratory, Riken)

--We have developed a novel in vitro assay system of nucleocytoplasmic transport. We aim at application of the method to new single-molecule experiments, imaging and nano- or force-measurement. Nuclear envelope was formed on a planar surface of a small agarose plate. Agarose plates were modified with glutation, and were coated with GST-RanGDP fusion protein. Nuclear envelope was formed onto the RanGDP-coated surface using extracts from Xenopus laevis frog eggs. Formation of Nuclear Pore Complexes was confirmed by observing import of fluorescently labeled proteins. Import assays using fluorescently labeled proteins showed that the reconstituted NPCs retain the transport activity. The dependence of the import activity on RanGTP concentration showed cooperativity.
--Import and export of importin a was examined in the absence of RanGTP and other soluble factors using the present cell-free in vitro assay system. Asymmetry between import and export was discovered in the interaction of importin a and the NPC in the absence of Ran-GTP and other soluble factors. In the apparent equilibrium, import of importin a showed a difference in the concentration between at the cytoplasmic side and at the nucleoplasmic side, whereas export of importin a showed no difference. The concentration difference of importin a is found to correlate with the amount of binding of importin a with the NPC. The asymmetry between import and export was also found in the transport of importin a in the presence of NLS cargo molecules. In the presence of NLS-protein, importin a showed no biding with the NPC. The present finding shows that the NPC has an asymmetric feature in the interactions with cargo molecules and transport factors between import and export.

(6) Molecular Imaging of translation initiation factors in neuronal dendrites

Hiraku Miyagi, Nobuyuki Shiina and Makio Tokunaga

--Local protein synthesis in neuronal dendrites is required for synaptic plasticity, which is associated with long-term memory storage. This protein synthesis is reported to be induced at activated postsynaptic sites. In order to investigate when and where the translation is initiated locally in the dendrites, we visualized eukaryotic translation initiation factors (eIF) 4E and eIF4G in dendrites of rat hippocampal neurons.
--Immunostaning of rat hippocampal primary neurons showed that most of eIF4E and eIF4G were spreaded in distinct granular structures, and few of them were colocalized in some granular structures in the dendrites. Immunostaning of rat hippocampal slices showed essentially the same results as in the case of the primary cultures. Stimulation by brain-derived neurotrophic factor (BDNF) increased colocalization of eIF4E and eIF4G in both rat hippocampal slices and primary neurons. As the association of eIF4E and eIF4G is known to trigger translation initiation, the increase of their colocalization suggests that translation is induced. Immunostaning of rat hippocampal slices and primary neurons for eIF4G and PSD-95, a marker protein for postsynapses, showed that colocalization of eIF4G and PSD-95 was increased by BDNF stimulation. Colocalization of eIF4E and PSD-95 is also increased by same stimulation. In GFP-expressed primary neurons, colocalization of eIF4E and eIF4G in spines is increased by BDNF stimulation. These results indicate that translation in the neuronal dendrites is inhibited by separating spatially eIF4E and eIF4G, and translation is induced by BDNF stimulation at postsynaptic areas.

PUBLICATIONS

Papers
1. Kitamura, K., Tokunaga, M., Esaki, S., Iwane, A.H. and Yanagida, T. (2004). Mechanism of muscle contraction based on stochastic mechanical features of single actomyosin motors observed in vitro. BIOPHYSICS, in press.

ORAL PRESENTATIONS

1. Tokunaga M.: Single Molecule Imaging and Quantitative Analysis of Molecular Interactions Inside Cells. The 1st Pacific-Rim International Conference on Protein Science, Yokohama, Japan, April, 2004.
2. Hirakawa Y, Hasegawa T, Masujima T, Tokunaga M, Tsuyama N, Kawano M.: Single-molecular Imaging of Protein in living Cell by Pin-fiber Video-microscope. 13th International Symposium on Bioluminescence & Chemiluminescence, Yokohama, Japan, August, 2004.
3. Shiina, N., Shinkura, K. and Tokunaga, M.: RNG105: A novel RNA-binding protein in neuronal RNA granules, regulatory machinery for synaptic stimulation-dependent local translation. Symposium "RNA neurobiology: Posttranscriptional world in neurons" in joint meeting of the 27^th annual meeting of the Japan neuroscience society and the 47th annual meeting of the Japanese society for neurochemistry, Osaka, September, 2004.
4. Tokunaga M.: Single Molecule Imaging and Quantitative Analysis of Molecular Interactions Inside Cells. Kazusa International Workshop: "Beyond the Identification of Transcribed Sequences:Functional, Expression and Evolutionary Analysis", Kisarazu, Chiba, October, 2004.
5. Hiroshima, M., Fukuzawa, A., Maruyama, K., Kimura, S., and Tokunaga, M.: Single molecule measurement of elasticity of SEK-rich repeats of invertebrate connectin reveals that its elasticity is caused entropically by random coil structure. International Symposium on Muscle Elastic Proteins: Koscak Maruyama Memorial Meeting, Chiba, November, 2004.
6. 徳永万喜洋「見えなかったものを観る計る」特定領域・生命現象の1分子イメージング・公開シンポジウム,東京,2004年3月
7. 椎名伸之「シナプス刺激依存的な局所的翻訳における新規タンパク質RNG105の機能解析」生理学研究所研究会,岡崎市,2004年5月
8. 徳永万喜洋「1分子イメージングと定量解析」東京工業大学生命理工学研究科生命情報専攻セミナー,横浜,2004年8月
9. 徳永万喜洋「1分子イメージング」阪大・北大COE細胞生物学ワークショップ,神戸,2004年8月
10. 椎名伸之,新倉和美,徳永万喜洋「RNA結合タンパク質RNG105によるシナプス刺激依存的な局所的翻訳制御」第6回日本RNA学会年会,熊本市,2004年8月
11. 徳永万喜洋「生体分子の1分子イメージングと計測」東京大学大学院工学系研究科応用化学専攻講演会,東京都文京区,2004年9月
12. 徳永万喜洋「生きている細胞の1分子イメージングと分子定量解析」生理学研究所研究会,岡崎,2004年10月
13. 徳永万喜洋「生体分子相互作用の1分子計測と細胞内1分子イメージング」東京工業大学生命理工学研究科シンポジウム,横浜,2004年10月
14. 徳永万喜洋「細胞1分子イメージングと相互作用の分子定量」基礎生物学研究所研究会,岡崎,2004年12月
　
POSTER PRESENTATIONS

1. Sakata-Sogawa, K., Yamasaki, S., Saito, T. and Tokunaga, M.: Dynamics of lipid rafts revealed by molecular imaging. The 1st Pacific-Rim International Conference on Protein Science, Yokohama, April, 2004.
2. Hiroshima M., Fukuzawa A., Kimura S., Tokunaga M. & Maruyama K.: Single molecule measurement of elasticity of Serine-, Glutamate- and Lysine-rich repeats of invertebrate connectin: its elasticity is caused entropicall y by random coil structure. The 1st Pacific-Rim International Conference on Protein Science, Yokohama, Japan, April, 2004.
3. Shiina, N., Shinkura, K. and Tokunaga, M.: RNG105: A novel mRNA-binding protein in neuronal RNA granules for synaptic stimulation-dependent local translation. The 57^th annual meeting of Japan society for cell biology, Osaka, May26-28, 2004.
4. 池田和敏,廣島通夫,福澤淳,徳永万喜洋,木村澄子「I-コネクチンの一分子計測による弾性の解析」日本動物学会第74回大会,神戸市,2004年9月
5. 椎名伸之,新倉和美,徳永万喜洋「RNA結合タンパクRNG105によるシナプス刺激依存的な局所的翻訳制御」第42回日本生物物理学会年会,京都市,2004年12月
6. 廣島通夫,徳永万喜洋「DNA塩基対を形成する水素結合1個の力計測」日本生物物理学会第42回年会,京都市,2004年12月
7. 新倉和美,椎名伸之,十川久美子,徳永万喜洋「神経樹上突起におけるmRNA輸送複合体とミトコンドリアrRNAの局在性」日本生物物理学会第42回年会,京都市,2004年12月
8. 小此木孝仁,廣島通夫,椎名伸之,小瀬真吾,今本尚子,徳永万喜洋「新しいin vitro assay系を用いた細胞質-核間輸送の非対称性」日本生物物理学会第42回年会,京都,2004年12月
9. 宮城拓,椎名伸之,徳永万喜洋「神経樹上突起における局所的な翻訳制御のイメージング」日本生物物理学会第42回年会,京都市,2004年12月

EDUCATION

他大学／研究機関での講義やセミナー
1. Dr. M. Tokunaga gave a lecture at Tokyo Institute of Technology, Department of Bioscience and Biotechnology, August, 2004 (in Japanese).
2. Dr. M. Tokunaga gave a Seminar at the University of Tokyo, Department of Applied Chemistry, School of Engineering, September, 2004 (in Japanese).

SOCIAL CONTRIBUTIONS AND OTHERS

各種委員
1. 徳永万喜洋　バイオテクノロジー開発技術研究組合「細胞内ネットワークのダイナミズム解析技術開発」研究開発委員
2. 徳永万喜洋　科学技術振興調整費研究評価部会「細胞・生体システム研究評価ＷＧ」科学技術・学術審議会専門委員

集会／シンポジウムの主宰
1. Dr. M. Tokunaga organized a symposium entitled “Frontier of Single Molecule, GFP and Bioimaging" supported by Grant-in-Aid for Scientific Research on Priority Areas “Molecular Imaging" from MEXT, Tokyo International Forum, March, 2004.