軸索輸送、細胞骨格 Axonal Transport and Cytoskeleton
P1-1-41 海馬神経細胞軸索内におけるミトコンドリア動態の解析 Regulation of mitochondrial dynamics in the axon with multiple time scales ○小橋一喜1, 岡部繁男1 ○Kazuki Obashi1, Shigeo Okabe1 東京大院・医・神経細胞生物学1 Dept Cell Neurobiol, Univ of Tokyo, Tokyo1 The elaborate structure of the neuron requires a regulatory mechanism to allocate a sufficient number of organelles to its subcellular compartments, such as the soma, neurites and synapses. Maintaining proper distribution of axonal mitochondria is critical for multiple neuronal functions including energy production, calcium homeostasis, apoptosis, synaptic transmission and plasticity. To attain this, mitochondria should be bi-directionally transported along the axonal cytoskeleton and positioned appropriately in response to physiological demands. Therefore, distribution processes should be dependent on multiple dynamic factors involving fractions of mitochondria in stationary or mobile state, transition rates between these two states, and dynamic properties of mobile mitochondria. To study the regulation of long and short-term dynamics of axonal mitochondria in cultured hippocampal neurons, we performed live-cell imaging with multiple sampling frequencies ranging from seconds to days. We demonstrated that both stability of stationary mitochondria and behavior of mobile mitochondria were regulated spatially and temporally by multiple physiological conditions, including neuronal maturation, neuronal activity and synaptic positions. Quantitative analysis further indicated that transition process from mobile to stationary states should also be regulated by proximity to synaptic sites and neuronal activity. These results collectively indicate that mitochondrial distribution is regulated by multiple dynamic parameters in response to physiological demands.	P1-1-42 モーター分子KIF5AはGABAA 受容体の輸送に必須であり、KIF5Aの欠損はてんかんを引き起こす Molecular motor KIF5A is essential for GABAA receptor transport, and KIF5A deletion causes epilepsy ○中島一夫1, イジリン1, 武井陽介1, 石大賢3, 本間典子1, 廣川信隆1,2 ○Kazuo Nakajima1, Xilin Yin1, Yosuke Takei1, Dae-Hyun Seog3, Noriko Homma1, Nobutaka Hirokawa1,2 東京大院・医・細胞生物学解剖学1, King Abdulaziz 大学2, Inje 大学3 Dept Cell Biol & Anat, Univ of Tokyo, Tokyo1, King Abdulaziz University, Jeddah, Saudi Arabia2, Dept Biochem, Inje University, Busan, Korea3 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 GABAA receptor (GABAAR)-mediated synaptic transmission. We also identified reduced cell surface expression of GABAAR in knockout neurons.Importantly, we identified that KIF5A specifically interacted with GABAAR-associated protein (GABARAP) that is known to be involved in GABAAR trafficking. KIF5A regulated neuronal surface expression of GABAARs via an interaction with GABARAP. These results provide an insight into the molecular mechanisms of KIF5A, which regulate inhibitory neural transmission.
P1-1-43 小脳顆粒細胞の極性形成における分子ネットワークの解析 Molecular network analysis of neuronal polarity in cerebellar granule neurons ○瀬野岳史1, 久保田健太1, 栄成美1, 小西慶幸1,2 ○Takeshi Seno1, Kenta Kubota1, Narumi Sakae1, Yoshiyuki Konishi1,2 福井大院・工・知能1, 生命科学複合研究教育センター2 Dept technology,Univ of Fukui, Fukui1, Research and Education Program for Life Science, Univ of Fukui, Fukui2 Polarity formation that generates two different types of processes (i.e. dendrites and axons) is a critical event in neuronal morphology. One of the factors that contribute to maintain the neuronal polarity is considered to be a difference of molecules transported in each compartment. The question is how neurons distinguish these two different processes.Tubulin composing microtubules can be subjected to post-translational modifications. Among these modifications, tyrosinated tubulins are enriched in dendrites rather than axons. Previous studies suggest that kinesin motor domain binds more strongly to detyrosinated microtubules, and kinesin can transport materials to the axon by recognizing the modification of microtubules.The above study has been performed mainly by using hippocampal neurons, and it has not been elucidated whether other types of neurons use same system. In this study, we aimed to address this issue by examining whether the intracellular localization of kinesin is regulated by the modification of microtubules in cerebellar granule cells.By observing GFP fluorescence under the microscope, it is revealed that kinesin motor domain fused to GFP selectively localized to axons in cerebellar granule cells. When we inhibited the expression of the TTL (Tubulin tyrosine ligase) by RNA interference, kinesin motor domain was appeared to be localized to dendrites as well. From these results, we conclude that kinesin controls the polarized axonal transport by recognizing the modification of microtubules in cerebellar granule neurons. Thus, different type of neurons uses common mechanism for the polarized intracellular transport. In addition to the issue of neuronal polarity, in this study, we investigate whether similar mechanisms are involved in the axonal branch selection by kinesin.	P1-1-44 モーター分子KIF13Aトランスポートセロトニンレセプター1A A molecular motor, KIF13A, controls anxiety by transporting the serotonin type 1a receptor 丹羽伸介1広川信隆1 ○Ruyun Zhou1, Shinsukei Niwa1, Laurent Guillaud1, Ying Tong1, Nobutaka Hirokawa1 東京大学大学院医学系研究科　細胞生物1 Dept Medicine, Univ of Tokyo, Tokyo1 Molecular motors are fundamental to neuronal morphogenesis and function. However, it remains largely unknown to what extent molecular motors are involved in higher brain functions. In this study, we have shown that mice deficient in the kinesin family motor protein KIF13A exhibit elevated anxiety-related behavioral phenotypes, probably because of a reduction in 5HT1A receptor transport. The cell surface expression level of the 5HT1A receptor was reduced in KIF13A knockdown neuroblastoma cells and KIF13A knockout hippocampal neurons. Biochemical analysis showed that the forkhead associated (FHA) domain of KIF13A and an intracellular loop of the 5HT1A receptor are the interface between the motor and cargo vesicles. A mini-motor consisting of the motor and FHA domains is able to transport 5HT1A receptor-carrying organelles in in vitro reconstitution assays. Collectively, our results suggest a role for this molecular motor in anxiety control.
P1-1-45 ドレブリンはSpikarの細胞内局在とスパイン形成機能を制御している Drebrin is responsible for spine formation activity of spikar and its cytoplasmic localization ○山崎博幸1, 白尾智明1 ○Hiroyuki Yamazaki1, Tomoaki Shirao1 群馬大学大学院医学系研究科神経薬理学1 Dept. Neurobiol and Behav. Gunma Univ. Sch. Med, Maebashi, Japan1 Spikar is a novel drebrin-binding protein, which regulates spine formation. In early stage of spine formation, drebrin is responsible for recruitment of postsynaptic components into dendritic filopodia and governs spine formation. Spikar is a transcriptional co-activator isolated as a drebrin-binding protein by yeast two-hybrid screen using brain cDNA library. In cultured neurons, spikar is localized in dendritic spine as well as in nucleus. Overexpression of cytoplasmic spikar (mNLS-spikar), which is mutated in nuclear localization signal, increases the number of dendritic spine and filopodium. This indicates that spikar in dendritic spines is involved in spine formation. In this study, we investigated the role of the interaction between drebrin and spikar in dendritic spine formation. Although spikar was localized in mainly dendritic spines in control neurons, spikar was distributed diffusely throughout the dendrites in drebrin-knockdowned neurons. We then examined whether the spine-formation activity of mNLS-spikar would require the presence of drebrin. We co-transfected mNLS-spikar and drebrin-shRNA into cultured neurons and measured the numbers of spines and filopodia. The drebrin knockdown abolished the mNLS-spikar-induced increase in spine and filopodium density. The inhibition of mNLS-spikar function by drebrin knockdown was rescued by the co-expression of an RNAi-resistant drebrin mutant. These data indicate that drebrin is responsible for the formation of spines and filopodia facilitated by cytoplasmic spikar.	P1-1-46 アクチンのトレッドミルによって引き起こされる遅い軸索輸送 Slow axonal transport driven by directional actin treadmilling ○勝野弘子1,2, 鳥山道則1, 作村諭一1,2, 池田和司2, 水野健作3 ○Hiroko Katsuno1,2, Michinori Toriyama1, Yuichi Sakumura1,2, Kazushi Ikeda2, Kensaku Mizino3 奈良先端科学技術大学院大学　バイオサイエンス研究科1, 奈良先端科学技術大学院大学　情報科学研究科2, 東北大学大学院　生命科学研究科3 Grad. sch. of Biol. Sci., Nara Inst. Sci. and Technol., Nara, Japan1, Grad. sch. of Inform. Sci., Nara Inst. Sci. and Technol., Nara, Japan2, Grad. sch. of Life Sci., Tohoku Univ., Miyagi, Japan3 Axonal transport is essential for the growth, maintenance and regeneration of axons, and categorized into the fast component (2-5 μm/s), slow component a (0.002-0.01 μm/s) and slow component b (0.02-0.09 μm/s). Slow component b conveys more than 200 proteins including actin and actin binding proteins. The fast component and slow component a are thought to be transported by kinesins and dyneins along microtubules, however the mechanism for slow component b has been not well known since it was first reported more than 30 years ago. Previous report showed that an actin rich structure called "wave" moves along the axonal shaft at the rate of slow component b, thereby suggesting that the wave transports slow component b. Here we show that assemblies of actin filaments in wave move anterogradely along the axonal shaft at the rate of slow component b, depending on their directional polymerization/depolymerization, the process called treadmilling, and their anchoring to the substrate. We found that mass of actin filaments in waves undergo treadmilling in which the polymerizing ends are orientated toward the neurite tip. Inhibition of actin polymerization by cytochalasin B led to a decrease in the velocity of wave movement. Enhancement of the actin polymerization by promoting the cofilin activity led to an increase in the velocity of wave movement. We also found that the treadmilling F-actins are anchored to the substrate, through a clutch protein shootin1 and cell adhesion molecule L1-CAM. Inhibition of the interaction between F-actins and the substrate induced a slippage of treadmilling F-actins. Importantly, this slippage decreased the velocity of wave movement. We also confirmed the actin transport with the wave in vivo. We propose a novel type of axonal transport system driven by directional F-actin treadmilling anchored to the substrate which is different from motor protein dependent mechanism.

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