軸索輸送、細胞骨格 Axonal Transport and Cytoskeleton
P2-2-38 プルキンエ細胞特異的カテプシンD欠損およびAtg7欠損マウスの比較解析 Characteristic differences between Purkinje cells specifically deficient in cathepsin D and Atg7 ○小池正人1, 柴田昌宏2, 砂堀毅彦1, 小松雅明3, 崎村建司4, 内山安男1 ○Masato Koike1, Masahiro Shibata2, Takehiko Sunabori1, Masaaki Komatsu3, Kenji Sakimura4, Yasuo Uchiyama1 順天堂大学・医学研究科・神経生物学・形態学1, 新潟大院・医歯・肉眼解剖学2, 東京都医学総合研・蛋白質リサイクル3, 新潟大院・脳研・細胞神経生物4 Dept Cell Biol Neurosci, Juntendo Univ, Tokyo, Japan1, Div Gross Anat Morphogenesis, Niigata Univ, Niigata, Japan2, Tokyo Metropolitan Inst Med Sci, Tokyo, Japan3, Brain Res Inst, Niigata Univ, Niigata, Japan4 Neurological phenotypes of cathepsin D-deficient mice, a murine model of neuronal ceroid lipofuscinoses (NCLs), indicate the importance of CD in the maintenance of metabolism in central nervous system (CNS) neurons. To further understand the role of CD in CNS neurons, we generated conditional CD-deficient mice specifically in Purkinje cells (PC) (CTSDflox/flox;GluD2-Cre) and PC-selective Atg7-deficient (Atg7flox/flox;GluD2-Cre) mice. In both lines of mice, PCs underwent degeneration. Interestingly, CD-deficient PCs largely disappeared until 2 months of age, whereas Atg7-deficient PCs still survived at that time. Immunohistochemical observations exhibited that formation of calbindin-positive axonal spheroids and swelling of vesicular GABA transporter-positive in presynaptic terminals were more pronounced in Atg7-deficient PCs than in CD-deficient PCs. Electron microscopy also demonstrated that abnormal tubular vacuoles, nascent autophagosome-like structures, and membrane-bound and electron-dense granules accumulated within axons and presynaptic terminals of PCs in both lines of mice. In CD-deficient mice, immunosignals for p62 and Nbr1 were detected only in perikarya but not in axons of PCs, while those for ubiquitin were seen throughout neurons. Different from immunostaining of ubiquitin in PCs from both lines of mice, that of p62 and Nbr1 was not detected in axons and axon terminals. These results suggest that although axonal and synaptic degeneration was much more severe in Atg7-deficient PCs, CD-deficient PCs were more vulnerable than Atg7-deficient PCs. This further indicates that autophagy plays an essential role in the anabolic maintenance of axons, but degradation starts in cell soma where lysosomes work.	P2-2-39 脆弱X精神遅滞タンパク質の軸索・成長円錐における局在はセマフォリン3Aにより制御される Localization of Fragile X Mental Retardation Protein in axons and growth cones is regulated by Semaphorin3A ○佐々木幸生1,2, 高畠将1,3, 花岡和則3, 五嶋良郎1 ○Yukio Sasaki1,2, Masaru Takabatake1,3, Kazunori Hanaoka3, Yoshio Goshima1 横浜市立大学大学院医学研究科分子薬理神経生物学教室1, 横浜市立大学大学院生命医科学研究科機能構造部門機能構造科学研究室2, 北里大学理学部生物科学科3 Dept Mol Pharmacol & Neurobiol, Yokohama City Univ Grad Sch Med, Yokohama1, Funct Struct Biol Lab, Div Funct Struct Biol, Yokohama City Univ Grad Sch Med Life Sci, Yokohama2, Dept Biosci, Sch Sci, Kitasato Univ, Sagamihara3 There is increasing evidence that localized mRNAs in axons and growth cones play an important role in axon extension and pathfinding via local translation. Recently, we demonstrated that some of microRNAs (miRNAs), which are small non-coding RNAs to regulate translation, are also localized in axons and growth cones. Argonaute (Ago) proteins are known to bind miRNAs for suppression of translation, but Ago proteins have not been shown to interact motor proteins. Therefore, it remains unclear how miRNA localization is regulated in axons and growth cones. Because Fragile X Mental Retardation Protein (FMRP) family interacts with both motor proteins and Ago proteins, we have investigated role of FMRP family in miRNA localization in axons and growth cones. We found that all FMRP family (FMRP, Fxr1p, and Fxr2p) and Ago2, one of Ago proteins, exist in axons and growth cones in mouse hippocampal neurons. FMRP and Ago2 co-localizes in growth cones. FMRP-mCherry traffics along axons. Therefore, it is possible that FMRP-Ago complex is responsible for miRNA transport in axons. One of axon guidance factors, Semaphorin3A (Sema3A), induces growth cone collapse in dependent on protein synthesis. Sema3A-induced collapse response is suppressed in FMRP gene (Fmr1) knockout neurons, indicating involvement of FMRP-dependent protein synthesis in the growth cone collapse. We found that Sema3A stimulation leads to decrease in localized FMRP in growth cones. Because FMRP-Ago2 complex releases miRNA to unlock translation in dendrites after stimulation of metabotropic glutamate receptor, it is plausible that the Sema3A-stimulated decrease in FMRP induces local translation in axonal growth cones. This work would provide new insight into miRNA-mediated translational regulation in axons and growth cones.
P2-2-40 Cdk5-p39 のRac1依存的なアクチンフィラメント構造への局在とその構造に対する影響 Association of Cdk5-p39 with Rac1-dependent F-actin structure ○伊藤有紀1, 浅田明子1, 高野哲也1, 斉藤太郎1, 天野睦紀2, 貝淵弘三2, 久永眞市1 ○Yuki Ito1, Asada Akiko1, Tetsuya Takano1, Taro Saito1, Mutsuki Amano2, Kozo Kaibuchi2, Shin-ichi Hisanaga1 首都大学東京・理工学・神経分子機能1, 名大・医・神経情報薬理2 Dept. Biol. Sci., Tokyo Metro. Univ., Tokyo, Japan1, Dept. Cell. Pharm., Nagoya Univ. Grad. Sch. of Med., Aichi, Japan2 Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase, which plays a critical role in neuronal development, synaptic plasticity, and neurodegeneration. Cdk5 is activated by a neuron-specific activator, p35 or p39. p35 and p39 are isoforms with a high similarity (72%) in their amino acid sequences of the C-terminal Cdk5-activation domain. Although gene deletion mutants indicate that Cdk5-p39 has distinct functions from Cdk5-p35 with some overlaps, the biochemical and cell biological properties of Cdk5-p39 are known only a little, compared to Cdk5-p35. We have shown that the activators determine the cellular localization of active Cdk5. Myristoylated p35 and p39 anchor the active Cdk5 to membranes (Asada et al., 2008). p35 and p39 are also localized in lamellipodia-like structure. However, it was not known what these structures are and how Cdk5-p35 and Cdk5-p39 are targeted to them. Lamellipodia formation is regulated by Rac1, which is a member of Rho family small GTPases. Here, we investigated the interaction between Rac1 and Cdk5-p35 or Cdk5-p39. Overexpression of Rac1-V12, a constitutive active form of Rac1, in Neuro2A cells induced F-actin-rich structures at the cell periphery, which may correspond to lamellipodia. When p35 or p39 was coexpressed, p39 was predominantly localized to the peripheral F-actin-rich region while most of p35 was found in the prinuclear region. p39, but not p35, showed overlap distribution to Rac1-V12. In the case of p39 coexpression with Rac1-V12, cells with spiny protrusions were frequently observed, concomitant decrease in cells with lamellipodium. Those protrusions were attenuated by coexpression with knCdk5. Such morphological alternations were not observed in the case of p35 coexpression. These results suggest that Cdk5-p39, but not Cdk5-p35, interacts with Rac1 to regulate formation of lamellipodia-like F-actin structures. Cdk5-p39 may have a dominant role in actin-dependent cellular activities in neurons more than Cdk5-p35.	P2-2-41 成長円錐におけるアクチン再編に伴う小胞輸送 Vesicular transport with actin rearrangement in the growth cone ○野住素広1,2, 加藤薫3, 武内恒成1,2, 五十嵐道弘1,2 ○Motohiro Nozumi1,2, Kaoru Kato3, Kosei Takeuchi1,2, Michihiro Igarashi1,2 新潟大院・医歯学・分子細胞機能学1, 新潟大・超域学術院2, 産総研・バイオメディカル3 Div Mol Cell Biol, Niigata Univ Grad Sch Med, Niigata1, Trans-disciplinary Res Progr, Niigata Univ, Niigata2, Biomed Res Inst, AIST, Tsukuba3 The growth cone is a motile tip formed at the developing and the regenerating neuronal processes. Continuous rearrangement of cytoskeletons and recruitment of transport vesicles to plasma membrane are thought to be essential to the growth cone motility, however, how the membrane trafficking regulates the growth cone motility are not clarified well, and the relationship between cytoskeletons and transported vesicles is poorly understood. To understand regulating the machinery of vesicular trafficking in the growth cone, we analyzed dynamics of vesicle movements in the growth cone using GFP-synaptophysin expressed in NG108 cells. We found that there were at least three different types of movements in the peripheral region of the growth cone. The first type of vesicles showed slowly retrograde movement. Their average speed was similar to that of actin retrograde flow. Dual color imaging of GFP-synaptophysin and mCherry-actin showed that such vesicles were transported on the F-actin bundles. The dual color observation also showed the second type of vesicle movements. These vesicles slowly moved together with the actin comet tail. The number of vesicles moving in these two types was decreased by latrunculin B treatment. In addition, we found the third type of moving vesicles; they moved both anterogradely and retrogradely in alternative ways, and the movements appeared on the microtubules invading into the peripheral region. The anterograde speed was corresponding to that of the GFP-EB3/GFP-dynactin. These results indicated that the retrograde trafficking of vesicles is dependent upon the actin retrograde flow in the peripheral region. These vesicles may be associated with F-actin bundles. Discontinuous fast anterograde movements may be associated with the dynein and/or kinesin motor on the growing microtubules. We suspect that these three different types of vesicle transport in the growth cone are important to the regulation of neuronal growth.
P2-2-42 高分解能音響インピーダンス顕微鏡を用いた培養筋細胞およびグリオーマの細胞内構造変化の観察 Subcellular structure observation of glioblastoma and myoblast differentiation using high-resolution acoustic impedance microscope ○別段碧1, 山田ひかり1, 塩木康紀1, 古橋友秀2, 小林和人3, 山本清二4, 穂積直裕2, 吉田祥子1 ○Midori Betsudan1, Hikari Yamada1, Yasunori Shioki1, Tomohide Furuhashi2, Kazuto Kobayashi3, Seiji Yamamoto4, Naohiro Hozumi2, Sachiko Yoshida1 豊橋技術科学大学　環境生命工学系1, 豊橋技術科学大学　電気電子情報工学系2, 本多電子(株)3, 浜松医科大学4 Dept Environ & Life Sci, Toyohashi Univ of Technology, Toyohashi1, Electrical & Electronic Info Eng, Toyohashi Univ of Technology, Toyohashi2, Honda Electronics Co., Ltd., Toyohashi3, Hamamatsu Univ. Sch. of Med. Hamamatsu4 Two-dimensional acoustic impedance imaging is useful for observation on living organs with no invasion. We have reported that the high-resolution acoustic impedance microscopy with 320 MHz transducer could visualize intracellular conditions of cultured glial cells. In the present study, we observed the intracellular process of myoblast differentiation and glioma reaction to cytoskeletal agents using the high-resolution acoustic impedance microscopy.Murine C2C12 myoblasts maintain their differentiation potential and, upon incubation with differentiation media, the myoblasts fuse into each other to form myotubes that are responsible for muscle contraction. Cultured C2C12 on OptiCellTM, a film culture system, were observed with the acoustic microscopy through the OptiCellTM film. Fused C2C12 cells expressed fast-type myosin, and their intracellular impedance increased remarkably. Under the low-density culture condition, neither fast myosin expression nor high acoustic impedance was observed. Murine C6 glioma showed rather low acoustic impedance, and their impedance was more decreased to cytochalasin B application than normal astrocytes'. High-resolution acoustic impedance was enable to observe cytoskeletal change in living cells conveniently. We suggest that the acoustic impedance microscopy would useful to detect the cytoskeletal conditions.

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