突起伸展1 Neurite Growth 1
O1-6-3-1 カルシウムおよびサイクリックヌクレオチドはそれぞれ異なるv-SNAREを介して成長円錐の進路を決定する Calcium and cyclic nucleotides utilize distinct v-SNAREs for growth cone turning ○秋山博紀1, 福田徹子1, 戸島拓郎1,2, 上口裕之1 ○Hiroki Akiyama1, Tetsuko Fukuda1, Takuro Tojima1,2, Hiroyuki Kamiguchi1 理研・BSI1, JSTさきがけ2 RIKEN BSI, Saitama1, PRESTO, JST, Saitama2 Environmental guidance cues attract or repel developing axons through asymmetric generation of second messenger signals in the growth cones. While many studies have focused on Ca2+ as the polarizing signal inducing growth cone turning, less attention has been paid to the cyclic nucleotides, cAMP and cGMP. Here we found that cyclic nucleotides regulated the dynamics of microtubules (MTs) and vesicle-associated membrane protein (VAMP) 7-positive vesicles. This is in contrast to the mechanism of Ca2+-dependent turning, which utilizes VAMP2 vesicles. When cAMP concentration was artificially and locally increased by photolysis of caged compound in the growth cones of embryonic chick dorsal root ganglion neurons, the frequency of MT contact with the leading edge increased on the side with cAMP signals. The opposite was true for cGMP: MT contact decreased with cGMP increases. These changes in MT dynamics in turn led to spatial asymmetry in the frequency of centrifugal VAMP7 transport toward the leading edge, i.e. cAMP ipsilaterally increased, while cGMP decreased, VAMP7 transport. In contrast, VAMP2 vesicle dynamics was unchanged by cyclic nucleotide signals, and Ca2+ signals did not affect the dynamics of MTs and VAMP7 vesicles. Furthermore, the contact of MTs and VAMP7 vesicles with the leading edge caused localized lamellipodial protrusions. To assess the importance of VAMP7 in cyclic nucleotide-dependent turning, longin domain of VAMP7 that inhibit SNARE complex formation was used. The longin domain significantly reduced the distance of MT-dependent lamellipodial protrusion, and inhibited both cAMP-induced attraction and cGMP-induced repulsion. These results indicate that asymmetrical cAMP and cGMP signals determine the direction of growth cone turning through asymmetrical VAMP7 vesicle trafficking, and highlight the differential involvement of VAMP7 versus VAMP2 in cyclic nucleotide versus Ca2+ signaling cascades.	O1-6-3-2 ERK2によるPar3のリン酸化は神経細胞の極性形成を制御する ERK2-mediated phosphorylation of Par3 regulates neuronal polarization ○船橋靖広1, 中牟田信一1難波隆志1, 貝淵弘三1 ○Yasuhiro Funahashi1, Sinichi Nakamuta1, Xu Chundi1, Takashi Namba1, Kozo Kaibuchi1 名古屋大院・医・神経情報薬理学1 Dept pharmaco, Univ of Nagoya, Nagoya1 Axon formation is one of the important events for neuronal polarization, and it is regulated by signaling molecules involved in cytoskeletal rearrangement and protein transport. We previously found that Partition-defective 3 (Par3) is associated with KIF3A (kinesin-2) and is transported into the nascent axon in a KIF3A-dependent fashion. Par3 interacts with the Rac-specific guanine nucleotide-exchange factors (GEFs) Tiam1/2, which activate Rac1, and participates in axon formation in cultured hippocampal neurons. However, the regulatory mechanism of the Par3-KIF3A interaction is not well understood, and the role of Par3 in neuronal polarization in vivo remains elusive. Here, we found that extracellular signal-regulated kinase 2 (ERK2) directly interacts with Par3, ERK2 phosphorylates Par3 at Ser-1116 and the phosphorylated Par3 accumulates at the axonal tips in a manner dependent on ERK2 activity. The phosphorylation of Par3 by ERK2 canceled the interaction of Par3 with KIF3A but not with the other Par3 partners, including Par6 and aPKC. The phosphomimic mutant of Par3 (Par3-S1116D) showed less binding activity with the KIF3s and slower transport in the axons. Knockdown of Par3 by RNA interference impaired neuronal polarization, which was recovered with RNAi-resistant Par3 but not with the phosphomimic Par3 mutant in cultured hippocampal neurons and in cortical projection neurons in vivo. These results suggest that ERK2 phosphorylates Par3 and inhibits its binding with KIF3A, thereby controlling Par3 transport and neuronal polarity.
O1-6-3-3 M6a-M6BP-Rap2タンパク質複合体を介した大脳皮質におけるラミニン依存性の神経極性決定制御 Laminin-dependent regulation of the polarity determination through the M6a-M6BP-Rap2 complex in the cortical neuron ○本多敦子1, 武内恒成1,2, 五十嵐道弘1,2 ○Atsuko Honda1, Kosei Takeuchi1,2, Michihiro Igarashi1,2 新潟大院・医・分子細胞機能1, 新潟大学・超域学術院2 Div of Mol Cell Biol, Grad Sch of Med and Dent Sci, Niigata Univ, Niigata1, Cent. for Transdisciplinary Res., Niigata Univ2 Mechanisms of neuronal polarity determination has been intensively investigation in the hippocampus, however, in developmental studies of cerebral cortex, these mechanisms have not been established. Our previous proteomic analysis showed that glycoprotein M6a is an abundant membrane protein in the neuronal growth cone, and we identified an intracellular M6a-binding protein named M6BP, interacting with the active form of Rap2. M6a-M6BP-Rap2 formed a ternary complex, closely related with the polarity determination in the hippocampal neuron, and we found that M6a showed asymmetric localization in the cortical neurons on laminin substrate. Here, we examined the effects of laminin on the polarity determination in cortical neurons, and its relationship to the M6a-M6BP-Rap2 complex. After the culture plating, the process of their morphological changes more slowly progressed but similarly to that of the hippocampal neuron (Dotti CG., et al; 1988). On the laminin substrate, protrusion of a specific neurite was induced at 4-16h after the plating, and significantly earlier than that on poly-L-lysine. M6a and its binding proteins, M6BP and Rap2 also showed the asymmetric localization just after the plating on laminin, in spite of the symmetric localization of the well-known polarity determinants (e.g. Par3 or CRMP2). Laminin signals induced speedy accumulation of the polarity determinants at the tip of a selective neurite, where the M6a-M6BP-Rap2 complex was localized in advance. Inhibition of this complex formation suppressed the laminin-dependent accumulation of other polarity determinants and the axon formation. These results indicated that the extracellular laminin accelerated the polarity determination of the cortical neurons through the M6a-M6BP-Rap2 complex, leading to the sequential asymmetric localization of other polarity determinants.	O1-6-3-4 ダブルコルチン様キナーゼ1によるMAP7D1のリン酸化の軸索輸送における役割 A role for Doublecortin-like kinase 1-dependent phosphorylation of MAP7D1 in axonal transport ○古泉博之1, 藤岡洋美1,2, 富樫和也1, 岡田康志3榎本和生1 ○Hiroyuki Koizumi1, Hiromi Fujioka1,2, Kazuya Togashi1, Yasushi Okada3, Joseph G. Gleeson4,5, Kazuo Emoto1 大阪バイオサイエンス研究所・神経細胞生物学部門1, 奈良先端技術大学2, 理化学研究所　生命システム研究センター3 Dept Cell Biology, Osaka Bioscience Institute1, Nara Institute of Science and Technology2, Laboratory for Cell Polarity Regulation, Quantitative Biology Center RIKEN3, Neurogenetics Laboratory, Howard Hughes Medical Institute4, Department of Neuroscience, University of California San Diego5 Doublecortin-like kinase 1 (DCLK1) is a unique serine-threonine protein kinase that has a microtubule-binding domain in its N-teminal. N-terminal domain has 70% homology with Doublecortin (DCX) that was identified as a causative gene product of human cortical malformation due to defect in neuronal migration.Dcx knockout mice exhibit mild lamination defects in hippocampus and Dclk1 knockout mice exhibit defects in corpus callosum formation. In contrast, Dclk1/Dcx double knockout mice display severe lamination defects in the cortex and hippocampus and disruption in commissural axons extending across the midline. These results suggest that DCLK1 functions cooperatively with DCX to regulate neuronal migration and neural circuit formation during brain development. DCX has been shown to contribute to neuronal migration and neurite outgrowth through regulating microtubule bundling and stabilization, however the function of the DCLK1 kinase domain remains elusive. To further understand the role of the DCLK1 kinase domain during brain development, we previously searched for the substrates of DCLK1 by using GST-affinity column purification and identified MAP7D1 (microtubule-associated protein 7 domain containing 1) as a novel substrate of DCLK1. Both MAP7D1 knockdown and DCLK1 knockdown resulted in impaired axonal outgrowth in cortical neurons. We further identified that MAP7D1 interacted with kinesin heavy chain, KIF5. We currently examine how MAP7D1 phosphorylation by DCLK1 is related to KIF5-dependent axonal transport. We will discuss the functional mechanism of DCLK1 in kinesin-1-dependent axonal transport through MAP7D1 phosphorylation during axon outgrowth.

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