記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

RUN and FYVE domain-containing 3 (Rufy3) is an adapter protein for small GTPase proteins and is bound to activated Rap2, a Ras family protein in the developing neuron. Previously, we reported the presence of a rapid cell polarity determination mechanism involving Rufy3, which is likely required for in vivo neuronal development. However, the molecular details of this mechanism are unclear. To this end, here we produced Rufy3 knock-out (Rufy3-KO) mice to study the role of Rufy3 in more detail. Examining Rufy3-KO neurons, we found that Rufy3 is recruited via glycoprotein M6A to detergent-resistant membrane domains, which are biochemically similar to lipid rafts. We also clarified that Rufy3, as a component of a ternary complex, induces the assembly of Rap2 in the axonal growth cone, whereas in the absence of Rufy3, the accumulation of a Rac guanine nucleotide exchange factor, T-cell lymphoma invasion and metastasis 2 (Tiam2/STEF), is inhibited downstream of Rap2. We also found that Rufy3 regulates the cellular localization of Rap2 and Tiam2/STEF. Taken together, we conclude that Rufy3 is a physiological adapter for Rap2 and activates Tiam2/STEF in glycoprotein M6A-regulated neuronal polarity and axon growth.

DOI： 10.1074/jbc.M117.809541

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC NEUROSCIENCE  

Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. Toexaminehowsignaling protein complexes are clustered in rafts, we focused on the functions of glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing mouse neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a GPM6a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a, such as Rufy3, Rap2, and Tiam2/STEF, accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation in neuronal development. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of neuronal polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.

DOI： 10.1523/JNEUROSCI.3319-16.2017

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

Identification of proteins in the mammalian growth cone has the potential to advance our understanding of this critical regulator of neuronal growth and formation of neural circuit; however, to date, only one growth cone marker protein, GAP-43, has been reported. Here, we successfully used a proteomic approach to identify 945 proteins present in developing rat forebrain growth cones, including highly abundant, membrane-associated and actin-associated proteins. Almost 100 of the proteins appear to be highly enriched in the growth cone, as determined by quantitative immunostaining, and for 17 proteins, the results of RNAi suggest a role in axon growth. Most of the proteins we identified have not previously been implicated in axon growth and thus their identification presents a significant step forward, providing marker proteins and candidate neuronal growth-associated proteins.

DOI： 10.1073/pnas.0904092106

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

Endocytosis of synaptic vesicles follows exocytosis, and both processes require external Ca2+. However, it is not known whether Ca2+ influx through one route initiates both processes. At larval Drosophila neuromuscular junctions, we separately measured exocytosis and endocytosis using FM1-43. In a temperature-sensitive Ca2+ channel mutant, cacophony(TS2), exocytosis induced by high K+ decreased at nonpermissive temperatures, while endocytosis remained unchanged. In wild-type larvae, a spider toxin, PLTXII, preferentially inhibited exocytosis, whereas the Ca2+ channel blockers flunarizine and La3+ selectively depressed endocytosis. None of these blockers affected exocytosis or endocytosis induced by a Ca2+ ionophore. Evoked synaptic potentials were depressed regardless of stimulus frequency in cacophony(TS2) at nonpermissive temperatures and in wild-type by PLTXII, whereas flunarizine or La3+ gradually depressed synaptic potentials only during high-frequency stimulation, suggesting depletion of synaptic vesicles due to blockade of endocytosis. In shibire(ts1), a dynamin mutant, flunarizine or La3+ inhibited assembly of clathrin at the plasma membrane during stimulation without affecting dynamin function.

DOI： 10.1016/S0896-6273(03)00815-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The synaptic vesicle protein synaptotagmin I probably plays important roles in the synaptic vesicle cycle. However, the mechanisms of its action remain unclear. In this study, we have searched for cytoplasmic proteins that interact with synaptotagmin I. We found that the cytoskeletal protein tubulin directly and stoichiometrically bound to recombinant synaptotagmin L The binding depended on mm Ca2+, and 1 mol of tubulin dimer bound 2 mol of synaptotagmin I with half-maximal binding at 6.6 mum tubulin. The Ca2+ dependence mainly resulted from Ca2+ binding to the Ca2+ ligands of synaptotagmin I. The C-terminal region of beta-tubulin and both C2 domains of synaptotagmin I were involved in the binding. The YVK motif in the C2 domains of synaptotagmin I was essential for tubulin binding. Tubulin and synaptotagmin I were co-precipitated from the synaptosome extract with monoclonal antibodies to tubulin and SNAP-25 (synaptosome-associated protein of 25 kDa), indicating the presence of tubulin/synaptotagmin I complex and tubulin binding to synaptotagmin I in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Synaptotagmin I promoted tubulin polymerization and bundled microtubules in the presence of Ca2+. These results suggest that direct interaction between synaptotagmin I and tubulin provides a mechanism for attaching synaptic vesicles to microtubules in high Ca2+ concentrations.

DOI： 10.1074/jbc.M112080200

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記述言語：日本語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

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