3次元免疫電顕法を用いた小胞輸送過程の高解像解析

• Project/Area Number: 15079208
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Nagahama Institute of Bio-Science and Technology
• Principal Investigator: 山本 章嗣 Nagahama Institute of Bio-Science and Technology, バイオサイエンス学部, 教授 (30174775)
• Co-Investigator(Kenkyū-buntansha): 奈良 篤樹 長浜バイオ大学, バイオサイエンス学部, 講師 (60387959)
• Project Period (FY): 2003 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥106,400,000 (Direct Cost: ¥106,400,000); Fiscal Year 2007: ¥19,600,000 (Direct Cost: ¥19,600,000); Fiscal Year 2006: ¥19,600,000 (Direct Cost: ¥19,600,000); Fiscal Year 2005: ¥19,600,000 (Direct Cost: ¥19,600,000); Fiscal Year 2004: ¥23,800,000 (Direct Cost: ¥23,800,000); Fiscal Year 2003: ¥23,800,000 (Direct Cost: ¥23,800,000)
• Keywords: 3次元免疫電子顕微鏡法 / 銀増感法 / 小胞体 / ゴルジ体 / エンドソーム / オートファジー / 小胞輸送 / メンブレントラフィック / マルチベシキュラーボディー / SKD1 / 電子顕微鏡トモグラフィー / Hsp47 / シナプス / NMDA受容体 / ミクロソーム型アルデヒド脱水素酵素 / COPII / 3次元 / 免疫電顕法 / シスゴルジ網

Research Abstract

本研究は、免疫電顕法と電顕トモグラフィーなどの3D技術とを組み合わせ、オルガネラ間の小胞輸送過程を高解像力で立体観察することにより、複雑な形態を持つ輸送装置(小胞体-ゴルジ体、分泌顆粒、エンドソーム、オートファゴソームなど)における小胞輸送蛋白質分子の正確な局在と挙動を明らかにし、小胞輸送機構の全体像を明らかにすることを目的としている。本年度は、電顕トモグラフィーを用いた小胞輸送系の3次元解析を本格的に推進した。
1.オートファゴソーム形成の解析.オートファゴソームの形成過程を電子線トモグラフィーによって3次元的に解析し、隔離膜と他のオルガネラの位置関係を明らかにした。その結果、隔離膜と密着して小胞体が存在し、その伸張に何らかの作用を及ぼしていることが示唆された
2.マルチベシキュラーボディー(MVB)における内部小胞の形成機構。RNA干渉法により、エンドソーム系に働くAAA型-ATPase SKD1の発現を抑制すると内部小胞形成が阻害され、エンドソームからリソソームへの輸送も阻害されることを明らかにした。現在、電子線トモグラフィーによる解析を進めている。

Research Products

• [Journal Article] Pivotal role of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in apoptosis and autophagy. (2008)
  • Author(s): Fujiwara K
  • Journal Title: J. Biol. Chem 283 Pages: 388-397
  • Peer Reviewed
• [Journal Article] Identification of a vesicular nucleotide transporter. (2008)
  • Author(s): Sawada K
  • Journal Title: Proc Natl Acad Sci USA. 105 Pages: 5683-5686
  • Peer Reviewed
• [Journal Article] Bap31 Is an Itinerant Protein that Moves between the Peripheral ER and a Juxtanuclear Compartment Related to ER-associated Degradation. (2008)
  • Author(s): Wakana Y
  • Journal Title: Mol. Biol. Cell (In press)
  • Peer Reviewed
• [Journal Article] Type I collagen in Hsp47-null cells is aggregated in ER and deficient in N-propeptide processing and fibrillogenesis (2006)
  • Author(s): Ishida Y
  • Journal Title: Mol.Biol.Cell 17 Pages: 2346-2355
• [Journal Article] Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice (2006)
  • Author(s): Hara T
  • Journal Title: Nature 441 Pages: 885-889
• [Journal Article] Analysis of knock-out mice to determine the role of HPC-1/syntaxin 1A in expressing synaptic plasticity. (2006)
  • Author(s): Fujiwara T
  • Journal Title: J.Neurosci 26 Pages: 5767-5776
• [Journal Article] Deubiquitinating enzyme UBPY regulates the level of protein ubiquitination on endosomes. (2006)
  • Author(s): Mizuno E
  • Journal Title: Traffic 7 Pages: 1017-1031
• [Journal Article] Secretion of L-glutamate from osteoclasts through transcytosis. (2006)
  • Author(s): Morimoto R
  • Journal Title: EMBO J. 25 Pages: 4175-4186
• [Journal Article] The a3 isoform of V-ATPase regulates insulin secretion from pancreatic {beta}-cells. (2006)
  • Author(s): Sun-Wada D-H
  • Journal Title: J.Cell Sci. 119 Pages: 4531-4540
• [Journal Article] Type I collagen in Hsp47-null cells is aggregated in ER and deficient in N-propeptide processing and fibrillogenesis. (2006)
  • Author(s): Ishida Y
  • Journal Title: Mol.Biol.Cell in press
• [Journal Article] Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice (2006)
  • Author(s): Hara T
  • Journal Title: Nature (in press)
• [Journal Article] ABCA5 resides in lysosomes and ABCA5 knockout mice develop lysosomal desease-like symptonms. (2005)
  • Author(s): Kubo Y
  • Journal Title: Mol.Cell.Biol. 25 Pages: 4138-4149
• [Journal Article] Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain. (2005)
  • Author(s): Abe T
  • Journal Title: Eur.J.Neurosci. 22 Pages: 1445-1454
• [Journal Article] NSF/SNAPs and p97/p47/VCIP135 are sequentially required for cell cycle-dependent reformation of the ER network. (2005)
  • Author(s): Kano F
  • Journal Title: Genes Cells 10 Pages: 989-999
• [Journal Article] Vesicular storage and secretion of L-glutamate from glucagon-like peptide 1-secreting clonal intestinal L cells. (2005)
  • Author(s): Uehara S
  • Journal Title: J.Neurochem. 96 Pages: 550-560
• [Journal Article] p125 is localized in endoplasmic reticulum exit sites and involved in their organization (2005)
  • Author(s): Shimoi W
  • Journal Title: J Biol Chem. (印刷中)
• [Journal Article] Rabphilin and Noc2 are recruited to dense-core vesicles through specific interaction with Rab27A in PC12 cells (2004)
  • Author(s): Fukuda M
  • Journal Title: J.Biol.Chem 279 Pages: 13065-13075
• [Journal Article] In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker (2004)
  • Author(s): Mizushima N
  • Journal Title: Mol Biol Cell. 15 Pages: 1101-1111
• [Journal Article] LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation (2004)
  • Author(s): Kabeya Y
  • Journal Title: J Cell Sci. 117 Pages: 2805-2812
• [Journal Article] A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor (2004)
  • Author(s): Morino C
  • Journal Title: Exp.Cell Res. 297 Pages: 380-391
• [Journal Article] Involvement of BNIP1 in apoptosis and endoplasmic reticulum membrane fusion. (2004)
  • Author(s): Nakajima K
  • Journal Title: EMBO J. 23 Pages: 3216-3226
• [Presentation] The AAA-type ATPase SKD1 is required for inward vesiculation of endosomes. (2007)
  • Author(s): Nara A
  • Organizer: International symposium on membrane traffic.
  • Place of Presentation: 淡路夢舞台国際会議場
  • Year and Date: 2007-11-27
• [Book] Methods in Enzymology Vol.403, GTPases regulating membrane targeting and fusion. (2005)
  • Author(s): Fukuda M, Yamamoto A, 他多数(ed.Balch WE, Der CJ, Hall A)
  • Total Pages: 863
  • Publisher: Academic Press