Functional analyses of dynamin GTPase on early steps of endocytosis

2001 Fiscal Year Final Research Report Summary

• Project/Area Number: 10215202
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas (B)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Yamanashi Medical University
• Principal Investigator: BABA Takeshi Yamanashi Medical University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (90208710)
• Co-Investigator(Kenkyū-buntansha): UEDA Hideho Yamanashi Medical University, Faculty of Medicine, Research Associate, 医学部, 助手 (10242629) ; TERADA Nobuo Yamanashi Medical University, Faculty of Medicine, Research Associate, 医学部, 助手 (60293461) ; FUJII Yasuhisa Yamanashi Medical University, Faculty of Medicine, Research Associate, 医学部, 助手 (60126703) ; TAKEI Kohji University of Okayama Faculty of Medicine, Professor, 医学部, 教授 (40322226)
• Project Period (FY): 1998 – 2001
• Keywords: endocytosis / dynamin / clathrin-coated pit / clathrin-coated vesicle / synaptic vesicle / PIP2 / lysenin

Research Abstract

The following results were obtained in the term of current project. 1. Combined morphological and biochemical analyses of dynamin mutant cells : We have analyzed three dominant-negative mutants of dynamin, K44A, S45N, and T65F. All three mutants lacked binding capacity for both GTP and GDP, and biochemically indistinguishable. However, electron microscopic analyses revealed the each mutant showed the accumulation of unique morphological intermediates in transformed cells. These results indicated that these mutations in dynamin may inhibit the endocytosis through unknown dynamin's function not through well-known GTP/GDP-dependent fission steps. 2. Ultrastructural study of lysenin : Lysenin is a sphingomyelin-specific binding protein which induce hemolysis. We have identified the unique hexagonal oligomer on sphingomyelin-containing liposomes. The similar structures were found on the surface of lysenin-treated erythrocytes. These results indicated that lysenin induced hemolysis by pore-forming oligomer on the cell surface. 3. Analyses of in vitro reconstitution of synaptic vesicle formation : We have succeeded in reconstitution of the synaptic vesicle formation by incubating liposomes with brain cytosol, ATP and GTPγS. We then analyzed the effect of various lipid composion of liposomes on the rate of vesicle formation. We found that PIP2 accumulate the formation of vesicles. The accumulation of vesicle formation persisted after rapid degradation of PIP2. These results indicated that PIP2 may recruited various coat proteins to the liposomes required for vesicle budding.

Research Products

• [Publications] Lamaze C, Dujeancourt A, Baba T, Lo CG, Benmerah A, Dautry-Varsat A: "Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway"Molecular Cell. 7. 661-671 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Ueda H, Ueda K, Baba T, Ohno S: "δ-and γ-Sarcoglycan localization in the sarcoplasmic reticulum of skeletal muscle"J Histochem Cytochem. 49. 529-538 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Serizawa Y, Ueda H, Baba T, Ueno A, Takeda M, Ohno S: "Intravesicular Fas localization in epithelial cells of castrated rat prostate glands"Histol Histopathol. 16. 453-462 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Baba T, Rauch C, Xue M, Terada N, Fujii Y, Ueda H, Takayama I, Ohno S, Farge E, Sato SB: "Clathrin-dependent and clathrin-independent endocytosis are differentially sensitive to insertion of poly (ethylene glycol)-derivatized cholesterol in the plasma membrane"Traffic. 2. 501-512 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Damke H, Binns DD, Ueda H, Schmid SL, Baba T: "Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages"Mol Biol Cell. 12. 2578-2589 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kinuta M, Yamada H, Abe T, Watanabe M, Li SA, Kamitani A, Yasuda T, Matsukawa T, Kumon H, Takei K: "Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol"Proc Natl Acad Sci USA. 99. 2842-2847 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Lamaze C, Dujeancourt A, Baba T, Lo CG. Benmerah A, Dautry-Varsat A.: "Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway"Molecular Cell. 7. 661-671 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Ueda H, Ueda K, Baba T, Ohno S, d- and g-: "Sarcoglycan localization in the sarcoplasmic reticulum of skeletal muscle"J Histochem Cytochem.. 49. 529-538 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Serizawa Y, Ueda H, Baba T, Ueno A, Takeda M, Ohno S.: "Intravesicular Fas localization in epithelial cells of castrated rat prostate glands"Histol Histopathol. 16. 453-462 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Baba T, Rauch C, Xue M, Terada N, Fujii Y, Ueda H, Takayama I, Ohno S, Farge E, Sato SB.: "Clathrin-dependent and clathrin-independent endocytosis are differentially sensitive to insertion of poly (ethylene glycol)-derivatized cholesterol in the plasma membrane"Traffic. 2. 501-512 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Damke H, Binns DD, Ueda H, Schmid SL, Baba T.: "Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages"Mol Biol Cell. 12. 2573-2589 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kinuta M, Yamada H, Abe T, Watanabe M, Li SA, Kamitani A, Yasuda T, Matsukawa T, Kumon H, Takei K.: "Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol"Proc Natl Acad Sci USA. 99. 2842-2847 (2002)
  • Description: 「研究成果報告書概要(欧文)」より