| Project/Area Number |
01870106
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
医学一般
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| Research Institution | Osaka University |
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Principal Investigator |
SOBUE Kenji Osaka University Medical School, Department of Neurochemistry and Neuropharmacology, Professor, 医学部, 教授 (20112047)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Junya Osaka University Medical School, Department of Neurochemistry and Neuropharmacol, 医学部, 助手 (70217040)
INUI Makoto Osaka University Medical School, Department of Neurochemistry and Neuropharmacol, 医学部, 助手 (70223237)
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| Project Period (FY) |
1989 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥24,600,000 (Direct Cost: ¥24,600,000)
Fiscal Year 1991: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1990: ¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1989: ¥16,800,000 (Direct Cost: ¥16,800,000)
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| Keywords | Cytoskeletal Protein / Biochemical Analysis / Ultrastructural Analysis / Synapsin 1 / Calpactin I / Actin / Exocytosis / 細胞骨格 / 神経成長円錐 / カルスペクチン / αーアクチニン / src遺伝子産物 / 細胞骨格架橋蛋白質 / 急速凍結ディ-プエッチ法 / 免疫電顕法 |
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| Research Abstract |
To elucidate the molecular mechanisms by which the cell functions are regulated by the cytoskeletal and their related proteins, we developed a new analyzing method combining the biochemical techniques with ultrastructural analyses by electron microscopy. Using this method, we examined the molecular mechanisms of neurotransmitter release and hormone secretion.
In the process of neurotransmitter release, we exarffined the molecular structure of synapsin I and its molecular organization in presynaptic terminals using the low angle rotary shadowing technique, quick-freeze deep etch electron microscopy(QF-DE), immunoelectron microscopy. The high resolution provided by QFDE revealed that a single synapsin 1 cross-linked actin filaments and linked actin filaments with synaptic vesicles forming 30 nm short strands. Synapsin I also connected a microtubule to synaptic vesicles, forming 30 nm strands. These data suggest that synapsin I could be a main element of short bridge between actin filament
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s and synaptic vesicles, and between microtubules and synaptic vesicles, and between synaptic vesicles. Because phosphorylation of synapsin I by Ca^<2+>/calmodulin-dependentkinase detaches synapsin I from vesicles, it could release synaptic vesicles, and thus increase mobility of synaptic vesicles to. the presynaptic membrane upon depolarization-dependent Ca^<2+> flux into the presynaptic terminal.
In adrenal medulla, calpactin I was found to be a globular molecule with a diameter of I Inm on mica. When liposomes were aggregated by calpactin, QF-DE revealed a fine thin strand of 6.5 nm long cross-linking opposing membrane in addition to the globules on liposomes. In cultured chromaffin cells, similar crosslinking short strands(6-10 nm)were found between chromaffin vesicles and the plasma membrane after stimulation with acetylcholine. Plasma membranes also revealed numerous globular structure of 10 nm in diameter on their cytoplasmic surface. Immunoelectron microscopy showed that calpactin I was closely associated with the inner face of the plasma membrane and was especially conspicuous between plasma membrane and adjacent chromaffin vesicles. These data strongly suggest that calpactin I changes its conformation to cross-link vesicles and the plasma membrane after stimulation 6f cultured chromaffin cells and that it may play an important role in the binding of chromaffin vesicles to the plasma membrane during exocytosis.
Thus, the developed method presented here is quite powerful to elucidate a role of cytoskeletal and their related proteins in the processes of various cell functions. Less
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