| Project/Area Number |
08457001
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General anatomy (including Histology/Embryology)
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| Research Institution | Gunma University |
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Principal Investigator |
FUJIMOTO Toyoshi Gunma University School of Medicine, Department of Anatomy and Cell Biology, Professor, 医学部, 教授 (50115929)
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| Co-Investigator(Kenkyū-buntansha) |
KOGO Hiroshi ibid, 医学部, 助手 (20282387)
AOKI Takeo ibid, 医学部, 助手 (70150919)
HAGIWARA Haruo Gunma University School of Medicine, Department of Anatomy and Cell Biology, Pro, 医学部, 講師 (80189464)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥7,700,000 (Direct Cost: ¥7,700,000)
Fiscal Year 1997: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1996: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Keywords | Caveola / Plasma membrane / Calcium / Gi2a / Cholesterol / Freeze fracture / Immunoelectron microscopy |
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| Research Abstract |
1) A simple quick freezing method to observe the plasma membrane from monolayr cultured cells was invented. The metal sandwich method is simple, does not require any expensive equipment, and provides a large fracture plane of the plasma membrane for subsequent histochemical manipulation.
2) rheta-Toxin (perfringolysin O), a cholesterol-binding toxin, was partially proteolysed and biotinylated (BCrheta) and used as a cytochemical probe. Because the plasma membrane keeps integrity even after the binding of BCrheta, the probe can be used not only for cytochemical labeling of fixed cells, but for pursuing the behavior of cross-linked cholesterol molecules in living cells. By using this new probe, it was revealed that cross-linked cholesterol in the plasma membrane are sequestered to caveolae.
3) By immunoelectron microscopy, whether Gi2a is constitutively concentrated in caveolae was critically assessed. First, behavior of caveolin and Gi2a in density-equilibrium ultracentrifugation was reexamined. By collecting fractions efficiently, caveolin and Gi2a were found to distribute differently. Secondly, by novel immunocytochemical methods it was found that the labeling density of Gi2a was 2.29 times more in caveolae than in the non-caveolar plasma membrane. The results indicate that concentration of Gi2a in caveolae is less than deduced from most biochemical studies.
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