1996 Fiscal Year Final Research Report Summary
Studies on the ability of the centrosome to nucleate microtubules
| Project/Area Number |
06454680
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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 |
Cell biology
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| Research Institution | Japan Women's University |
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Principal Investigator |
SAKAI Hikoichi Japan Wamen's University, Faculty of Science, Department of Chemical and Biological Sciences, Professor, 理学部, 教授 (80011477)
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| Co-Investigator(Kenkyū-buntansha) |
TORIYAMA Masaru Shizuoka University, Department of Applied Biological Chemistry, Associate Profe, 農学部, 助教授 (60202206)
OSUMI Masako Japan Wamen's University, Faculty of Science, Department of Chemical and Biologi, 理学部, 教授 (60060646)
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| Project Period (FY) |
1994 – 1996
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| Keywords | centrosome / microtubules / tubulin / G protein / mitotic apparatus / aster formation / mitosis / sea urchin egg |
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| Research Abstract |
(1) The mitotic apparatuses (MAs) were isolated from sea urchin eggs and the centrosomes were separated by chilling and homogenizing the MA suspension. Upon addition of tubulin purified from porcine brain, many asters were formed, GTP,GTP-gammaS,or GMP-PNP promoted the reconstruction of asters initiated from the isolated centrosomes. In contrast, GDP was found to inhibit the activity of the centrosome to nucleate microtubules.
(2) The regulatory mechanism of bound GTP*GDP interconversion in the G protein was suggested by the action of 45K/30K complex which was newly isolated by affinity chromatograpy using fixed anti-G protein antibody conjugated with the G protein. The complex was found to enhance the affinity of the G protein to GTP.
(3) Upon addition of the G protein to sperm axonemes, the entire surface of the axonemes was stained with anti-G protein antibody, suggesting that the C-terminal of alpha-tubulin is protruded from the microtubule wall. When the G protein fraction was incubated with the spem axoneme or tubulin dimer fraction and cross-linked with EDAC (a zero lenghth cross linker) followed by processing for SDS-PAGE,alpha-tubulin was found to be preferentially cross-linked with the G protein. The polarity of the spindle microtubules is such that the minus end is toward proximal. Therefore, the G protein will bind to alpha-tubulin at the centrosome.
(4) A model system fo the centrosome has newly established using latex beads (polystirene beads), G protein and tubulin. In the presence of GTP,latex beads were coated with the G protein fraction, followed by additon of brain tubulin. An aggregate of latex beads favored the formation of asters.
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