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Studies on the ability of the centrosome to nucleate microtubules

中心体成核微管能力的研究

基本信息

批准号：
06454680
负责人：
SAKAI Hikoichi
金额：
$ 4.48万
依托单位：
Japan Women's University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454680/
关键词：
centrosome microtubules tubulin G protein mitotic apparatus aster formation mitosis sea urchin egg 微小管形成中心分裂装置ウニ卵 G蛋白質分裂中心

项目摘要

(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.

(1)从海胆卵中分离出有丝分裂器（MAs），用冷冻和离心法分离中心体。加入纯化的微管蛋白后，形成大量的星形细胞，GTP、GTP-gammaS或GMP-PNP均能促进中心体的星形细胞的重建。与此相反，GDP被发现抑制中心体的活性，微管成核。(2)用亲和层析法分离得到的45 K/30 K复合物，推测了G蛋白中GTP与GDP相互转化的调节机制。发现该复合物增强G蛋白对GTP的亲和力。(3)在精子轴丝中加入G蛋白后，轴丝的整个表面被抗G蛋白抗体染色，表明α-微管蛋白的C-末端从微管壁突出。当G蛋白级分与精子轴丝或微管蛋白二聚体级分孵育并与EDAC（零长度交联剂）交联，随后进行SDS-PAGE处理时，发现α-微管蛋白优先与G蛋白交联。纺锤体微管的极性是负端朝向近端。因此，G蛋白将在中心体与α-微管蛋白结合。(4)用乳胶珠（聚苯乙烯珠）、G蛋白和微管蛋白建立了一个新的中心体模型系统。在GTP存在下，乳胶珠用G蛋白级分包被，然后加入脑微管蛋白。乳胶珠的聚集有利于紫苑的形成。