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Microtubule-Nucleation Sites on Nuclei in Higher Plant Cells

高等植物细胞核上的微管成核位点

基本信息

批准号：
02640521
负责人：
MIZUNO Koichi
金额：
$ 1.02万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The nucleation and the elongation of microtubules from isolated nuclei of higher plant cells were investigated. Isolated intact nuclei failed to nucleate microtubules from their surface when they were incubated with purified tubulin from plant or animal sources. However, frozen-thawed nuclei or nuclear particles obtained by gentle homogenization of nuclei nucleated microtubules and nucleated microtubules elongated radially from the surface of nuclei or from the nuclear materials. Microtubules radiating from the nuclear particles were extremely shorter than those radiating from frozen-thawed nuclei. The washing of the nuclear particles diminished the ability of the particles to nucleate microtubules. The ability of the washed nuclear particles to nucleate microtubules was restored by the addition of the soluble fraction of a nuclear homogenate. The staining with a monoclonal antibody specific for plant tubulin of the complexes of radiating microtubules-prepared by successive polymerization of animal tubulin and plant tubulin revealed that microtubules in the complex incorporated tubulin at their proximal ends. This result indicates that the mode of incorporation of tubulin in frozen-thawed nuclei or the nuclear particles is different from that in pericentriolar bodies in animal cells. The partially purified microtubulenucleation particles contained a peptide having an apparent molecular weight of 50 kd as a main constitution, which cross-reacted specifically with monoclonal antibody against alpha-tubulin. 2-D PAGE analysis showed the 50 kd peptide to be separated into two peptides, one of which was assumed to be phosphorylated. Therefore, the essential factor in the soluble fraction is possible to be estimated to be protein kinase or phosphoprotein phosphatase. The action of such essential factor may enable the 50 kd peptide to change from inactive form to active form.

本文研究了高等植物细胞核中微管的成核和伸长。当分离的完整细胞核与来自植物或动物来源的纯化微管蛋白一起孵育时，它们未能从其表面使微管成核。然而，通过温和均质化核获得的冻融核或核颗粒使有核微管和有核微管从核表面或从核材料径向伸长。从核颗粒辐射的微管比从冻融核辐射的微管短得多。核颗粒的洗涤降低了颗粒使微管成核的能力。通过添加核匀浆的可溶性部分，恢复洗涤的核颗粒使微管成核的能力。用对植物微管蛋白特异的单克隆抗体对通过动物微管蛋白和植物微管蛋白的连续聚合制备的辐射微管复合物进行染色，发现复合物中的微管在其近端掺入微管蛋白。这一结果表明，微管蛋白在冻融细胞核或核颗粒中的掺入方式与在动物细胞的中心粒周围体中的掺入方式不同。部分纯化的微管成核颗粒含有表观分子量为50 kd的肽作为主要成分，其与抗α-微管蛋白的单克隆抗体特异性交叉反应。2-D PAGE分析表明，50 kd的肽被分离成两个肽，其中一个被认为是磷酸化的。因此，可溶性组分中的必需因子可能是蛋白激酶或磷蛋白磷酸酶。这种必需因子的作用可使50 kd肽从无活性形式转变为活性形式。