Opposing motor activities are required for the organization of the mammalian mitotic spindle pole.
Opposing motor activities are required for the organization of the mammalian mitotic spindle pole.
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DOI：
10.1083/jcb.135.2.399
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发表时间：
1996-10
期刊：
影响因子：
--
通讯作者：
Compton DA
中科院分区：
文献类型：
--
作者：
Gaglio T;Saredi A;Bingham JB;Hasbani MJ;Gill SR;Schroer TA;Compton DA
我们使用体外和体内方法研究了Eg5（动力蛋白相关蛋白）、细胞质动力蛋白和动力蛋白在哺乳动物有丝分裂纺锤体极性末端的微管组织和NuMA（与有丝分裂装置相关的核蛋白）定位中的作用。通过无细胞系统中用于装配有丝分裂紫母细胞的免疫耗竭或将抗体微量注射到培养细胞中，对Eg5的功能进行扰动，导致有组织的星状微管阵列具有扩展的极性区域，其中微管的负端从含有NuMA的环状结构中发出。相反，细胞质dynein或dynactin的功能受到细胞游离系统的特异性免疫消耗或dynactin在培养细胞中的显性阴性亚基的表达的扰动，导致微管的组织完全缺乏，尽管NuMA蛋白与微管相关，但仍不能有效地浓缩。在有丝分裂aster组装过程中，这些蛋白从无细胞系统中同时免疫缺失表明，Eg5的正端定向活性可以拮抗细胞质动力蛋白的负端定向活性和微管组织形成形态极点过程中与NuMA相关的负端定向活性。综上所述，这些结果表明，微管负端的独特组织和NuMA在哺乳动物有丝分裂纺锤体极性末端的定位可以通过正负端定向马达的相反活动以中心体独立的方式完成。
We use both in vitro and in vivo approaches to examine the roles of Eg5 (kinesin-related protein), cytoplasmic dynein, and dynactin in the organization of the microtubules and the localization of NuMA (Nu-clear protein that associates with the Mitotic Apparatus) at the polar ends of the mammalian mitotic spindle. Perturbation of the function of Eg5 through either immunodepletion from a cell free system for assembly of mitotic asters or antibody microinjection into cultured cells leads to organized astral microtubule arrays with expanded polar regions in which the minus ends of the microtubules emanate from a ring-like structure that contains NuMA. Conversely, perturbation of the function of cytoplasmic dynein or dynactin through either specific immunodepletition from the cell free system or expression of a dominant negative subunit of dynactin in cultured cells results in the complete lack of organization of microtubules and the failure to efficiently concentrate the NuMA protein despite its association with the microtubules. Simultaneous immunodepletion of these proteins from the cell free system for mitotic aster assembly indicates that the plus end- directed activity of Eg5 antagonizes the minus end-directed activity of cytoplasmic dynein and a minus end-directed activity associated with NuMA during the organization of the microtubules into a morphologic pole. Taken together, these results demonstrate that the unique organization of the minus ends of microtubules and the localization of NuMA at the polar ends of the mammalian mitotic spindle can be accomplished in a centrosome-independent manner by the opposing activities of plus end- and minus end-directed motors.