Gaglio T, Saredi A, Bingham J B, Hasbani M J, Gill S R, Schroer T A, Compton D A
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
J Cell Biol. 1996 Oct;135(2):399-414. doi: 10.1083/jcb.135.2.399.
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.
我们采用体外和体内两种方法，来研究驱动蛋白相关蛋白Eg5、胞质动力蛋白和动力蛋白激活蛋白在微管组织以及核有丝分裂器蛋白（NuMA，一种与有丝分裂器相关的核蛋白）于哺乳动物有丝分裂纺锤体两极定位中的作用。通过从用于组装有丝分裂星状体的无细胞体系中进行免疫去除，或者向培养细胞中显微注射抗体，来干扰Eg5的功能，会导致有组织的星状微管阵列出现，其极性区域扩大，微管的负端从含有NuMA的环状结构发出。相反，通过从无细胞体系中进行特异性免疫去除，或者在培养细胞中表达动力蛋白激活蛋白的显性负亚基，来干扰胞质动力蛋白或动力蛋白激活蛋白的功能，会导致微管完全缺乏组织性，并且尽管NuMA蛋白与微管相关联，但却无法有效地集中。从用于有丝分裂星状体组装的无细胞体系中同时免疫去除这些蛋白，表明在将微管组织成形态学上的极的过程中，Eg5的正端定向活性拮抗了胞质动力蛋白的负端定向活性以及与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以及NuMA在哺乳动物有丝分裂纺锤体两极的定位，可以通过正端和负端定向马达的相反活性，以一种不依赖中心体的方式实现。
