McNally F J, Okawa K, Iwamatsu A, Vale R D
Section of Molecular and Cellular Biology, University of California at Davis 95616, USA.
J Cell Sci. 1996 Mar;109 ( Pt 3):561-7. doi: 10.1242/jcs.109.3.561.
The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. Here we determine the subcellular localization of katanin by immunofluorescence as a preliminary step in determining whether katanin mediates the poleward flux of tubulin. We find that katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin's localization is different from that of gamma-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and gamma-tubulin reveals that katanin is localized in a region surrounding the gamma-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux.
有丝分裂纺锤体的组装和功能涉及微管动态特性的特定变化。其中一种变化导致微管蛋白向极运动，即纺锤体微管在其与动粒相连的正端聚合，而在其与中心体相连的负端缩短。由于游离的微管负端在体内不会解聚，微管蛋白的向极运动表明纺锤体微管在其中心体附着点或附近被主动拆卸。微管切割ATP酶——katanin，具有主动切割和拆卸微管的能力，因此是介导微管蛋白向极运动的蛋白质角色的候选者。在这里，我们通过免疫荧光确定katanin的亚细胞定位，作为确定katanin是否介导微管蛋白向极运动的初步步骤。我们发现，在整个细胞周期中，katanin都高度集中在中心体。katanin的定位与γ-微管蛋白不同，因为微管需要维持katanin在中心体的定位。对katanin和γ-微管蛋白定位的直接比较表明，在去垢剂提取的有丝分裂纺锤体中，katanin定位在含有γ-微管蛋白的中心粒周围区域。katanin在中心体的定位与katanin在向极运动过程中介导微管负端拆卸的假设一致。
