Beach D L, Thibodeaux J, Maddox P, Yeh E, Bloom K
Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA.
Curr Biol. 2000 Nov 30;10(23):1497-506. doi: 10.1016/s0960-9822(00)00837-x.
Two genetic 'pathways' contribute to the fidelity of nuclear segregation during the process of budding in the yeast Saccharomyces cerevisiae. An early pathway, involving Kar9p and other proteins, orients the mitotic spindle along the mother-bud axis. Upon the onset of anaphase, cytoplasmic dynein provides the motive force for nuclear movement into the bud. Loss of either pathway results in nuclear-migration defects; loss of both is lethal. Here, to visualize the functional steps leading to correct spindle orientation along the mother-bud axis, we imaged live yeast cells expressing Kar9p and dynein as green fluorescent protein fusions.
Transport of Kar9p into the bud was found to require the myosin Myo2p. Kar9p interacted with microtubules through the microtubule-binding protein Bim1p and facilitated microtubule penetration into the bud. Once microtubules entered the bud, Kar9p provided a platform for microtubule capture at the bud cortex. Kar9p was also observed at sites of microtubule shortening in the bud, suggesting that Kar9p couples microtubule shortening to nuclear migration.
Thus, Kar9p provides a key link between the actin cytoskeleton and microtubules early in the cell cycle. A cooperative mechanism between Kar9p and Myo2p facilitates the pre-anaphase orientation of the spindle. Later, Kar9p couples microtubule disassembly with nuclear migration.
在酿酒酵母出芽过程中，两条遗传“途径”有助于核分离的准确性。一条早期途径，涉及Kar9p和其他蛋白质，使有丝分裂纺锤体沿母细胞-芽轴定向。在后期开始时，细胞质动力蛋白为核向芽内移动提供动力。任何一条途径的缺失都会导致核迁移缺陷；两条途径都缺失则是致命的。在这里，为了可视化导致纺锤体沿母细胞-芽轴正确定向的功能步骤，我们对表达Kar9p和作为绿色荧光蛋白融合体的动力蛋白的活酵母细胞进行了成像。
发现Kar9p向芽内的运输需要肌球蛋白Myo2p。Kar9p通过微管结合蛋白Bim1p与微管相互作用，并促进微管向芽内穿透。一旦微管进入芽内，Kar9p就在芽皮质提供了一个微管捕获平台。在芽内微管缩短的部位也观察到了Kar9p，这表明Kar9p将微管缩短与核迁移联系起来。
因此，Kar9p在细胞周期早期在肌动蛋白细胞骨架和微管之间提供了关键联系。Kar9p和Myo2p之间的协同机制促进了纺锤体在后期前的定向。后来，Kar9p将微管解聚与核迁移联系起来。
