Sun G H, Hirata A, Ohya Y, Anraku Y
Department of Biology, Faculty of Science, University of Tokyo, Japan.
J Cell Biol. 1992 Dec;119(6):1625-39. doi: 10.1083/jcb.119.6.1625.
Yeast calmodulin (CaM) is required for the progression of nuclear division (Ohya, Y. and Y. Anraku. 1989. Curr. Genet. 15:113-120), although the precise mechanism and physiological role of CaM in this process are unclear. In this paper we have characterized the phenotype caused by a temperature-sensitive lethal mutation (cmdl-101) in the yeast CaM. The cmdl-101 mutation expresses a carboxyl-terminal half of the yeast CaM (Met72-Cys147) under the control of an inducible GAL1 promoter. Incubation of the cmdl-101 cells at a nonpermissive temperature causes a severe defect in chromosome segregation. The rate of chromosome loss in the cmdl-101 mutant is higher than wild-type cell even at permissive temperature. The primary visible defect observed by immunofluorescence and electron microscopic analyses is that the organization of spindle microtubules is abnormal in the cmdl-101 cells grown at nonpermissive temperature. Majority of budded cells arrested at the high temperature contain only one spindle pole body (SPB), which forms monopolar spindle, whereas the budded cells of the same strain incubated at permissive temperature all contain two SPBs. Using the freeze-substituted fixation method, we found that the integrity of the nuclear morphology of the cmdl-101 mutant cell is significantly disturbed. The nucleus in wild-type cells is round with smooth contours of nuclear envelope. However, the nuclear envelope in the mutant cells appears to be very flexible and forms irregular projections and invaginations that are never seen in wild-type cells. The deformation of the nuclear becomes much more severe as the incubation at nonpermissive temperature continues. The single SPB frequently localizes on the projections or the invaginations of the nuclear envelope. These observations suggest that CaM is required for the functions of SPB and spindle, and the integrity of nucleus.
酵母钙调蛋白（CaM）是核分裂进程所必需的（Ohya，Y.和Y. Anraku. 1989. 《当代遗传学》15:113 - 120），尽管CaM在此过程中的精确机制和生理作用尚不清楚。在本文中，我们对酵母CaM中一个温度敏感型致死突变（cmdl - 101）所导致的表型进行了表征。cmdl - 101突变在可诱导的GAL1启动子控制下表达酵母CaM的羧基末端一半（Met72 - Cys147）。在非允许温度下培养cmdl - 101细胞会导致染色体分离出现严重缺陷。即使在允许温度下，cmdl - 101突变体中的染色体丢失率也高于野生型细胞。通过免疫荧光和电子显微镜分析观察到的主要明显缺陷是，在非允许温度下生长的cmdl - 101细胞中纺锤体微管的组织异常。大多数在高温下停滞的出芽细胞仅含有一个纺锤极体（SPB），形成单极纺锤体，而在允许温度下培养的同一菌株的出芽细胞都含有两个SPB。使用冷冻置换固定方法，我们发现cmdl - 101突变体细胞的核形态完整性受到显著干扰。野生型细胞中的细胞核呈圆形，核膜轮廓光滑。然而，突变体细胞中的核膜似乎非常柔韧，形成不规则的凸起和内陷，这在野生型细胞中从未见过。随着在非允许温度下培养的持续，核的变形变得更加严重。单个SPB经常定位在核膜的凸起或内陷处。这些观察结果表明，CaM是SPB和纺锤体功能以及细胞核完整性所必需的。
