MUTATIONS IN YEAST CALMODULIN CAUSE DEFECTS IN SPINDLE POLE BODY FUNCTIONS AND NUCLEAR INTEGRITY

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 (cmd1-101) in the yeast CaM. The cmd1-101 mutation expresses a carboxyl-terminal half of the yeast CaM (Met72-Cys147) under the control of an inducible GAL1 promoter. Incubation of the cmd1-101 cells at a nonpermissive temperature causes a severe defect in chromosome segregation. The rate of chromosome loss in the cmd1-101 mutant is higher than wild-type cell even at permissive temperature. The primary visible defect observed by inununofluorescence and electron microscopic analyses is that the organization of spindle microtubules is abnormal in the cmd1-101 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 cmd1-101 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.