SPINDLE DYNAMICS AND CELL-CYCLE REGULATION OF DYNEIN IN THE BUDDING YEAST, SACCHAROMYCES-CEREVISIAE

We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIG, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle, Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length, This stage is protracted in the dynein mutants and devoid of oscillatory motion, Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndc1), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex; with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.