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

We have used time-lapse digital- and video-enhanced differential inter ference contrast (DE-DIG, VE-DIC) microscopy to study the role of dyne in in spindle and nuclear dynamics in the yeast Saccharomyces cerevisi ae. The real-time analysis reveals six stages in the spindle cycle, An aphase 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 an d dynein mutants. In the absence of dynein the nucleus does not migrat e as close to the neck as in wild-type cells and initial spindle elong ation 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 reach es its maximal length, This stage is protracted in the dynein mutants and devoid of oscillatory motion, Thus dynein is required for rapid pe netration 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 b ud. Immunofluorescent localization of dynein-beta-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies a nd the cell cortex; with spindle pole body localization dependent on i ntact microtubules. A kinetic analysis of nuclear movement also reveal ed that cytokinesis is delayed until nuclear translocation is complete d, indicative of a surveillance pathway monitoring nuclear transit int o the bud.