We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, 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 (ndcl), 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.

Dynein motor isoforms have been implicated as potential kinetochore-associated motors that power chromosome-to-pole movements during mitosis. The recent identification and sequence determination of genes encoding dynein isoforms has now permitted the in vivo analysis of dynein function in mitosis. In this report we describe the identification and mutational analysis of the gene, DHC1, encoding a dynein heavy chain isoform in Saccharomyces cerevisiae. Sequence analysis of a 9-kb genomic fragment of the DHC1 gene predicts a polypeptide highly homologous to dynein sequences characterized from sea urchin, Dictyostelium, Drosophila, and rat. Mutations in the yeast dynein gene disrupt the normal movement of the spindle into budding daughter cells but have no apparent effect on spindle assembly, spindle elongation, or chromosome segregation. Our results suggest that, in yeast, a dynein microtubule motor protein has a nonessential role in spindle assembly and chromosome movement but is involved in establishing the proper spindle orientation during cell division.

Cse4 is the budding yeast homologue of CENP-A, a modified histone H3 that specifies the base of kinetochores in all eukaryotes. Budding yeast is unique in having only one kinetochore microtubule attachment site per centromere. The centromere is specified by CEN DNA, a sequence-specific binding complex (CBF3), and a Cse4-containing nucleosome. Here we compare the ratio of kinetochore proximal Cse4-GFP fluorescence at anaphase to several standards including purified EGFP molecules in vitro to generate a calibration curve for the copy number of GFP-fusion proteins. Our results yield a mean of ∼5 Cse4s, ∼3 inner kinetochore CBF3 complexes, and ∼20 outer kinetochore Ndc80 complexes. Our calibrated measurements increase 2.5–3-fold protein copy numbers at eukaryotic kinetochores based on previous ratio measurements assuming two Cse4s per budding yeast kinetochore. All approximately five Cse4s may be associated with the CEN nucleosome, but we show that a mean of three Cse4s could be located within flanking nucleosomes at random sites that differ between chromosomes.