Abstract. We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle. Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division. These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC). In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of lO-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed. Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth. In contrast, nocodazole profoundly affected the behavior of the nucleus. Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked. Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together. Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there. Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal ceils. Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division.T HE budding yeast Saccharomyces cerevisiae offers an opportunity to study the mechanisms of cellular morphogenesis in an experimentally tractable eukaryotic system. In the course of the yeast cell cycle, a series of morphogenetic events results in the establishment of cell polarity, the appropriate positioning of organelles, and the localized incorporation of new cell-surface material. These events include the following (4,7,49,50,64). (a) Selection of a nonrandom budding site. Early in the cell cycle, a single bud emerges near a pole of the ellipsoidal cell; the choice of poles is determined by the mating type of the cell. (b) Organization of the budding site. Just before bud emergence, a small ring of chitin is formed in the largely nonchitinous cell wall; the bud then emerges within the confines of this chitin ring. At about the same time, there appears a ring of filaments of ~10 nm diameter, of unknown biochemical nature. These filaments encircle the mother-bud neck in close proximity to the plasma membrane; they remain in place as the bud grows, then disappear just before cytokinesis. however, new cell-surface material is incorporated almost exclusively in the growing bud, while the mother cell rema...

Haploid sporidia of the dimorphic phytopathogen Ustilago maydis (D.C.) Corda reproduce by budding once each cell cycle. Homogeneous log-phase sporidial cultures were generated by serial passage in liquid culture, and the growth characteristics and percentages of budded cells were determined for the cultures. The characteristics of budding were determined for individual cells by light and electron microscopy. Buds emerged only from the poles of mother cells, and cells could select either a previously used bud site, or a new bud site, each cycle. Time-lapse photomicroscopy indicated that, on solid medium, the first two buds emerged from new cells at a point distal to the site of attachment to the mother cell. In subsequent cell cycles, the buds tended to emerge from alternate poles of the mother cell. The cells used multiple bud sites at each pole. In addition, transmission and scanning electron microscopy revealed a series of annulations (bud scars) at the base of some buds, indicating that cells also used the same budding site many times. This versatility in selecting bud sites indicates that budding likely depends on complex regulatory pathways for determining cellular polarity. Key words: Ustilago maydis, bud, polarity, cell cycle, morphogenesis, yeast.

No abstract

The microtubule inhibitor nocodazole (methyl-5-[2-(thienylcarbonyl)-1H-benzimidazol-2-yl]-carbamate) prevented nuclear migration and nuclear division in yeasts and developing multicellular forms of the polymorphic fungus Wangiella dermatitidis. It did not prevent yeast bud formation during at least two or three budding cycles, and caused yeasts to accumulate as premitotic forms with one to three buds. The effects of the drug suggested that at least three control pathways were involved in the yeast cell cycle; that the nocodazole block point was separate from the execution points of two temperature-sensitive mutations which lead to multicellularity; and that microtubules were controlling neither the yeast budding process nor the development of multicellular forms.

CDC3, CDC25 and CDC42 were localized to chromosome XII by hybridizing the cloned genes to Southern blots of chromosomes separated by orthogonal-field-alternation gel electrophoresis. Meiotic tetrad analyses further localized these genes to the region distal to the RDN1 locus on the right arm of the chromosome. The STE11 gene, which had previously been mapped to chromosome XII (Chaleff and Tatchell, 1985), was found to be tightly linked to ILV5. The data suggest a map order of CEN12-RDN1-CDC42-(CDC25-CDC3)-(ILV5- STE11)-URA4. Certain oddities of the data set raise the possibility that there may be constraints on the patterns of recombination in this region of chromosome XII.