Abstract. We have used in vitro mutagenesis and gene replacement to construct five new cold-sensitive mutations in TUB2, the sole gene encoding 13-tubulin in the yeast Saccharomyces cerevisiae. These and one previously isolated tub2 mutant display diverse phenotypes that have allowed us to define the functions of yeast microtubules in vivo. At the restrictive temperature, all of the tub2 mutations inhibit chromosome segregation and block the mitotic cell cycle. However, different microtubule arrays are present in these arrested cells depending on the tub2 allele. One mutant (tub2-401) contains no detectable microtubules, two (tub2-403 and tub2-405) contain greatly diminished levels of both nuclear and cytoplasmic microtubules, one (tub2-104) contains predominantly nuclear microtubules, one (tub2-402) contains predominantly cytoplasmic microtubules, and one (tub2-404) contains prominent nuclear and cytoplasmic microtubule arrays.Using these mutants we demonstrate here that cytoplasmic microtubules are necessary for nuclear migration during the mitotic celt cycle and for nuclear migration and fusion during conjugation; only those mutants that possess cytoplasmic microtubules are able to perform these functions. We also show that microtubules are not required for secretory vesicle transport in yeast; bud growth and invertase secretion occur in cells which contain no microtubules.M ICROTUBULES are found in an array of morphologically distinct structures in eukaryotes and have been implicated in a wide range of motile processes, including chromosome separation, intracellular transport of organelles, and maintenance of cell shape (reviewed in McIntosh, 1982;Roberts and Hyams, 1979). The precise mechanisms by which cells regulate the temporal and spatial assembly of microtubules, establish interactions between microtubules and other cell structures, and generate the force required for microtubule functions are not known. The key to understanding these processes is likely to reside in both the tubulin subunits that assemble to form microtubules and the nontubulin "associated" proteins that influence and mediate microtubule function. Much is known about the assembly properties of tubulin in vitro (reviewed in Dustin, 1984;Kirschner and Mitchison, 1986;McKiethan and Rosenbaum, 1984;Purich and Kristofferson, 1984) and several proteins have been identified which promote tubulin polymerization in vitro (reviewed in Olmsted, 1986). However, the relationship between these in vitro properties and the in vivo function of microtubules is largely uncertain. For this reason, we have chosen a genetic system that allows us to associate molecular characterizations with cellular functions.The yeast Saccharomyces cerevisiae is a particularly trac-T. C. Huffaker's present address is Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853. table organism for such studies. It contains relatively simple microtubule arrays (Byers, 1981;Kilmartin and Adams, 1984;Peterson and Ris, 1976) that participate ...

Abstract. In the yeast Saccharomyces cerevisiae, before the onset of anaphase, the spindle apparatus is always positioned with one spindle pole at, or through, the neck between the mother cell and the growing bud. This spindle orientation enables proper chromosome segregation to occur during anaphase, allowing one replicated genome to be segregated into the bud and the other to remain in the mother cell. In this study, we synchronized a population of cells before the onset of anaphase such that >90% of the cells in the population had spindles with the correct orientation, and then disrupted specific cytoskeletal elements using temperature-sensitive mutations. Disruption of either the astral microtubules or actin function resulted in improper spindle orientation in "~40-50% of the cells. When cells with disrupted astral microtubules or actin function entered into anaphase, there was a 100-200-fold increase in the frequency of binucleated cell bodies. Thus, the maintenance of proper spindle orientation by these cytoskeletal elements was essential for proper chromosome segregation. These data are consistent with the model that proper spindle orientation is maintained by directly or indirectly tethering the astral microtubules to the actin cytoskeleton.After nuclear migration, but before anaphase, bulk chromosome movement occurs within the nucleus apparently because the chromosomes are attached to a mobile spindle. The frequency and magnitude of bulk chromosome movement is greatly diminished by disruption of the astral microtubules but not by disruption of the nonkinetochore spindle microtubules. These results suggest that astral microtubules are not only important for spindle orientation before anaphase, but they also mediate force on the spindle, generating spindle displacement and in turn chromosome movement. Potential roles for this force in spindle assembly and orientation are discussed. CELL division in the yeast, Saccharomyces cerevisiae, is an asymmetric process in which a bud emerges from a discrete region of the periphery of the parental, "mother" cell. Before anaphase the nucleus migrates from a location in the center of the mother cell to a position at the isthmus, or neck, between the mother cell and the growing bud (Pringle and Hartwell, 1981), and the nuclear envelope becomes extended between the mother cell and bud. Embedded in the nuclear envelope are the two separated spindle pole bodies (SPBs) ~ (Byers and Goetsch, 1974;Byers and Goetsch, 1975). Emanating from the lumenal face of each SPB into the nucleus are microtubules that form the bipolar spindle (Byers and Goetsch, 1974). Radiating away from the cytoplasmic face of each SPB towards the cell periphery are astral or cytoplasmic microtubules. Before the onset of anaphase, the bipolar spindle is always positioned with one SPB at, or through, the neck (Byers 1. Abbreviations used in this paper: DIM, digital imaging microscopy; HU, hydroxyurea; SPB, spindle pole body.and Goetsch, 1975;Pringle and Hartwell, 1981). At the onset of anaphase, the spin...

Coordination of spindle orientation with the axis of cell division is an essential process in all eukaryotes. In addition to ensuring accurate chromosomal segregation, proper spindle orientation also establishes differential cell fates and proper morphogenesis. In both animal and yeast cells, this process is dependent on cytoplasmic microtubules interacting with the cortical actin-based cytoskeleton, although the motive force was unknown. Here we show that yeast Myo2, a myosin V that translocates along polarized actin cables into the bud, orientates the spindle early in the cell cycle by binding and polarizing the microtubule-associated protein Kar9 (refs 7-9). The tail domain of Myo2 that binds Kar9 also interacts with secretory vesicles and vacuolar elements, making it a pivotal component of yeast cell polarization.

Stu2p is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast. Here, we report the first in vivo analysis of microtubule dynamics in cells lacking a member of this protein family. For these studies, we have used a conditional Stu2p depletion strain expressing alpha-tubulin fused to green fluorescent protein. Depletion of Stu2p leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells. The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing. These changes have significant consequences for the cell because they impede the ability of cytoplasmic microtubules to orient the spindle. In addition, recovery of fluorescence after photobleaching indicates that kinetochore microtubules are no longer dynamic in the absence of Stu2p. This deficiency is correlated with a failure to properly align chromosomes at metaphase. Overall, we provide evidence that Stu2p promotes the dynamics of microtubule plus-ends in vivo and that these dynamics are critical for microtubule interactions with kinetochores and cortical sites in the cytoplasm.

Previously we isolated tub2-423, a cold-sensitive allele of the Saccharomyces cerevisiae gene encoding β-tubulin that confers a defect in mitotic spindle function. In an attempt to identify additional proteins that are important for spindle function, we screened for suppressors of the cold sensitivity of tub2-423 and obtained two alleles of a novel gene, STU2. STU2 is an essential gene and encodes a protein whose sequence is similar to proteins identified in a variety of organisms. Stu2p localizes primarily to the spindle pole body (SPB) and to a lesser extent along spindle microtubules. Localization to the SPB is not dependent on the presence of microtubules, indicating that Stu2p is an integral component of the SPB. Stu2p also binds microtubules in vitro. We have localized the microtubule-binding domain of Stu2p to a highly basic 100-amino acid region. This region contains two imperfect repeats; both repeats appear to contribute to microtubule binding to similar extents. These results suggest that Stu2p may play a role in the attachment, organization, and/or dynamics of microtubule ends at the SPB.