Diverse effects of beta-tubulin mutations on microtubule formation and function.

Abstract

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 ...

Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae.

“…Perhaps the preferential segregation of the nucleus to the bud in espl cells reflects the behavior of a mechanism for nuclear motility, such as that demonstrated by Palmer et al (1989), but operating aberrantly in espl cells because of the absence of a rigid spindle. Alternatively, the segregation of the nucleus to the daughter cell might result from a defect in the behavior of cytoplasmic microtubules, which have been shown to control positioning of the nucleus in the bud neck (Huffaker et al, 1988). In contrast to the way division proceeds in budding yeast, nuclear division and cytokinesis of the fission yeast S. pombe take place in a symmetric manner.…”

Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly.

“…In arrested cells, virtually all nuclei appeared to be located adjacent to or in the neck dividing the mother and the bud cell bodies. Nuclear migration to the neck, a microtubule-dependent motility event (Huffaker et al, 1988;Jacobs et al, 1988), was therefore not noticeably affected by the cin8 and kip1 mutations.…”

β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

“…The budding yeast S. cerevisiae contains few microtubules compared with most other eukaryotic cells (Huffaker et al, 1988), allowing one to dissect the contribution of microtubules and their dynamic properties to specific cellular events. The cytoplasmic microtubules are required for positioning the nucleus to the neck before anaphase onset, and contribute to spindle elongation in anaphase.…”