Alanine-scanning Mutagenesis ofAspergillusγ-Tubulin Yields Diverse and Novel Phenotypes

Abstract

We have created 41 clustered charged-to-alanine scanning mutations of the mipA, ␥-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of ␥-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that ␥-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of ␥-tubulin and these data give a good initial indication of the functionally important regions of the molecule.

TheDrosophilaγ-Tubulin Small Complex Subunit Dgrip84 Is Required for Structural and Functional Integrity of the Spindle Apparatus

“…Long microtubules could also result from an increase of the concentration of free tubulin in consequence of low microtubule assembly. Similar phenotypes displaying long microtubules have been observed after mutations in the ␥-tubulin encoding gene both in budding yeast and A. nidulans and in ␥-TuSC components in S. pombe (Marschall et al, 1996;Spang et al, 1996;Vardy and Toda, 2000;Jung et al, 2001). In S2 cells, this effect could be hidden because of the density and the complexity of microtubule arrays.…”

γ-Tubulin Is Essential for Microtubule Organization and Development inArabidopsis

“…The abnormally large nuclei observed in female tubg1-1 tubg2-1 gametophytes (Figure 8) could correspond to such polyploid nuclei that went through multiple cycles of replication without division. Inhibition of nuclear division and increased ploidy have already been reported for g-tubulin mutants in A. nidulans (Oakley et al, 1990;Martin et al, 1997;Jung et al, 2001), S. pombe (Paluh et al, 2000), and Drosophila (Sunkel et al, 1995) as well as in S. pombe cells expressing mutant forms of human g-tubulin (Hendrickson et al, 2001).…”

“…This finding is in agreement with a role of g-tubulin in cortical microtubule nucleation on extant microtubules, as evidenced by Murata et al (2005), but do not exclude additional effects on polymerization, organization, or dynamics of microtubules. Indeed, in S. pombe and A. nidulans, g-tubulin is involved in the spatial arrangement of cytoplasmic microtubules in addition to its documented role in nucleation (Paluh et al, 2000;Jung et al, 2001). Nucleation, dynamics, and spatial organization of microtubules are tightly coupled processes in plant cells and seem all affected in tubg1-1 tubg2-2 mutants.…”

“…Recent genetic and molecular studies in fission and budding yeast, in Aspergillus nidulans, and in Drosophila melanogaster have revealed that g-tubulin could be involved in microtubule dynamics or organization (Paluh et al, 2000;Jung et al, 2001;Vogel et al, 2001) and in the control of mitotic checkpoint and coordination of late mitotic events (Hendrickson et al, 2001;Sampaio et al, 2001;Prigozhina et al, 2004). Evidence for such roles is needed for plant g-tubulin.…”

Function of γ-Tubulin in Plants

“…The mutant allele was originally isolated using the innovative approach of charged-to-Ala scanning mutagenesis (Jung et al, 2001), based on the principle that charged regions tend to be on the outside of proteins and that replacement with Ala can alter regions important for protein interactions without causing gross structural changes. Hendrickson et al (2001) used a similar approach to analyze g-tubulin function in Saccharomyces pombe, pointing to roles in the coordination of postmetaphase events, anaphase, and cytokinesis.…”