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The Saccharomyces cerevisiae kinesin-related motor Kar3p acts at preanaphase spindle poles to limit the number and length of cytoplasmic microtubules.

Saunders W, Hornack D, Lengyel V, Deng C - J. Cell Biol. (1997)

Bottom Line: We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner.Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization.These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA. wsaund@vms.cis.pitt.edu

ABSTRACT
The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

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The short spindle phenotype from cin8 kip1 mutations  is sensitive to tubulin mutations. Cells with the indicated genotypes were arrested with hydroxyurea at 26°C (a permissive temperature for growth of these strains), fixed, stained with antitubulin antibody, and the length of the spindles measured (see  Materials and Methods). Mutational inhibition of the Cin8p and  Kip1p motors produced a decrease in spindle length. This defect  could be corrected by the tub2-150 mutation and was worsened  by the tub3-Δ mutation. The numbers given represent the combined averages for two separate strains containing the indicated  allele(s). Complete data for the individual strains are given in Table I. Each value represents at least 200 spindles counted during  two to four separate experiments.
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Figure 6: The short spindle phenotype from cin8 kip1 mutations is sensitive to tubulin mutations. Cells with the indicated genotypes were arrested with hydroxyurea at 26°C (a permissive temperature for growth of these strains), fixed, stained with antitubulin antibody, and the length of the spindles measured (see Materials and Methods). Mutational inhibition of the Cin8p and Kip1p motors produced a decrease in spindle length. This defect could be corrected by the tub2-150 mutation and was worsened by the tub3-Δ mutation. The numbers given represent the combined averages for two separate strains containing the indicated allele(s). Complete data for the individual strains are given in Table I. Each value represents at least 200 spindles counted during two to four separate experiments.

Mentions: If Kar3p acts antagonistically to Cin8p and Kip1p and Kar3p function is associated with microtubule destabilization, then we should be able to show directly that microtubule depolymerization acts antagonistically to Cin8p and Kip1p. To investigate the effect of changing tubulin polymerization on cin8 kip1 mutants, two related mutant phenotypes were considered. We have observed previously that cin8 kip1 mutants had spindles slightly shorter than wild-type cells (Saunders and Hoyt, 1997). We were able to use the short spindle cin8 kip1 phenotype to investigate the relationship between loss of Cin8p/Kip1p activity and tubulin depolymerization. Two different tubulin mutations were used, the previously described TUB3 deletion and a mutation in the sole β-tubulin gene of S. cerevisiae, tub2-150 (Neff et al., 1983). The tub2-150 mutation acts in an opposite manner as TUB3 deletion, raising the resistance of mutant cells to microtubule depolymerizing agents, most likely by mutationally inhibiting depolymerization. The tub2-150 allele corrected the short spindle defect in cin8 kip1 cells while deletion of TUB3 made the phenotype worse (Fig. 6). These results suggest that spindle length is dependent on rates of microtubule polymerization, and at least this one defect in cin8 kip1 mutants could be corrected by stabilizing microtubules with the tub2-150 mutation.


The Saccharomyces cerevisiae kinesin-related motor Kar3p acts at preanaphase spindle poles to limit the number and length of cytoplasmic microtubules.

Saunders W, Hornack D, Lengyel V, Deng C - J. Cell Biol. (1997)

The short spindle phenotype from cin8 kip1 mutations  is sensitive to tubulin mutations. Cells with the indicated genotypes were arrested with hydroxyurea at 26°C (a permissive temperature for growth of these strains), fixed, stained with antitubulin antibody, and the length of the spindles measured (see  Materials and Methods). Mutational inhibition of the Cin8p and  Kip1p motors produced a decrease in spindle length. This defect  could be corrected by the tub2-150 mutation and was worsened  by the tub3-Δ mutation. The numbers given represent the combined averages for two separate strains containing the indicated  allele(s). Complete data for the individual strains are given in Table I. Each value represents at least 200 spindles counted during  two to four separate experiments.
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Related In: Results  -  Collection

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Figure 6: The short spindle phenotype from cin8 kip1 mutations is sensitive to tubulin mutations. Cells with the indicated genotypes were arrested with hydroxyurea at 26°C (a permissive temperature for growth of these strains), fixed, stained with antitubulin antibody, and the length of the spindles measured (see Materials and Methods). Mutational inhibition of the Cin8p and Kip1p motors produced a decrease in spindle length. This defect could be corrected by the tub2-150 mutation and was worsened by the tub3-Δ mutation. The numbers given represent the combined averages for two separate strains containing the indicated allele(s). Complete data for the individual strains are given in Table I. Each value represents at least 200 spindles counted during two to four separate experiments.
Mentions: If Kar3p acts antagonistically to Cin8p and Kip1p and Kar3p function is associated with microtubule destabilization, then we should be able to show directly that microtubule depolymerization acts antagonistically to Cin8p and Kip1p. To investigate the effect of changing tubulin polymerization on cin8 kip1 mutants, two related mutant phenotypes were considered. We have observed previously that cin8 kip1 mutants had spindles slightly shorter than wild-type cells (Saunders and Hoyt, 1997). We were able to use the short spindle cin8 kip1 phenotype to investigate the relationship between loss of Cin8p/Kip1p activity and tubulin depolymerization. Two different tubulin mutations were used, the previously described TUB3 deletion and a mutation in the sole β-tubulin gene of S. cerevisiae, tub2-150 (Neff et al., 1983). The tub2-150 mutation acts in an opposite manner as TUB3 deletion, raising the resistance of mutant cells to microtubule depolymerizing agents, most likely by mutationally inhibiting depolymerization. The tub2-150 allele corrected the short spindle defect in cin8 kip1 cells while deletion of TUB3 made the phenotype worse (Fig. 6). These results suggest that spindle length is dependent on rates of microtubule polymerization, and at least this one defect in cin8 kip1 mutants could be corrected by stabilizing microtubules with the tub2-150 mutation.

Bottom Line: We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner.Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization.These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA. wsaund@vms.cis.pitt.edu

ABSTRACT
The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle-specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential alpha-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.

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