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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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Temperature sensitivity of strains containing the kar3-Δ  allele was mostly corrected by addition of benomyl to the medium or deletion of TUB3. Cells of the indicated genotypes were  suspended in water and serial dilutions placed on YPD plates  with or without 5 μg/ml benomyl, at 30° or 37°C, for 2–3 d. Growth  of kar3-Δ cells was observed to be partially inhibited at 37°C. This  temperature sensitivity was eliminated by addition of benomyl to  the medium or by deletion of TUB3. The benomyl sensitivity  caused by the tub3-Δ allele was not rescued by kar3-Δ (not  shown). The kar3-Δ, tub3-Δ, and tub3-Δ kar3-Δ double mutants  are all from the same tetrad. (Growth of kar3-Δ mutants at 37°C  did not cause a noticeable cell cycle arrest phenotype, results not  shown.) In contrast, deletion of CIN8, which also causes slight  temperature sensitivity, could not be rescued by benomyl.
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Figure 5: Temperature sensitivity of strains containing the kar3-Δ allele was mostly corrected by addition of benomyl to the medium or deletion of TUB3. Cells of the indicated genotypes were suspended in water and serial dilutions placed on YPD plates with or without 5 μg/ml benomyl, at 30° or 37°C, for 2–3 d. Growth of kar3-Δ cells was observed to be partially inhibited at 37°C. This temperature sensitivity was eliminated by addition of benomyl to the medium or by deletion of TUB3. The benomyl sensitivity caused by the tub3-Δ allele was not rescued by kar3-Δ (not shown). The kar3-Δ, tub3-Δ, and tub3-Δ kar3-Δ double mutants are all from the same tetrad. (Growth of kar3-Δ mutants at 37°C did not cause a noticeable cell cycle arrest phenotype, results not shown.) In contrast, deletion of CIN8, which also causes slight temperature sensitivity, could not be rescued by benomyl.

Mentions: The kar3-Δ phenotypes tested were the abnormal spindle morphology introduced above, the kar3-Δ–induced mitotic delay described previously (Meluh and Rose, 1990), and an observed temperature sensitivity caused by KAR3 deletion. Benomyl at 10 μg/ml or deletion of TUB3 mostly corrected the observed microtubule defect of kar3-Δ mutants (Figs. 1 and 2; benomyl at around 12–15 μg/ml is lethal). KAR3 deletion was found to cause a slight temperature sensitivity that was completely suppressed by benomyl at 5 μg/ml (Fig. 5). In contrast, cells with a deletion of CIN8, which also causes temperature sensitivity (Hoyt et al., 1992), were not rescued by benomyl (Fig. 5; even at higher concentrations not shown). To test the effect of tub3-Δ on temperature sensitivity, we crossed kar3-Δ and tub3-Δ strains. The kar3-Δ tub3-Δ double mutants were less temperature sensitive than the kar3-Δ parent (Fig. 5), but there was much variability in temperature sensitivity among the progeny. The cause of this variation is unknown, but both the tub3 and kar3 mutations can cause high inviability among meiotic spores (Schatz et al., 1986; and Saunders, W., unpublished observations), a phenotype often caused by aneuploidy, which may also contribute to the observed variation in temperature sensitivity. To confirm the genetic suppression by tub3-Δ, eight kar3-Δ tub3-Δ double mutants were transformed with the TUB3 gene (a gift of D. Botstein, Stanford University, Palo Alto, CA). In each example, addition of the wild-type TUB3 gene significantly increased the temperature sensitivity of the kar3 tub3 double mutants. It is notable that addition of the wild-type allele made the double mutants more temperature sensitive and strongly suggests that loss of TUB3 improved the growth rates of kar3 mutants. The kar3-Δ allele did not change the benomyl sensitivity of cells containing the tub3-Δ allele (results not shown).


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)

Temperature sensitivity of strains containing the kar3-Δ  allele was mostly corrected by addition of benomyl to the medium or deletion of TUB3. Cells of the indicated genotypes were  suspended in water and serial dilutions placed on YPD plates  with or without 5 μg/ml benomyl, at 30° or 37°C, for 2–3 d. Growth  of kar3-Δ cells was observed to be partially inhibited at 37°C. This  temperature sensitivity was eliminated by addition of benomyl to  the medium or by deletion of TUB3. The benomyl sensitivity  caused by the tub3-Δ allele was not rescued by kar3-Δ (not  shown). The kar3-Δ, tub3-Δ, and tub3-Δ kar3-Δ double mutants  are all from the same tetrad. (Growth of kar3-Δ mutants at 37°C  did not cause a noticeable cell cycle arrest phenotype, results not  shown.) In contrast, deletion of CIN8, which also causes slight  temperature sensitivity, could not be rescued by benomyl.
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Figure 5: Temperature sensitivity of strains containing the kar3-Δ allele was mostly corrected by addition of benomyl to the medium or deletion of TUB3. Cells of the indicated genotypes were suspended in water and serial dilutions placed on YPD plates with or without 5 μg/ml benomyl, at 30° or 37°C, for 2–3 d. Growth of kar3-Δ cells was observed to be partially inhibited at 37°C. This temperature sensitivity was eliminated by addition of benomyl to the medium or by deletion of TUB3. The benomyl sensitivity caused by the tub3-Δ allele was not rescued by kar3-Δ (not shown). The kar3-Δ, tub3-Δ, and tub3-Δ kar3-Δ double mutants are all from the same tetrad. (Growth of kar3-Δ mutants at 37°C did not cause a noticeable cell cycle arrest phenotype, results not shown.) In contrast, deletion of CIN8, which also causes slight temperature sensitivity, could not be rescued by benomyl.
Mentions: The kar3-Δ phenotypes tested were the abnormal spindle morphology introduced above, the kar3-Δ–induced mitotic delay described previously (Meluh and Rose, 1990), and an observed temperature sensitivity caused by KAR3 deletion. Benomyl at 10 μg/ml or deletion of TUB3 mostly corrected the observed microtubule defect of kar3-Δ mutants (Figs. 1 and 2; benomyl at around 12–15 μg/ml is lethal). KAR3 deletion was found to cause a slight temperature sensitivity that was completely suppressed by benomyl at 5 μg/ml (Fig. 5). In contrast, cells with a deletion of CIN8, which also causes temperature sensitivity (Hoyt et al., 1992), were not rescued by benomyl (Fig. 5; even at higher concentrations not shown). To test the effect of tub3-Δ on temperature sensitivity, we crossed kar3-Δ and tub3-Δ strains. The kar3-Δ tub3-Δ double mutants were less temperature sensitive than the kar3-Δ parent (Fig. 5), but there was much variability in temperature sensitivity among the progeny. The cause of this variation is unknown, but both the tub3 and kar3 mutations can cause high inviability among meiotic spores (Schatz et al., 1986; and Saunders, W., unpublished observations), a phenotype often caused by aneuploidy, which may also contribute to the observed variation in temperature sensitivity. To confirm the genetic suppression by tub3-Δ, eight kar3-Δ tub3-Δ double mutants were transformed with the TUB3 gene (a gift of D. Botstein, Stanford University, Palo Alto, CA). In each example, addition of the wild-type TUB3 gene significantly increased the temperature sensitivity of the kar3 tub3 double mutants. It is notable that addition of the wild-type allele made the double mutants more temperature sensitive and strongly suggests that loss of TUB3 improved the growth rates of kar3 mutants. The kar3-Δ allele did not change the benomyl sensitivity of cells containing the tub3-Δ allele (results not shown).

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.

Show MeSH
Related in: MedlinePlus