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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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Measurements of cytoplasmic microtubule numbers  and lengths in α-factor–arrested and released cells. Wild-type and  kar3 mutants were arrested and released as in Fig. 3. The numbers  and lengths of the cytoplasmic microtubules were counted as in  Fig. 2. For this analysis, short spindles were defined as those  where no chromatin separation was visible (typically 2.5 μm or  less). Medium length spindles were those with spindles between  ∼2.5 and 7.0 μm in length and at least some chromatin separation  visible. Longer spindles were those greater than ∼7.0 μm in  length with well separated chromatin masses. 100 spindles were  examined for each category. As shown, the number and length  of cytoplasmic microtubules in wild-type cells (black) increased  slightly with spindle length. kar3 mutants (gray) with short spindles had many more and longer cytoplasmic microtubules than  wild-type cells. Late anaphase spindles had similar numbers of  cytoplasmic microtubules in wild-type and kar3 mutants.
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Figure 4: Measurements of cytoplasmic microtubule numbers and lengths in α-factor–arrested and released cells. Wild-type and kar3 mutants were arrested and released as in Fig. 3. The numbers and lengths of the cytoplasmic microtubules were counted as in Fig. 2. For this analysis, short spindles were defined as those where no chromatin separation was visible (typically 2.5 μm or less). Medium length spindles were those with spindles between ∼2.5 and 7.0 μm in length and at least some chromatin separation visible. Longer spindles were those greater than ∼7.0 μm in length with well separated chromatin masses. 100 spindles were examined for each category. As shown, the number and length of cytoplasmic microtubules in wild-type cells (black) increased slightly with spindle length. kar3 mutants (gray) with short spindles had many more and longer cytoplasmic microtubules than wild-type cells. Late anaphase spindles had similar numbers of cytoplasmic microtubules in wild-type and kar3 mutants.

Mentions: Considering only microtubules that were either too long to be entirely nuclear or that pointed away from the nucleus, the cytoplasmic microtubules in α-factor–arrested and released kar3-Δ mutants were measured and counted (Fig. 4). As observed for hydroxyurea-arrested cells, both the length and number of cytoplasmic microtubules associated with short spindles were higher in α-factor–arrested and released kar3-Δ mutants than in wild-type cells treated in the same manner. As cells entered anaphase, the cytoplasmic microtubules were seen to increase in wild-type cells but to decrease in kar3 mutants. By late anaphase the numbers were very similar.


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)

Measurements of cytoplasmic microtubule numbers  and lengths in α-factor–arrested and released cells. Wild-type and  kar3 mutants were arrested and released as in Fig. 3. The numbers  and lengths of the cytoplasmic microtubules were counted as in  Fig. 2. For this analysis, short spindles were defined as those  where no chromatin separation was visible (typically 2.5 μm or  less). Medium length spindles were those with spindles between  ∼2.5 and 7.0 μm in length and at least some chromatin separation  visible. Longer spindles were those greater than ∼7.0 μm in  length with well separated chromatin masses. 100 spindles were  examined for each category. As shown, the number and length  of cytoplasmic microtubules in wild-type cells (black) increased  slightly with spindle length. kar3 mutants (gray) with short spindles had many more and longer cytoplasmic microtubules than  wild-type cells. Late anaphase spindles had similar numbers of  cytoplasmic microtubules in wild-type and kar3 mutants.
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Related In: Results  -  Collection

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Figure 4: Measurements of cytoplasmic microtubule numbers and lengths in α-factor–arrested and released cells. Wild-type and kar3 mutants were arrested and released as in Fig. 3. The numbers and lengths of the cytoplasmic microtubules were counted as in Fig. 2. For this analysis, short spindles were defined as those where no chromatin separation was visible (typically 2.5 μm or less). Medium length spindles were those with spindles between ∼2.5 and 7.0 μm in length and at least some chromatin separation visible. Longer spindles were those greater than ∼7.0 μm in length with well separated chromatin masses. 100 spindles were examined for each category. As shown, the number and length of cytoplasmic microtubules in wild-type cells (black) increased slightly with spindle length. kar3 mutants (gray) with short spindles had many more and longer cytoplasmic microtubules than wild-type cells. Late anaphase spindles had similar numbers of cytoplasmic microtubules in wild-type and kar3 mutants.
Mentions: Considering only microtubules that were either too long to be entirely nuclear or that pointed away from the nucleus, the cytoplasmic microtubules in α-factor–arrested and released kar3-Δ mutants were measured and counted (Fig. 4). As observed for hydroxyurea-arrested cells, both the length and number of cytoplasmic microtubules associated with short spindles were higher in α-factor–arrested and released kar3-Δ mutants than in wild-type cells treated in the same manner. As cells entered anaphase, the cytoplasmic microtubules were seen to increase in wild-type cells but to decrease in kar3 mutants. By late anaphase the numbers were very similar.

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