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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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Cytoplasmic microtubule numbers in α-factor–arrested and released kar3-Δ mutants. Wild-type and mutant cells were arrested with α-factor, washed, and released in fresh medium. Samples were fixed and processed for anti-tubulin immunofluorescence for  0 (A) and 90 min (B–D) of release. α-Factor–arrested kar3 mutants had more and longer microtubules than arrested wild-type cells. For  the 90 min release samples, selected spindles are shown from the same sample which are short (B), medium (C), or longer in length (D).  Short spindles most likely represent cells that have not yet begun or are in the earliest stages of anaphase. kar3 mutant cells at this stage  have markedly abnormal microtubule arrays, similar to those seen in the hydroxyurea-arrested cultures (compare to Fig. 1). Medium  length spindles are typically from cells that have started but not completed anaphase, and in the kar3 mutants are closer in appearance  to those from wild-type cells. Cells with the longest spindles are presumed to be in late anaphase or early telophase. These spindles are  almost indistinguishable between wild-type cells and kar3 mutants. Bar, 2 μm.
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Figure 3: Cytoplasmic microtubule numbers in α-factor–arrested and released kar3-Δ mutants. Wild-type and mutant cells were arrested with α-factor, washed, and released in fresh medium. Samples were fixed and processed for anti-tubulin immunofluorescence for 0 (A) and 90 min (B–D) of release. α-Factor–arrested kar3 mutants had more and longer microtubules than arrested wild-type cells. For the 90 min release samples, selected spindles are shown from the same sample which are short (B), medium (C), or longer in length (D). Short spindles most likely represent cells that have not yet begun or are in the earliest stages of anaphase. kar3 mutant cells at this stage have markedly abnormal microtubule arrays, similar to those seen in the hydroxyurea-arrested cultures (compare to Fig. 1). Medium length spindles are typically from cells that have started but not completed anaphase, and in the kar3 mutants are closer in appearance to those from wild-type cells. Cells with the longest spindles are presumed to be in late anaphase or early telophase. These spindles are almost indistinguishable between wild-type cells and kar3 mutants. Bar, 2 μm.

Mentions: To determine if the abnormal microtubule array in kar3 mutants was specific for cells in mitosis, we next compared kar3-Δ and wild-type cells arrested at another point in the cell cycle. Cells exposed to mating pheromone reversibly arrest with a monopolar microtubule array at the START point of the cell cycle, in late G1 (Pringle and Hartwell, 1981). Cultures were treated with the pheromone α-factor for 3 h at 26°C and fixed and stained with anti-tubulin antibodies as above. As observed with hydroxyurea-arrested cells, α-factor–arrested kar3 mutants had microtubules that were more numerous and longer than those found in arrested wild-type cells (Fig. 3 A). These results indicate that loss of KAR3 leads to an increase in cytoplasmic microtubules at both START and S phase of the cell cycle.


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)

Cytoplasmic microtubule numbers in α-factor–arrested and released kar3-Δ mutants. Wild-type and mutant cells were arrested with α-factor, washed, and released in fresh medium. Samples were fixed and processed for anti-tubulin immunofluorescence for  0 (A) and 90 min (B–D) of release. α-Factor–arrested kar3 mutants had more and longer microtubules than arrested wild-type cells. For  the 90 min release samples, selected spindles are shown from the same sample which are short (B), medium (C), or longer in length (D).  Short spindles most likely represent cells that have not yet begun or are in the earliest stages of anaphase. kar3 mutant cells at this stage  have markedly abnormal microtubule arrays, similar to those seen in the hydroxyurea-arrested cultures (compare to Fig. 1). Medium  length spindles are typically from cells that have started but not completed anaphase, and in the kar3 mutants are closer in appearance  to those from wild-type cells. Cells with the longest spindles are presumed to be in late anaphase or early telophase. These spindles are  almost indistinguishable between wild-type cells and kar3 mutants. Bar, 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139775&req=5

Figure 3: Cytoplasmic microtubule numbers in α-factor–arrested and released kar3-Δ mutants. Wild-type and mutant cells were arrested with α-factor, washed, and released in fresh medium. Samples were fixed and processed for anti-tubulin immunofluorescence for 0 (A) and 90 min (B–D) of release. α-Factor–arrested kar3 mutants had more and longer microtubules than arrested wild-type cells. For the 90 min release samples, selected spindles are shown from the same sample which are short (B), medium (C), or longer in length (D). Short spindles most likely represent cells that have not yet begun or are in the earliest stages of anaphase. kar3 mutant cells at this stage have markedly abnormal microtubule arrays, similar to those seen in the hydroxyurea-arrested cultures (compare to Fig. 1). Medium length spindles are typically from cells that have started but not completed anaphase, and in the kar3 mutants are closer in appearance to those from wild-type cells. Cells with the longest spindles are presumed to be in late anaphase or early telophase. These spindles are almost indistinguishable between wild-type cells and kar3 mutants. Bar, 2 μm.
Mentions: To determine if the abnormal microtubule array in kar3 mutants was specific for cells in mitosis, we next compared kar3-Δ and wild-type cells arrested at another point in the cell cycle. Cells exposed to mating pheromone reversibly arrest with a monopolar microtubule array at the START point of the cell cycle, in late G1 (Pringle and Hartwell, 1981). Cultures were treated with the pheromone α-factor for 3 h at 26°C and fixed and stained with anti-tubulin antibodies as above. As observed with hydroxyurea-arrested cells, α-factor–arrested kar3 mutants had microtubules that were more numerous and longer than those found in arrested wild-type cells (Fig. 3 A). These results indicate that loss of KAR3 leads to an increase in cytoplasmic microtubules at both START and S phase of the cell cycle.

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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