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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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Change in spindle structure with loss of Kar3p. kar3-Δ  or wild-type (WT) cells were arrested with hydroxyurea (hu) or  grown to log phase without arrest, and samples were fixed and processed for anti-tubulin immunofluorescence. Each panel is a composite image of representative preanaphase spindles from a single  sample. As shown, loss of KAR3 led to an increase in cytoplasmic  microtubules in unarrested cultures (kar3-Δ). This phenotype was  greatly exaggerated after mitotic arrest with hydroxyurea (kar3-Δ  + hu). Short spindles in wild-type cells appeared essentially the  same with or without hydroxyurea treatment (shown with hydroxyurea treatment). Addition of 10 μg/ml benomyl during the  hydroxyurea arrest prevented the abnormal spindle structure in  kar3 mutants. Bar, 2 μm.
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Figure 1: Change in spindle structure with loss of Kar3p. kar3-Δ or wild-type (WT) cells were arrested with hydroxyurea (hu) or grown to log phase without arrest, and samples were fixed and processed for anti-tubulin immunofluorescence. Each panel is a composite image of representative preanaphase spindles from a single sample. As shown, loss of KAR3 led to an increase in cytoplasmic microtubules in unarrested cultures (kar3-Δ). This phenotype was greatly exaggerated after mitotic arrest with hydroxyurea (kar3-Δ + hu). Short spindles in wild-type cells appeared essentially the same with or without hydroxyurea treatment (shown with hydroxyurea treatment). Addition of 10 μg/ml benomyl during the hydroxyurea arrest prevented the abnormal spindle structure in kar3 mutants. Bar, 2 μm.

Mentions: To investigate the consequence of loss of KAR3 on mitotic spindle microtubules we carefully compared spindle structure between cells with KAR3-deleted (kar3-Δ) and wildtype cells. (The kar3-Δ strain was a gift of Meluh, P., and M. Rose, Princeton University, Princeton, NJ). Cultures were grown to log phase in YPD medium, fixed, and stained with anti-tubulin antibodies (see Materials and Methods). Spindles in kar3-Δ cells had more microtubules that were not associated with the nuclear spindle and appeared to be cytoplasmic (Fig. 1; kar3-Δ) than in wild-type cells (not shown; see control below). The abnormal microtubule numbers in the kar3 mutants could result from the reported mitotic cell cycle delay (Meluh and Rose, 1990), which may induce an accumulation of extra microtubules. To allow comparison with a mitotically arrested control, we examined kar3-Δ and wild-type cultures treated with the DNA synthesis inhibitor hydroxyurea (Pringle and Hartwell, 1981). Under these conditions most cells arrest in S phase with an assembled spindle but are unable to begin anaphase. Mitotic arrest of the kar3-Δ cells resulted in a marked exaggeration of the abnormal microtubule numbers seen in the asynchronous culture (Fig. 1; kar3-Δ + hu). Strikingly, large microtubule arrays could now be seen radiating from the spindle poles. The majority of cells under these conditions had separated spindle poles as determined by anti–spindle pole body staining (results not shown). Hydroxyurea arrest of the wild-type cells caused little if any change in the numbers of cytoplasmic microtubules (Fig. 1; WT + hu). Many of the extra microtubules seen in the kar3-Δ mutants were clearly cytoplasmic, because they were either too long to be nuclear or pointed away from the nuclear envelope. Others could be either nuclear or cytoplasmic. We carefully counted the numbers of microtubules that appeared to be cytoplasmic by the above criteria and found a marked increase in kar3-Δ cells compared to the wild-type control (Fig. 2; note that all cells are hydroxyurea arrested unless indicated otherwise). Cytoplasmic microtubule numbers in cin8 kip1 mutants were not counted, but they did not show the large microtubule arrays found in kar3-Δ cells following hydroxyurea arrest (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)

Change in spindle structure with loss of Kar3p. kar3-Δ  or wild-type (WT) cells were arrested with hydroxyurea (hu) or  grown to log phase without arrest, and samples were fixed and processed for anti-tubulin immunofluorescence. Each panel is a composite image of representative preanaphase spindles from a single  sample. As shown, loss of KAR3 led to an increase in cytoplasmic  microtubules in unarrested cultures (kar3-Δ). This phenotype was  greatly exaggerated after mitotic arrest with hydroxyurea (kar3-Δ  + hu). Short spindles in wild-type cells appeared essentially the  same with or without hydroxyurea treatment (shown with hydroxyurea treatment). Addition of 10 μg/ml benomyl during the  hydroxyurea arrest prevented the abnormal spindle structure in  kar3 mutants. Bar, 2 μm.
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Figure 1: Change in spindle structure with loss of Kar3p. kar3-Δ or wild-type (WT) cells were arrested with hydroxyurea (hu) or grown to log phase without arrest, and samples were fixed and processed for anti-tubulin immunofluorescence. Each panel is a composite image of representative preanaphase spindles from a single sample. As shown, loss of KAR3 led to an increase in cytoplasmic microtubules in unarrested cultures (kar3-Δ). This phenotype was greatly exaggerated after mitotic arrest with hydroxyurea (kar3-Δ + hu). Short spindles in wild-type cells appeared essentially the same with or without hydroxyurea treatment (shown with hydroxyurea treatment). Addition of 10 μg/ml benomyl during the hydroxyurea arrest prevented the abnormal spindle structure in kar3 mutants. Bar, 2 μm.
Mentions: To investigate the consequence of loss of KAR3 on mitotic spindle microtubules we carefully compared spindle structure between cells with KAR3-deleted (kar3-Δ) and wildtype cells. (The kar3-Δ strain was a gift of Meluh, P., and M. Rose, Princeton University, Princeton, NJ). Cultures were grown to log phase in YPD medium, fixed, and stained with anti-tubulin antibodies (see Materials and Methods). Spindles in kar3-Δ cells had more microtubules that were not associated with the nuclear spindle and appeared to be cytoplasmic (Fig. 1; kar3-Δ) than in wild-type cells (not shown; see control below). The abnormal microtubule numbers in the kar3 mutants could result from the reported mitotic cell cycle delay (Meluh and Rose, 1990), which may induce an accumulation of extra microtubules. To allow comparison with a mitotically arrested control, we examined kar3-Δ and wild-type cultures treated with the DNA synthesis inhibitor hydroxyurea (Pringle and Hartwell, 1981). Under these conditions most cells arrest in S phase with an assembled spindle but are unable to begin anaphase. Mitotic arrest of the kar3-Δ cells resulted in a marked exaggeration of the abnormal microtubule numbers seen in the asynchronous culture (Fig. 1; kar3-Δ + hu). Strikingly, large microtubule arrays could now be seen radiating from the spindle poles. The majority of cells under these conditions had separated spindle poles as determined by anti–spindle pole body staining (results not shown). Hydroxyurea arrest of the wild-type cells caused little if any change in the numbers of cytoplasmic microtubules (Fig. 1; WT + hu). Many of the extra microtubules seen in the kar3-Δ mutants were clearly cytoplasmic, because they were either too long to be nuclear or pointed away from the nuclear envelope. Others could be either nuclear or cytoplasmic. We carefully counted the numbers of microtubules that appeared to be cytoplasmic by the above criteria and found a marked increase in kar3-Δ cells compared to the wild-type control (Fig. 2; note that all cells are hydroxyurea arrested unless indicated otherwise). Cytoplasmic microtubule numbers in cin8 kip1 mutants were not counted, but they did not show the large microtubule arrays found in kar3-Δ cells following hydroxyurea arrest (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.

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