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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 increased cytoplasmic microtubule number in hydroxyurea-arrested  kar3-Δ cells can be corrected  by addition of benomyl or  deletion of TUB3. All samples were arrested with hydroxyurea (except as indicated) for 4 h at 26°C, fixed,  and stained with anti-tubulin  antibodies by indirect immunofluorescence (see Materials and Methods). Cells were  examined by epifluorescence microscopy, and the  approximate number of cytoplasmic microtubules was determined. Cytoplasmic microtubules were defined as  those that were too long to be  nuclear and/or that clearly  pointed away from the nuclear envelope. Loss of  Kar3p resulted in an increase  in cytoplasmic microtubules,  especially in the hydroxyurea-arrested culture. This  defect could be mostly corrected by addition of 10 μg/ ml benomyl to the culture  medium or by deletion of the  TUB3 genomic locus. tub3-Δ  single mutants had fewer cytoplasmic microtubules than  wild-type cells, while the  tub3-Δ kar3-Δ double mutants had a number intermediate between that of tub3-Δ and kar3-Δ single mutants. The number of  cytoplasmic microtubules from 100 cells was determined for each  sample. Note that these numbers may represent an underestimate of the total, as some short microtubules (∼25% of the total) could not be determined to be nuclear or cytoplasmic. Also,  the most abnormal spindles in the kar3-Δ populations were probably not recognized as spindles and not included in the sample.
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Figure 2: The increased cytoplasmic microtubule number in hydroxyurea-arrested kar3-Δ cells can be corrected by addition of benomyl or deletion of TUB3. All samples were arrested with hydroxyurea (except as indicated) for 4 h at 26°C, fixed, and stained with anti-tubulin antibodies by indirect immunofluorescence (see Materials and Methods). Cells were examined by epifluorescence microscopy, and the approximate number of cytoplasmic microtubules was determined. Cytoplasmic microtubules were defined as those that were too long to be nuclear and/or that clearly pointed away from the nuclear envelope. Loss of Kar3p resulted in an increase in cytoplasmic microtubules, especially in the hydroxyurea-arrested culture. This defect could be mostly corrected by addition of 10 μg/ ml benomyl to the culture medium or by deletion of the TUB3 genomic locus. tub3-Δ single mutants had fewer cytoplasmic microtubules than wild-type cells, while the tub3-Δ kar3-Δ double mutants had a number intermediate between that of tub3-Δ and kar3-Δ single mutants. The number of cytoplasmic microtubules from 100 cells was determined for each sample. Note that these numbers may represent an underestimate of the total, as some short microtubules (∼25% of the total) could not be determined to be nuclear or cytoplasmic. Also, the most abnormal spindles in the kar3-Δ populations were probably not recognized as spindles and not included in the sample.

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)

The increased cytoplasmic microtubule number in hydroxyurea-arrested  kar3-Δ cells can be corrected  by addition of benomyl or  deletion of TUB3. All samples were arrested with hydroxyurea (except as indicated) for 4 h at 26°C, fixed,  and stained with anti-tubulin  antibodies by indirect immunofluorescence (see Materials and Methods). Cells were  examined by epifluorescence microscopy, and the  approximate number of cytoplasmic microtubules was determined. Cytoplasmic microtubules were defined as  those that were too long to be  nuclear and/or that clearly  pointed away from the nuclear envelope. Loss of  Kar3p resulted in an increase  in cytoplasmic microtubules,  especially in the hydroxyurea-arrested culture. This  defect could be mostly corrected by addition of 10 μg/ ml benomyl to the culture  medium or by deletion of the  TUB3 genomic locus. tub3-Δ  single mutants had fewer cytoplasmic microtubules than  wild-type cells, while the  tub3-Δ kar3-Δ double mutants had a number intermediate between that of tub3-Δ and kar3-Δ single mutants. The number of  cytoplasmic microtubules from 100 cells was determined for each  sample. Note that these numbers may represent an underestimate of the total, as some short microtubules (∼25% of the total) could not be determined to be nuclear or cytoplasmic. Also,  the most abnormal spindles in the kar3-Δ populations were probably not recognized as spindles and not included in the sample.
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Related In: Results  -  Collection

Show All Figures
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Figure 2: The increased cytoplasmic microtubule number in hydroxyurea-arrested kar3-Δ cells can be corrected by addition of benomyl or deletion of TUB3. All samples were arrested with hydroxyurea (except as indicated) for 4 h at 26°C, fixed, and stained with anti-tubulin antibodies by indirect immunofluorescence (see Materials and Methods). Cells were examined by epifluorescence microscopy, and the approximate number of cytoplasmic microtubules was determined. Cytoplasmic microtubules were defined as those that were too long to be nuclear and/or that clearly pointed away from the nuclear envelope. Loss of Kar3p resulted in an increase in cytoplasmic microtubules, especially in the hydroxyurea-arrested culture. This defect could be mostly corrected by addition of 10 μg/ ml benomyl to the culture medium or by deletion of the TUB3 genomic locus. tub3-Δ single mutants had fewer cytoplasmic microtubules than wild-type cells, while the tub3-Δ kar3-Δ double mutants had a number intermediate between that of tub3-Δ and kar3-Δ single mutants. The number of cytoplasmic microtubules from 100 cells was determined for each sample. Note that these numbers may represent an underestimate of the total, as some short microtubules (∼25% of the total) could not be determined to be nuclear or cytoplasmic. Also, the most abnormal spindles in the kar3-Δ populations were probably not recognized as spindles and not included in the sample.
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.

Show MeSH